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... www.nature.com/scientificreports OPEN Received: 8 April 2019 Accepted: 27 July 2019 Published: xx xx xxxx Endothelial RhoA GTPase is essential for in vitro endothelial functions but dispensable for physiological in vivo angiogenesis FatemaTuz Zahra1, Md Sanaullah Sajib1, Yusuke Ichiyama2,3, Racheal Grace Akwii1, PaulE. Tullar4, Christopher Cobos1, Shelby A. Minchew1, Colleen L. Doi5, Yi Zheng6, Yoshiaki Kubota 2, J. Silvio Gutkind7 & Constantinos M. Mikelis1 Imbalanced angiogenesis is a characteristic of several diseases. Rho GTPases regulate multiple cellular processes, such as cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction and gene expression. Among the Rho GTPases, RhoA, Rac1 and Cdc42 are best characterized. The role of endothelial Rac1 and Cdc42 in embryonic development and retinal angiogenesis has been studied, however the role of endothelial RhoA is yet to be explored. Here, we aimed to identify the role of endothelial RhoA in endothelial cell functions, in embryonic and retinal development and explored compensatory mechanisms. In vitro, RhoA is involved in cell proliferation, migration and tube formation, triggered by the angiogenesis inducers Vascular Endothelial Growth Factor (VEGF) and Sphingosine-1 Phosphate (S1P). In vivo, through constitutive and inducible endothelial RhoA deficiency we tested the role of endothelial RhoA in embryonic development and retinal angiogenesis. Constitutive endothelial RhoA deficiency, although decreased survival, was not detrimental for embryonic development, while inducible endothelial RhoA deficiency presented only mild deficiencies in the retina. The redundant role of RhoA in vivo can be attributed to potential differences in the signaling cues regulating angiogenesis in physiological versus pathological conditions and to the alternative compensatory mechanisms that may be present in the in vivo setting. The Rho family of GTPases is part of the Ras superfamily, which comprises over 150 members in human, with evolutionarily conserved orthologs found in yeast and plants1,2. Rho GTPases are present in all eukaryotes, and play important role in the regulation of actin and microtubule cytoskeleton, cell migration and invasion, cell polarity, vesicle trafficking, regulation of gene expression and cell cycle progression1,3. The 20 known members of the Rho GTPase family are characterized as typical and atypical, based on whether their regulation depends on the interaction with the Rho-specific guanine nucleotide exchange factors (GEFs) and the GTPase-activating proteins (GAPs) for activation and inactivation respectively3. The best-known members of the typical GTPase family are Ras homolog gene family, member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), which are known regulators of the actin cytoskeleton and whose timely and spatially coordinated activation leads to cell migration and morphology3,4. Typically, RhoA regulates stress fiber formation and Rac1 and Cdc42 control lamellipodia and filopodia formation respectively5, whereas more recent findings highlight their role in less common cytoskeletal structures, such as podosome and invadopodia formation in cancer cells6. Rho GTPase activation is stimulated through activation of a variety of cell surface 1 Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, 79106, USA. 2Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. 3Department of Ophthalmology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga, 520-2192, Japan. 4Department of Obstetrics and Gynecology, School of Medicine, Texas Tech University Health Sciences Center, Amarillo, Texas, 79106, USA. 5College of Arts and Sciences, Marian University Indianapolis, Indianapolis, Indiana, 46222, USA. 6Cancer and Blood Diseases Institute, Cincinnati Childrens Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45229, USA. 7Department of Pharmacology, UCSD, San Diego, California, 92093, USA. Correspondence and requests for materials should be addressed to C.M.M. (email: constantinos.mikelis@ttuhsc.edu) Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 1 www.nature.com/scientificreports/ www.nature.com/scientificreports receptors, including G protein-coupled receptors (GPCRs) such as lysophosphatidic acid (LPA) and bombesin receptors, tyrosine kinase growth factor receptors such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors, as well as cell adhesion molecules such as integrins, cadherins and immunoglobulin superfamily members7. Angiogenesis is a well-coordinated process, important for the vascular development in all higher organisms, and its deregulation initiates or augments the development of many pathological conditions8. Small GTPases are actively involved in many steps of the angiogenic process, as the distinct role of RhoA, Rac1 and Cdc42 and others has been reported for endothelial cell migration, proliferation, basement membrane degradation, morphogenesis, capillary survival and barrier function9. Furthermore, they have all been demonstrated as downstream effectors of potent angiogenesis inducers, such as vascular endothelial growth factor (VEGF)10 and sphingosine-1-phosphate (S1P)11,12. Global deficiency of each Rho GTPase leads to embryonic lethality, indicative of their significance on embryonic development: Cdc42-knockout mice die prior to embryonic day 7.5 (E7.5)13, Rac1-knockout mice die prior to E9.5 due to germ-layer formation deficiencies14, whereas viable RhoA-knockout mice have not been reported15. The development of conditional deficient mice provides a better tool to evaluate the significance and function of the protein of interest on a specific tissue. Endothelial-specific deficiency of Cdc42 (through the Tie2-Cre promoter) led to embryonic lethality by E9-10 due to deficiencies in lumen formation and failed blood circulation. Inducible endothelial Cdc42 deficiency during mid-gestation (through the Cad5-CreERT2 promoter), also led to embryonic lethality, demonstrating the importance of Cdc42 during vessel formation and endothelial cell polarity during angiogenesis16. In the same mouse model, it was also shown that Cdc42 deletion affects retinal angiogenesis16,17. Rac1 endothelial-specific deficiency also led to embryonic lethality during midgestation (E9.5) due to defective development of major vessels and complete lack of small vessel branching18. Inducible homozygous endothelial deletion of Rac1 at E10 led to remarkable hemorrhage at E15.5 and to the reduction of the vascular area and number of branching points in the embryonic back skin, while inducible deficiency in the developing retina resulted in reduction of vascular area and branching points at P819. Despite the detailed knowledge on the role of Cdc42 and Rac1 on developmental angiogenesis, the precise role of RhoA has yet to be elucidated. We previously showed that combined global deficiency of two known RhoA GEFs, PDZ-RhoGEF and leukemia-associated Rho GEF (LARG) blocked RhoA activation downstream of the G12/13 GPCRs, leading to embryonic lethality during midgestation due to branching deficiencies of the cranial vessels and in the embryonic vascular network in the placenta20. The above prompted us to explore the biological role of endothelial RhoA during embryonic development and retinal angiogenesis. We engineered constitutive and inducible endothelial-specific RhoA deficiency through the Tie2-Cre and Cdh5-CreERT2 promoters respectively, studied the impact of endothelial RhoA deletion in embryonic survival and retinal angiogenesis, and compared the in vivo data with the in vitro outcome of RhoA deficiency in endothelial cell functions, under stimulation by potent angiogenesis inducers. Results Inhibition of endothelial RhoA expression affects angiogenesis in vitro. The participation of RhoA signaling pathway downstream of VEGF-induced angiogenesis has been previously reported10,21,22. To investigate the role of endothelial RhoA in angiogenesis induced by stimuli activating diverse signaling pathways, we selected VEGF and S1P as representative angiogenesis inducers through tyrosine kinase receptor23,24 and G protein-coupled receptor signaling respectively25. Both VEGF and S1P stimulation induced RhoA activation in primary endothelial cells (HUVECs) and this induction was blocked by C3 toxin (exoenzyme C3) treatment (Fig. 1A,B). To identify whether RhoA inhibition affects VEGF- and S1P-induced angiogenesis in vitro, RhoA expression was knocked down (Fig. 1C) with siRNAs. RhoA knockdown in the endothelial cells abrogated mitogenic activity in 24 h induced by both VEGF and S1P (Fig. 1D). VEGF-induced cell migration, measured through the Boyden chamber assays, was completely abrogated by RhoA knockdown (Fig. 1E), whereas S1P-induced cell migration was partially inhibited, demonstrating that although RhoA is involved in the downstream signaling cascade, S1P but not VEGF may also elicit cell migration by additional mechanisms (Fig. 1E). The diverse significance of RhoA in VEGF- versus S1P-induced angiogenesis was more obvious in the 2-D sprouting assay (Fig. 2A,B). RhoA deficiency led to abrogation of VEGF-induced tube formation, as assessed by the number of nodes, number of junctions and total sprout length, whereas it did not affect S1P-induced tube formation (Fig. 2A,B), demonstrating that the signaling circuits governing endothelial proliferation, migration and tube formation during angiogenesis are not identical. To better clarify the role of endothelial RhoA during tube formation, a 3-D sprouting assay was introduced, where endothelial cells form spheroids and the sprouting potential is identified in a more controlled (collagen type I and methocel) environment26,27. In this model, RhoA knockdown abolished VEGF-induced number of sprouts, average sprout length and total sprout length, although it did not block S1P-induced sprout formation (Fig. 2C,D). On the other hand, C3 toxin treatment completely abrogated both VEGF- and S1P-induced sprouting in the same model (Suppl. Fig. 1). Since C3 toxin has been shown to ADP-ribosylate RhoA and RhoB and to a lesser extent RhoC28, this opens up the possibility of a compensatory mechanism of the other members of the Rho family downstream of S1P-induced sprout formation, since S1P has also been shown to activate RhoB and RhoC apart from RhoA11. Overall, the above data demonstrate that RhoA affects several stages of endothelial cell behavior during the angiogenic process, however its significance is variable and depends on the signaling context. Endothelial RhoA deficiency is not detrimental for embryonic development. The above data prompted us to investigate the role of endothelial RhoA during physiological angiogenesis in vivo. To identify whether endothelial RhoA deficiency affects vasculogenesis and angiogenesis during development, we generated mice with sustained RhoA deficiency in the endothelial cells, under the control of the Tie2 promoter. RhoA was deleted in endothelial cells (ECs), by crossing a conditional allele of RhoA (RhoAf/f ) with the Tie2-Cre driver Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Involvement of endothelial RhoA in VEGF- and S1P-induced cell proliferation and migration. (A,B) Representative images (upper panel) and quantification (lower panel) of RhoA activation in HUVECs. Cells were stimulated with VEGF (100 ng/ml) (n = 5) (A) and S1P (500 nM) (n = 9) (B) in the presence or absence of C3 toxin (20 ng/ml) treatment. Full-length blots are presented in Supplementary Fig. 4. (C) Representative image of western blot analysis (upper panel) and quantification (lower panel) of RhoA expression after transfection with siRNA control (siNEG) and two sequences of siRNA for RhoA (siRhoA#1 and siRhoA#2) (50 nM) (n = 3). Full-length blots are presented in Supplementary Fig. 5. (D) Quantification of VEGF- (100 ng/ ml) and S1P-induced (500 nM) cell proliferation (n = 6) and cell migration (n = 6) (E) of HUVECs treated with the corresponding siRNAs. *P < 0.05; **P < 0.01; ***P < 0.001. line29, to obtain RhoAf/f Tie2-Cre+ mice (Fig. 3A). Tie2 is expressed in endothelial and hematopoietic cells emerging from the mesoderm30 and thus Tie2-promoter-driven activity is expected to occur as early as E7.531,32. When the RhoAf/+ Tie2-Cre+ mice were backcrossed with the RhoAf/f to obtain the final RhoAf/f Tie2-Cre+ line, we obtained viable pups at weaning, although in a smaller than expected ratio (Fig. 3B). Even though the difference in the number of obtained versus expected RhoAf/f Tie2-Cre+ pups was significant (Fig. 3C), the obtained RhoAf/f Tie2-Cre+ pups did not present morphological or behavioral abnormalities from their littermate controls (RhoAf/+ Tie2-Cre+). Since the efficiency of the Tie2 promoter has been previously demonstrated16,18,29 and lung endothelial cells isolated from 56 week old mice showed almost complete RhoA deficiency in the RhoAf/f Tie2-Cre+ mice (Fig. 3D), it is highly likely that the RhoA gene is efficiently deleted during embryogenesis. Embryos with endothelial RhoA deficiency do not present gross vascular abnormalities. The decreased number of RhoAf/f Tie2-Cre+ survivors, led us perform timed-matings to identify potential vascular deficiencies. Tie2-driven deficiency of key angiogenesis mediators is known to lead to vascular abnormalities after E10, therefore potential embryonic lethality should be visible at E12.5. To identify potential vascular deficiencies in embryos and yolk sacs, the following timed-matings were performed: RhoAf/+ Tie2Cre+ X RhoAf/f, the embryos were dissected at E12.5 and the RhoAf/f Tie2-Cre+ embryos were compared with the RhoAf/+ Tie2-Cre+ littermate controls (Fig. 4A; Suppl. Fig. 2). Macroscopically, the embryos did not present gross morphological or size differences (Fig. 4B), and further vascular analysis with CD31 staining did not reveal gross vascular abnormalities in cranial vessel branching (Fig. 4C), in vessel branching in the trunk (Fig. 4D), or the limbs (Fig. 4E). Yolk sac analysis did not show gross vascular deficiencies either (Fig. 4F), which was not surprising, since no size difference in the embryos was observed. Breeding potential and phenotypic analysis of endothelial RhoA-deficient mice. To identify whether the viable mice with endothelial RhoA deficiency were able to breed normally, we backcrossed the RhoAf/f Tie2-Cre+ mice with the RhoAf/f (Fig. 5A). If no deficiencies occur, 50% of the offspring should carry the Tie2 promoter, thus should be deficient for endothelial RhoA. Indeed, the offspring were tested at weaning age and almost 50% of the offspring carried the Tie2 promoter (Fig. 5B). Male versus female RhoAf/f Tie2-Cre+ Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 3 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 2. Involvement of endothelial RhoA in VEGF- and S1P-induced sprout formation in vitro. (A,B) Quantification of number of nodes, number of junctions and total junction length (n = 8) (A) and representative images (B) of the 2-D matrigel tube formation assay of siRNA-transfected HUVECs in response to VEGF and S1P stimulation. (C,D) Quantification of average sprout length, number of sprouts per spheroid and total sprout length (n = 3) (C) and representative images (D) of the 3-D spheroid sprouting assay of siRNAtransfected HUVECs in response to VEGF and S1P stimulation. *P < 0.05; **P < 0.01; ***P < 0.001. mice were obtained at normal Mendelian ratios (Fig. 5C), and mutant offspring were both males and females (not shown) denoting no sex-linked deficiencies. The endothelial RhoA-deficient mice did not present any abnormality in physical characteristics, behavior or growth rate, as also denoted from their weight measurements (Fig. 5D). Effect of endothelial RhoA deficiency in retinal angiogenesis. The early postnatal mouse retina is a well-developed model for developmental angiogenesis study3335. To avoid potential secondary consequences of embryonic vascular deficiencies due to RhoA deletion and to achieve endothelial cell-specific RhoA deletion at will, we used an endothelial promoter with inducible (Cdh5-BAC-CreER+), instead of sustained (Tie2-Cre+) activation. Therefore, we generated mice with inducible endothelial RhoA deficiency, driven by the VE-cadherin Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 4 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 3. Endothelial RhoA deficiency is not detrimental for embryonic development. (A) Schematic diagram of RhoA transgenic mice carrying the Tie2-Cre endothelial-specific promoter and the RhoA floxed construct to generate RhoA-deficient endothelial cells upon endogenous promoter activation in mouse embryo. (B) Mating strategy to generate the RhoAf/f Tie2-Cre+ line, with ratio of expected and obtained genotypes. (C) Number of mouse pups with the corresponding genotypes. (D) Western blot analysis of RhoA expression in isolated mouse lung endothelial cells from endothelial RhoA-deficient mice and the corresponding littermate controls (n = 3). Full-length blots are presented in Supplementary Fig. 6. ***P < 0.001. tamoxifen-inducible promoter Cdh5-BAC-CreER +36 (Fig. 6A). After 4-hydroxytamoxifen (4-OHT) administration during P2-P5 (Fig. 6B), as previously described36, we examined the developing retinas on day P6 (Fig. 6C; Suppl. Fig. 3). The pattern of retinal vascular sprouting of the endothelial RhoA-deficient retinas was mildly disrupted, resulting in an uneven growing front, characterized by ragged and caved edges. However, no significant difference was observed in the radial growth or in the number of filopodia in the retinas upon endothelial RhoA deficiency (Fig. 6D), except from the number of caved lesions per retina, which were consistently present only in the endothelial RhoA-deficient retinas (Fig. 6D). The mild, although reproducible, phenotype suggested that either RhoA in the endothelial cells is dispensable for retinal angiogenesis or that RhoA deletion did not result in significant reduction of RhoA protein levels at this point. To exclude the second scenario, we repeated the analysis in P9 retinas, after 4-hydroxytamoxifen (4-OHT) administration during P2-P6.5 (Fig. 6E). At P9 retinas, the remodeling of the superficial layer is almost complete, whereas the plexus formation of the deep layers is still ongoing33. No difference was observed in the superficial plexus (Fig. 6F), nor in the deep plexus coverage (Fig. 6F,G), demonstrating that endothelial RhoA seems to be dispensable for retinal angiogenesis. Study of compensatory mechanisms and effect on downstream signaling pathway. Several studies have demonstrated compensatory roles of RhoB and RhoC, the other two members of the Rho GTPase family, upon RhoA deficiency3741. To identify whether RhoA deficiency in human and mouse endothelial cells initiates compensatory mechanisms, we analyzed the expression of the RhoB and RhoC in HUVECs treated with siRNAs for RhoA and mouse lung endothelial cells from endothelial RhoA deficient mice and littermate controls (Fig. 7). In HUVECs, we saw that RhoA knockdown led to a significant upregulation of RhoB expression, whereas RhoC expression was not affected. We hypothesize that RhoB upregulation should compensate for RhoA deficiency, since the basal phosphorylation levels of the downstream signaling molecules, such as Myosin Light Chain 2 (MLC)42 and cofilin43 not only were not blocked, but were slightly elevated in HUVECs (Fig. 7A,B). Interestingly, in the RhoA-deficient mouse endothelial cells we did not observe RhoB or RhoC upregulation. This was verified also from the complete inhibition of the basal activation levels of MLC and cofilin upon RhoA deficiency in the murine endothelial cells (Fig. 7A,B). We further checked whether RhoA deficiency affected the basal expression levels of Rac1 and Cdc42 (Fig. 7A,C). The expression of Cdc42 was not affected upon RhoA deficiency in either human or mouse endothelial cells, whereas Rac1 levels were slightly inhibited in HUVEC and presented an inhibitory tendency in mouse endothelial cells, however that inhibition was not significant (Fig. 7A,C). Evaluation of RhoA-deficient mouse endothelial cells on angiogenesis in vitro. The difference in compensatory mechanisms upon RhoA deficiency in human versus mouse endothelial cells prompted us to Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 5 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 4. Endothelial RhoA-deficient embryos present no gross vascular abnormalities. (A) Schematic presentation of mating strategy for embryonic analysis experiments. (B) Side view of endothelial RhoAdeficient embryos and corresponding controls. (CF) Whole mount CD31 staining of cranial (C), trunk (D), limb (E) and yolk sac (F) vessels from littermate controls (RhoAf/+ Tie2-Cre+ and endothelial RhoA-deficient (RhoAf/f Tie2-Cre+) embryos (n = 4). Figure 5. Endothelial RhoA-deficient mice present normal breeding profile and phenotypic characteristics. (A) Schematic representation of mating strategy for identification of breeding capability of endothelial RhoA knock out (RhoAf/f Tie2-Cre+) versus RhoA f/f mice. (B) Number of mice with the two different genotypes. (C) Percentage of male versus female offspring. (D) Weight values between endothelial RhoA-deficient and littermate controls of male and female mice. Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 6 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 6. Endothelial RhoA deficiency does not affect postnatal retinal angiogenesis. (A) Schematic diagram of inducible endothelial-RhoA deficient mice with the Cdh5 tamoxifen-inducible promoter Cdh5-BAC-CreER+. (B) Experimental outline for retinal angiogenesis assay on postnatal day 6 (P6). (C,D) Representative images (C) and radial growth, number of filopodia quantifications and number of caved lesions per retina (D) of P6 CD31-stained retinal vessels between endothelial RhoA-deficient mice and littermate controls (n = 4). Empty arrowheads represent caved edges of the vascular fronts, observed in RhoA-deficient mice. (E) Experimental outline for retinal angiogenesis assay on P9. (F,G) Representative images (F) and quantification (G) of retinal vessels, distinguishing superficial and deep plexus stained with biotinylated isolectin B4 (IB4) (n = 3). **P < 0.01 evaluate the response of the murine endothelial cells upon S1P- and VEGF-induced angiogenesis in vitro (Fig. 8). RhoA deficiency completely abrogated both S1P- and VEGF-induced cell migration (Fig. 8A) and tube formation (Fig. 8B,C), assessed by the number of nodes, junctions and total sprout length. Furthermore, in the tube formation experiments (Fig. 8C) the basal angiogenesis levels upon RhoA deficiency were significantly lower, which was significant in all quantification parameters (number of nodes, junctions and total sprout length), and consistent with the reduced activation levels of the downstream signaling pathway upon RhoA deficiency in the murine endothelial cells. Discussion It has been demonstrated that small GTPases regulate biological functions in endothelial cells and are considered key mediators of the angiogenic process1619. In the present study we aimed to delineate the precise role of RhoA in angiogenesis in vitro and in vivo. Here we identified that RhoA is activated by both tyrosine kinase and G protein-coupled receptor ligands and regulates some of the downstream in vitro angiogenic functions. RhoA knockdown blocked endothelial cell proliferation induced by VEGF and S1P, as well as VEGF-induced cell migration and tube formation, whereas it partially blocked S1P-induced cell migration and did not affect S1P-induced tube formation in either 2-D or 3-D sprouting assays. Endothelial-specific RhoA deletion in vivo through the Cre-lox system was not detrimental for embryonic development. The endothelial-specific RhoA-deficient mice, although fewer than expected, did not present anatomic or behavioral deficiencies and gave rise to mutant offspring in the expected Mendelian ratios. Inducible endothelial RhoA deletion led to a mild phenotype, without significantly affecting retinal angiogenesis. These findings provide direct evidence that although endothelial RhoA is important for endothelial cell functions triggered by angiogenic stimuli in vitro, its loss can be compensated during physiological in vivo angiogenesis. Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 7 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 7. Study of compensatory mechanisms and downstream signaling pathway analysis. (AC) Representative images of western blot analysis (A) and quantification (B,C) of RhoB, RhoC expression, MLC and cofilin activation (B) and Rac1 and Cdc42 expression (C) in HUVECs after transfection with siRNA control (siNEG) and two sequences of siRNA for RhoA (siRhoA#1 and siRhoA#2) (50 nM) (n = 3) and mouse lung ECs from RhoAEC mice and littermate controls (n = 2). Full-length blots are presented in Supplementary Fig. 7. *P < 0.05; **P < 0.01; ***P < 0.001. Our in vitro data on VEGF-induced RhoA activation is in accordance with previous findings10,21,22. RhoA knockdown in HUVECs demonstrated that RhoA blockade abrogates VEGF-induced migration, confirming previous data with dominant negative RhoA mutant (RhoA-19N) overexpression21,44. VEGF-induced cell migration was potently blocked with siRNA treatment, similarly to the blockade caused by RhoA-19N overexpression21, or by inhibition of Rho kinase or ROCK, the direct downstream RhoA target10,22. Similarly, RhoA knockdown in HUVECs efficiently blocked VEGF-induced tube formation. Similar findings had been previously obtained in Human Microvascular Endothelial Cells and in the MS1 endothelial-like cell line after treatment with the ROCK inhibitor Y-27632 or by knockdown experiments of Rho kinases respectively10,22. However, previous studies demonstrating the effect of ROCK inhibition on angiogenesis may not reflect the effect of RhoA depletion. RhoA controls other downstream pathways45, that may be required for angiogenesis, so it is not necessarily expected that ROCK inhibition would phenocopy RhoA inhibition in vitro or in vivo. S1P is a bioactive lipid mediator, abundant in plasma, which participates in several physiological processes, including angiogenesis, through GPCR activation25. S1P binds and signals through five receptors, S1PR15, all GPCRs, from which S1PR13 are expressed in endothelial cells, with S1PR1 presenting higher expression levels than S1PR2 and S1PR346. S1PR1 couples mostly with Gi, S1PR2 with G12/13 and S1PR3 with Gq25. The significant role of S1P on angiogenesis in vivo is evident from the fact that S1PR1 deficiency caused embryonic lethality due to vascular deficiencies and severe hemorrhage47. This phenomenon was exacerbated in the S1PR1/2/3 triple deficient mice48, whereas the single deficiency of S1PR2 or S1PR3 did not present viability issues49,50. Previous studies have shown that S1P induces RhoA activation in endothelial cells11,51,52 and this activation has been studied in the context of endothelial barrier regulation. Here, we verified the activation of RhoA downstream of S1P signaling, S1Ps stimulating effect on endothelial cell proliferation and migration, and the RhoA knockdown experiments revealed the critical role of RhoA in both biological functions. Participation of RhoA on S1P-induced angiogenesis was previously shown with exoenzyme C3 toxin treatment, where RhoA activation was found to be downstream of S1PR1 and S1PR353, and our data confirmed this finding. Regarding the role of RhoA on S1P-induced sprouting, it has been reported that RhoA has no effect on endothelial 3-D sprouting54, which coincides with our findings in our 2-D and 3-D sprouting systems. S1P has also been reported to exert an inhibitory role on angiogenesis, which is attributed mainly to RhoC activation, since exoenzyme C3 treatment increased sprouting formation in the 3-D sprouting assay54. Our data, however, showed that C3 treatment abolished the S1P-induced sprouting efficiency, without inducing basal sprouting levels. This discrepancy could be Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 8 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 8. Effect of endothelial RhoA deficiency on VEGF- and S1P-induced cell migration and sprout formation in vitro. Quantification of mouse VEGF- (50 ng/ml) and S1P-induced (250 nM) cell migration (n = 3) (A) and sprout formation (n = 3) (B,C) of mouse lung ECs from RhoAEC mice and littermate controls. (B) Representative images and (C) quantification of number of nodes, number of junctions and total junction length. *P < 0.05; **P < 0.01; ***P < 0.001. attributed to the difference of C3 toxin efficiency, as higher concentration is routinely being used for our experiments, guided by the efficiency to block RhoA activation. Global deficiency of RhoA is not compatible with life15. Tie2 (tunica intima endothelial kinase 2) is a tyrosine kinase receptor, expressed in endothelial and some hematopoietic cells30,31. Although Tie2 is not considered critical for fetal hematopoiesis55, Tie2-deficient mice have been reported to die at E10.5 due to vascular and hematopoietic deficiencies30,56. Similarly, Tie2 promoter-driven deficiency leads to embryonic lethality around E9-10.516,18,57. Therefore, potential deficiencies due to Tie2-driven excision should be evident prior to E12.5, where the embryo dissection took place. We had previously demonstrated that global RhoA activation deficiency, through the combined global deficiency of two RhoGEFs, PDZ-RhoGEF and LARG, led to embryonic lethality before E10.5, due to partial branching failure of the cranial vessels and deficient embryonic vascular network in the placenta20. Here, however, we found that although RhoA deficiency in the endothelial cells could partially affect the viability of the mutant embryos, is not detrimental for life. A possible explanation may be that RhoA deficiency in the endothelium may not be strictly responsible for embryonic lethality due to vascular abnormalities, but RhoA expression in the surrounding tissues should play a significant role. Although the precise mechanism of RhoA functions in each cell type has not been described in detail, nor their role on paracrine signaling between different cell types, that scenario cannot be excluded, since RhoA regulates the activity of several transcription factors, such as the (SRF)/MAL, the AP-1 or the NF-kB58. RhoA activation in the surrounding tissues seems to play compensatory role for endothelial cell functions in the absence of endothelial RhoA, which when lost, either in the in vivo20 or in vitro settings10,21,22 (and our data), exacerbates the vascular deficiencies. The mouse retina is an established post-embryonic angiogenesis model59. Retinal vasculature develops postnatally through sprouting angiogenesis60, following a well-defined sequence of events. From birth (P0) till post-natal day 6 (P6) the retinal vessels grow from the optic disc to the peripheral margin forming a superficial vascular plexus, and around that point they also start invading the retina to form the deeper vascular layers33,36. Our experiments revealed that endothelial RhoA deficiency led to mild phenotype, characterized by uneven retinal vascular front with ragged and caved edges, however the major quantitative parameters were not affected. Similar mild phenotype has been previously reported for sema3E, plexin-D1 and neuronal VEGFR2 deficiencies36,61, and here could suggest that RhoA is mainly dispensable during retinal angiogenesis or that RhoA gene deletion may not result in significant reduction of RhoA protein levels, although the efficiency of the inducible promoter has been previously demonstrated36. Our findings are generally in accordance with previous experiments with retinal explants where treatment with RhoA pathway inhibitors did not present differences in the basal levels of capillary vessel outgrowth, although exogenous VEGF-induced outgrowth was significantly inhibited22. In the absence of endothelial RhoA in vivo, compensation from the other members of the family of Rho GTPases cannot be excluded. RhoC shares ~88% amino acid homology with RhoA, both are equally regulated by most RhoGEFs and they both regulate actin cytoskeleton62,63. Although they have different spatial localization patterns, suggesting distinct roles64, there are studies demonstrating functional redundancy. RhoA-deficient cells present no deficiencies in forming actin-rich protrusions, RhoA-deficient mouse fibroblasts present no significant Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 9 www.nature.com/scientificreports/ www.nature.com/scientificreports actin cytoskeleton abnormalities, suggesting functional compensation from RhoC39,40, while similar compensatory role has also been reported in lung adenoma formation in vivo41. Our data are in line with a previous study reporting the compensatory role of RhoB upon RhoA deficiency, leading to activation of downstream targets, such as MLC and cofilin38. MLC and cofilin phosphorylation could also be endothelial type-specific, rather than species-specific. Although RhoA controls MLC phosphorylation in mouse lung ECs, this may not be the case for retinal mouse ECs, which could explain the lack of phenotype with RhoA depletion, and which warrants further investigation. Both in vivo models included in this study are well described models for physiological angiogenesis35. Although physiological and pathological angiogenesis share many common characteristics, they also have significant differences65. Vessels driven by pathological angiogenesis, such as tumor-induced angiogenesis, are disorganized and leaky66. The signaling cascades in pathological angiogenesis have different origin and are more persistent, although not fully elucidated to date67. Moreover, the same receptors or signaling pathways may induce diverse outcome, based on the microenvironmental context. An example is the S1P2R pathway, which although induces pathological neovascularization, at the same time blocks hypoxia-triggered revascularization in the retina68. The transcriptomic profile of endothelial cells between physiological and pathological angiogenesis differs: tumor endothelial cells overexpress certain proteins, such as Doppel or Vscp, which present limited or no expression during developmental angiogenesis69. Some angiogenesis mediators, such as VEGF, basic Fibroblast Growth Factor (bFGF) or angiopoietins are known to mediate both physiological and pathological angiogenesis, whereas others, such as Cox2, Placental Growth Factor (Plgf), v3 integrin, nitric oxide and TSP-2 mediate pathological angiogenesis without affecting the developmental one70,71. Even in the case of VEGF, blockade of VEGF164 potently suppressed pathological neovascularization, while it had no effect on physiological neovascularization72, highlighting the differences in the molecular mechanisms regulating physiological versus pathological angiogenesis. In summary, we report here that although the small GTPase RhoA regulates in vitro angiogenesis, its role during developmental angiogenesis may not be as significant. Additional work will be necessary to elucidate the compensatory mechanisms upon RhoA deficiency in the context of physiological versus pathological angiogenesis, which may help expose the potential of RhoA as an anti-angiogenic therapeutic target. Methods Antibodies and reagents. Cell culture reagents were purchased from GibcoTM (Life technologies, Carlsbad, CA). ECGS (Cat#356006) was purchased from Corning (San Jose, CA), 5000U/ml Heparin solution was purchased from Hospira (NDC#63739-920-11; Lake Forest, IL). Exoenzyme C3 from Clostridium botulinum (Cat#CT03) was purchased from Cytoskeleton (Denver, CO). The following pre-designed siRNAs: Silencer Select Negative Control #1 (Cat No: 4390846), siRhoA (Cat Nos: s758 and s759), fluorescent streptavidin conjugates (Molecular Probes), Alexa 488 fluorescence-conjugated IgGs (Molecular Probes) and the Halt Protease and Phosphatase Inhibitor Cocktail (Cat#PI78445) were purchased from Thermo Fisher Scientific (Waltham, MA). DharmaFECT 1 (Cat# T-2001-02) transfection reagent was purchased from Dharmacon (Lafayette, CO). Reduced Growth Factor (RGF)-Basement Membrane Extract was purchased from Trevigen (Gaithersburg, MD). Sphingosine-1-Phosphate (S1P) (Cat# NC9978856), rat anti-mouse CD31 primary antibody (Clone MEC13.3, Cat#553370, BD Pharmingen) and donkey anti-rat Alexa488 secondary antibody (Cat#A-21208, Life Technologies) were purchased from Fisher (Hampton, NH). Glutathione SepharoseTM 4B beads (Cat#45-000-139), Collagen type I (Cat#CB354249), rat anti-mouse CD102 (ICAM2) antibody (Clone 3C4, mlC2/4 Cat#553325), 4-Hydroxytamoxifen (Cat#50-136-5306), Alexa Fluor 594 Phalloidin (Cat#A12381), Hoechst Trihydrochloride Trihydrate (Cat#33342), Triton X-100 (Cat#BP151-100), Bovine Serum Albumin (BSA), Tris-HCl, NaCl, phenylmethylsulfonyl fluoride, aprotinin, leupeptin and other chemicals were also purchased from Fisher (Hampton, NH). Rabbit anti-RhoA (Cat# 21017) was purchased from NewEast Biosciences (King of Prussia, PA). Hamster anti-CD31 antibody (2H8 clone; Cat# ENMA3105) was purchased from Chemicon (Temecula, CA). Cy3/ Cy5 DyLight549/DyeLight649-conjugated IgGs were obtained from Jackson ImmunoResearch (West Grove, PA). Human (Cat# SRP3182-10UG) and mouse (Cat#V4512) VEGF, Gelatin 2% (Cat#G1393-100ML), biotinylated isolectin B4 (IB4; Cat#L2140) and Methyl Cellulose (Cat#M0512-250G), Mayers Hematoxylin Solution (Cat# MHS32-1L), JumpStart REDTaq ReadyMix Reaction Mix (Cat#P0982) were purchased from Sigma Aldrich (St. Louis, MO). 32% Paraformaldehyde (formaldehyde) aqueous solution (Cat#15714-S) was purchased from Electron Microscopy Systems (Hatfield, PA). Primary antibodies against RhoA (Cat#2117; 1:1000), RhoB (Cat#63876; 1:1000), RhoC (Cat#3430; 1:1000), Phospho-Myosin Light Chain (Cat#3674; 1:1000), Phospho-Cofilin (Cat#3313; 1:1000), Cdc42 (Cat#2462; 1:1000), -actin (Cat#3700; 1:2000) and GAPDH (Cat#5174; 1:2000) were purchased from Cell Signaling Technology (Beverly, MA). Mouse anti-Rac1 primary antibody was purchased from BD Biosciences (San Jose, CA). Goat anti-rabbit (Cat#4010-05, 1:50,000) and anti-mouse (Cat#1010-05, 1:50,000) secondary antibodies were from Southern Biotech (Birmingham, AL). Immobilon Western Chemiluminescent HRP substrate (Cat# WBKLS0500) was from Millipore (Burlington, MA). Cell lines and culture procedures. Cells were maintained at 37 C with 5% CO2 in a humidified environment, following standard protocols73,74. Human Umbilical Vein Endothelial Cells (HUVECs) were isolated from human umbilical cords under Institutional Review Board (IRB)-approved protocol A15-3891 (Texas Tech University Health Sciences Center Institutional Review Board) in accordance with relevant guidelines and informed consent was obtained from all donors. HUVECs were used between passages 1 and 6 and all experiments were performed in HUVECs from at least three different donors, unless stated otherwise. They were routinely cultured in M199 medium (Corning) (Cat#MT10060CV), supplemented with 15% Fetal Bovine Serum (FBS) (GIBCOTM) (Cat#10438026), 150 g/ml Endothelial Cell Growth Supplement (ECGS), 5 U/ml heparin sodium and 1X Antibiotic-Antimycotic solution (GIBCOTM) (Cat#15240-062) (EC complete medium). Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 10 www.nature.com/scientificreports/ www.nature.com/scientificreports Mice. Animal studies were carried out according to Texas Tech University Health Sciences Center (TTUHSC) Institutional Animal Care and Use Committee (IACUC)-approved protocols, in compliance with the Guide for the Care and Use of Laboratory Animals. All mice used were maintained on a C57BL/6 background and both males and females were used for experiments. The generation of the floxed alleles of the genes encoding RhoA has been described previously40. Endogenously-regulated endothelial-specific RhoA knockouts were obtained by crossing the RhoA floxed mice with mice carrying a Cre-mediated recombination system, driven by the Tie2 promoter (Tie2-Cre)29. Conditionally-regulated endothelial-specific RhoA knockouts were obtained by crossing the RhoA floxed mice with mice carrying a tamoxifen-inducible Cre-mediated recombination system, driven by the Cdh5 promoter (Cdh5-BAC-CreER+)36. 4-hydroxytamoxifen (40 g) was subcutaneously injected at postnatal days P2, P3.5, P5 and P6.5. Schemes of the protocol for gene deletion are presented in Fig. 6B,E. Genotyping assay for RhoAf/f Cdh5-CreERT2 and Tie2-Cre mutants was performed by Polymerase Chain Reaction (PCR) on mouse genomic DNA extracts from tail biopsies. The JumpStart REDTaq ReadyMix Reaction Mix was used for PCR, along with the following primers: RhoAf/f Forward: 5-TCTCTGCACTGAGGGAGTTAGG-3, RhoAf/f Reverse: 5-GTACATACAGGGAATGGAAACAAGG-3, Cre Forward: 5-GCGGTCTGGCAGTAAAAACTATC-3, Cre Reverse: 5-GTGAAACAGCATTGCTGTCACTT-3, Tie2-Cre Forward: 5-CGATGCAACGAGTGATGAGG-3, Tie2-Cre Reverse: 5-CGCATAACCAGTGAAACAGC-3. Cell transfection. HUVECs were transfected with siNEG, siRhoA#1 or siRhoA#2, using DharmaFect1, following manufacturers instructions. Briefly, the cells were cultured in a 6-well till 80% confluency, then the cells were starved for 1 h with M199 medium without antibiotics (starvation medium). Meanwhile, 500 nM siRNA solution in 200 l of M199 starvation medium was prepared from the stock solution and Dharmafect was also diluted 20X in M199 starvation medium in another vial to reach the volume of 200 l. After 5 min incubation in room temperature the siRNA vial content was transferred to the Dharmafect one, the solution was briefly vortexed and incubated for 15 min more in room temperature. Then the 400 ul of the final solution was added dropwise in the 1600 ul of M199 starvation medium per well (50 nM final concentration) and the cells were incubated for 6 h. After the incubation period the medium was replaced with EC complete medium. Cell proliferation assay. Cell proliferation of HUVECs was evaluated through the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-dimethyltetrazolium bromide) colorimetric assay, as previously described75. HUVECs were seeded at a density of 2 104 cells/well in gelatin-coated 24-well plates and grown in complete medium (500 l/well) for 24 h, prior to siRNA transfection. After 32 h of incubation in EC complete media, the medium was replaced with M199 containing 0.1% BSA. After 16 h the medium was replaced again with fresh M199 containing 0.1% BSA, as well as the tested agents and the cells were further incubated for 24 h. At the end of the incubation period, 50 l of MTT stock (5 mg/ml in PBS) was added per well and the plates were incubated at 37 C for 2 h. The medium was removed, the cells were washed with PBS pH 7.4 and 100 l acidified isopropanol (0.33 ml HCl in 100 ml isopropanol) was added to each well and the plate was agitated thoroughly to solubilize the dark blue formazan crystals, formed by metabolically active cells. The solution was transferred to a 96-well plate and immediately read on a microplate reader, at a wavelength of 570 nm. Results were confirmed by direct measurement of the cells using a standard hemocytometer. Cell migration assay. Cell migration was performed as previously described20, using a 48-well Boyden chamber with an 8-m pore size polyvinyl pyrrolidone-free polycarbonate membrane (NeuroProbe) coated with collagen. Transfected HUVECs were added to the upper chamber 48 h post-transfection, and M199 with 0.1% BSA with or without human (100 ng/ml) or mouse (50 ng/ml) VEGF or S1P (250 or 500 nM) was added to the lower chamber. After incubation for 6 h at 37 C, the cells on the upper surface of the membrane were removed, and the cells at the lower surface were fixed with methanol and stained with hematoxylin. The cells were manually counted using a bright-field microscope (Microscoptics, IV-900). Tube formation assay. Matrigel tube formation assay was performed with transfected HUVECs 48 h post-transfection, as previously described74,76. Briefly, wells of a 96-well culture plate were coated with 40 l/well RGF-Basement Membrane Extract (Trevigen, Cat #3433) and were left to polymerize for 20 min at 37 C. After polymerization, 104 cells suspended in 100 l of M199 0.1% BSA were added to the respective wells. Human (100 ng/ml) or mouse (50 ng/ml) VEGF and S1P (250 or 500 nM) were added in the medium and after 6 h incubation at 37 C, the medium was removed, the cells were fixed, and pictures of the wells were captured using a bright-field microscope (Microscoptics, IV-900) connected with a digital camera (AmScope FMA050) at 4X magnification and later analyzed for number of nodes, number of junctions and total sprout length using the Angiogenesis analyzer plug-in77 in ImageJ software (National Institutes of Health). Immunoblot analysis. The immunoblot analysis was performed as described previously73. The cells were lysed on ice in RIPA buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 0.5 mmol/L EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS and 140 mmol/L NaCl), supplemented with protease and phosphatase inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail; Thermo Scientific). Cell lysates were centrifuged at 13,000 rpm for 10 min at 4 C and each supernatant was mixed with the appropriate volume of 5x SDS loading buffer, heated to 95100 C for 5 min and briefly centrifuged. Equal amounts of proteins were subjected to SDS-PAGE and transferred onto an Immobilon P, polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membranes were then incubated with the appropriate primary antibodies: RhoA (1:1000), p-cofilin (1:1000), p-MLC (1:1000), RhoB (1:1000), RhoC (1:1000), Cdc42 (1:1000), -actin (1:2000) GAPDH (1:2000) (all from Cell Signaling Technology, Beverly, MA), or Rac1 (1:1000) (from BD Biosciences). As a secondary antibody, goat anti-rabbit was used (1:50000). The antigens were visualized using the Immobilon Western Chemiluminescent Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 11 www.nature.com/scientificreports/ www.nature.com/scientificreports HRP substrate (Millipore), according to manufacturers instructions. The protein levels that corresponded to immunoreactive bands were quantified using the Image PC image analysis software (Scion Corp., Frederick, MD) and ImageJ image analysis software (National Institutes of Health). Rho GTPase pull-down assay. Rho activation in cultured cells was assessed as follows20,42: after serum starvation for 3 h, the cells were treated as indicated and lysed on ice in lysis buffer, containing 20 mM Hepes, pH 7.4, 0.1 M NaCl, 1% Triton X-100, 10 mM EGTA, 40 mM b-glycerophosphate, 20 mM MgCl2, 1 mM Na3VO4, 1 mM dithiothreitol, 10 g/ml aprotinin, 10 g/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. The lysates were incubated with the glutathione S-transferase-rhotekin-Rho-binding domain previously bound to glutathione-Sepharose beads (Amersham Biosciences) and washed three times with lysis buffer. Associated GTP-bound forms of Rho were released with SDSpolyacrylamide gel electrophoresis loading buffer and analyzed by western blot analysis using a monoclonal antibody against RhoA, as described above. Endothelial spheroid sprouting assay. The sprouting assay was performed as previously described26,78. Methocel preparation: Methyl Cellulose (Cat#M0512-250G, Sigma Aldrich) was autoclaved and gradually dissolved in M199 (6 g in 500 ml) under stirring at 4 C overnight. The supernatant from a 3 h centrifugation at 3,500 g was collected and termed methocel from now on26. Sprouting assay: HUVECs with or without siRNA transfection (above) were resuspended in EC complete medium (described above), containing 20% methocel (Sigma) or alternatively in the presence or absence of 20 ng/ml C3 toxin. 25 l cell suspension drops were pipetted on non-adherent plastic plates and then the plates were turned upside-down to form hanging drops in which HUVEC spheroids were formed. The plates were incubated for 24 h at 37 C and spheroids were harvested by washing the plates with 10% FBS/PBS. Next, spheroids were centrifuged at 200 g for 5 min and resuspended in 20% FCS and 80% methocel. The collagen matrix was prepared on ice using Collagen type I (Cat#CB354249, Fisher), Medium 199 and NaOH (1 M) in 8:1:1 ratio. Additionally, 1X HEPES buffer was added to the mix for pH adjustment. The Collagen solution and the spheroid solution were mixed in 1:1 ratio and transferred to a 24-well plate. For polymerization, gels were incubated for 30 min at 37 C. Stimulation took place with VEGF (100 ng/ ml) and S1P (500 nM) in M199 (starvation medium). Pictures of the spheroids were obtained with a bright-field microscope (Microscoptics, IV-900) connected with a digital camera (AmScope FMA050) at 4X magnification and number of sprouts and sprout length were quantified with the ImageJ software (National Institutes of Health). Spheroid immunostaining. To obtain immunofluorescence images, the spheroids were stained for actin with phalloidin staining, according to the manufacturers instructions. Briefly, the spheroids were fixed by addition of 4% PFA overnight in 4 C. After two 5-min washes with PBS, the wells were incubated with 0.2% Triton X-100 in PBS for 10 min at room temperature, followed by two 5-min washes with PBS. Then the spheroids were blocked with 3% BSA in PBS for 1 h at room temperature and incubated with Alexa 594 Phalloidin 1:100 in PBS with 3% BSA overnight at 4 C. The extra Phalloidin was washed out with two 10-min washes with PBS and the spheroids were incubated with Hoechst 1:2000 in PBS for 10 min at room temperature. After two more 10-min washes with PBS, PBS was added in each well and the 24-well plates were covered in aluminum foil and stored at 4 C till pictures were obtained through confocal microscopy (see respective paragraph). Lung endothelial cell isolation. Isolation of mouse lung microvascular endothelial cells was performed as previously described42. Lung endothelial cell isolation took place after weaning. Lungs were removed from two or more mice, washed in 10% FBS-DMEM, minced into 12 mm2 pieces and digested with Collagenase Type I (2 mg/ ml, Cat#17-100-017, Fisher) at 37 C for 2 h with occasional agitation. The cellular digest was filtered through a 70 m cell strainer, centrifuged at 1,500 rpm and the cells were plated (day 0) on gelatin-coated dishes, containing endothelial cell full media (see above). On day 1, floating cells (including red blood cells) were removed and washed with PBS and fresh culture medium was added. Sheep anti-rat IgG Dynabeads (Invitrogen) were incubated with a rat anti-mouse ICAM-2 mAb (3C4) (22.5 l Ab per 150 l Dynabead solution) at 4 C overnight and washed three times with PBS supplemented with 0.1% BSA and 2 mM EDTA. First purification using the pre-coated beads was done on day 5. Cells were incubated for 10 min with ICAM-2-coated beads at room temperature, under continuous agitation. This was followed by two washes with PBS and the cells were trypsinized. After trypsinization, the bead-bound cells were recovered with a magnet, washed four times, resuspended in full growth medium and plated on fresh gelatin-coated dishes. On day 10, the cells were subjected to a second purification, following the same procedure. Purity (>85%) of endothelial cells was verified by PECAM-1 staining in FACS analysis. Preparation of whole-mount retinas. Enucleated eyes were fixed for 20 min in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and then dissected. Retinal cups were post-fixed for 30 min and then stained as described below. Retinal immunostaining and in situ hybridization. Immunohistochemistry (IHC) of whole-mount samples or tissue sections was performed as previously described79. At P6, the primary monoclonal antibodies used were anti-RhoA (21017; NewEast Biosciences, King of Prussia, PA), hamster anti-CD31 (2H8; Chemicon, Temecula, CA), Secondary antibodies used were Alexa 488 fluorescence-conjugated IgGs (Molecular Probes) or Cy3/Cy5 DyLight549/DyeLight649-conjugated IgGs (Jackson ImmunoResearch, West Grove, PA). At P9, blood vessels were visualized using biotinylated isolectin B4 (IB4) (Sigma), followed by fluorescent streptavidin conjugates (Molecular Probes). Whole mount embryo immunohistochemistry. Embryos were collected at embryonic day (E) 11.5, as previously reported20, with some modifications80. Noon of the plug day was E0.5. Embryos were dissected from their deciduas and were fixed in 4% paraformaldehyde/PBS at 4 C overnight. They were rinsed twice with PBS, Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 12 www.nature.com/scientificreports/ www.nature.com/scientificreports 5 min each time, and were dehydrated through immersion in the following solutions: 25% Methanol in PBS with 0.5% Triton X-100, 50% Methanol in PBS with 0.5% Triton X-100, 75% Methanol in PBS with 0.5% Triton X-100 and 100% Methanol twice, each step for 5 min with gentle shaking, at room temperature. Embryos were bleached with 5% hydrogen peroxide in methanol for 4 h at room temperature and rehydrated through immersion in the following solutions: 100% Methanol twice, 75% Methanol in PBS with 0.5% Triton X-100, 50% Methanol in PBS with 0.5% Triton X-100, 25% Methanol in PBS with 0.5% Triton X-100, and three times in PBS with 0.5% Triton X-100, each step for 5 min with gentle shaking at room temperature. Embryos were blocked in PBS with 0.1% Triton X-100 and 3% instant skim milk (blocking solution) for 2 hours and incubated with the rat anti-mouse CD31 primary antibody (Clone MEC13.3, Cat#553370, BD Pharmingen) in 1:100 dilution in the blocking solution in 4 C for 2 days with gentle shaking. Embryos were then washed in blocking solution for 5 times, 1 h each with gentle shaking and were incubated with donkey anti-rat Alexa 488 secondary antibody (Cat#A-21208, Life Technologies) in the blocking solution in 4 C overnight. After 5 consecutive washes of 1 h each at 4 C, each embryo was transferred in PBS-containing wells and was imaged by confocal microscopy (below). Confocal microscopy. For spheroids. Fluorescent Images were obtained using a multiphoton microscope (A1R; Nikon, NY, USA) in the confocal mode, using a 10x objective. Each spheroid image was the projection of merging of 510 images of different Z focus covering an approximate 10 m Z distance. For embryos. Fluorescent Images were obtained using a multiphoton microscope (A1R; Nikon, NY, USA) in the confocal mode. Each image analyzing the vasculature of embryonic parts was the projection of merging of 800 images of different Z focus covering an 800 m Z distance. For embryo size comparison, a fluorescent dissecting microscope (MVX10; Olympus, Pennsylvania, USA) was used. For retinas. Fluorescent Images were obtained using a confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan). Quantification of cells or substances of interest was conducted on eight 500 m x 500 m fields of view per sample in scanned images, and numbers obtained from each of the eight fields were averages using an FV10-ASW Viewer (Olympus). Statistical analysis. All experiments were repeated at least three times with similar results. Statistical analy- sis for embryo viability (difference from expected ratio) was performed with Chi Square test, whereas for the rest of the experiments statistical analysis was performed by an unpaired two-tailed Students t-test. Data analysis was performed using GraphPad Prism version 7.00 for Windows (GraphPad Software, San Diego, CA). The asterisks in the figures denote statistical significance (NS: not significant, *P < 0.05; **P < 0.01; ***P < 0.001). Ethical approval and informed consent. All experimental protocols were approved by the TTUHSC IACUC committee and the experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals. For HUVEC isolation, informed consent was obtained from all participants. Data Availability All data generated or analyzed in this study are included in the article (and the Supplementary Information Files). References 1. Haga, R. B. & Ridley, A. J. Rho GTPases: Regulation and roles in cancer cell biology. Small GTPases 7, 207221, https://doi.org/10.1 080/21541248.2016.1232583 (2016). 2. Wennerberg, K., Rossman, K. L. & Der, C. J. The Ras superfamily at a glance. J Cell Sci 118, 843846, https://doi.org/10.1242/ jcs.01660 (2005). 3. Lawson, C. D. & Ridley, A. J. Rho GTPase signaling complexes in cell migration and invasion. J Cell Biol 217, 447457, https://doi. org/10.1083/jcb.201612069 (2018). 4. Ridley, A. J. Rho GTPase signalling in cell migration. 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Author Contributions F.T.Z., M.S.S., Y.I., R.G.A., P.T., C.C., S.A.M., C.L.D. and C.M.M. performed experiments; F.T.Z., M.S.S., Y.I., Y.K. and C.M.M. analyzed results and made the figures; F.T.Z., Y.Z., Y.K., J.S.G., C.M.M. designed the research and wrote the paper. All authors have read and approved the submitted version. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-48053-z. Competing Interests: The authors declare no competing interests. Publishers note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the articles Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. The Author(s) 2019 Scientific Reports | (2019) 9:11666 | https://doi.org/10.1038/s41598-019-48053-z 15  ...

... Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research ABT-510 Is an Effective Chemopreventive Agent in the Mouse 4-Nitroquinoline 1-Oxide Model of Oral Carcinogenesis Rifat Hasina,1 Leslie E. Martin,1 Kristen Kasza,2 Colleen L. Jones,1 Asif Jalil1 and Mark W. Lingen1 Abstract Despite numerous advances, the 5-year survival rate for head and neck squamous cell cancer (HNSCC) has remained largely unchanged. This poor outcome is due to several variables, including the development of multiple primary tumors. Therefore, it is essential to supplement early detection with preventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we sought to define an appropriate dose and duration of administration that would predict the histologic timeline of HNSCC progression. Additionally, we sought to determine the timing of the onset of the angiogenic phenotype. Finally, using ABT-510 as a proof-of-principle drug, we tested the hypothesis that inhibitors of angiogenesis can slow/delay the development of HNSCC. We determined that 8 weeks of 100 g/mL 4-NQO in the drinking water was the optimal dosage and duration to cause a sufficient incidence of hyperkeratoses, dysplasias, and HNSCC over a period of 32 weeks with minimal morbidity and mortality. Increased microvessel density and vascular endothelial growth factor expression in hyperkeratotic lesions provided evidence that the initiation of the angiogenic phenotype occurred before the development of dysplasia. Importantly, ABT-510 significantly decreased the overall incidence of HNSCC from 37.3% to 20.3% (P = 0.021) as well as the combined incidence of dysplasia and HNSCC from 82.7% to 50.6% (P < 0.001). These findings suggest that our refinement of the 4-NQO model allows for the investigation of the histologic, molecular, and biological alterations that occur during the premalignant phase of HNSCC. In addition, these data support the hypothesis that inhibitors of angiogenesis may be promising chemopreventive agents. At current rates, approximately 400,000 cases of head and led Slaughter et al. (4) to propose the concept of field cancerization. This theory suggests that multiple individual primary tumors may develop independently in the upper aerodigestive tract as a result of years of chronic exposure to carcinogens. The occurrence of these new primary tumors can be particularly devastating for individuals whose initial lesions are small. Their 5-year survival rate for the first primary tumor is considerably better than patients with late-stage disease. However, second primary tumors are the most common cause of treatment failure and death among early-stage HNSCC patients (5). Therefore, it is insufficient treatment to address only the initial lesion. To improve the outcome of such patients, some form of chemopreventive treatment is essential. Chemoprevention can be defined as the systemic use of natural or synthetic agents to reverse or halt the progression of premalignant lesions. Chemopreventive agents are being tested for their efficacy in the preclinical and clinical settings for several malignancies, including HNSCC (6). However, the initial promising responses have not been consistently reproduced and toxicity was often a significant issue. Therefore, more effective and better tolerated therapy is needed for premalignant oral disease. Angiogenesis, the growth of new blood vessels from preexisting ones, is an essential phenotype in several physiologic and pathologic processes, including growth and development, wound healing, reproduction, arthritis, and tumor formation neck squamous cell cancer (HNSCC) will be diagnosed worldwide this year (1). Despite numerous advances in therapy, the long-term survival for these patients has remained largely unchanged. Several factors contribute to this poor outcome. First, oral cancer is often diagnosed in an advanced stage. The 5-year survival rate of early-stage oral cancer is approximately 80%, whereas the survival drops to 19% for late-stage disease (2). Second, the development of multiple primary tumors has a major effect on survival. The rate of second primary tumors in these patients has been reported to be 3% to 7% per year, higher than for any other malignancy (3). The observation of frequent second primary tumors in oral cancer Authors' Affiliations: Departments of 1Pathology, Medicine, and Radiation and Cellular Oncology and 2Health Studies, The University of Chicago, Chicago, Illinois Received 11/13/08; revised 2/7/09; accepted 3/4/09; published OnlineFirst 3/31/09. Grant support: Abbott Laboratories and NIH grant DE012322. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Mark W. Lingen, Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6101, Chicago, IL 60637. Phone: 773-702-5548; Fax: 773-702-9903; E-mail: mark.lingen@uchospitals. edu. 2009 American Association for Cancer Research. doi:10.1158/1940-6207.CAPR-08-0211 www.aacrjournals.org 385 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research (7). Whether active neovascularization occurs is dependent on the relative concentrations of inducers and inhibitors of angiogenesis present in a given tissue microenvironment. Therefore, the inhibition of tumor-associated angiogenesis, using natural or synthetic inhibitors of angiogenesis, is an attractive target for therapy that has been gaining traction in the field of oncology. Like all solid tumors, HNSCCs must develop multiple direct and indirect ways to induce angiogenesis. Importantly, the expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human HNSCC (811), suggesting that inhibitors of angiogenesis may also hold promise as chemopreventive agents. In addition to their main biological/molecular effects, some of the drugs currently under investigation in the chemopreventive setting have potential antiangiogenic activity. However, to date, no pure inhibitors of angiogenesis have been tested for their ability to act as chemopreventive agents in HNSCC. Animal models that faithfully recapitulate the human condition are critical to further our understanding of the molecular, biological, and clinical aspects of various diseases, including cancer. The hamster buccal pouch and the rat tongue models of oral carcinogenesis are well-established surrogate models for the human condition. However, although the hamster buccal pouch and rat tongue models have been extensively investigated, they have several limitations, resulting in the recent development of mouse oral cancer models that have several advantages (1218). In particular, mouse models enable the development and testing of new approaches to prevention and treatment, identification of early diagnostic markers, and an understanding of the biology and genetics of tumor initiation, promotion, and progression in an animal model whose genome is most similar to humans (1922). Although the 4-nitroquinoline 1-oxide (4-NQO) mouse model of oral carcinogenesis has gained increased attention as an alternative model, several parameters require further investigation. For example, although various treatment protocols have been described, the optimal administration, timing, and dosage of 4-NQO required to develop lesions that clinically mimic the human condition (singular or synchronous neoplasms) have not been fully established. In addition, a detailed and systematic investigation of the histologic changes during progression in the 4-NQO mouse model before the development of HNSCC has not been done. Finally, the timing and mechanisms of the development of various tumor-related phenotypes have not been determined in this model. Each of these issues is of critical importance and must be addressed to determine how closely this model system mimics the human condition such that it can be used effectively for future preclinical studies. Therefore, the purpose of this work was 4-fold. First, we sought to establish a dosage and treatment schedule that resulted in the development of singular or occasionally synchronous HNSCC rather than innumerable lesions throughout the oral cavity over a protracted period of time. Second, we established a predictable timeline for the histologic progression of mucosal lesions in mice treated with 4-NQO. Third, we sought to clarify when the expression of the angiogenic phenotype can be first observed in this model. Finally, ABT510, a mimetic peptide of thrombospondin-1 (Fig. 1; refs. 23, 24), was administered to 4-NQOtreated mice as a proof of principle to test the hypothesis that inhibitors of angiogenesis can be successfully used as chemopreventive agents for HNSCC. Materials and Methods Administration of 4-NQO Two hundred thirty male CBA mice, 6 to 8 wk of age, were purchased from The Jackson Laboratory and housed in the Animal Resource Facility under controlled conditions and fed normal diet and autoclaved water. All animal procedures were carried out in accordance with Institutional Animal Care and Use Committee approved protocols. Mice were administered 4-NQO in their drinking water on a continuous basis at the required dose for the required duration. 4-NQO powder (Sigma) was first dissolved in DMSO at 50 mg/mL as a stock solution and stored at 20C until used. On the days of 4-NQO administration, the stock solution was dissolved in propylene glycol (Sigma) and added to the drinking water bottles containing autoclaved tap water to obtain a final concentration of either 50 or 100 g/mL. A fresh batch of water was prepared every week for each of the 8 or 16 wk of carcinogenic treatment. Normal autoclaved drinking water was resumed at the end of this period. Control mice not receiving 4-NQO were given water containing vehicle only. Treatment with ABT-510 ABT-510, a synthetic peptide that mimics the antiangiogenic activity of the naturally occurring protein thrombospondin-1, was provided by Abbott Laboratories. The peptide was dissolved in sterile Fig. 1. Chemical structure of ABT-510. ABT-510 is a nonapeptide derived from the antiangiogenic fragment of the second type 1 repeat of thrombospondin-1. The key structures in the synthesis and the chemical structure from which this figure was derived can be found in the original publication by Haviv et al. (23). Cancer Prev Res 2009;2(4) April 2009 386 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 2. Histopathology of 4-NQOinduced oral lesions in the mouse tongue. Photomicrographs show the histopathologic progression in this model system: histologically normal (control; A), hyperkeratosis (B), epithelial dysplasia (C), and squamous cell carcinoma (D). 5% dextrose, immediately filter sterilized, and stored at 4C. Mice receiving ABT-510 treatment were given a daily i.p. injection of 50 mg/kg body weight for the required duration of 4, 8, 12, 16, 20, or 24 wk. The route of administration and dosage given was determined based on previously published studies (2429). polymer-labeled horseradish peroxidasebound secondary reagent (EnVision+, DAKO). CD31 antigen retrieval was done using the Dako Target Retrieval System (pH 9.0) in a decloaking chamber. The primary antibody PECAM (Santa Cruz Biotechnology) was applied at 1:200 dilution in PBS for 1 h at room temperature. Antibody binding was visualized using the LSAB kit (DAKO). For determination of cell proliferation, sections were treated in ET buffer using decloaking chamber, incubating at 1:300 dilution using a Ki-67 antibody (NeoMarkers) for 1 h at room temperature. This was followed by anti-rabbit polymer-labeled horseradish peroxidasebound secondary reagent (EnVision+). All three immunohistochemistry stains were developed with 3,3-diaminobenzidine chromogen and counterstained with hematoxylin. Corresponding negative control experiments were done by omitting the incubation step with the primary antibody. Histologic examination Mice were sacrificed in accordance with Institutional Animal Care and Use Committee recommendations. Specifically, cervical dislocation was done after anesthesia by i.p. injection of xylazine and ketamine. Immediately following death, the tongues were excised, longitudinally bisected, and processed in 10% buffered formalin and embedded in paraffin. Fifty 5-m sections from each specimen were then cut and the 1st, 10th, 20th, 30th, 40th, and 50th slides were stained with H&E for histopathologic analysis. Histologic diagnoses were rendered using established criteria. Hyperkeratoses were characterized by a thickened keratinized layer, with or without a thickened spinous layer (acanthosis), and an absence of nuclear or cellular atypia. Dysplasias were characterized as lesions that showed various histopathologic alterations, including enlarged nuclei and cells, large and/or prominent nucleoli, increased nuclear to cytoplasmic ratio, hyperchromatic nuclei, dyskeratosis, increased and/or abnormal mitotic figures, bulbous or teardrop-shaped rete ridges, loss of polarity, and loss of typical epithelial cell cohesiveness. Because of the subjective nature of grading of epithelial dysplasia and its limited ability to predict biological progression (30, 31), we chose to not assign descriptive adjectives of severity to the dysplastic lesions. Rather, we grouped all lesions showing cytologic atypia but lacking evidence of invasion into the single category of dysplasia. HNSCCs were characterized by lesions that showed frank invasion into the underlying connective tissue stroma. Scoring of immunohistochemistry A combined scoring method that accounts for intensity of staining as well as percentage of cells stained was used for the evaluation of VEGF as previously described (32). Strong, moderate, weak, and negative staining intensities were scored as 3, 2, 1, and 0, respectively. For each of the intensity scores, the percentage of cells that stained at that level was estimated visually. The resulting combined score consisted of the sum of the percentage of stained cells multiplied by the intensity scores. For example, a case with 10% weak staining, 10% moderate staining, and 80% strong staining would be assigned a score of 270 (10 1 + 10 2 + 80 3 = 270) out of a possible score of 300. The determination of microvessel density (MVD) using CD31 as a marker was done as previously described (33). Briefly, using low-power magnification, the region containing the most intense area of tumor neovascularization was chosen for counting in each of the tumors. For the normal control tissue, MVD was determined by finding the most intense area of neovascularization directly below the overlying mucosa. Individual microvessels were counted using a 100 field (10 objective lens and 10 ocular lens). Any brown staining endothelial cells that were clearly separate in appearance were counted as individual vessels. Ten random fields within this hotspot area were viewed and counted at 100. Results were expressed as the total number of microvessels observed in the hotspot region of each individual tumor. For Ki-67, the labeling indices were determined by randomly analyzing at least 500 nuclei in 10 high-powered fields (400 magnification) for Immunohistochemistry Antigen retrieval was achieved on deparaffinized sections and endogenous peroxidase activity was quenched in 3% hydrogen peroxide and blocked in milk peroxidase. For vascular endothelial growth factor (VEGF) detection, slides were treated in ET buffer in a decloaking chamber and mouse primary antibody (Santa Cruz Biotechnology) was applied at 1:50 dilution in PBS for 1 h at room temperature. Antibody binding was visualized with anti-mouse www.aacrjournals.org 387 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research each tissue section. Labeling indices were expressed as a percentage of the total number of cells. quent deaths were observed in study A. At week 4 (n = 4), 75% of the tongues showed hyperkeratotic lesions, whereas 25% contained dysplasia. In the week 8 mice (n = 4), 25% had hyperkeratosis, 25% had dysplasia, and 50% had carcinoma. At 12 weeks (n = 4), 75% contained dysplasia and 25% had squamous cell carcinoma. Finally, by 24 weeks (n = 4), 25% showed dysplasia and 75% contained squamous cell carcinoma. Pilot study B (50 g/mL for 16 weeks) was terminated early because of an excessive number of deaths. Of the initial mice, 40% (8 of 20) died either during carcinogen treatment or within 6 weeks after the completion of the carcinogen treatment. In pilot studies C and D, mice were treated with 100 g/mL 4-NQO for either 8 weeks (pilot study C) or 16 weeks (pilot study D). In pilot study C (100 g/mL for 8 weeks), there were no deaths during the study. At week 4 (n = 5), 100% of animals showed hyperkeratotic lesions (Fig. 2B). At week 8 (n = 5), 60% showed hyperkeratosis and 40% contained epithelial dysplasia (Fig. 2C). By 12 weeks (n = 5), 100% of animals had dysplasia. Finally, at week 24 (n = 5), 40% had dysplasia and 60% showed squamous cell carcinoma (Fig. 2D). Similar to pilot study B, unacceptable mortality rates were observed in pilot study D after animals were treated with 100 g/mL for 16 weeks, resulting in the premature termination of this arm of the study as well. Although this was a relatively small sample size, these results showed that a sequential histologic progression could be observed using this type of carcinogenic induction protocol. It further suggested that the majority of the dysplastic and cancerous lesion were likely to be found starting at 12 weeks after carcinogen treatment. Therefore, to expand on the pilot studies, a cohort of 55 additional mice was treated with 100 g/mL 4-NQO for 8 weeks and tongues were subsequently harvested at weeks 16, 20, and 24. At week 16 (n = 20), 15% contained hyperkeratosis, 55% had dysplasia, and 30% showed squamous cell carcinoma. At 20 weeks (n = 15), 13% had hyperkeratosis, 60% contained dysplasia, and 23% showed squamous cell carcinoma. By 24 weeks after carcinogen treatment (n = 20), 25% of specimens contained dysplasia and 75% of the tissues showed squamous cell carcinoma. Collectively, the data for the 100 g/mL 4-NQO administered for 8 weeks show a reproducible timeline of histologic progression. Specifically, the data show that hyperkeratotic Data analysis For comparison of immunohistochemical scores across groups, ANOVA was done. A transformation of the data (square root or natural log) was used, as needed, to stabilize the variance across groups. If a significant overall difference was found by ANOVA, then pairwise comparisons were done with a Bonferroni adjustment for multiple comparisons. A test for trend was also done using ANOVA with the appropriate linear contrasts, and these results were confirmed using a nonparametric trend test as described by Cuzick (34). For comparison of ABT-510 treatment groups, Fisher's exact test was done by collapsing data across the six sacrifice times. All analyses were done using Stata version 10 (StataCorp). Results Histopathologic progression of 4-NQOtreated mice Articles describing the mouse 4-NQO model have typically reported the incidence of dysplasia and/or cancer at the end of the prescribed treatment protocols. However, these articles have not systematically characterized the sequential timing of histologic atypia development after carcinogen treatment over a protracted period of time. This is an important aspect of the model because a more thorough understanding of the histologic and molecular progression in this system is required if it is to be used to model oral premalignancy as well as preclinical chemoprevention studies. To carefully characterize the development of the histopathologic changes in this model, a series of pilot studies were done to identify the optimal carcinogen dosage and duration of exposure required to develop a predictable time line of progression. The animals were sacrificed at planned intervals after completion of carcinogen treatment, and their tongues were excised and examined histologically. No histopathologic changes were noted in the tongue mucosa from the control mice (Fig. 2A). Mice were treated with 50 g/mL 4-NQO for 8 weeks (pilot study A) and 16 weeks (pilot study B). They were subsequently randomly assigned to groups and sacrificed at 4, 8, 12, and 24 weeks after 4-NQO treatment. From the initial 20 animals in pilot study A (50 g/mL for 8 weeks), 4 mice died during treatment from undetermined causes. However, no subse- Fig. 3. Incidence of each histologic diagnosis of control and ABT-510 treatment groups at each sacrifice time. The number located at the top of each bar indicates the total sample size of each group. ABT-510 significantly decreased the incidence of HNSCC from 37.3% to 20.3% (P = 0.021) as well as the combined incidence of dysplasia and HNSCC from 82.7% to 50.6% (P < 0.001). Cancer Prev Res 2009;2(4) April 2009 388 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 4. Immunohistochemical staining of Ki-67 in the 4-NQO mouse model of HNSCC. A, tissue sections containing areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for Ki-67 and labeling indices were quantified (B and C). Ki-67 labeling indices in the specimens containing hyperkeratosis, dysplasia, and HNSCC were all significantly greater when compared with the normal mucosa (P < 0.001). to the mouse tongue epithelium from untreated control mice (n = 5) contained only occasional capillaries that were relatively evenly dispersed throughout the connective tissue stroma (Fig. 5A). However, the number and distribution of microvessels at the mucosal/connective tissue interface increased in the hyperplastic, dysplastic, and malignant mucosa (Fig. 5A). With increasing histologic atypia, the vessels were more densely packed directly adjacent to the basal cell layer. Quantification of MVD during histologic progression revealed a statistically significant difference (P < 0.001 by ANOVA) in CD31 scores when normal epithelium (mean SD: 23.2 4.3) was compared with hyperplasia (90.2 6.7), dysplasia (191.6 10.6), and squamous cell carcinoma (309.3 17.9). Pairwise comparisons indicated that each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Furthermore, there was a significant linear trend in CD31 levels across the four naturally ordered groups (i.e., increasing CD31 levels with increasing disease severity; P for trend < 0.001; Fig. 5B and C). lesions predominate at weeks 4 and 8, dysplasias are the most common diagnosis at weeks 12, 16, and 20, and carcinoma is the predominant histologic finding at week 24 (Fig. 3). Ki-67 Expression during histologic progression in 4NQOtreated mice Expression of Ki-67, a nuclear proliferation-associated antigen that is specific for cells in the active phases of the cell cycle, was determined via immunohistochemistry to quantify the relative proliferative rates of normal, hyperkeratotic, dysplastic, and malignant mouse tongue mucosa. Cells positive for Ki-67 expression showed distinct nuclear staining. In normal mucosa, Ki-67 expression was limited to basilar and occasional parabasilar cells (Fig. 4A), whereas greater suprabasilar labeling was observed with increasing histologic atypia (Fig. 4A). There was a statistically significant difference (P < 0.001 by ANOVA) in labeling indices when normal epithelium (mean SD: 16.4 3.0) was compared with hyperplasia (23.5 2.6), dysplasia (32.3 3.5), and squamous cell carcinoma (47.8 3.6). Subsequent pairwise comparisons indicated that each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Additionally, there was evidence for an increasing linear trend in Ki-67 levels across the four ordered groups (P for trend < 0.001; Fig. 4B and C). Expression of VEGF during the histologic progression in 4-NQOtreated mice Like all solid tumors, HNSCC must develop direct and indirect mechanisms to induce the production of new blood vessels. Several dozen candidate angiogenic molecules are produced by oral keratinocytes, and VEGF is an important angiogenic factor in both physiologic and pathologic settings, including HNSCC (35). Therefore, we did immunohistochemistry for VEGF to quantify its expression at various stages of histologic progression in the mouse 4-NQO model. Expression of VEGF by tongue keratinocytes from untreated control mice (n = 5) was rare and limited to the basilar and parabasilar cells (Fig. 6A). In addition, occasional stromal cells as well as endothelial lined vascular channels stained positively. The intensity of VEGF expression as well as the overall expression Increased MVD occurs before histologic atypia in 4NQOtreated mice The induction of blood vessel growth is an early phenotypic change in both human HNSCC as well as in the hamster buccal pouch and rat tongue models of oral carcinogenesis (811). To determine the timing of the expression of the angiogenic phenotype in the 4-NQO mouse model, we did immunohistochemistry for CD31 to quantify MVD as a surrogate for in vivo angiogenesis activity. The connective tissue stroma adjacent www.aacrjournals.org 389 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research of the cytokine by various keratinocytes present in the different layers of the epithelium increased in the hyperplastic, dysplastic, and malignant mucosa (Fig. 6A). Quantification of VEGF expression among histologic stages revealed a statistically significant difference (P < 0.001 by ANOVA) when normal epithelium (mean SD: 17.0 5.7) was compared with hyperplasia (70.6 14.0), dysplasia (144.9 35.4), and squamous cell carcinoma (237.6 41.4). Each group was significantly different from all other groups (Bonferroni adjusted P < 0.001 in all cases). Most importantly, there also was evidence for an increasing linear trend in VEGF levels with disease progression (P for trend < 0.001; Fig. 6B and C). normal, 40% contained hyperkeratosis, and 10% had dysplasia. At week 8 (n = 10), 10% were normal, 70% contained hyperkeratosis, and 20% showed dysplasia. By week 12 (n = 10), 10% were normal, 30% contained hyperkeratosis, and 60% had dysplasia. At week 16 (n = 12), 58% had hyperkeratosis, 25% had dysplasia, and 17% showed carcinoma. Progressing to week 20 (n = 16), 25% had hyperkeratosis, 25% had dysplasia, and 50% showed carcinoma. Finally, at week 24 (n = 21), 33% had hyperkeratosis, 38% had dysplasia, and 29% showed carcinoma. With respect to overall incidence of cancer, a 46% reduction in HNSCC was observed, with the 4-NQO group having an incidence rate of 37.3% and the ABT-510 treatment group having an incidence of 20.3% (P = 0.021). In addition, the combined incidence of dysplasia and HNSCC was 82.7% in the 4-NQO control group and 50.6% in the ABT-510 treatment group. This difference was highly statistically significant (P < 0.001). Effects of ABT-510 administration in 4-NQOtreated mice The expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human HNSCC (811), suggesting that inhibitors of angiogenesis may also hold promise as chemopreventive agents. However, to date, no pure inhibitors of angiogenesis have been tested for their ability to act as chemopreventive agents in HNSCC. Therefore, using ABT-510 as a proof-of-principle drug, we tested the hypothesis that inhibitors of angiogenesis can be used as chemopreventive agents in the realm of HNSCC. Based on our findings described above, mice were administered 4-NQO (100 g/mL) in their drinking water for 8 weeks. At the completion of this initiation phase, the mice were returned to normal water and given daily i.p. injections of ABT-510 (50 mg/kg/d) and sacrificed at regular intervals over the next 24 weeks. During the 24-week chemopreventive period, no significant differences in food or fluid consumption among the groups were observed. Similarly, there was no difference in body weight or activity between the control and the treatment group mice (data not shown). Data for the incidence of tongue lesions are shown in Fig. 3. At week 4 (n = 10), 50% of the tongues were histologically Discussion Animal models of HNSCC have traditionally used either 7,12-dimethylbenz(a)anthracene or 4-NQO as the carcinogenic agent. The induction of HNSCC using 4-NQO has been achieved in several different rodent species, including mice, hamsters, and rats, and has been generally found to be preferable for several reasons. First, in contrast to the topical application of 7,12-dimethylbenz(a)anthracene, 4-NQO can be delivered via the drinking water, thereby making the outcomes more predictable. Second, the molecular alterations induced in mouse mucosa by 4-NQO closely mimic the human disease. For example, similar to the human condition, epidermal growth factor receptors are overexpressed in this model (36). Similar to humans, altered expression of p53 as well as mutation of p53 have been shown in 4-NQO models (37). In addition, 4-NQO induces activating point mutations in the H-ras oncogenes (38). Although H-ras mutations are infrequent events in U.S. HNSCC (39), they are common in the Fig. 5. Expression of the angiogenic phenotype in the 4-NQO mouse model of HNSCC. A, tissue sections containing areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for CD31 and MVD was quantified (B and C). MVD in the specimens containing hyperkeratosis, dysplasia, and HNSCC was all significantly greater when compared with the normal mucosa (P < 0.001). Cancer Prev Res 2009;2(4) April 2009 390 www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Chemoprevention of Oral Cancer Fig. 6. Expression of VEGF in the 4-NQO mouse model of HNSCC. A, tissue sections containing representative areas of normal, hyperkeratosis, dysplasia, and squamous cell carcinoma immunohistochemically stained for VEGF and expression levels were quantified (B and C). Expression of VEGF in the specimens containing hyperkeratosis, dysplasia, and HNSCC was significantly greater when compared with normal mucosa (P < 0.001). opment of singular or occasionally synchronous oral lesions. This aspect was critical because we wanted to refine the model to ensure that the mice developed lesions in a fashion that was most similar to the human condition rather than forming innumerable ones. In addition, the prescribed dosage and timing of treatment resulted in a predictable histologic progression over the 24-week period of time. Specifically, the data show that after the completion of carcinogen treatment, the predominant histopathologic diagnoses are hyperkeratosis at weeks 4 and 8, dysplasia at weeks 12, 14, and 16, and HNSCC at week 24 (Fig. 3). Importantly, the establishment of the progression timeline in this model will allow for future studies aimed at investigating the molecular and biological alterations that occur during the premalignant phase of HNSCC as well as for the validation of potential diagnostic biomarkers. The induction of new blood vessel growth is a critical tumor phenotype in all malignancies, including HNSCC. There is considerable interest in determining how cells, progressing from normal to tumorigenic, make this switch. In some animal models, a distinct switch to the angiogenic phenotype is seen (44). In other cases, the cells developing into tumors sequentially become more angiogenic in a stepwise fashion (8, 45). Although the phenotype has been studied using animal models as well as human cells and tissue, the mechanisms about how this occurs in HNSCC are unknown. However, although the phenotypic changes in these models are similar to the human condition, the specific mechanisms involved in the induction of new blood vessel growth can be quite different. For example, although the major angiogenic factor in the hamster buccal pouch model is transforming growth factor (8), this growth factor has not been found to be a significant participant in the induction of angiogenesis in human HNSCC. Rather, a different subset of growth factors, including interleukin-8 and VEGF, seems to play predominant roles (35). As there were no data about the angiogenic phenotype in the 4NQO mouse model, we sought to determine the timing of the rest of the world (40). Therefore, because the majority of the 400,000 cases of HNSCC are outside of the United States/Europe and may therefore harbor H-ras mutations (in conjunction with epidermal growth factor receptor and p53 alterations), we believe that this is an excellent model from an experimental, histologic, and molecular perspective. Finally, 4-NQOinduced lesions develop in the absence of nonspecific inflammatory changes. This is a critical point because substances such as 7,12-dimethylbenz(a)anthracene can be significant irritants, resulting in chronic inflammation, necrosis, sloughing of tissue, and the formation of organizing granulation tissue (41). The etiology, type of inflammatory cell infiltrate, and therefore perhaps mechanisms in this type of injurious situation are likely to be different from what one sees in the human condition. Furthermore, these factors make it difficult to study premalignant lesions because inflammation itself can cause cytologic and/or morphologic changes that can be confused with dysplasia. Because 4-NQO treatment does not result in this type of injurious nonspecific inflammation, it is more likely to reflect the events that occur during human HNSCC. Although 4-NQO has been used as a carcinogenic agent to induce tumors in animal models since the 1950s, and in the oral cavity since the 1970s (42, 43), a thorough review of the literature failed to reveal studies that were specifically designed to characterize the histopathologic changes that develop over time in the 4-NQO mouse model. Furthermore, the route of administration, the amount of carcinogen used, and the duration of treatment schedules have been highly variable. Therefore, we sought to characterize the optimal dosage and timing of 4-NQO treatment and to establish a timeline for the reproducible development of lesions showing hyperkeratosis, dysplasia, and HNSCC. Using two different doses and two different durations of treatment, we determined that 100 g/mL in the drinking water for 8 weeks provided the most preferable results. This decision was based on the fact that this treatment protocol resulted in the devel- www.aacrjournals.org 391 Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 Cancer Prevention Research cumulative decrease of 39% in the incidence of both dysplasia and HNSCC between the control and ABT-510 treatment groups (82.7% versus 50.6%; P < 0.001), the rates being higher in the control groups at each time point except for week 4 where there was only one case of dysplasia. Overall, these cumulative data strongly support the contention that ABT-510 was effective at decreasing the incidence of dysplasia and HNSCC in the mouse 4-NQO model of oral carcinogenesis. The integration of inhibitors of angiogenesis into the treatment regimens of various diseases is increasing in frequency (47, 48). However, although there has been considerable discussion about the potential role of antiangiogenic agents in the area of chemoprevention, there are limited data to support the role of this class of drugs in this clinical setting (49, 50). ABT-510 is a mimetic peptide of thrombospondin-1 and acts as an inhibitor of angiogenesis by modulating the ability of endothelial cells to respond to various angiogenic factors. Specifically, ABT-510 binds to CD36, thereby inducing caspase-8 mediated endothelial cell apoptosis. This mechanism has been shown to block angiogenesis in vitro and in vivo as well as slow tumor growth in preclinical studies (2429). Further, it has been tested clinically for the treatment of inflammatory bowel disease and in cancer therapy as a single agent or in combination with chemotherapeutic agents (5155). Because it directly abrogates the ability of endothelial cells to respond to angiogenic factors, rather than altering the expression of these factors by tumor and/or stromal cells, one does not typically observe the direct modulation of various angiogenic factors in tumor cells. In keeping with these previous observations, we did not observe an alteration in the expression of VEGF by the oral keratinocytes in the treatment group animals (data not shown). However, the nearly 2-fold decrease in the incidence of HNSCC in the ABT-510 group at 24 weeks shows that the drug has a potent effect in vivo in this animal model. One of the long-term goals of chemoprevention must be the development of treatments that can be easily taken by at-risk individuals for prolonged periods of time with minimal side affects to achieve widespread acceptance and long-term compliance. This would be particularly important in the case of high-risk patients who have not yet developed their first malignancy. As such, because ABT-510 is not an orally administered agent, it is unlikely that it would be found acceptable in its current form. Nonetheless, our proof-of-principle findings support the hypothesis that inhibitors of angiogenesis may have activity as chemopreventive agents. Furthermore, many of the chemopreventive agents currently under investigation, such as epidermal growth factor receptor tyrosine kinase inhibitors and cyclooxygenase-2 inhibitors, have multiple activities, including the inhibition of angiogenesis. However, the toxicities observed at the current prescribed dosages may preclude them from being used widely as chemopreventive agents. Therefore, the combination of one or both of these agents at lower doses in concert with other antiangiogenic agents may reduce toxicities and improve efficacy. The data presented here show proof of principle that the induction of new blood vessel growth by premalignant cells may be one such phenotype that could be targeted in a chemoprevention cocktail. appearance of this phenotype as well as identify the angiogenic factors that might be involved. The determination of MVD in tissue sections, using endothelial cell markers such as factor VIII, CD31, and/or CD34, is an accepted surrogate marker for in vivo angiogenesis. In the 4-NQOtreated animals, we observed a statistically significant increase in MVD as early as the hyperkeratotic stage, thereby showing that, much like other models as well as the human condition, the expression of the angiogenic phenotype is an early phenotypic change (Fig. 6). Furthermore, we observed sequentially increasing levels of VEGF expression at the stages of hyperkeratosis, dysplasia, and HNSCC (Fig. 6B and C). The mechanisms for this sequential increase are not known at this time. However, one could hypothesize that the progressive increase in VEGF expression might in part be the result of potential tumor-stroma paracrine interactions (46). The design of the study did not allow us to functionally validate whether VEGF was the key angiogenic factor produced in this animal model. However, the concordant increased expression of both MVD and VEGF supports the hypothesis that VEGF plays a central role in the expression of the angiogenic phenotype in the mouse 4-NQO model. We have shown that daily treatment with ABT-510 for 24 weeks resulted in limited toxicity and significantly decreased the incidence of dysplasias and carcinomas in the mouse 4-NQO model of HNSCC (Fig. 3). The rationale for the 24-week treatment schedule was based on the observed cancer incidence and histologic diagnosis in our preliminary studies. As a result of these findings, animals were subsequently sacrificed at the same regular intervals over the 24-week time course to synchronize the treatment arm results with the initial histologic observations. Specifically, we observed most HNSCC (72%) at week 24, whereas dysplasia was the predominant histologic diagnosis at weeks 16 (55%) and 20 (60%). Because we were testing the hypothesis that ABT-510 would reduce the incidence of HNSCC, we believe it was most appropriate to carry out the prevention study to a time point where the predominant histologic diagnosis in the control group would be expected to be HNSCC. Overall, we observed a 46% cumulative reduction in the incidence of HNSCC between the control and treatment groups (37.3% versus 20.3%; P = 0.021). We did observe a small increase of HNSCC in the treatment group at week 20 when compared with the 20-week control group. However, this difference was not statistically significant (P = 0.273). The reasons for this observation are unknown but may be the result of uneven cohort sizes between the control and treatment groups within and at different sacrifice points. For example, the week 20 treatment group contained fewer animals (n = 16) than some of the other time points, such as week 24 (n = 21). The differences in cohort sizes at the given time points were a reflection of the fact that our initial experiments determined that week 24 should be the major cancer end point of this study. As such, although we were interested in synchronizing the treatment arm results with the histologic studies, we also felt it was most important to have the largest n at the major end point of the study. Further, it should be noted that the 16- and 24-week time points showed significant differences in the incidence of HNSCC between the control and treatment groups. 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Hoekstra R, de Vos FY, Eskens FA, et al. Phase I study of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 with 5-fluorouracil and leucovorin: a safe combination. Eur J Cancer 2006; 42:46772. 55. Hoekstra R, de Vos FY, Eskens FA, et al. Phase I safety, pharmacokinetic, and pharmacodynamic study of the thrombospondin-1-mimetic angiogenesis inhibitor ABT-510 in patients with advanced cancer. J Clin Oncol 2005;23:518897. Cancer Prev Res 2009;2(4) April 2009 Downloaded from cancerpreventionresearch.aacrjournals.org on March 8, 2021. 2009 American Association for Cancer Research. Published OnlineFirst March 31, 2009; DOI: 10.1158/1940-6207.CAPR-08-0211 ABT-510 Is an Effective Chemopreventive Agent in the Mouse 4-Nitroquinoline 1-Oxide Model of Oral Carcinogenesis Rifat Hasina, Leslie E. Martin, Kristen Kasza, et al. Cancer Prev Res 2009;2:385-393. Published OnlineFirst March 31, 2009. 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... Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 RESEARCH ARTICLE Open Access Identification and functional analysis of NOL7 nuclear and nucleolar localization signals Guolin Zhou, Colleen L Doi, Mark W Lingen* Abstract Background: NOL7 is a candidate tumor suppressor that localizes to a chromosomal region 6p23. This locus is frequently lost in a number of malignancies, and consistent loss of NOL7 through loss of heterozygosity and decreased mRNA and protein expression has been observed in tumors and cell lines. Reintroduction of NOL7 into cells resulted in significant suppression of in vivo tumor growth and modulation of the angiogenic phenotype. Further, NOL7 was observed to localize to the nucleus and nucleolus of cells. However, the mechanisms regulating its subcellular localization have not been elucidated. Results: An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using sequence homology and prediction algorithms, four putative nuclear localization signals (NLSs) were identified. NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins confirmed the functionality of three of these NLSs. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the minimal functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The nucleolar occupancy of NOL7 revealed a complex balance of rapid nucleoplasmic shuttling but low nucleolar mobility, suggesting NOL7 may play functional roles in both compartments. In support, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7. Conclusions: These results identify the minimal sequences required for the active targeting of NOL7 to the nucleus and nucleolus. Further, this work characterizes the relative contribution of each sequence to NOL7 nuclear and nucleolar dynamics, the subnuclear constituents that participate in this targeting, and suggests a functional role for NOL7 in both compartments. Taken together, these results identify the requisite protein domains for NOL7 localization, the kinetics that drive this targeting, and suggest NOL7 may function in both the nucleus and nucleolus. Background NOL7 is a predicted 29 kDa, 257 amino acid protein with no significant homologies to other characterized proteins that localizes to the nucleus and nucleoli of cells. NOL7 localizes to 6p23, a region with frequent loss of heterozygosity (LOH) in a number of cancers, including hormone-refractory breast carcinoma, leukemia, lymphoma, osteosarcoma, retinoblastoma, nasopharyngeal carcinoma and cervical cancer (CC) [1-19]. Using CC as a model for investigation, where LOH of * Correspondence: Mark.Lingen@uchospitals.edu Contributed equally Departments of Pathology, Medicine and Radiation and Cellular Oncology, The University of Chicago, Chicago, IL, USA 6p23 is the most common allelic loss in this neoplasm [20-25], we demonstrated that reintroduction of NOL7 suppresses in vivo tumor growth by 95% [26]. This suppression is due in part to the induction of an anti-angiogenic phenotype via decreased expression of the angiogenic factor Vascular Endothelial Growth Factor (VEGF) and increased expression of the inhibitor of angiogenesis Thrombospondin-1 (TSP-1). One of the important features that differentiate eukaryotic from prokaryotic cells is the presence of intracellularly distinct compartments and organelles such as the nucleus, nucleolus and mitochondria. The rapid exchange of proteins between the cytoplasm and the nucleus is a vital process in eukaryotic cells, and this 2010 Zhou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 occurs through the nuclear pore complex (NPC), a large macromolecular structure embedded in the double membrane of the nuclear envelope [27-29]. Small molecules such as ions and some small proteins can move from the cytoplasm to the nucleus by passive diffusion through the NPC. However, proteins larger than ~20 kDa typically cross the NPC in a carrier-mediated fashion [30]. This active nuclear transport of proteins is mediated by specific amino acids sequences, which are referred to as nuclear localization signals (NLS) and nuclear export signals (NESs). The classical NLSs contain a cluster of basic amino acids, while classical NESs contain stretches of hydrophobic, leucine-rich residues. The best-described nuclear import pathway is driven by the so-called classical NLS (cNLS). This signal is typically lysines (K) or arginines (R), that are organized as a single-stretch monopartite NLS: (K/R) 4-6 , or as a bipartite NLS in which there are two small clusters typically separated by ten to twelve amino acids (K/R)2X10-12(KR)3. The SV40 large-T antigen (PKKKRKV) and nucleoplasmin (KRPAATKKAGQAKKKK) cNLS are the prototypical mono- and bipartite cNLS [31,32]. Recently, a tripartite NLS, consisting of three clusters of basic residues separated by two spacer peptides, has also been described [33-38]. Finally, a fourth type of NLS contains two to four dispersed basic residues contiguous to hydrophobic amino acids [39,40]. The active transport of proteins between the cytoplasm and nucleus is facilitated by the karyopherin/importin family of carrier proteins. During classical nuclear import, NLSs are typically recognized in the cytoplasm by a heterodimeric complex consisting of importin a and b with the a-subunit providing the NLS binding site. The NLS protein-receptor complex docks to the nuclear pore complex via importin b and is subsequently translocated through the pore by an energydependent mechanism [41]. Once the import complex reaches the nucleus, it is dissociated by RanGTP. Binding of RanGTP to importin b can cause a conformational change, resulting in the release of the importin a/cargo complex [42]. Recent modeling studies have shown that some cargo proteins can also bind directly to b -karyopherins [43]. While active transport mechanisms are required for nuclear localization, targeting to the nucleolus has been shown to depend on interactions with nucleolar constituents. Nucleolar localization sequences (NoLSs) have been shown to represent binding domains with resident nucleolar proteins, rRNA, and other nucleolar components and function more as retention rather than targeting signals [44-50]. The affinity and stability of these interactions among nucleolar proteins has been shown to have functional consequences that are reflected in the dynamics of their nucleolar localization. Many ribosomal proteins show higher immobility and slower recovery Page 2 of 20 compared to processing and transcriptional factors, and these differences have been attributed to the stability and duration of their nucleolar functions [51,52]. Mutations, truncations, and changes in posttranslational modifications that have functional consequences have also been shown to affect the immobility and recovery of classic nucleolar proteins such as NPM [53]. Together, these observations suggest the nucleolar localization is a factor of the affinity, stability, and abundance of nucleolar interactions and that the dynamics of nucleolar occupancy are a reflection of potential functions within the nucleus and nucleolus. The purpose of this work was to determine the mechanism by which NOL7 was transported into the nucleus, identify the minimal functional sequences required for NOL7 nuclear translocation, and the relative influence each of these sequences may have on the rate and efficiency of localization. We further wished to define the elements responsible for the nucleolar localization of NOL7 and characterize the dynamics of this targeting. Together, the aim of this paper was to define the mechanism responsible for localization of NOL7 within the cell and the functional consequences of the different components that comprise that mechanism. Results NOL7 is imported into the nucleus via an energydependent, nucleoporin-mediated mechanism NOL7 is predicted to have a molecular weight of approximately 29 kDa. Some proteins of this size have been reported to enter the nucleus through passive diffusion, while others require active NLS-mediated transport. To distinguish between an active transport and a passive diffusion mechanism of nuclear localization for NOL7, we subjected GFP-tagged NOL7 to an in vitro transport assay using permeabilized HeLa cells [54]. In this assay, the cytoplasmic membranes are permeabilized with digitonin, which depletes the cells of their soluble endogenous cytosolic factors while leaving the nuclear membrane intact. In this fashion, the nuclear import of GFP-tagged proteins can be studied under various conditions. In these experiments, digitonin-permeabilized HeLa cells were incubated with GFP-tagged NOL7 under various conditions to assay the mechanism of its localization. NOL7-GFP alone was not sufficient for nuclear import (Figure 1, first panel). Addition of cytosolic extract or ATP alone was also insufficient for transport (Figure 1, second and third panels), suggesting that NOL7 localization is energy and complex dependent. At 4C or in the presence of heat-inactivated cytosol, NOL7-GFP was restricted to the cytoplasm (Figure 1, fourth and fifth panels), demonstrating that NOL7 requires cytosolic proteins and is localized via an active transport mechanism. At physiologic temperature, Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 3 of 20 Figure 1 NOL7 requires cytosolic factors for efficient nuclear localization. HeLa cells permeabilized with digitonin were incubated at 4C or 37C as indicated with (+) or without (-) full length NOL7 expressing a C-terminal GFP tag, cytosol, heat-inactivated cytosol, ATP, or WGA. Localization of NOL7 was confirmed by visualization of GFP. combination of cytosol and ATP was sufficient for NOL7 nuclear localization (Figure 1, sixth panel). However, this was blocked by pretreatment with wheat germ agglutinin (WGA) (Figure 1, seventh panel). Together, these controls demonstrate that NOL7 is actively targeted to the nucleus and nucleolus via an energy-dependent, nucleoporin-mediated mechanism. Protein prediction programs identify distinct biochemical domains and putative NLSs in NOL7 Database analysis of the full length sequence of human NOL7, utilizing the protein domain prediction programs PSORT [55], TargetP [56], SAPS [57] and NetNES [58], identified five distinct biochemical domains and four putative NLSs within NOL7 (Figure 2). SAPS analysis revealed the existence of four highly basic regions and one acidic domain which correlates with its high pI of 9.7. Stretches of highly basic residues have been shown to participate in nucleic acid binding, nuclear transport, and may contribute to the tumor suppressive function and localization of NOL7 (Figure 2A). While NOL7 lacks homology to other known proteins and domains, the significant sequence conservation among its orthologs suggests a consistent evolutionary role. In particular, four long stretches of basic amino acids are particularly conserved throughout evolution (Figure 2B). Not coincidently, the prediction programs also identified potential NLSs within these basic biochemical domains. However, NOL7 was not predicted to contain any NESs. Putative Sequence (PS) 1, amino acids 1-10 appeared to be a monopartite NLS, while PS2, amino acids 88-112 was predicted to be an example of the recently described tripartite NLS. PS3, amino acids 144-162 appeared to be bipartite NLS and PS4, amino acids 242257, was predicted to be a bipartite NLS (Figure 2B). Analysis of potential sequence conservation of each of the four putative NOL7 NLSs among its orthologs was performed using BLAST and aligned by the ClustalW method (Figure 2B). Significant evolutionary conservation between mammalian species Homo sapiens, Pan troglodytes, Macaca mulatta, Bos taurus, Canis familiaris, Rattus norvegicus, and Mus musculus was observed not only for the NLS regions for the full-length protein as well. Less striking but still significant homology was also seen in Gallus gallus, Tetraodon nigroviridis, Danio rerio, and Saccharomyces cerevisiae. While some stretches of the full-length protein showed typical divergence of the sequence, the basic stretches of residues that comprised the putative NLSs were remarkably conserved. In particular, PS1, which is composed of three basic residues contiguous to hydrophobic amino acids, showed highly significant homology between all of the NOL7 orthologs. This suggests that PS1 may target NOL7 to the nucleus despite its dispersed amino acid sequence. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 4 of 20 Figure 2 NOL7 is composed of distinct biochemical domains and multiple putative NLSs that show evolutionary conservation. (A) Multiple sequential analysis programs confirmed the existence of four basic (blue) and one acidic (green) region in the full-length sequence of NOL7. Putative NLSs identified in sequence analysis programs are shown in red. (B) Sequence conservation between human NOL7 and its putative orthologs was analyzed for each of the putative NLSs and the alignment is shown. Black shaded boxes indicate identical amino acid conservation, while grey boxes signify similar amino acids to Homo sapiens. Numbers correspond to residues within the RefSeq sequences listed in the Materials and Methods 2.7. Interestingly, no homology could be detected between NOL7 and any other proteins or domains. Further, despite the significant conservation between human NOL7 and its putative orthologs, no functional role can be extrapolated for NOL7. With the exception of S. cerevisiae ortholog Bud21, no functional data exists for any of these proteins. In the case of the putative yeast ortholog, initial studies suggest that the U3 snoRNA function of Bud21 is not conserved, as NOL7 is incapable of interacting with major Bud21 cofactors that regulate its activity (unpublished data). Further, no similarity could be detected between NOL7 and other characterized Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 proteins of any species, either in the context of the fulllength NOL7 or for shorter stretches of the protein. Taken together, this data suggests that NOL7 is a critical protein in higher eukaryotes that may function in a specialized manner. Furthermore, the correlation between the regions of strongest sequence conservation and predicted NLSs suggests the localization and function of NOL7 may be linked. NOL7 contains three functional NLSs that translocate cytoplasmic PK into the nucleus In order to determine the functionality of the four putative NLSs, we first generated a series of N- and C- terminal deletion mutants of NOL7 with an HA tag that were transiently expressed in HeLa and 293T cells (Figure 3A). The subcellular localization of the truncations was visualized by immunofluorescence with DAPI costaining of the nucleus. While the majority of the deletion constructs retained nuclear localization, a deletion construct lacking all four PSs [1-10, 88-257] was cytoplasmic (Figure 3B). Further, PS1 and PS2 were shown to function individually as NLSs, as constructs missing PS2, PS3, and PS4 [88-257] or PS1, PS3, and PS4 [1-10, 113-257] were nuclear localized. While these results demonstrated that PS1 and PS2 were sufficient to target NOL7 to the nucleus, we sought to further clarify the role of PS3 and PS4. Therefore, five additional truncations were cloned together with inactivating mutations in the region of PS1 [PS1 (-)]. PS4 was found to be sufficient for nuclear localization of NOL7 [PS1(-),88-216 and PS1(-),88-241] but a construct containing only an intact PS3 [PS1(-),88112,217-257] remained in the cytoplasm, suggesting that the putative sequence of PS3 is not a functional NLS. To specifically determine if the three candidate NLSs were sufficient to target proteins for nuclear import and further confirm that PS3 was not a functional NLS, the localization of a series of fusion constructs containing each putative NLS sequence and the cytoplasmic protein PK was evaluated. PS1, 2, 3 and 4 were cloned in frame with the C- terminus of the PK bearing an N-terminal myc tag and transiently transfected into HeLa cells (Figure 4A). The subcellular localization of the chimeric proteins was visualized using an a-myc monoclonal antibody and Cy3-conjugated secondary, with DAPI costaining for visualization of the nucleus. Both wild-type Myc-tagged PK protein and the PS3-PK fusion were seen exclusively in the cytoplasm (Figure 4B). In contrast, PK-PS1, PKPS2, and PK-PS4 localized predominately to the nucleus (Figure 4B). Taken together, these results from the truncation and PS-PK fusion experiments confirm that PS3 is not a functional NLS while PS1, PS2, and PS4 are functional nuclear localization signals. Furthermore, the data Page 5 of 20 demonstrate that each NLS is capable of translocating a cytoplasmic protein into the nucleus independently. From this point forward, we will therefore refer to PS1, PS2, and PS4 as NLS1, NLS2, and NLS3, respectively. Identification of the amino acids required for functionality of each NLS The deletion and PK fusion constructs demonstrated the regions within NOL7 that are individually capable of driving nuclear localization, but do not define the specifc sequence elements that comprise the individual NLSs. To specifically define the minimal amino acids required for functionality of each NLS, site-directed mutagenesis was performed to convert the basic amino acids of interest (arginine and lysine) to the nonpolar, electrically neutral amino acid alanine in each of the NLSs. Within each sequence, three individual basic amino acids (NLS1) or clusters of basic amino acids (NLS2 and NLS3) were identified and mutated individually to determine their relative contribution to the functionality of the NLS. Each intact NLS and the mutant NLSs were cloned in-frame with PK bearing an N-terminal myc tag (Figure 5A). As before, fusion with all three wild type NLSs resulted in nuclear localization of PK (Figure 5B-D). Mutation of any one of the three basic residues of NLS1 abolished nuclear localization, suggesting that each of these amino acids is critical for functionality of NLS1 (Figure 5B). For NLS2, loss of the second or third cluster of basic residues resulted in cytoplasmic localization, while loss of the first basic cluster had no effect on nuclear localization of PK (Figure 5C). These mutations suggest that the minimal region required for nuclear localization directed by NLS2 resides within residues 95-112. Consistent with our predictions, both basic clusters of NLS3 were required for nuclear localization (Figure 5D), suggesting that NLS1 is a monopartite sequence while both NLS2 and NLS3 are bipartite sequences. Taken together, these data confirm the functionality of the NLSs and define the specific amino acids present in each of the individual NLS that are required for the nuclear import of PK. While experiments using individual NLS fused to PK are useful, there are several limitations to these types of studies. For example, Burgess et al [59] demonstrated that EBNA3B has three functional NLSs when investigated in truncation experiments but only two were found to be functional in the context of the full-length protein. To determine the contributions of each NLS within full-length GFP-tagged NOL7, the arginine and lysine residues in each NLS were mutated to alanine (Figure 6A). The subcellular localization of the constructs was visualized using by GFP fluorescence, with DAPI costaining of the nucleus. Mutation of all three Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 6 of 20 Figure 3 NOL7 contains three separate NLSs that are necessary for nuclear localization. (A) Schematic representation of deletion constructs of NOL7 used to determine which regions of NOL7 are required for nuclear localization. Results as demonstrated in (B) are summarized in the column to the right, where No designates nucleolar localization, Np designates nucleoplasmic localization, and C designates cytoplasmic localization. (B) Localization of the constructs was confirmed in HeLa cells by immunofluorescence using an a-HA primary and FITC-coupled secondary antibody. NLSs resulted in cytoplasmic localization of NOL7 but retention of only one NLS was sufficient for nuclear localization (Figure 6B). Taken together, these results demonstrate that NOL7 has three functional NLS that can independently cause translocation of full length NOL7. Each NLS contributes differentially to the rate and efficiency of NOL7 nuclear import While each NLS was shown to be independently capable of directing nuclear transport of NOL7, it was unclear what the relative contribution of each signal to this function might be. To address this question, we Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 A Page 7 of 20 PK-WT Myc PS1 Myc PK PS2 Myc PK PS3 Myc PK PS3 C PS4 Myc PK PS4 Np B PK-WT PK PS1 C PS1 PS2 PS2 PS3 Np Np PS4 DAPI a-Myc Merge Figure 4 NOL7 contains three NLSs that are sufficient for nuclear localization. (A) Schematic representing the three different NLSs cloned in-frame with the cytoplasmic protein PK bearing a c-myc tag. Results demonstrated in (B) are summarized in the column to the right, where Np designates nucleoplasmic localization and C designates cytoplasmic localization. (B) Localization of the constructs in Hela cells was confirmed by immunofluorescence using an a-myc primary and Cy-3 conjugated secondary antibody. Costaining of the nucleus with DAPI is shown in blue. determined the rate and efficiency of nuclear import for each NLS construct. While fluorescence recovery after photobleaching (FRAP) has been employed previously for measuring the rate of import, this method is limited to small bleaching areas and measures a combination of active nuclear import and nucleoplasmic diffusion, the magnitude of which can vary greatly between proteins [60-62]. In most cases, the nuclear diffusion can be considered equivalent among different constructs of the same protein. However, subnuclear targeting of proteins within the nucleus affects their nuclear diffusion and can no longer be discounted in the calculation of rate by FRAP. As mutant constructs of NOL7 are differentially localized to the nucleoplasm and nucleolus, a different approach needed to be applied to investigate the role of different NLSs in the rate of nuclear import that would not be influenced by mobility within the nuclear compartment. Therefore, two complementary methods were adapted to measure both rate and efficiency of NOL7 nuclear import, based on quantitative immunofluorescence methods previously established in the literature [63-67]. In both cases, HA-tagged NOL7 constructs were transfected into HeLa cells and imaged by immunofluorescence against the HA tag. Using ImageJ software, the fluorescence intensity was measured and reported as a ratio of nuclear to total fluorescence. While previous reports typically utilize the nuclear to cytoplasmic ratio, we normalized to total cell fluorescence to accommodate differences in subnuclear localization and expression level between the different mutant constructs. For the efficiency experiments, data was collected twenty hours after transfection, when import had reached steady-state equilibrium (Figure 7A). It was found that WT NOL7 was most efficiently localized to the nucleus, and the strictly nucleoplasmic mutant N23(-), was least efficiently targeted. The single mutants demonstrated nearly 10% more efficient nuclear targeting than the double mutants, with a p-value of 2.1310-7. The most dramatic loss in targeting efficiency was observed upon the Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 8 of 20 Figure 5 Basic residues within each of the NLSs are required for nuclear localization of PK. (A) Schematic representing the three different NLSs bearing neutralizing mutations in the basic residues were cloned in-frame with the cytoplasmic protein PK bearing a c-Myc tag. Results demonstrated in (B-D) are summarized in the column to the right, where Np designates nucleoplasmic localization and C designates cytoplasmic localization. (BD) Subcellular localization was determined by immunofluorescence in HeLa cells using an a-myc primary and either a HRP or Cy-3 conjugated secondary antibody. Costaining of the nucleus with DAPI is shown in blue. (B) Mutations in either the first (NLS1-sub1), second (NLS1-sub2), or third (NLS1-sub3) basic cluster of NLS1 were mutated in alanine and visualized for nuclear localization. (C) Expression and localization of NLS2 mutants lacking either the first (NLS2-sub1), second (NLS2-sub2), or third (NLS2-sub3) basic cluster. (D) Expression and localization of the constructs bearing mutations in the first (NLS3-sub1) or second (NLS3-sub2) basic cluster of NLS3. combined loss of NLS2 and NLS3, with over a 15% decrease in efficiency for N23(-) compared to WT NOL7 and over 10% decrease compared to all other mutants. This decrease was highly significant, with a p-value of 9.4510 -9 compared to WT NOL7 and 2.6810 -5 and 1.2410-4 compared to the other double mutants N12(-) and N13(-), respectively. Together, these observations suggest that NLS2 and NLS3 are the major sequences involved in the efficient targeting of NOL7 to the nucleus. To determine the rate of nuclear import, a similar approach was used, this time measuring the relative nuclear fluorescence intensity over a four-hour time course. To ensure that this rate represented strictly nuclear import, the increase in fluorescence intensity was measured prior to the establishment of steady-state NOL7 levels. To determine the time frame for measurement, we transfected cells with GFP-tagged wild type NOL7 and measured the time posttransfection when fluorescent signal can first be detected until it accumulates to a steady-state level. It was determined that NOL7 protein is detectable 5 hours posttransfection, and its accumulation reaches steady-state levels approximately 10 hours after transfection (Additional file 1: Supplementary Movie 1). Therefore, the rate was calculated as the change in fluorescence intensity at 5, 6, 7, and 8 hours posttransfection (Figure 7B). Loss of even one NLS had a significant effect on the change in rate, regardless of identity. The single NLS mutants had a significantly higher rate of import than the double mutants (4.94 0.34 versus 3.05 0.40, p = 6.1910-8). The most dramatic decrease was observed with the N23(-) mutant, which was imported at a rate nearly 80% less than WT NOL7, with a p-value of 6.1310-17. This suggests that each NLS plays a unique Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 9 of 20 Figure 6 Basic residues within each of the NLSs are required for nuclear localization of full-length NOL7. (A) Schematic representing the different mutant constructs used to evaluate nuclear localization in the context of the full length protein. Results demonstrated in (B) are summarized in the column on the right, where No designates nucleolar localization, Np designates nucleoplasmic localization, and C designates cytoplasmic localization. (B) Localization of the GFP-tagged constructs in HeLa cells was confirmed by fluorescent microscopy and costaining of the nucleus with DAPI is shown in blue. role in the targeting of NOL7. Together with efficiency, this suggests that NLS2 and NLS3 in combination are critical for efficient and rapid targeting of NOL7 to the nucleus. NLS2 and NLS3 of NOL7 comprise domains that are required for nucleolar localization It has been previously demonstrated that NOL7 localizes to the nucleolus via colocalization with the nucleolar protein NPM [26]. In the analysis of the NLSs, it was noted that loss of NLS2 and NLS3 together abolished the nucleolar but not nuclear localization of NOL7 (Figure 6B). Interestingly, NLS23(-) also significantly decreased both the rate and efficiency of NOL7 nuclear transport (Figure 7A and 7B). To identify the possible NoLS(s) within these signals, systematic mutation of the basic residues within NLS2 or NLS3 was undertaken (Figure 8A). Restoration of any basic cluster in NLS2 or in NLS3 was sufficient to restore nucleolar localization, suggesting that these regions are capable of individually functioning as NoLSs (Figure 8B). Thus, NOL7 contains at least four separate NoLSs within its nuclear targeting sequences that are individually capable of directing nucleolar localization. NOL7 demonstrates rapid recovery but low mobility within the nucleolus Protein occupancy and complex assembly in subnuclear bodies has been shown to relate to function for a majority of proteins [68-70]. Therefore, the nucleolar occupancy of NOL7 was evaluated by FRAP. The occupancy was described by the recovery half life (t1/2) and mobile fraction (Mf) of GFP-fusion constructs. In order to define an upper and lower limit for nucleolar protein mobility, NOL7 was evaluated with the controls NCL, a freely diffusing nuclear/nucleolar shuttle with functions in both the Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 10 of 20 Figure 7 Each NLS contributes differently to the rate and efficiency of NOL7 nuclear localization. The steady-state efficiency and rate of import for NLS mutants was evaluated to determine their relative contribution to the subcellular localization of NOL7 in HeLa cells. (A) Twenty hours after transfection, mutants were imaged by immunofluorescence against the HA tags and costained with DAPI and WGA to delineate the nucleus and cytoplasm. Using ImageJ, the nuclear-to-total cell fluorescence ratio was calculated for twenty cells per construct. Error bars represent standard error. (B) Cells were transfected with the different NOL7 NLS constructs and imaged at 5, 6, 7, and 8 hours post-transfection. The nuclear accumulation was measured by a-HA immunofluorescence and the rates were calculated as the change in nuclear signal over time. Bars represent the average rate for ten cells and error bars are representative of the standard error. nucleus and nucleolus, and RPS5, a low-mobility resident nucleolar protein (Figure 9A). These proteins represent typical controls within the literature and allow for comparison to other dynamic studies [71]. The t1/2 of NOL7 was found to be most similar to a shuttling protein such as NCL, suggesting that NOL7 can freely exchange with the nucleoplasm (Figure 9B). Conversely, the immobile fraction (Mf) of NOL7 was found to be most similar to an immobile, complexed nucleolar protein like RPS5 (Figure 9B). This is consistent with previous reports describing the nucleolar occupation of a number of nucleolar proteins, including NPM, NCL, and RPS5 [71,72]. This suggests that a large pool of nucleolar NOL7 is functionally occupied in a nucleolar complex, while the free protein is able to shuttle rapidly between subnuclear compartments. Compared to literature reports, this data indicates that NOL7 is most similar to proteins with multiple nuclear and nucleolar roles like NPM, which is both a nucleolar shuttle and associates in functional nucleolar complexes, than either NCL or RPS5. Further, these data suggest that NOL7 shuttles between the nucleolus and nucleoplasm and may play a functional role in both compartments. NOL7 localization is dynamically regulated by changes in RNA composition The shuttling of NOL7 between the nucleus and nucleolus suggested that specific interactions within these compartments may regulate the nucleolar occupancy of Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 11 of 20 Figure 8 Basic residues within NLS2 and NLS3 are required for nucleolar localization of NOL7. Basic residues within each of the NLSs are required for nucleolar localization of full length NOL7. A) Schematic representation of the different mutant constructs used to evaluate nucleolar localization. Results demonstrated in (B) are summarized in the column on the right, where No designates nucleolar localization and Np designates nucleoplasmic localization. B) Localization of the constructs in HeLa cells was confirmed by GFP visualization. Costaining of the nucleus with DAPI is shown in blue. NOL7. Due to the highly basic nature of the protein, it was hypothesized that subnuclear localization of NOL7 may be due to interactions with nucleic acids. To investigate, various cell treatments were employed to change the abundance of different nucleic acid species (Figure 10). Cells overexpressing GFP-tagged NOL7 were treated with RNase, DNase, actinomycin D (ActD), or a-amanitin and visualized by fluorescence microscopy for changes in subcellular localization. RNase treatment resulted in nucleolar loss and nucleoplasmic accumulation of NOL7, while cells treated with DNase did not show any significant change. Culture of mammalian cells in low doses of ActD selectively inhibit rRNA synthesis while having no effect on tRNA, 5S rRNA, nuclear RNA and mRNA synthesis [73,74]. Similarly, treatment with low doses of a-amanitin inhibits RNAPII and subsequent mRNA synthesis without affecting the abundances of other RNA species. Loss of these specific RNA species has been shown to selectively deplete their RNA-binding protein counterparts from different cellular compartments, enabling visualization of binding activities that may participate in protein localization [75-77]. Upon treatment with ActD, NOL7 was found to translocate to the nucleoplasm. Upon treatment with Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 12 of 20 Figure 9 FRAP analysis of NOL7 nucleolar occupancy demonstrates rapid recovery but low mobility within the nucleolus. (A) The fluorescence recovery within the nucleolus was measured over time for HeLa cells transfected with GFP-tagged NOL7, the high mobility shuttle NCL, and the low mobility resident protein RPS5. Measurements represent thirteen different cells per protein. The curves were fit to the line curve F(t) = F(1-et). (B) The nucleolar occupancy was plotted as a function of recovery versus mobility. The mobile fraction was used as a measurement of free versus complexed protein within the nucleolus and calculated from the regression values in (A) using the formula Mf = (F-F0)/(Fi-F0). The half-time to maximal recovery was calculated using the formula t1/2 = ln(0.5)/ and used as a measurement of shuttling between the nucleolus and nucleoplasm. All error bars represent the standard error of the measurement. a-amanitin, no change in the nucleolar localization of NOL7 was observed. However, the nucleoplasmic localization of NOL7 previously observed was absent. This data suggests that targeting of NOL7 to both the nucleus and nucleolus results in multiple RNA-dependent interactions. Discussion Active nuclear transport involves complex interactions between the transport machinery and protein cargo, mediated in part through NLSs. Typically composed of discrete patterns of basic residues, these sequences are recognized by the transport machinery and can vary in their affinity, rate, and efficiency of localization, which in turn can influence the function and biologic relevance of the cargo protein in different physiologic settings. Here, we have shown that NOL7 is targeted to the nucleus via an energy- and nucleoporin-dependent mechanism. This transport is mediated by three evolutionarily conserved but distinct NLSs. In addition, each NLS was found to be independently capable of directing the nuclear localization of the cytoplasmic protein PK or full length NOL7. Each NLS individually and additively contributed to the rate and efficiency of NOL7 nuclear targeting, suggesting that each of the NLSs has differential effects in driving the localization kinetics, likely reflecting differences in the regulation of import. Taken together, these data indicate that NOL7 localization is tightly regulated and may contribute to functions in various cellular compartments. Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 Page 13 of 20 Figure 10 NOL7 subnuclear localization is dynamically regulated by changes in RNA composition. 293T cells were stably transfected with NOL7-GFP and treated with RNase A (100 g/ml, 2 hours), DNase I (100 g/ml, 2 hours), actinomycin D (0.05 g/ml, 4 hours), or a-amanitin (50 g/ml, 4 hours) to specifically deplete individual nucleic acid species. Treatment with DNase (total DNA), RNase (total RNA), ActD (rRNA), or aamanitin (mRNA) was performed and localization of NOL7 was confirmed by fluorescent microscopy of the GFP tag. The transport of proteins and RNAs into the nucleus occurs through the NPC and is an important step in regulating the subcellular location of a number of different proteins, including transcription factors, signalling proteins, and various enzymes. Although alternative mechanisms exist, the classic nuclear import pathway appears to be the predominate method of transport into the nucleus. A recent survey of Saccharomyces cerevisiae screened over 5800 genomic sequences and found that 45% contained classic NLSs and nearly 60% of nuclear proteins contained monopartite or bipartite sequences [78]. This is likely true across species, as a number of studies have found that the nuclear transport machinery for essential proteins is highly conserved between Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 animals, yeast and plants [30-32]. This observation is certainly true for the three NLSs present in NOL7, where sequence alignment of the three NLSs demonstrated significant evolutionary conservation and aided in the identification of putative targeting sequences. It further suggested that targeting may play a significant role in the regulation and function of NOL7, as these sequences were highly conserved across species but demonstrated little similarity to other proteins or domains. NOL7s three functional NLSs are located in the N terminus, middle, and C terminus of the protein. While a single functional NLS is sufficient for most proteins, the presence of multiple functional NLSs is seen frequently among proteins whose function is critically determined by its localization. Proteins such as p53 [79], E2F1 [80], c-Abl [81], p14 ARF , HPV E6 [82], BRCA2 [83], most ribosomal proteins including RPS7 [84,85], b-myb [86], ATF2/c-jun heterodimer [87], PAK-1 [88] and others have been demonstrated to contain more than one NLS. Interestingly, many of these proteins are also implicated in cancer, and aberrant or mislocalized protein plays a significant role in the development and progression of the disease. As such, nuclear localization, and the rate and efficiency at which it occurs, has been shown to have many downstream functional consequences for proteins [89-92]. Terry, et al, have proposed a hierarchical regulation to classical nuclear transport via NLSs, with multiple mechanisms acting at the level of the cargo, receptors, and NPC [93]. The existence of multiple NLSs within a single protein may therefore provide a mechanism to exploit these different targeting controls for proteins whose nuclear localization is critical for function [93]. The first level of regulation involves the NPC, and the permeability, stability, and expression of the proteins that comprise this complex can affect the efficiency and targeting of cargo. The existence of multiple NLSs within NOL7 may therefore be used to achieve nuclear localization despite cellular conditions where NPC is less accessible. The next level of regulation involves the transport receptors. Here, differing accessibility, affinity, competition, and expression of the importins in various cell types and under different cellular conditions can affect transport [40,94-99]. In this case, the existence of multiple NLSs can increase likelihood of transporter interaction regardless of environment, coordinate for better efficiency and rate of localization, or outcompete other NLS-bearing proteins for these receptors. Indeed, combined loss of NLS2 and NLS3 significantly impact both the rate and efficiency of NOL7 localization, and the presence of more than one NLS results in a statistically significant increase in NOL7 nuclear accumulation (Figure 7). Finally, at the Page 14 of 20 level of the cargo, modifications and interactions of the cargo protein itself regulates its own localization. Inter- and intramolecular interactions can provide or preclude access to NLSs, and modifications within NLSs can also affect transport, either inhibiting or promoting import to the nucleus [79,100]. The differential rate and efficiency of localization observed among NOL7 mutants, particularly in the N23(-) mutant, suggests each NLS may participate in different levels of this regulation. In addition, many NLSs have also been shown to harbor subnuclear targeting sequences such as NoLSs. NoLSs typically represent interaction motifs between nucleolar constituents, making nucleolar localization a dynamic, multidirectional process compared to nuclear targeting [44-50,101,102]. Our results have shown that NLS2 and NLS3 include four NoLSs. These sequences are composed of basic clusters and each is capable of individually driving nucleolar localization of NOL7. Whether these regions represent unique binding domains or are functionally redundant to ensure efficient interaction with nucleolar cofactors is unknown at this time. Investigation of the nucleolar occupancy of proteins under various cellular conditions has demonstrated that the kinetics are often highly similar for functionally related proteins [75]. In particular, FRAP analysis of the recovery and mobility of proteins within this compartment has been shown to reflect their functional roles. The nucleolar mobility is typically viewed as a reflection of the stability of the interactions and size of the interaction complex within that compartment, while the recovery reflects the shuttling characteristics of a protein between the nucleoplasm and nucleolus. While many ribosomal proteins are highly immobile within the nucleolus, proteins such as NCL, UBF, and NPM have higher mobility and rapid recovery, due to their multiple functional roles in the nucleus and nucleolus [60,62,71,72]. FRAP analysis of the nucleolar occupancy of NOL7 demonstrates that a large fraction of nucleolar NOL7 is involved in a relatively stable complex, as evidenced by its small Mf. Interestingly, free NOL7 protein rapidly shuttles between compartments. These dynamics, with low Mf and high t1/2, have been demonstrated in the literature to be unique to proteins that functionally interact with ribonucleoproteins (RNPs) in both the nucleus and nucleolus such as NPM [60-62,68-72]. Together, this suggests that NOL7 may interact in RNP complexes in both compartments. Further support for the potential nuclear and nucleolar interactions of NOL7 can be observed by the changes in localization for NOL7 upon specific depletion of nucleic acid species. The pattern of NOL7 expression is significantly altered by loss of RNA but not loss of DNA, suggesting that NOL7 is an RNA-associated protein, either Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 directly or through RNP complexes. Further, changes in rRNA and mRNA abundance affected the abundance of NOL7 in the nucleolus and nucleoplasm, respectively, suggesting that NOL7 may be participating in distinct functional complexes within each compartment. Whether this is a direct effect of rRNA and mRNA interaction, or an indirect consequence of changes in the transcriptome of the cell remains to be investigated. However, together these observations indicate that the RNA abundance within the cell can influence the localization of NOL7 protein, and the dynamics of this localization is similar to the kinetics of proteins that play functional roles in nuclear and nucleolar RNP complexes. While it is unknown what, if any, function NOL7 may have in either compartment, it suggests that its localization is actively regulated and this differential targeting may influence its role in cancer development and progression. Localization and function within multiple cellular compartments has previously been observed for many proteins. In addition, regulation of protein function through localization mechanisms is known to be employed in multiple cancer signaling pathways, including the Wnt, TGFb, and Hh pathways. Oncogenes and tumor suppressors such as Rb, c-Myc, p53, VHL, and p14Arf have multiple, different functions depending on their localization or sequestration [103-117]. Our evidence suggests that like many of these oncogenes and tumor suppressors, NOL7 may have be regulated through its subcellular localization, and its targeting may be critically linked to its tumor suppressive activity. Conclusions In summary, we have found that NOL7 requires cytosolic proteins for active transport into the nucleus, consistent with a classical import mechanism. We have identified three functional NLSs within NOL7, each of which is independently capable of directing the nuclear localization of the cytoplasmic protein PK or full length NOL7 and contribute to different degrees to the rate and efficiency of NOL7 nuclear import. Further, these sequences harbor at least four NoLSs that are independently capable of mediating nucleolar localization. The nucleolar occupancy of NOL7 is balanced by its rapid recovery and low mobility, similar to other proteins that play multiple functional roles in both the nucleus and nucleolus. Further, the nucleolar localization of NOL7 is dependent upon the presence of rRNA, while the nucleoplasmic localization of NOL7 is mediated by the abundance of mRNA. This work provides the basis for further investigation into the levels, activity, and mechanism of regulation for NOL7 and elucidation of its role in tumor growth suppression. Page 15 of 20 Methods Deletion Mutant Constructs Constructs were cloned as described. Template and primer sequences are listed in Additional file 2: Supplementary Table 1. NOL7 Deletion Constructs Each deletion construct was cloned by PCR and inserted into the pcDNA3.1/Hygro(+) vector (Invitrogen, Carlsbad CA). For the mutants, residues were mutated using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla CA). NLS-PK Fusion Proteins Myc-tagged chicken muscle PK expression DNA was obtained from Gideon Dreyfuss (University of Pennsylvania, Howard Hughes Medical Institute) [118]. It was cloned in frame with individual NLSs to create fusion constructs. GFP-fusion Constructs Fusion constructs were generated using the GFP Fusion TOPO TA Cloning kit (Invitrogen, Carlsbad CA). Briefly, the full-length NOL7 DNA fragments were TOPO cloned into the plasmid vector pcDNA3.1/NTGFP-TOPO, and the cloning reaction was transformed into chemically competent cells provided in the kit. The plasmids were purified with Qiagen Plasmid Mini kit (Qiagen, Valencia CA), and sequenced for verification of insert orientation. Mutation of individual residues within the NLSs were constructed by using Quikchange XL site-directed mutagenesis kit (Stratagene, La Jolla CA). NOL7-GFP Purification Construct For nuclear import assays, NOL7-GFP was cloned with tandem C-terminal GFP-V5-His6 tags using the Gatweway cloning system from Invitrogen (Carlsbad, CA). All TOPO and LR cloning reactions were performed as described by the manufacturer. First, wild-type NOL7 was PCR amplified and TOPO cloned into pENTR-SDD-TOPO. The pENTR-NOL7 construct was transferred to the pcDNA-DEST47 vector, resulting in a C-terminal GFP tag. The NOL7-GFP fusion was PCR amplified and TOPO cloned into the pENTR-SD-D-TOPO vector and this time transferred to the pcDNA-DEST40 vector, thereby expressing NOL7 in frame with a tandem Cterminal GFP-V5-His6 tag. Tissue Culture HeLa cells were grown in minimum essential medium supplemented with 10% fetal bovine serum (FBS), 100 g/ml penicillin and streptomycin. 293T cells were grown in DMEM supplemented with 10% FBS, 100 g/ ml penicillin and streptomycin. Transfections for HeLa, and 293T cells were done using Lipofectin following the manufacturers directions (Invitrogen, Carlsbad CA) in 75 mm2 dishes when the cells were approximately 80% Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 confluent. Five hours after addition of the DNA precipitate, cells were washed and refed with minimum essential medium or Dubeccos Modified Essential media plus 10% FBS. For stable cell lines, cells were selected in 400 g/ml G418 (Invitrogen, Carlsbad CA) for three weeks. For transient expression experiments, cell extracts were prepared 20-36 hours after transfection. Immunofluorescence For immunofluorescence staining, cells were plated on 4-well chamber slides and were transfected using Lipofectin for HeLa and 293T cells according to the manufacturers instructions. Expression of all constructs was validated by western blot. Cells were fixed and stained as previously described [119] thirty-six hours post-transfection unless otherwise stated. Immunostaining was performed using the following primary antibodies: Rabbit a-HA (Invitrogen, Carlsbad CA), 1:4000; Mouse a-cMyc (Ab-1) (Calbiochem, Gibbstown NJ), 1:500. Secondary antibodies were fluorescein isothiocyanate (FITC) AffiniPure F(ab) 2 Fragment Goat Anti-Rabbit IgG (H +L) (Jackson ImmunoResearch Labs, West Grove PA), 1:500; Cy3 AffiniPure F(ab)2 Fragment Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Labs, West Grove PA), 1:500. Cells were mounted in DAPI-containing media (Vector Labs, Burlingame CA) according to the manufacturers instructions. WGA staining (Invitrogen, Carlsbad CA) was performed at a concentration of 5.0 g/ml according to the manufacturers instructions. Protein Purification 293T cells were transfected with the NOL7-GFP purification construct and positive clones were selected and maintained as described. For purification, approximately 1 10 8 cells were collected by trypsinization and washed twice with ice cold PBS. Cells were pelleted by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate, pH 7.4; 300 mM NaCl; 1% Triton X100; Roche Complete EDTA-free protease inhibitor tablet). Cell pellets were sonicated 6 30 s at 30% power. Lysates were then cleared by centrifugation and the supernatant was collected and filtered through a 0.45 m filter. Size-exclusion chromatography was performed using a 0.7 cm 50 cm Econo-column (BioRad, Hercules CA) that was packed with 5-100 kDA polyacrylamide beads (Bio-gel P-100, 45-90 M, BioRad, Hercules CA) according to manufacturers instructions. Fractions of approximately 300 l were collected and tested for the presence of NOL7 by SDS-PAGE followed by silver staining and western blot using mouse a-V5 monoclonal antibody (Invitrogen, Carlsbad CA). Positive fractions were further purified by affinity chromatography against the His6 tag of NOL7 using the ProPur IMAC Kit (Nunc, Rochester NY) under native Page 16 of 20 conditions with 30 mM imidazole washes. The column was washed five times and five elution fractions were collected. NOL7-containing fractions were verified by silver stain and western blot against the V5 tag of NOL7. Positive fractions were concentrated and dialyzed against transport buffer (20 mM Hepes-KOH, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 1 mM EGTA, Roche complete mini protease inhibitor) for use in the import assay. Preparation of Cytosol Fractions Exponentially growing cultures of HeLa cells were collected by low speed centrifugation and washed twice with cold PBS, pH 7.4, by resuspension and centrifugation. The cells were then washed with 10 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT and pelleted. The cell pellet was gently resuspended in 1.5 volumes of lysis buffer (5 mM Hepes, pH 7.3, 10 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 20 M cytochalasin B, 1 mM PMSF, and 1 g/ml each aprotinin, leupeptin, and pepstatin) and swelled for 10 min on ice. The cells were lysed with a homogenizer. The resulting homogenates were centrifuged at 1,500g for 15 min to remove nuclei and cell debris. The supernatants were then sequentially centrifuged at 15,000g for 20 min and 100,000g for 30 min. The final supernatants were dialyzed against transport buffer (20 mM HEPES, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, and 1 g/ml each aprotinin, leupeptin, and pepstatin) and frozen in aliquots in liquid nitrogen before storage at -80C. Cell Permeabilization and In Vitro Transport Assay Import assays was performed essentially as previously described [54]. Cells plated on 4-well chamber slides were rinsed in cold transport buffer (20 mM Hepes, pH 7.3, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM DTT, 1.0 mM EGTA, and 1 g/ml each aprotinin, leupeptin, and pepstain). Wells were immersed in ice cold transport buffer containing 40 g/ml digitonin (Calbiochem, Gibbstown NJ). The cells were allowed to permeabilize for 5 min, after which the digitonin-containing buffer was removed and replaced with cold transport buffer. For each assay, 150 l of transport buffer was supplemented with 10 g/ml NOL7-GFP and incubated for 30 minutes at either 37C or 4C. Where indicated, assays were supplemented with 1 mM ATP and 15 mg/ml cytosol. For heat-inactivated cytosol, extracts were boiled at 95C for 5 min, chilled, and then added to the import assay. For WGA treatment, cells were pre-incubated with 50 ug/ml WGA for 15 minutes at 20C, washed, and then the import assay was performed as described above. After the 30 min incubation, Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 all slides were washed, fixed with 4.0% paraformaldehyde and analyzed directly by fluorescence microscopy. Protein analysis, Domain prediction, and sequence alignment Protein analysis of NOL7 was carried out using a variety of prediction programs on the following accession sequences: Homo sapiens, NP_057251.2; Pan troglodytes, XP_518245.2; Macaca mulatta, XP_001092572.1; Bos taurus, NP_001029556.1; Canis familiaris, XP_535892.2; Rattus norvegicus, XP_573999.2; Mus musculus, NP_076043.2; Gallus gallus, XP_418926.1; Tetraodon nigroviridis, CAF97792.1; Danio rerio, XP_687281.1; Saccharomyces cerevisiae, NP_014721.1. Alignment of sequences was doing using MegAlign software under the ClustalW parameters. Page 17 of 20 Transport Rate Experiments HeLa cells were fixed and stained with rabbit a-HA primary (Invitrogen, Carlsbad CA, 1:4000 dilution) and FITC AffiniPure F(ab)2 Fragment Goat Anti-Rabbit IgG (H+L) secondary (Jackson ImmunoResearch Labs, West Grove PA, 1:500 dilution) as described 5, 6, 7, and 8 hours after transfection. Immunofluorescent images were captured by using Zeiss Axiovert 200 M microscope system. Image analysis was performed using Image J to quantify the nuclear staining intensity per unit of area. Ten high power fields were selected for analysis of each construct. The rate of import was calculated as the slope of the fluorescent intensity versus time and the statistical significance of this data was evaluated using Students t-test. Drug treatment and fluorescence microscopy FRAP Photobleaching, Imaging, and Quantitation Approximately forty-eight hours after transfection, HeLa cells were maintained in MEM supplemented with 30 mM Hepes, pH 7.1, to stabilize the pH of the medium during imaging. FRAP was performed on a DM4000 microscope (Leica Microsystems, Wetzlar Germany) equipped with a MicroPoint Laser System (Photonic Instruments, St. Charles, IL), a Roper Coolsnap HQ camera (Princeton Instruments, Trenton NJ), and a Leica 63X HCX PL APO L U-V-I aqueous immersion objective (Molecular Devices, Sunnyvale CA). Fluorescence intensity was measured using Metamorph imaging software (Universal Imaging Corp, West Chester PA). The average intensities of the areas of interest, including before, immediately after, and a series of time points after bleaching, were measured under the same condition for each data set. Data was analyzed using SigmaPlot software and fit to the curve F(t) = F(1-et). From the regression values, the half-maximal recovery [t1/2 = ln (0.5)/] and mobile fraction [M f = (F -F 0 )/(F i -F 0 )] were calculated for each replicate and statistical significance was determined using Students t-test. Transport Efficiency Experiments HeLa cells were fixed and stained with rabbit a-HA primary (Invitrogen, Carlsbad CA, 1:4000 dilution) and FITC AffiniPure F(ab)2 Fragment Goat Anti-Rabbit IgG (H+L) secondary (Jackson ImmunoResearch Labs, West Grove PA, 1:500 dilution) as described twenty hours after transfection. Immunofluorescent images were captured by using Zeiss Axiovert 200 M microscope system. Image analysis was performed using Image J to quantify per unit area staining intensity in the total cell and nucleus. Twenty high power fields were selected for analysis of each stain. The efficiency was calculated as the ratio of nuclear to total intensity and Statistics were evaluated using Students t-test. HeLa cells were stably transfected with wild-type NOL7GFP and plated on 2-well chamber slides. When cells were approximately 70% confluent, media was replaced with serum-free DMEM containing 0.05 g/ml actinomycin D (Sigma-Aldrich, St. Louis MO) or 50 g/ml aamanitin (Sigma-Aldrich, St. Louis MO) and incubated for 4 hours at 37C in 5% CO 2. Cells were then fixed with 4% paraformaldehyde, washed with PBS, and mounted with DAPI-containing media (Vector Labs, Burlingame CA). For nuclease treatment, cells were first washed with PBS and permeabilized with ice-cold methanol for 10 min and then incubated with 100 g/ ml RNase A (Sigma-Aldrich, St. Louis MO) or 100 g/ ml DNase I (Sigma-Aldrich, St. Louis MO) at 37C for 2 hours. Cells were then fixed and mounted in the same manner. All cells were imaged on a Zeiss Axioplan microscope. Additional material Additional file 1: Supplementary Movie 1 - Detection of NOL7-GFP Posttransfection. HeLa cells were transfected with WT NOL7 in frame with a GFP fusion tag and imaged every fifteen minutes from the time fluorescent signal can first be detected, approximately five hours after transfection, until it reaches steady-state intensity, approximately nine hours after transfection. Time zero corresponds to five hours posttransfection. Additional file 2: Supplementary Table 1 - Primers used to clone the constructs used in this study. Each construct is listed, along with the forward and reverse PCR primers and template for cloning PCR reaction able legend text Abbreviations NLS(s): nuclear localization signal(s); CC: cervical cancer; HA: hemagglutinin; PK: pyruvate kinase; CHX: cycloheximide; VEGF: vascular endothelial growth factor; TSP-1: thrombospondin-1; NPM: nucleophosmin; NCL: nucleolin; RPS5: ribosomal protein 5; ribonucleoprotein: RNP; NPC: nuclear pore comples; NES: nuclear export signal; cNLS: classical nuclear localization signal; NoLS: nucleolar localization signal; WGA: wheat germ agglutinin; FBS: fetal bovine Zhou et al. BMC Cell Biology 2010, 11:74 http://www.biomedcentral.com/1471-2121/11/74 serum; FITC: fluorescein isothiocyanate; FRAP: (fluorescence recovery after photobleaching). Acknowledgements We would like to thank Dr. Jerrold Turner for his assistance with the FRAP experiments. We would also like to acknowledge Dr. Vytas Bindokas for his advice regarding the nuclear transport rate and efficiency experiments. This work was supported in part by Illinois Department of Public Health Penny Severns Cancer Research Fund and the National Institutes for Health grant DE00470 (MWL). Authors contributions GZ performed plasmid construction, localization immunofluorescence, import assays, transport rate and efficiency experiments, and FRAP. CLD performed plasmid construction, sequence analysis, protein purification, drug treatment fluorescent microscopy, statistical analysis, and participated in the design of the study and manuscript preparation. MWL conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Received: 22 April 2010 Accepted: 27 September 2010 Published: 27 September 2010 References 1. 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... Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Cancer Prevention Research Research Article Dual Inhibition of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor is an Effective Chemopreventive Strategy in the Mouse 4-NQO Model of Oral Carcinogenesis Guolin Zhou1, Rifat Hasina1, Kristen Wroblewski2, Tanmayi P. Mankame1, Colleen L. Doi1, and Mark W. Lingen1 Abstract Despite recent therapeutic advances, several factors, including field cancerization, have limited improvements in long-term survival for oral squamous cell carcinoma (OSCC). Therefore, comprehensive treatment plans must include improved chemopreventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we tested the hypothesis that ZD6474 (Vandetanib, ZACTIMA) is an effective chemopreventive agent. CBA mice were fed 4-NQO (100 g/mL) in their drinking water for 8 weeks and then randomized to no treatment or oral ZD6474 (25 mg/kg/d) for 24 weeks. The percentage of animals with OSCC was significantly different between the two groups (71% in control and 12% in the ZD6474 group; P 0.001). The percentage of mice with dysplasia or OSCC was significantly different (96% in the control and 28% in the ZD6474 group; P 0.001). Proliferation and microvessel density scores were significantly decreased in the ZD6474 group (P 0.001 for both). Although proliferation and microvessel density increased with histologic progression in control and treatment cohorts, epidermal growth factor receptor and vascular endothelial growth factor receptor-2 phosphorylation was decreased in the treatment group for each histologic diagnosis, including mice harboring tumors. OSCC from ZD6474-treated mice exhibited features of epithelial to mesenchymal transition, as shown by loss E-cadherin and gain of vimentin protein expression. These data suggest that ZD6474 holds promise as an OSCC chemopreventive agent. They further suggest that acquired resistance to ZD6474 may be mediated by the expression of an epithelial to mesenchymal transition phenotype. Finally, the data suggests that this model is a useful preclinical platform to investigate the mechanisms of acquired resistance in the chemopreventive setting. Cancer Prev Res; 3(11); 1493502. 2010 AACR. Introduction With an annual incidence of nearly 600,000 cases, oral and pharyngeal squamous cell carcinoma is the sixth most common malignancy in the world today (1). There will be over 35,000 new cases in the United States in 2010 with nearly 8,000 deaths from the disease (2). When focusing specifically on the oral cavity squamous cell carcinoma (OSCC), it is estimated that there will be over 23,000 new cases and more than 5,300 deaths (3). Despite advances in diagnosis and treatment, improved long-term survival for OSCC patients has remained modest. Several factors contribute to this relatively poor outcome. First, OSCC is often diagnosed at an advanced stage. The 5-year Authors' Affiliations: Departments of 1 Pathology, Medicine, and Radiation and Cellular Oncology, and 2Health Studies, The University of Chicago, Chicago, Illinois Corresponding Author: Mark W. Lingen, Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, MC 6101, Chicago, IL 60637. Phone: 773-702-5548; Fax: 773-702-9903; E-mail: mark. lingen@uchospitals.edu. doi: 10.1158/1940-6207.CAPR-10-0135 2010 American Association for Cancer Research. survival rate of early stage disease is approximately 80%, although the survival drops to approximately 20% for late stage disease (2). Second, as a result of field cancerization, the development of multiple primary tumors has a major effect on survival. For patients with early stage disease, second primary tumors are their most common cause of treatment failure and death (4, 5). Therefore, to improve outcomes, a comprehensive treatment plan must include both improved early detection and secondary prevention. Chemoprevention can be defined as the use of natural or synthetic agents to reverse or halt the progression of premalignant lesions. Chemopreventive agents are currently being tested for their efficacy in preclinical and clinical settings for many malignancies including OSCC (6, 7). However, initial promising results for OSCC chemoprevention have not been consistently reproduced and toxicity has often been a significant complication. The issue of toxicity is particularly important in the realm of chemoprevention as it is conceivable that patients may require therapy for prolonged periods of time. Angiogenesis is an essential phenotype in both physiologic and pathologic settings including growth and development, www.aacrjournals.org Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1493 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. wound healing, reproduction, arthritis, and tumor formation (8). Because of its critical role in cancer biology, the inhibition of tumor angiogenesis is an attractive target for cancer therapy. The induction of the angiogenic phenotype in OSCC is mediated by the direct and indirect production of various factors capable of inducing blood vessel growth (9). Among these, the vascular endothelial growth factor (VEGF) family is thought to play an important role. The biological effects of the VEGF ligands are mediated through their binding to members of the VEGF receptor family (VEGFR-1, VEGFR-2, and VEGFR-3). This interaction leads to the autophosphorylation of specific tyrosine residues and subsequent downstream activation of intracellular signaling pathways, such as the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways. Importantly, the expression of the angiogenic phenotype is one of the first recognizable phenotypic changes observed in both experimental models as well as in human OSCC (1013), suggesting that inhibitors of angiogenesis may also hold promise in the field of chemoprevention. The development, growth, and survival of OSCC are also highly dependent on the epidermal growth factor receptor (EGFR) signaling pathway. EGFR is a transmembrane glycoprotein that is a member of the ErB/HER receptor tyrosine kinase family. Upon ligand binding, EGFR signaling is mediated by the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt pathways. Increased expression of EGFR and its ligand transforming growth factor- (TGF-) are observed in most OSCC and premalignant oral lesions, and this expression correlates with poor prognosis (14). In addition to directly influencing tumor cell growth, members of the EGFR pathway can contribute to the expression of the angiogenic phenotype. For example, the expression of either TGF- or EGFR results in increased expression of VEGF (15, 16). Because of its importance in epithelial malignancies, there is considerable interest in targeting the EGFR pathway in the realm of chemoprevention. ZD6474 (Vandetanib, ZACTIMA) is an orally available tyrosine kinase inhibitor with direct activity against multiple signal transduction pathways including VEGFR-2 and EGFR (1719). ZD6474 has an IC50 of 0.04 mol/L for VEGFR-2 and an IC50 of 0.5 mol/L for EGFR (18, 20). In preclinical studies, ZD6474 was found to be a potent inhibitor of tumor angiogenesis and the proliferation of a number of different tumor cell types including OSCC xenografts (2130). Furthermore, it is currently under active investigation in clinical trials for the treatment of various malignant neoplasms (31). To date, it has been found to have greatest activity in nonsmall cell lung cancer and recurrent medullary thyroid cancer (3234). However, the clinical utility of this agent in the realm of chemoprevention, particularly for OSCC, is unknown. Because it has the potential to inhibit two pathways that are essential for the development of OSCC, we tested the hypothesis that ZD6474 is an effective chemopreventive agent in the 4-NQO model. 1494 Cancer Prev Res; 3(11) November 2010 Materials and Methods Administration of 4-NQO and treatment with ZD6474 CBA mice, 6 to 8 weeks of age, were purchased from The Jackson Laboratory and housed in the Animal Resource Facility under controlled conditions and fed normal diet and autoclaved water. All animal procedures were carried out in accordance with Institutional Animal Care and Use Committeeapproved protocols. Mice were given 4-NQO in their drinking water on a continuous basis at the required dose for the required duration as previously described (35). Briefly, 4-NQO powder (Sigma) was first dissolved in DMSO at 50 mg/mL as a stock solution and stored at 20C until used. On the days of 4-NQO administration, the stock solution was dissolved in propylene glycol (Sigma) and added to the drinking water bottles containing autoclaved tap water to obtain a final concentration of 100 g/mL. A fresh batch of water was prepared every week for each of the 8 weeks of carcinogenic treatment. Normal autoclaved drinking water was resumed at the end of this period. Control mice not receiving 4-NQO were given water containing vehicle only. ZD6474 was provided by Astra Zeneca and dissolved in Tween 80 solution (P8192-5X10ML, Sigma). Mice receiving ZD6474 treatment were given a daily dosage of 25 mg/kg/d for 24 weeks via oral gavage. Histologic examination Mice were sacrificed in accordance with Institutional Animal Care and Use Committee recommendations. Specifically, cervical dislocation was done subsequent to anesthesia by i.p. injection of xylazine and ketamine. Immediately following death, the tongues were excised, longitudinally bisected, and processed in 10% buffered formalin and embedded in paraffin. Fifty 5-m sections from each specimen were then cut and the 1st, 10th, 20th, 30th, 40th, and 50th slides were stained with H&E for histopathologic analysis. Histologic diagnoses were rendered as previously described (35). Briefly, hyperkeratoses were characterized by a thickened keratinized layer, with or without a thickened spinous layer (acanthosis), and an absence of nuclear or cellular atypia. Dysplasias were characterized as lesions that showed various histopathologic alterations including enlarged nuclei and cells, large and/or prominent nucleoli, increased nuclear to cytoplasmic ratio, hyperchromatic nuclei, dyskeratosis, increased and/or abnormal mitotic figures, bulbous or teardrop-shaped rete ridges, loss of polarity, and loss of typical epithelial cell cohesiveness. Because of the subjective nature of grading of epithelial dysplasia and its limited ability to predict biological progression (36, 37), we chose not to assign descriptive adjectives of severity to the dysplastic lesions. Rather, we grouped all lesions demonstrating cytologic atypia but lacking evidence of invasion into the single category of dysplasia. HNSCC were characterized by lesions that showed frank invasion into the underlying connective tissue stroma. Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer Immunohistochemistry For detection of phosphorylated EGFR (pEGFR) and phosphorylated VEGFR-2 (pVEGFR-2), antigen retrieval was achieved on deparaffinized 5 m sections using Immuno/ DNA retriever with citrate (Bio SB). Endogenous peroxidase activity was quenched with mouse/rabbit ImmunoDetector Peroxidase Block Kit. Sections were incubated using primary antibody to pVEGFR-2 1:300 (Abcam) or pEGFR 1:250 (Cell Signaling) for 1 hour at room temperature. Antibody binding was visualized by using mouse/rabbit ImmunoDetector HRP/DAB Detection System (Bio SB). For detection of CD31 and vimentin, antigen retrieval was achieved by using 10 mmol/L of citrate buffer (pH 6.0) on 5 m deparaffinized sections. Endogenous peroxidase activity was quenched with 1% hydrogen/methanol. The primary antibody for vimentin (Epitomics) was applied at 1:250 dilution for a 1-hour incubation at room temperature. For CD31 (Abcam), a 1:50 dilution was applied, followed by anti-rabbit polymer-labeled horseradish peroxidase (HRP)bound secondary reagent (DAKO EnVision+ System, HRP). For detection of E-cadherin and Ki67, antigen retrieval was achieved on 5-m deparaffinized sections using 10 mmol/L of Tris-base and 1 mmol/L of EDTA (pH 9.0). Endogenous peroxidase activity was quenched with 1% hydrogen peroxide/methanol. The primary antibody for E-cadherin (Zymed) was applied at a 1:25 dilution for 1 hour at room temperature. This was followed by antirabbit polymer-labeled HRP-bound secondary reagent (DAKO, EnVision+ System, HRP). For Ki67 (NeoMarkers), sections were incubated at a 1:300 dilution at room temperature for 1 hour followed by anti-rabbit polymer-labeled HRP-bound secondary reagent (EnVision+ System, HRP). All immunohistochemistry stains were developed with DAB chromogen and counterstained with hematoxylin. Corresponding negative control experiments were done by omitting the incubation step with the primary antibody. Scoring of immunohistochemistry Scoring of immunohistochemical staining was done using the Automated Cellular Imaging System (Chroma Vision). Stained sections were scanned and acquired using Automated Cellular Imaging System. Proliferation was measured by calculating the average labeling percentage of the epithelial compartment for Ki67 for each specimen. For determination of microvessel density (MVD), the total number of CD31-stained clusters or single cells, with or without a lumen, was quantified for each specimen. For pVEGFR-2 and pEGFR, quantification was done as previously described (38, 39). Briefly, an index of staining was calculated and expressed as the percentage of staining multiplied by staining intensity after subtracting the index staining of corresponding negative controls. Data analysis Fisher's exact test was done for the comparison of cancer and cancer + dysplasia rates between groups. Two-sample t tests, assuming unequal variances, were used for com- www.aacrjournals.org parison of MVD, Ki67, pEGFR, and pVEGFR-2 levels between groups. The nonparametric Wilcoxon rank-sum test was also done to confirm the results from the t tests. For pEGFR and pVEGFR-2, the average of five measurements for each mouse was first calculated, and this summary measure was used in the analyses. P 0.05 was considered statistically significant. All analyses were done using Stata version 11 (Stata Corp.). Results Effects of ZD6474 administration on the development of dysplasia and OSCC Mice were given 4-NQO (100 g/mL) in their drinking water for a period of 8 weeks, returned to normal water, and then randomized to observation or daily oral gavage of ZD6474 (25 mg/kg/d) for 24 weeks. We have previously shown that following the 8 weeks of 4-NQO administration, mice developed histologically identifiable hyperkeratotic and/or dysplastic lesions (35). Therefore, initiation of ZD6474 treatment at this time point was chosen because it closely mimics the clinical setting in which one would consider initiating chemopreventive therapy in patients. During the 24-week chemoprevention regimen, no significant differences in food and fluid consumption or activity were observed between the groups. At the completion of the 32-week study, there was a significant difference in the incidence of dysplasia and OSCC in the ZD6474 treatment group compared with the control group (Table 1). Overall, 71% (17 of 24) of the control mice and 12% (3 of 25) of the ZD6474-treated mice showed histologic evidence of OSCC (P 0.001). Similarly, the proportion of mice with dysplasia or OSCC was significantly different between the two treatment groups. In the control group, 96% (23 of 24) of the animals showed dysplasia or OSCC, whereas 28% (7 of 25) of the ZD6474 treatment group had dysplasia or OSCC (P 0.001). In total, this represented a 71% decrease in OSCC or dysplasia and an 83% decrease in OSCC. Effects of ZD6474 administration on proliferation and MVD ZD6474 has been shown to inhibit both tumor cell proliferation and angiogenesis via its dual activity against EGFR and VEGFR-2 (1719). Therefore, we performed Table 1. Effect of ZD6474 treatment on the development of OSCC in the mouse 4-NQO model Hyperkeratosis Dysplasia OSCC Total Control ZD6474 Total 1 6 17 24 18 4 3 25 19 10 20 49 Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1495 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. immunohistochemistry for Ki67 and CD31 as surrogate markers for cell proliferation and angiogenesis, respectively. Overall, the Ki67 proliferative index (PI) for the ZD6474treated animals was significantly decreased when compared with the control mice (Table 2). The control group had a PI of 46 10, whereas the ZD6474 treatment group had a PI of 29 10 (P 0.001). Proliferation increased with histologic progression in both control and treatment cohorts (Fig. 1). Of note, the OSCC that arose in the ZD6474 treatment group (n = 3) had a mean PI (54.3) that was similar to the PI of the control animals (n = 17) who developed OSCC (51.6), suggesting that the ZD6474-associated tumors were still actively proliferating. Overall, there was a significant decrease in MVD in the ZD6474-treated mice when compared with controls. The control group showed a MVD score of 265 60, although the ZD6474 treatment group had a MVD score of 106 73 (P 0.001). However, there was no difference in vascularity when comparing the MVD between similar histologic diagnoses (hyperkeratosis or dysplasia or OSCC) from different treatment groups (control versus ZD6474-treated; Fig. 1). The OSCC that arose in the ZD6474 treatment group had a mean MVD (253.7) that was similar to the MVD of the OSCC control group (300.2) suggesting that the tumor was still actively inducing angiogenesis. ZD6474) there was no difference in the intensity scores between the hyperkeratotic, dysplastic, or OSCC specimens (Fig. 2). Interestingly, the pEGFR intensity scores for the OSCC from the ZD6474 treatment group were much lower than the intensity scores for the control OSCC cohort (Fig. 2). Overall, tissue from control mice had a significantly higher expression of pVEGFR-2 when compared with the ZD6474-treated mice (Table 2; Fig. 3). The combined mean intensity score for the control tissue was 106 11, whereas the combined intensity score of the tissue from the ZD6474-treated animals was 32 3 (P 0.001). When comparing expression between histologic groups within the same experimental group (control or ZD6474), the intensity scores between hyperkeratotic, dysplastic or OSCC specimens were very similar (Fig. 3). Like the pEGFR findings, expression of pVEGFR-2 in the OSCC from the ZD6474-treated group was much lower in intensity when compared with the OSCC from the control group (Fig. 3). These data show that ZD6474 was pharmacologically active in the 4-NQO model. In addition, the data suggests the OSCC that arose in the ZD6474-treated group may have developed acquired drug resistance, as ZD6474 was still actively inhibiting the phosphorylation of both EGFR and VEGFR-2. Effects of ZD6474 administration on EGFR and VEGFR-2 activation In an effort to identify the potential mechanism(s) of acquired resistance to ZD6474 treatment, immunohistochemistry for pEGFR and pVEGFR-2 was done to determine if ZD6474 was still inhibiting the activation of these receptors. Overall, tissue from the control cohort of mice showed significantly stronger cytoplasmic membrane staining for pEGFR when compared with ZD6474-treated mice. The control group had a mean intensity score of 97 5, whereas the mean intensity score was 33 3 (P 0.001) for the ZD6474-treated cohort (Table 2). When comparing pEGFR expression between histologic groups within the same treatment scheme (control or ZD6474-resistant OSCC express epithelial to mesenchymal markers ZD6474 was able to significantly reduce the incidence of OSCC when compared with the control group (Table 1). Although the inhibition of tumor development was statistically significant, 12% of the animals in the ZD6474 group developed OSCC. Furthermore, our data suggests that ZD6474 was still pharmacologically active because low levels of both pEGFR and pVEGFR-2 were still observed after 24 weeks of treatment (Figs. 2 and 3). In addition, the PI and MVD data from the OSCC arising in ZD6474-treated mice were similar to the PI and MVD data in the control animals harboring OSCC (Fig. 1). Overall, these data suggest that the OSCC in the ZD6474-treated mice had developed a form of acquired drug resistance. Similar acquired resistance has been associated with the expression of an epithelial to mesenchymal (EMT) phenotype, an intricate process that can be both physiologic and pathologic in nature (3943). For example, the induction of EMT may be a novel mechanism of acquired resistance to chemotherapy and radiation in cancer therapy (40, 44). To address the possibility that the development of resistance to ZD6474 treatment in the mouse 4-NQO model of OSCC was driven by the expression of an EMT phenotype, we did immunohistochemistry for the EMT markers E-cadherin and vimentin (45). Each of the tumors from the control group expressed high levels of E-cadherin and undetectable levels of vimentin protein (Fig. 4). Conversely, the tumors from ZD6474-treated mice lost expression of E-cadherin and expressed high levels of vimentin protein (Fig. 4). Although the sample size is small (n = 3), these data show a correlation between resistance to ZD6474 Table 2. Modulation of surrogate biomarkers for angiogenesis, proliferation, and activation of the EGFR and VEGFR-2 pathways by ZD6474 Control, N = 24 MVD Ki67 pEGFR pVEGFR-2 265 46 97 106 60 10 5 11 ZD6474, N = 25 106 29 33 32 73* 10* 3* 3* *P 0.001 for comparison with the control group. All numbers indicate mean SD. 1496 Cancer Prev Res; 3(11) November 2010 Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer Fig. 1. ZD6474-treated animals show lower proliferative indices and microvessel densities compared with control animals. A, tissue sections were immunohistochemically stained for Ki67 and labeling indices were quantified. The overall labeling indices of the ZD6474 specimens were all significantly lower when compared with the control specimens (P 0.001). B, tissue sections were immunohistochemically stained for CD31 and MVD quantified. MVD of the ZD6474 specimens were significantly lower when compared with the control specimens (P 0.001). The total group was used for all statistical analysis. and the expression of EMT markers. They further suggest that the expression of an EMT phenotype may be a novel mechanism of acquired resistance to chemoprevention therapy for OSCC. Discussion The induction of cell proliferation and blood vessel growth are two critical phenotypes that are necessary for the development of malignant neoplasms. Aberrant EGFR tyrosine kinase activity plays an important role in a number of different tumor phenotypes including proliferation, apoptosis, angiogenesis, and metastasis. Furthermore, because www.aacrjournals.org EGFR has such a critical role in the development of OSCC, this signaling pathway has considerable therapeutic potential in the areas of cancer therapy and chemoprevention. Similarly, the activation of the VEGFR-2 pathway by VEGF is a critical component for the induction of angiogenesis in both physiologic and pathologic settings including OSCC. Therefore, because ZD6474 has the ability to inhibit both EGFR and VEGFR-2 activation, it has the potential to inhibit two critical signal transduction pathways and phenotypes involved in the development of OSCC. In this study, we show that ZD6474 was pharmacologically active in the 4-NQO model of OSCC. Animals treated with 25 mg/kg/d had significantly lower expression Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1497 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. Fig. 2. ZD6474 inhibits the phosphorylation of EGFR in the 4-NQO model of OSCC. A, tissue sections from control and ZD6474-treated animals were immunohistochemically stained for pEGFR and quantified. B, expression of pEGFR was significantly lower in the ZD6474-treated specimens when compared with the control specimens (P 0.001). levels of pEGFR and pVEGFR-2 when compared with controls (Figs. 2 and 3). We also report for the first time that daily treatment with ZD6474 decreased the incidence of dysplasias and carcinomas in the mouse 4-NQO model of OSCC (Table 1). The rationale for the 24-week treatment schedule was based on our previous work, which showed that the majority of control animals harbor OSCC by week 24, while dysplasia was the predominant histologic diagnosis at weeks 16 and 20 (35). Because we were testing the hypothesis that ZD6474 would reduce the incidence of OSCC, we believe that it is most appropriate to carry out the prevention study to a time point where the predominant histologic diagnosis in the control group would be expected to be OSCC. Overall, we observed an 83% reduction in the incidence of OSCC when comparing the control and treatment groups (71% versus 12%, P 0.001). We also observed a 71% decrease in the incidence of both dysplasia and OSCC when comparing the control and ZD6474 treatment groups (96% versus 28%, P 0.001). These data strongly support the 1498 Cancer Prev Res; 3(11) November 2010 hypothesis that ZD6474 may be an effective chemopreventive agent for OSCC. In preclinical studies, ZD6474 has been shown to be a potent inhibitor of tumor angiogenesis and proliferation for several different tumor cell types (2130). It is also under active investigation in clinical trials for the treatment of various malignant neoplasms (31), with the greatest activity observed in nonsmall cell lung cancer and medullary thyroid carcinoma (3234). However, published data regarding the potential utility of ZD6474 in the realm of chemoprevention is limited. In one study, ZD6474 markedly reduced the number and the size of intestinal polyps, resulting in a 75% decrease in tumor burden in a mouse model of colon cancer (46). The data from the colonic polyp study and our current work suggests that further studies are warranted to evaluate the potential utility of ZD6474 as a chemopreventive agent. One of the long-term goals of chemoprevention must be the development of treatments that can be easily taken by at-risk individuals for prolonged periods of time with Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer minimal toxicities to achieve widespread acceptance and long-term compliance. This would be particularly important in the case of high-risk patients who have not yet developed their first OSCC. We have previously shown that ABT-510, a mimetic peptide of thrombospondin-1, significantly decreased the incidence of dysplasia and OSCC in the 4-NQO model (35). However, because there is no oral formulation of the drug, the translation of this agent into clinical trials for prevention seems unlikely. Conversely, because ZD6474 is an orally available drug, it is potentially more feasible for prolonged use in human prevention studies. The maximum tolerated dose as well as toxicity profile of ZD6474 when used in cancer therapy is well described. ZD6474 has been well-tolerated at doses of 100 to 300 mg/d, with the most common adverse events being rash, diarrhea, fatigue, and asymptomatic QTc prolongation (31). However, because the drug may be initiated at a lower dose range in a chemoprevention setting, one might anticipate a lesser degree of side effects. Furthermore, treatment with higher doses of ZD6474 (50 and 100 mg/kg) than the current study (25 mg/kg/d) resulted in only a modest delay, but not inhibition, of cutaneous wound healing in a mouse model (47). Taken together, these data suggest that the toxicity profile of ZD6474, when used as a chemopreventive agent, might be acceptable when lower doses of the agent are used. This might be particularly true in the context of OSCC, in which the modest long-term survival is due in part to the frequent development of multiple additional primary tumors in individuals with a previous SCC. The rate of second primary tumors in these patients has been reported to be 3% to 7% per year, which is higher than for any other malignancy (48). This observation led Slaughter et al. to propose the concept of field cancerization. This theory suggests that multiple individual primary tumors develop independently in the upper aerodigestive tract as a result of years of chronic exposure of the mucosa to carcinogens (49). As a result of field cancerization, an individual who is fortunate to live 5 years after the initial primary tumor has up to a 35% chance of developing at least one new primary tumor within that time period. The occurrence of new primary tumors can be particularly devastating for individuals whose Fig. 3. ZD6474 inhibits the phosphorylation of VEGFR-2 in the 4-NQO model of OSCC. A, tissue sections from control and ZD6474-treated animals were immunohistochemically stained for pVEGFR-2 and quantified. B, expression of pVEGFR-2 was significantly lower in the ZD6474-treated specimens when compared with the control specimens (P 0.001). www.aacrjournals.org Cancer Prev Res; 3(11) November 2010 Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. 1499 Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Zhou et al. Fig. 4. OSCC arising in ZD6474-treated mice express EMT markers. Tumor samples from control mice show strong epithelial expression of E-cadherin and stromal expression vimentin. Histologically normal epithelium from the ZD6474-treated animals show strong expression of E-cadherin and no expression of vimentin (arrows). Conversely, tumor cells from ZD6474 mice show loss of expression of E-cadherin and strong expression of vimentin. initial lesions are small. Their 5-year survival rate for the first primary tumor is considerably better than late stage disease, but second primary tumors are their most common cause of treatment failure and death (4, 5). Resistance to cytotoxic chemotherapy and radiation therapy is well appreciated in the context of cancer therapy. In addition, mechanisms of resistance in response to targeted therapies have also been described. For example, several types of intrinsic and acquired resistance to inhibitors of angiogenesis have been postulated (50, 51). Similarly, several mechanisms of resistance related to antiEGFR therapy have been reported for nonsmall cell lung cancer, although the mechanisms for EGFR resistance in the context of OSCC seem to be different and remain unclear (52). Conversely, there are limited data concerning the potential mechanisms of acquired resistance in response to long-term chemoprevention therapy using targeted agents (5355). In the 4-NQO model of OSCC, 12% of the mice chronically treated with ZD6474 developed a form of acquired resistance to the drug. This resistance correlated with a loss of E-cadherin and a gain in vimentin protein expression, suggesting that these tumors began to express an EMT phenotype (44). Conversely, none of the control group OSCC expressed EMT markers. This correlation between resistance to ZD6474 and the expression of an EMT phenotype suggests a novel mechanism of acquired resistance in the chemopreventive 1500 Cancer Prev Res; 3(11) November 2010 setting. The expression of EMT transitions is well appreciated in embryology and various types of pathophysiology (40). Recently, there has been an increased interest in the role of EMT in areas of cancer progression as well as resistance to chemotherapy and radiation therapy (40, 44). At this time, we do not know if there is a causal link between the expression of EMT markers and resistance to ZD6474. However, the fact that EMT markers were not expressed in control OSCC provides compelling preliminary evidence worthy of further investigation. In addition, we do not know the timing of the gain of expression of the EMT phenotype. As designed, this prevention study harvested all tissues after 24 weeks of ZD6474 therapy. Therefore, to investigate the dynamics of EMT marker expression, one could sacrifice subsets of mice at specified intervals after the initiation of ZD6474 treatment to determine the incidence and timing of EMT marker expression at the stages of hyperkeratosis, dysplasia, and OSCC. It is also important to determine if one or both receptor pathways are mediating the expression of the EMT phenotype. Resistance to EGFR inhibitors erlotinib, gefitinib and cetuximab has been reported to induce an EMT transition (4143). Similarly, the induction of hypoxia has also been shown to induce EMT (40). Therefore, it is possible that ZD6474 may drive the expression of EMT via both pathways. In addition, the downstream mechanisms of the expression of the EMT phenotype are unknown. The transcription Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 ZD6474 Chemoprevention of Oral Cancer factors Twist, Snail, and Slug are major mediators of EMT and have been shown to repress E-cadherin expression (40). Further investigation into the altered expression of these and other EMT-related regulatory factors may aid in our understanding of how the expression of an EMT phenotype occurs in the setting of chemoprevention. In addition, the biological and clinical implications of EMT expression in ZD6474-resistant tumors requires further investigation, as the expression of the EMT phenotype can lead to resistance to multiple drugs and potentially lead to the progression of tumors (30). However, the potential for altered clinical behavior following a tyrosine kinase inhibitorbased chemopreventive treatment is not limited to this class of drugs, as resistance towards other types of chemopreventive agents has also been described (3941). Finally, if the pattern of EMT development can be modeled, one could envision using EMT markers as diagnostic beacons to herald the expression of acquired resistance. Such beacons may be useful as they could be used as indicators for when it would be most efficacious to switch to an alternative chemopreventive agent. For example, the expression of EMT markers might dictate a switch to a histone deacetylase inhibitor, as these have been shown to reverse the EMT phenotype (56). By doing so, one might hypothesize that the histone deacetylase could thereby restore sensitivity to ZD6474s and prolong its chemopreventive activity. We believe that the mouse 4-NQO model is an excellent model system to pursue each of these important preclinical questions. In conclusion, our data provides novel evidence that ZD6474, a combined inhibitor of the EGFR and VEGFR-2 pathways, holds promise as a chemopreventive agent for OSCC. They further suggest that the development of acquired resistance to ZD6474 may be mediated by the expression of an EMT phenotype. Finally, the data suggests that the 4-NQO model of OSCC is a useful preclinical platform to investigate the mechanisms of acquired resistance in the chemopreventive setting. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Grant Support In part by the NIH (DE012322). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received 06/16/2010; revised 08/12/2010; accepted 09/02/2010; published OnlineFirst 10/26/2010. References 1. Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:7196. 2. In: Ries LAG, Melbert D, Krapcho M, et al, editors. SEER cancer statistics review, 19752005. Bethesda, MD: National Cancer Institute; 2008. 3. Murphy GP LW, Lenhhardt RE. American cancer society textbook of clinical oncology. 2nd ed. Atlanta: American Cancer Society; 1995. 4. Lippman SM, Hong WK. Second malignant tumors in head and neck squamous cell carcinoma: the overshadowing threat for patients with early-stage disease. 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Cancer Prevention Research Downloaded from cancerpreventionresearch.aacrjournals.org on March 5, 2021. 2010 American Association for Cancer Research. Published OnlineFirst October 26, 2010; DOI: 10.1158/1940-6207.CAPR-10-0135 Dual Inhibition of Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor is an Effective Chemopreventive Strategy in the Mouse 4-NQO Model of Oral Carcinogenesis Guolin Zhou, Rifat Hasina, Kristen Wroblewski, et al. Cancer Prev Res 2010;3:1493-1502. Published OnlineFirst October 26, 2010. Updated version Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: doi:10.1158/1940-6207.CAPR-10-0135 This article cites 53 articles, 22 of which you can access for free at: http://cancerpreventionresearch.aacrjournals.org/content/3/11/1493.full#ref-list-1 This article has been cited by 4 HighWire-hosted articles. 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... International Journal of Gynecological Pathology 31:1524, Lippincott Williams & Wilkins, Baltimore r 2012 International Society of Gynecological Pathologists Original Article Characterization of NOL7 Gene Point Mutations, Promoter Methylation, and Protein Expression in Cervical Cancer Downloaded from http://journals.lww.com/intjgynpathology by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdtwnfKZBYtws= on 03/08/2021 Colleen L. Doci, Ph.D., Tanmayi P. Mankame, M.S., Alexander Langerman, M.D., Kelly R. Ostler, B.A., Rajani Kanteti, Ph.D., Timothy Best, B.S., Kenan Onel, M.D., Ph.D., Lucy A. Godley, M.D., Ph.D., Ravi Salgia, M.D., Ph.D., and Mark W. Lingen, D.D.S., Ph.D. Summary: NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulte sequencing and mutations were identied through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 50 untranslated region and rst exon of NOL7. However, bisulte sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may aect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were signicantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23. Key Words: NOL7HypermethylationMutation. Cervical cancer (CC) is the most common gynecological malignancy and the third most common cancer among women worldwide (1). CC development is strongly associated with human papillomavirus (HPV) infection. However, additional genetic alterations are required for malignant transformation (26). Therefore, the successful screening, prevention, diagnosis, and treatment of CC require further characterization of the key genetic alterations required for CC development. NOL7 is a putative tumor suppressor gene (TSG) localized to 6p23, a region with frequent loss of heterozygosity (LOH) in a number of cancers, including hormone-refractory breast carcinoma, leukemia, lymphoma, osteosarcoma, From the Departments of Pathology, Medicine and Radiation and Cellular Oncology (C.L.D., T.P.M., M.W.L.); Surgery (A.L.), Section of Otolaryngology-Head and Neck Surgery; Medicine (K.R.O., R.K., L.A.G., R.S.), Section of Hematology/Oncology; and Pediatrics (T.B., K.O.), University of Chicago, Chicago, IL. This study was supported in part by the Illinois Department of Public Health Penny Severns Cancer Research Fund (M.W.L.) and National Institutes for Health 5R01CA100750-07 (R.S.) and 5R01CA129501-03 (R.S.). The authors declare no conict of interest. Address correspondence and reprint requests to Mark W. Lingen, Department of Pathology, University of Chicago, 5841 S. Maryland Avenue MC6101, Chicago, IL 60637. E-mail: mark.lingen@uchospitals.edu Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the Journals Website, www.intjgynpathology.com. 15 DOI: 10.1097/PGP.0b013e318220ba16 16 C. L. DOCI ET AL. retinoblastoma, nasopharyngeal carcinoma, and CC (725). Using CC as a model, in which LOH of 6p23 is one of the most common allelic losses in this neoplasm (2631), we demonstrated that NOL7 expression is regulated through genomic instability. Fluorescent in-situ hybridization experiments using BAC clones and an 8-kb NOL7 genomic probe demonstrated consistent loss of one NOL7 allele in CC cell lines and in CC tumor samples (32). Reintroduction of NOL7 into CC cells with decreased NOL7 expression modulated the angiogenic phenotype by decreased expression of the proangiogenic cytokine vascular endothelial growth factor and increased expression of the antiangiogenic factor TSP-1. Importantly, reintroduction of NOL7 suppressed tumor growth in vivo by 95% (32). These studies suggest that NOL7 plays a signicant role in the suppression of tumor growth. However, according to the Knudson 2-hit hypothesis, inactivation of a TSG requires the functional loss of both alleles through various genetic or epigenetic mechanisms (33,34). Although numerous studies, including those within our own lab, have documented the LOH of 6p23 and NOL7, an additional hit has not been identied. In addition to the LOH, mutation and methylation represent 2 common methods of genetic and epigenetic inactivation (35,36). A recent study identied mutations within NOL7 in 25% of the CC specimens examined. However, the limited number of samples investigated precludes denitive conclusions (37). Promoter methylation of genes localized to 6p is associated with cancer, and methylation of ID4 and POU2F3 has been reported on 6p23 (38,39). However, no studies have specically determined the methylation status of NOL7. MATERIALS AND METHODS Identication of GC-rich Genomic Regions and CpG Islands The EMBOSS-Isochore program was used to identify regions within the NOL7 gene that were GC rich (4042). The program was input with base pairs 13,614,790 to 13,621,437 of the Chr6 Genbank accession NC_000006.11. This identied a region from 13,614,790 to 13,616,240, which was subsequently analyzed for CpG islands using the EMBOSS-CpGPlot program (43,44). Default settings for both programs were used (GC40.5 and CpG40.7 thresholds). Int J Gynecol Pathol, Vol. 31, No. 1, January 2012 Cell Lines All cell lines were obtained from the ATCC (Manassas, VA) and were cultured at 371C in a 5% CO2 in humidied incubators. Media and reagents were purchased from Invitrogen (Carlsbad, CA). HEK293 T was used as a positive control for NOL7 expression. All media were supplemented with 10% FBS, penicillin (100 mg/mL), and streptomycin. Media were as follows: SiHa, Ca Ski, and ME180 RPMI; HeLa and HEK293 TDMEM; AN3CA, C-33A, MS751, and CC-IMEM; HEC-1-A and HT-3McCoys; C-4I and C-4IIWaymouths; and SW756Leibovitzs. Genomic DNA from cell lines was extracted using the Gentra Puregene Kit (Qiagen, Valencia, CA) as per the manufacturers instructions. Genomic DNA from normal adult cervix was obtained from the Biochain Institute Incorporated (Hayward, CA). Tissue Specimens CC tissue specimens were collected and used under the approval of the University of Chicago Institutional Review Board. Paran blocks of diagnosed CC specimens were obtained and representative hematoxylin and eosin-stained sections were reviewed. Matched normal and cancer specimens were collected by laser capture microdissection) using the Leica AS LMD system. Genomic DNA from laser capture microdissection sections was extracted as described previously (45). Bisulte Treatment and Polymerase Chain Reaction Amplication Genomic DNA from the cell lines, normal cervical mucosa, and 23 CC samples were treated with sodium bisulte as described previously (46). The bisultetreated DNA was used as template for polymerase chain reaction (PCR), using AmpliTaq Gold PCR Master Mix (Applied Biosystems, Foster City, CA) and primers region A (50 -GTGGTAGTAGGGTTG ATTGG-30 and 50 -AATAAACCCCACTAAAAAT ACTCTAC-30 ) and region B (50 -GTAGAGTATTTT TAGTGGGGTTTATT-30 and 50 -AAACTACACCA TAACCCA-30 ) (Figure 4). The PCR program used was 941C for 10 minutes, 35 cycles of 941C for 30s, 591C for 45s, 721C for 45s, and 721C for 10 minutes. The PCR products were resolved and visualized on a 1% agarose gel. GENOMIC REGULATION OF NOL7 TOPO TA Cloning and Sequencing PCR products from bisulte-treated DNA extracted from cell lines and tissue samples were cloned into the pCR4-TOPO vector by TOPO TA cloning according to the manufacturers instructions (Invitrogen, Carlsbad, CA). Ten colonies per transformation were sequenced using T7 and T3 primer sites within the pCR4-TOPO vector. Sequences were compared with the genomic template (accession number: NC_000006.11) and the CpGs were analyzed using MegAlign software (DNASTAR, Inc., Madison, WI). Mutational Sequencing Genomic DNA from cell lines and tissues was amplied by PCR using AmpliTaq Gold PCR Master Mix (Applied Biosystems). Primers are listed in Supplementary Table 1 (Table, Supplemental Digital Content 1; http://links.lww.com/IJGP/A6). Cycling conditions were 941C for 5 minutes, 35 cycles of 941C for 20 seconds, varied Tm for 30 seconds, 721C for 2 minutes, and 721C for 10 minutes. PCR products were puried using the Wizard SV Gel and PCR Cleanup System (Promega, Madison, WI) and sequenced using amplication primers. Sequences were compared with bases 13,614,022 to 13,621,437 of NC_000006.11 using MegAlign software (DNASTAR, Inc.) and ChromasLite (Technelysium Pty Ltd, Eden Prairie, MN). Potential biologic signicance of the mutations was evaluated using Genomatix MatInspector (47), Human Splicing Finder (48), CRYP-SKIP (4951), MicroInspector Prediction Software (52), and miRBase (5357). Real-Time Quantitative PCR Endogenous NOL7 mRNA expression was determined by quantitative real-time PCR as described previously (58). Relative fold change for mRNA expression was quantied using the DDCT relative quantication method and normalized to 293T mRNA levels. Statistical signicance was determined using the Student t test. Western Blotting Western blotting was performed as described (58). Antibody conditions were as follows: NOL7 (12 ng/mL) (Sigma-Aldrich, St. Louis, MO); b-actin (0.75 ng/mL) (Abcam, Cambridge, MA); and goat a-rabbit-HRP (12.5 ng/mL) (Cell Signaling Technology, Danvers, MA). Results were quantied using 17 the Bio-Rad QuantityOne Software (Bio-Rad, Hercules, CA) and normalized to b-actin. Tissue Microarray and Immunohistochemistry Immunohistochemistry for NOL7 was performed on cervical tissue obtained with the approval of the University of Chicago Institutional Review Board. A tissue microarray (TMA) was generated using a Beecher Instruments ATA-27 Automated Tissue Arrayer. The TMA consisted of normal cervical mucosa (n 70), cervical intraepithelial neoplasia (CIN) I (n 22), CIN II (n 20), CIN III (n 23), and cervical squamous cell carcinoma (n 88). Deparanized sections were microwaved in ET buer, a-NOL7 primary (Sigma-Aldrich) was applied at 1:120 dilution for 1 hour at room temperature, and the rabbit EnVision kit (DAKO, Carpinteria, CA) was used for detection. All sections were counterstained with hematoxylin and scored on a 0 to 2 scale, with 0 being the lowest staining and 2 the greatest. The Fisher exact test was performed for the comparison of NOL7 expression among the histologic groups. Unless otherwise stated, NOL7 expression was ranked as present (staining intensity 1 and 2) or absent (staining intensity 0) against normal CIN I versus CIN II CIN III squamous cell carcinoma (SCC) groups. RESULTS NOL7 is downregulated in CC cell lines and its expression decreases with progression in CC. We have previously demonstrated decreased mRNA expression and allelic loss of NOL7 in a limited number of CC cell lines and tumor samples by Northern blot and uorescent in-situ hybridization (32). To further characterize the expression of endogenous NOL7 in CC, we assessed mRNA and protein levels in an expanded set of CC cell lines. Using a polyclonal antibody against NOL7, we performed western blotting (Fig. 1A) and compared this with mRNA levels as determined by real-time quantitative PCR (Fig. 1B), normalized to 293 T cells for which previous analyses have indicated as containing high levels of endogenous normal NOL7. NOL7 protein expression was decreased by half in 6 of 13 (46%) cell lines compared with 293 T control (Fig. 1A). We also found that 6 of 13 (46%) CC cell lines demonstrated signicantly decreased NOL7 mRNA expression (o50% of 293 T control, Int J Gynecol Pathol, Vol. 31, No. 1, January 2012 18 C. L. DOCI ET AL. FIG. 1. Analysis of endogenous NOL7 expression in CC cell lines. (A) Western blotting was performed on 25 mg of whole cell lysate from a panel of CC cell lines. Expression was quantied in (B) using QuantityOne software from Bio-Rad and normalized to b-actin. (B) Real-time quantitative polymerase chain reaction was performed on total RNA isolated from 13 CC cell lines and compared with 293T controls. Po2  104; Fig. 1B). Four of these cell lines, Ca Ski, ME-180, SiHa, and SW756, demonstrated consistently downregulated NOL7 mRNA and protein. Interestingly, 5 of the 13 (40%) cell lines, C-4 I, Ca Ski, CC-1, HEC-1-A, and HeLa, showed dierential expression between NOL7 mRNA and protein levels, suggesting that NOL7 may also be posttranscriptionally and posttranslationally regulated. To assess endogenous NOL7 expression during the histologic progression of CC, immunohistochemistry against NOL7 was performed on TMAs consisted of specimens from normal, CIN I, CIN II, CIN III, and SCC cervical tissue (Fig. 2A). Tissues were scored 0, 1, or 2 based on staining intensity, with 0 being the lowest and 2 being the highest expressing tissue. Although the majority (95%) of normal and CIN I samples Int J Gynecol Pathol, Vol. 31, No. 1, January 2012 demonstrated staining for NOL7, o23% of samples from CIN II, CIN III, and SCC had a staining intensity of 1 or 2 (Po0.001) (Fig. 2B). Conversely, 77% of CIN II, CIN III, and SCC tissues did not express endogenous NOL7, whereas only 5% of normal and CIN I tissues lacked NOL7 expression (Po0.001) (Fig. 2B). These data suggest that NOL7 expression decreases signicantly with histologic progression in CC, and that loss of NOL7 occurs after CIN I. CC Cell Lines and Tumor Samples Do Not Have Methylated NOL7 Promoter The signicant loss of NOL7 expression in CC cell lines and cervical tissue suggests that NOL7 may function as a TSG. In accordance with the Knudson GENOMIC REGULATION OF NOL7 19 FIG. 3. Prediction and analysis of NOL7 genomic elements. (A) The genomic region of chromosome 6 (NC_000006.11) from bases 13,614,790 to 13,621,437 was analyzed using EMBOSS Isochore program to predict regions of high GC content. Dashed lines indicate the designated threshold of 0.5. (B) The 1451 bp region of NOL7 demonstrating that 450% GC content was analyzed for the presence of a CpG island using EMBOSS CpGPlot. Dashed lines indicate a threshold of 0.7. FIG. 2. Immunohistochemical expression of NOL7 in normal (A) CIN I (B) CIN III (C), and malignant cervical mucosa (D). Decreased nucleoplasmic NOL7 protein expression is observed with increasing histologic atypia. Original magnication: 200  and 1000  . E, Quantication of NOL7 expression in cervical mucosa. Normal (n 70), CIN I (n 22), CIN II (n 20), CIN III (n 23), and SCC (n 88). Data are expressed as percentage of tumor specimens demonstrating an intensity of staining ranging from 0 to 2 . 2-Hit hypothesis, loss of the rst allele of NOL7 by LOH must be followed by loss or silencing of the second allele through genetic or epigenetic mechanisms, likely methylation or mutation. Epigenetic regulation by methylation in a gene-specic manner is dictated by the presence of a CpG island in the promoter of these genes (59). To determine whether NOL7 might be regulated by this mechanism, the NOL7 genomic region was analyzed for high GC content that might indicate the presence of a CpG island. The region upstream of the NOL7 start codon averages 460% GC across the region of interest (Fig. 3A) (4042). To determine whether this GC-rich region contained a CpG island, the genomic region sequence was analyzed using the EMBOSS CpG Plot program (44,46). The analysis predicted a large CpG island approximately 1120 nucleotides in length, containing 111 CpG dinucleotides (Figs. 3B). Promoter hypermethylation is a common mechanism by which many TSGs such as p16 and APC are silenced in CC (60). The promoter of NOL7 spans the 560 bp region upstream of the start codon, which contains 47 CpG dinucleotides (58). To specically assess NOL7 promoter methylation, bisulte PCR primers were designed such that the promoter region was split into 2 fragments, region A and B (Fig. 4A). To assess the methylation status of NOL7 in cell lines that express a wide spectrum of endogenous NOL7, genomic DNA from 293 T, HeLa, and SiHa cell lines that express high, moderate, and low levels of endogenous NOL7 was bisulte treated, PCR amplied, and TOPO TA cloned (Fig. 4B). There was no methylation observed in the 3 cell lines (Fig. 4C). To determine whether the lack of methylation was an artifact due to cell culture, 23 consecutive CC tissue samples were analyzed and compared with normal cervix for methylation status. No signicant methylation pattern was observed in either the normal or CC tissue samples (Fig. 4C). These data suggest that methylation is not a mechanism of inactivation of the NOL7 gene. Int J Gynecol Pathol, Vol. 31, No. 1, January 2012 20 C. L. DOCI ET AL. FIG. 4. Bisulte PCR-mediated methylation analysis. (A) Schematic of the NOL7 genomic region with exons (light gray box), promoter region (white box), and CpG Island (dark gray box). (A) and (B) indicate the bisulte primer location. (B) Representative bisulte polymerase chain reaction products for regions A and B from HEK293 T, HeLa, and SiHa cell lines. (C) Analysis of the methylation pattern of 47 individual CpG dinucleotides in the NOL7 promoter region. Ten clones were sequenced for the 3 cell lines, normal, and 23 cervical cancer samples (CC-1 to 23), which is represented in parentheses. Open circles represent unmethylated CpG dinucleotides. Ten clones are represented per circle, with the methylated CpGs corresponding to the black shaded fraction. NOL7 is Mutated in CC Lack of NOL7 methylation in the promoter region suggested that there may be inactivation through mutation. To determine whether the genomic region of NOL7 harbors inactivating mutations, a series of primers were designed to cover the length of the NOL7 gene. Genomic DNA from cell lines (Table 1) and 23 consecutive matched normal and CC tumor specimens (Table 2) were collected and the NOL7 genomic region was sequenced. Four consistent variations from the genomic sequence of NOL7 were identied within the 13 cell lines. Fifteen of the 23 TABLE 1. Mutational analysis of NOL7 genomic region in CC cell lines Coding change SNP Frequency Predicted biologic consequence G38R rs2841524 61.54% 61.54% Intron 5 38.46% Intron 5 7.69% HIF1a-binding site lost May aect functionality of adjacent acidic domain Destroys splicing silencer motifs and creates new enhancer motifs; alternative splicing pattern Destroys splicing silencer motifs and creates new enhancer motifs; alternative splicing pattern Variance Genomic NOL7 region A-C G-A 13615401 13615702 Promoter Exon 1 G-A 13618408 T-C 13620097 The results of direct mutational sequencing of the NOL7 genomic region within CC cell lines are listed. The location of the mutation corresponding to Genbank accession NC_000006.11 is listed, along with the region of NOL7 aected and frequency of the mutation within the 13 cell lines examined. The mutation, SNP status, and predicted eect are included. CC indicates cervical cancer. Int J Gynecol Pathol, Vol. 31, No. 1, January 2012 GENOMIC REGULATION OF NOL7 21 TABLE 2. Mutational analysis of NOL7 genomic region in CC tumor samples Variance Genomic NOL7 region SNP Frequency G -A 13614430 5 UTR 4.35% A-C C-T G-A 13615401 13615444 13617979 Promoter Promoter Intron 3 rs2841524 39.13% 4.35% 4.35% A-G 13618177 Intron 4 4.35% G-A 13618408 Intron 5 8.70% T-C 13620207 Intron 5 4.35% A-G 13620386 Intron 5 4.35% rs10695200 4.35% 4.35% 4.35% AACT Deletion G-A 13621391 13621627 13621698 0 0 3 UTR 30 UTR 30 UTR Predicted biologic consequence Loss of hsa-miR-1225-3p, -145, -331-3p, -1274a, -500, and -326; Gain of hsa-miR-134 HIF1a-binding site lost AP-2-binding site lost Destroys splicing silencer and enhancer motifs; splice site broken Destroys splicing silencer motifs and creates new enhancer motifs; alternative splicing pattern Destroys splicing silencer motifs and creates new enhancer motifs; alternative splicing pattern Destroys splicing silencer and enhancer motifs; splice site broken Destroys splicing silencer and enhancer motifs; splice site broken Change in 30 UTR cis-elements Loss of hsa-miR-19-1b and -182;Gain of hsa-miR-1184 Loss of hsa-miR-106b;Gain of hsa-miR-548m The results of direct mutational sequencing of the NOL7 genomic region in CC tumor samples are listed. The location of the mutation corresponding to Genbank accession NC_000006.11 is listed, along with the region of NOL7 aected. The mutation, SNP status, and predicted eect are included. The total mutation frequency is indicated. samples showed variation from the National Center for Biotechnology Information reference sequence, and 40% of these represented somatic tumor-associated mutations. The majority of these mutations are clustered in intron 5 and the 30 untranslated region (UTR) of NOL7. One mutation in intron 5 was identied in 2 of the tumor samples and in 5 of the 13 cell lines examined (G13618408A). Using software prediction programs, the majority of the intronic mutations are within splicing regions that may inuence alternative splicing of the NOL7 gene, whereas the 30 UTR mutations are associated with multiple miRNA sites (Tables 1, 2). In addition to somatic tumor-associated mutations, some cell lines and tumor samples also demonstrated single-nucleotide polymorphism (SNP) variations. In particular, SNP rs2841524 is located in the promoter region of NOL7. The variation at this locus was identied in 61% of CC cell lines and 39% of tumor samples. Variation of this particular allele from A-C is predicted to change the consensus HIF-1a-binding element within the promoter. Taken together, this demonstrates that NOL7 harbors tumor-associated mutations and SNP variations, and suggests these genomic alterations may potentially contribute to NOL7 inactivation in conjunction with LOH. DISCUSSION HPV infection has a well-characterized role in the pathogenesis of CC (6163). However, this event is insucient for oncogenesis, and the genetic and epigenetic changes that contribute to the development of CC are poorly understood. Chromosomal instability and LOH were among the rst nonviral mechanisms identied that contribute to CC (26 29,64). However, a limited number of putative TSGs, and their functional role in the development of CC, have been adequately described. One of the most common allelic losses in CC occurs at 6p23 (2631). Within this chromosomal region, there are several potential cancer-associated genes, including CD83 and NOL7. Evidence of genetic alterations of CD83 in CC has been demonstrated (37,65). However, functional demonstration of its tumor suppressive capacity is lacking. Conversely, we have previously shown that NOL7 displays allelic loss in both CC cell lines and tumor samples. Furthermore, reintroduction of NOL7 into CC tumor cell lines suppresses in vivo tumor growth by 95% in part by the modulation of the angiogenic phenotype (32). In this study, we investigated the genetic and epigenetic regulation of NOL7 and provide evidence of both mutations and LOH, further supporting the role of NOL7 as a TSG in CC. CC development represents a continuum of cytologic and molecular alterations that occurs over decades. The interrogation of normal cervical mucosa and various grades of CIN and CC demonstrated a decrease in the number of samples demonstrating NOL7 protein expression at the juncture between CIN I and CIN II (77.3% and 50.0%; Fig. 2B). A further decrease was observed in CIN III (13%, Int J Gynecol Pathol, Vol. 31, No. 1, January 2012 22 C. L. DOCI ET AL. Fig. 2B) and vast majority of CC lacked NOL7 expression (19.3%; Fig. 2B). This mirrors the progressive gain of expression of the angiogenic phenotype, as measured by the surrogate of microvessel density, during CC development (6672). Interestingly, the dramatic loss of NOL7 expression in CIN III also correlates with the stable integration of the HPV E6 and E7 oncoproteins (62,73,74). Therefore, decreased NOL7 expression concurrent with increased microvessel density and HPV integration supports the hypothesis that loss of NOL7 plays a critical role in the expression of the angiogenic phenotype during CC development and suggests that NOL7 expression may be additionally regulated through HPV oncoproteins. Although it is currently unknown whether there is a direct correlation between HPV type and NOL7 expression levels, the impact of HPV oncoproteins on upstream regulation of NOL7 is being investigated. It will also be of interest to determine whether other cancers whose etiology is correlated with HPV infection, such as head and neck cancers, also demonstrate dierential expression of NOL7 during histologic progression as has been observed in our CC model system. Both global DNA hypomethylation and regional DNA hypermethylation have been observed in CC, suggesting that methylation is likely a key mechanism for regulation of gene expression in CC (75). The identication of a CpG island within the promoter region of NOL7 suggested that the expression of the NOL7 gene might be epigenetically regulated. However, no methylation was detected in cell lines or tumor samples, suggesting that methylation is not a mechanism of NOL7 regulation. In addition to the promoter region CpG island, an additional CpG island within the fth intron of the NOL7 gene was detected. Although nonpromoter methylation has been described in gene bodies and intergenic regions, their functional signicance is not well understood (76,77). Nonpromoter methylation can enhance transcription in a protein-specic and tissue-specic manner (78). Enhanced methylation in intergenic regions is also associated with splicing eciency (79). A number of mutations were also detected in this region, suggesting that intron 5 may be an epigenetic hotspot that is targeted during CC development. The mutations identied within intron 5 and other introns are predicted to aect splicing patterns of the anking exons. Alternative splicing may account for up to 75% of the diversity in the human proteome and it is estimated that as much as 15% of the Int J Gynecol Pathol, Vol. 31, No. 1, January 2012 somatic mutations in cancer are attributable to alternative splicing (80,81). These alternative splicing patterns can manifest as truncated, frameshifted, or unstable transcripts. Alternative or truncated protein products would likely demonstrate altered functionality in vivo, as critical localization domains of NOL7 are coded within the carboxy terminus of the protein (82). Aberrant splicing patterns can also negatively impact the stability and expression of mutant transcripts, triggering nonsense-mediated decay and other nuclear surveillance mechanisms (8385). In addition, coupled splicing mechanisms can lead to altered cotranscriptional processing, leading to dierential expression of an mRNA at multiple levels (8688). Mutations within the 30 UTR of genes have also been shown to have signicant eects on mRNA polyadenylation, stability, export, and subsequent translation (8991). Although the eect of the specic mutations on NOL7 expression and function cannot be determined from this study alone, the number of mutations within a small genomic locus suggests that NOL7 may play a critical role in CC development and progression. It will be critical to experimentally determine the eect of these genetic alterations on NOL7 isoform expression, and to assess their functional consequences in the context of the second NOL7 allele. In addition, some of the identied nucleotide changes correlate with characterized SNPs NOL7. In particular, the expression of the A versus C allele identied in the promoter region of NOL7 corresponds to the consensus for an HIF-1a-binding site. Although the role of HIF-1a in NOL7 expression must be validated experimentally, this is particularly compelling when considered in conjunction with data showing that loss of NOL7 correlates with the onset of angiogenesis in CC clinical progression. In conclusion, NOL7 is a putative TSG in CC and perhaps other malignancies with loss of 6p23. In this study, we sought to characterize the expression of endogenous NOL7 in CC and to identify the mechanism of inactivation of its other allele. NOL7 was found to be signicantly downregulated in 6 of 13 CC cell lines. Furthermore, NOL7 protein expression decreased with histologic progression. Through bisulte and mutational sequencing of CC cell lines and tumor samples, we demonstrated that NOL7 is not methylated, but contains numerous tumor-associated somatic mutations and potentially deleterious SNPs in CC cell lines and tissue specimens. This provides additional evidence for the role of NOL7 as a bona de TSG in CC and suggests a mechanism by which NOL7 may contribute to the pathogenesis of cancer. GENOMIC REGULATION OF NOL7 REFERENCES 1. Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011;61:6990. 2. Narisawa-Saito M, Yoshimatsu Y, Ohno S, et al. An in vitro multistep carcinogenesis model for human cervical cancer. Cancer Res 2008;68:5699705. 3. Branca M, Giorgi C, Ciotti M, et al. Down-regulated nucleoside diphosphate kinase nm23-H1 expression is unrelated to high-risk human papillomavirus but associated with progression of cervical intraepithelial neoplasia and unfavourable prognosis in cervical cancer. J Clin Pathol 2006;59:104451. 4. 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... REVIEW Cell Research (2012) 22:23-32. 2012 IBCB, SIBS, CAS All rights reserved 1001-0602/12 $ 32.00 www.nature.com/cr Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer Atsuko Sakurai1, Colleen Doci1, J Silvio Gutkind1 1 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Rm. 211, Bethesda, MD 20892, USA Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for many physiological processes, and aberrant angiogenesis contributes to some of the most prevalent human diseases, including cancer. Angiogenesis is controlled by delicate balance between pro- and anti-angiogenic signals. While pro-angiogenic signaling has been extensively investigated, how developmentally regulated, naturally occurring anti-angiogenic molecules prevent the excessive growth of vascular and lymphatic vessels is still poorly understood. In this review, we summarize the current knowledge on how semaphorins and their receptors, plexins and neuropilins, control normal and pathological angiogenesis, with an emphasis on semaphorin-regulated anti-angiogenic signaling circuitries in vascular and lymphatic endothelial cells. This emerging body of information may afford the opportunity to develop novel anti-angiogenic therapeutic strategies. Keywords: semaphorin; signaling; angiogenesis; lymphangiogenesis; cancer Cell Research (2012) 22:23-32. doi:10.1038/cr.2011.198; published online 13 December 2011 Introduction Angiogenesis, the formation of new blood vessels from preexisting vasculature, is essential for many physiological processes, such as wound healing, and also plays a critical role in many pathological conditions including diabetic retinopathy, age-related macular degeneration, and tumor growth. Angiogenesis is controlled by delicate balance between pro- and anti-angiogenic signals, thus elucidating the molecular mechanisms underlying normal and aberrant blood vessel growth may provide new therapeutic options for many human diseases [1]. Among the pro-angiogenic molecules, vascular endothelial growth factor (VEGF) has received a considerable amount of attention as a promising target for anti-angiogenic therapy. Anti-angiogenic drugs targeting the VEGF pathway, such as a humanized monoclonal antibody against VEGF, were developed rapidly and used in the clinical settings. However, anti-VEGF treatments often result in only modest improvement in progression-free Correspondence: J Silvio Gutkind Tel: +1-301-496-3695, Fax: +1-301-402-0823 E-mail: sg39v@nih.gov survival, and tumors can eventually acquire resistance to anti-angiogenic therapies. To overcome these problems, a better understanding of the molecular mechanisms responsible for angiogenesis is necessary, which may afford the opportunity to develop new anti-angiogenic therapeutic strategies. The most widely investigated angiogenesis inhibitors are the proteolytic cleavage products of extracellular matrix or serum components, such as endostatin, angiostatin, arresten, and tumstatin (reviewed in [2, 3]). Multiple cytokines can also exert anti-angiogenic properties, including interferons and certain interleukins, which often act indirectly by limiting the expression of pro-angiogenic mediators or inducing anti-angiogenic molecules (reviewed in [2, 3]). In contrast, there are very few known developmentally regulated, naturally occurring anti-angiogenic molecules, which include platelet factor 4 [4], thrombospondin-1 [5], and pigment epithelium-derived factor [6], whose precise mechanism of action is not fully understood. In this regard, emerging evidence suggests that proteins involved in transmitting axonal guidance cues, including members of the netrin, slit, eph and semaphorin families, also play a critical role in blood vessel guidance during physiological and pathological blood vessel development [7, 8]. In this review, npg npg Semaphorin signaling in angiogenesis 24 we summarize the current knowledge on semaphorin family proteins in angiogenesis, with an emphasis on semaphorin-regulated anti-angiogenic signaling circuitries in endothelial cells. The possible role of semaphorin signaling in supressing lymphangiogenesis will also be discussed. Semaphorins Semaphorins are a family of cell surface and soluble proteins originally identified as axon guidance factors that control the development of central nervous system [9]. All semaphorins are characterized by an aminoterminal 500-amino acid Sema domain that is essential for signaling. Semaphorins are grouped into eight classes based on their structural domains, with classes 3-7 comprising the vertebrate semaphorins (Figure 1). Although initially identified as potent axon chemorepellents, several semaphorins can provide bifunctional guidance cues, functioning as repulsive or attractive molecules depending on the cell types and biological context [10, 11]. The common targets of semaphorins are the actin cytoskeleton and focal adhesions; the latter are dynamic cell-to-extracellular matrix adhesive structures that are assembled upon integrin engagement. Semaphorin signaling affects focal adhesion assembly/disassembly and induces cytoskeletal remodeling, thus consequently af- fecting cell shape, attachment to the extracellular matrix, cell motility, and cell migration [12, 13]. Semaphorin receptors: plexins and neuropilins (Nrps) Semaphorins signal through two major receptor families, plexins and Nrps. In vertebrates, two Nrps (Nrp1 and Nrp2) and nine plexins have been identified [14] (Figure 1). Membrane-bound semaphorins bind directly to plexins, whereas secreted type of semaphorins (class 3 semaphorins; Sema3s) bind to a holoreceptor complex consisting of Nrps as ligand binding and plexins as signal transducing subunit. An exception to this rule is Sema3E, which binds and signals directly through plexin-D1, independently of Nrps [15]. Plexins are single-pass transmembrane receptors and subdivided into four groups, type A, B, C, and D. Similar to semaphorins, plexins have extracellular Sema domains. In addition, plexins have PSI (plexins, Sema, integrins) and IPT (Ig-like, plexins, transcription factors) domains, and share homology in their extracellular segment with the Met family tyrosine kinase receptors [14]. The intracellular domains of plexins have weak sequence similarity to GTPase activating proteins (GAPs) and display GAP activity towards the small GTPase R-Ras [16]. Type A, B, and D plexins require the association of the Figure 1 Human Plexins, Nrps, and Semaphorins: Vertebrates express semaphorin classes 3 through 7, plexins A, B, C, D, and Nrps 1 and 2. Semaphorins and plexins are comprised of a Sema domain and variable repeats of the PSI domain, as indicated. Members of the semaphorin 3 (Sema3, A-G) class are secreted, while the semaphorins 4-7 (Sema4-7) are membrane bound. Plexins contain cytoplasmic tails that contain both GAP and GTPase-binding domains. Additionally, plexins B1, B2, B3, and D1 contain a PDZ binding domain. Nrp1 and Nrp2 are comprised of extracellular complement binding, FV/FVIII, and MAM domains, but have only a short cytoplasmic tail, requiring their association with plexin A1-4 to facilitate signaling. Cell Research | Vol 22 No 1 | January 2012 Atsuko Sakurai et al. npg 25 Rnd family of Rho-related GTPases to function as R-Ras GAP, while plexin-C1 displays GAP activity without Rnd [17]. Plexin-B1 also possesses GAP activity for RRas3/M-Ras [18]. Nrps are transmembrane proteins with a short cytoplasmic domain of about 40 amino acids, and their three C-terminal amino acids (S-E-A) constitute a PDZbinding motif [19]. In addition to Sema3s, Nrps also bind to structurally unrelated molecules, such as VEGF family proteins, and serve as their co-receptors [20, 21]. The extracellular domains of Nrps contain two complementbinding domains (a1/a2), two coagulation factor V/VIII homology domains (b1/b2), and a MAM domain (c). Sema3s principally bind to a1/a2 domains, and VEGFs bind to b1/b2 domains [14]. Genetic studies have shown that Nrp1 is required for vascular morphogenesis. Nrp1knockout mice are embryonically lethal due to vascular remodeling and branching defects [22, 23], whereas Nrp2-knockout mice are viable and their vasculature is grossly normal. However, Nrp2 null mice show absence of or reduced lymphatic vessel sprouting during development [24], suggesting that Nrp2 plays a key role in lymphangiogenesis (discussed below). 37]. The molecular mechanisms underlying the antiangiogenic effects of Sema3A are complex, as its receptor Nrp1 also controls VEGFR2 signaling by binding VEGF165 [38, 39]. Hence, it was initially suggested that Sema3s compete for the binding to Nrp1 with VEGF, thus inhibiting VEGF-induced angiogenesis. However, recent reports have shown that Sema3s and VEGF use different domains on Nrp1 for binding [40]. Consistent with a separate function of Sema3A on Nrp1, Sema3A increases vascular permeability, inhibits endothelial cell proliferation, and induces apoptosis even in the absence of VEGF [34, 41], suggesting Sema3A activates its own signaling routes. Interestingly, Sema3A impairs endothelial cell adhesion and migration by inhibiting integrin function [32]. The molecular mechanisms by which Sema3A regulates integrins are not fully understood. From the studies in neuronal cells, it is likely that activation of plexin-A1 by Sema3A induces the intrinsic R-Ras GAP activity of Plexin-A1, thus resulting in R-Ras inhibition [16, 42]. As R-Ras is known to sustain integrin activation, the inactivation of R-Ras by plexin-A1 may lead to the inactivation of integrins, thereby inhibiting integrinmediated cell adhesion. Anti-angiogenic semaphorins Sema3B Sema3B and Sema3F were identified as tumor suppressors that are deleted or inactivated in lung cancer [43, 44]. Consistently, overexpression of Sema3B suppresses tumorigenesis in adenocarcinoma cell lines [45], and Sema3B also decreases proliferation of lung and breast cancer cell lines [30], suggesting Sema3B exerts direct effects on cancer cells. In addition to the inhibitory effect on cancer cells, Sema3B can repel endothelial cells mainly through Nrp1, and therefore functions as an angiogenesis inhibitor [46]. Interestingly, Sema3B activity is abrogated as a result of proteolytic cleavage by furin-like pro-protein convertases, which is associated with enhanced invasion and proliferation in many cancers [46, 47]. Together, Sema3B may function as a tumor suppressor by working on both tumor cells and endothelial cells. Certainly, more work is necessary to define the precise signaling events that mediate this effect of Sema3B. Sema3s are the only secreted type of semaphorins in vertebrates. Seven Sema3s have been identified (designated by the letters A-G) and multiple Sema3s are reported to control physiological and pathological angiogenesis [25, 26]. Nrps and the type A/D family plexins (plexinA1, -A2, and -A3, and plexin-D1) act as receptors for Sema3s, and each Sema3 family member shows distinct binding preference for Nrps. For example, Sema3A binds to Nrp1 [27, 28], while Sema3F binds exclusively to Nrp2 [29]. Other Sema3s, such as Sema3B, can bind to both Nrps [30, 31]. Each Sema3-Nrp complex associates with specific plexins to mediate downstream signaling. Some Sema3s, such as Sema3A and Sema3F, are expressed in endothelial cells, suggesting an endothelialinitiated autocrine regulation of angiogenesis [32]. Sema3A Sema3A regulates endothelial cell migration and survival in vitro [33, 34], and tumor-induced angiogenesis in vivo [35]. The information emerging from the analysis of Sema3A null mice and mutant mice lacking Sema3A-Nrp1 signaling suggests that Sema3A may not be required for the early stages of developmental angiogenesis, but rather that Sema3A contributes to the reshaping of the vasculature to form a mature vascular network, such as that in the heart and brain [32, 36, www.cell-research.com | Cell Research Sema3E As described above, Sema3E binds directly to and activates plexin-D1 receptor. Plexin-D1 is highly expressed in endothelium during development [48] and also in tumor-associated blood vessels [49]. Interestingly, the expression of plexin-D1 is dynamically regulated and increased in tip cells, which extend numerous filopodia and respond to attractive and repulsive guidance cues at npg Semaphorin signaling in angiogenesis 26 the leading edge of the new branching blood vessels [50]. Sema3E controls vascular patterning during development by inhibiting the expansion of intersomitic vessels into the somites [15, 51], and causes endothelial-tip cell filopodial retraction in growing blood vessels [52]. These data suggest that Sema3E is a potent chemorepellent for plexin-D1-expressing endothelial cells. Indeed, Sema3E displays anti-angiogenic properties in several different in vivo angiogenesis models [52, 53]. Furthermore, Sema3E is highly expressed in metastatic tumors, where a cleaved form of Sema3E may enhance tumor cell motility. Conversely, when wild-type Sema3E is overexpressed in cancer cell lines, Sema3E decreases tumor vessel density and tumor growth [54]. The interplay between cleaved products of Sema3E and potential receptors in cancer cells is an area of active investigation. More progress has been recently made on the study of how Sema3E prevents angiogenesis in endothelial cells, given the obligatory role of plexin-D1 but not of Nrps in this biological response. Sema3E may counteract the pro-angiogenic effects of VEGF by promoting the expression and release of a soluble VEGF receptor, thus inhibiting VEGF function in developing vessels [55]. In a more direct fashion in endothelial cells, upon stimulation with Sema3E, plexin-D1 initiates a two-pronged mechanism involving R-Ras inactivation and Arf6 activation, thereby affecting the activation status of 1 integrin and its intracellular trafficking, respectively [52] (Figure 2). At the molecular level, Sema3E binding to plexin-D1 induces the activation of type I phosphatidylinositol-4-phosphate5-kinase (PIP5K) , which may stimulate the production of phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) locally [56]. PI(4,5)P2 then binds to the PH domain of a guanine nucleotide exchange factor (GEF) for Arf6, guanine nucleotide exchange protein 100, thus increasing its GEF activity and activating Arf6. Sema3E-induced Arf6 activation induces rapid focal adhesion disassembly and endothelial cell collapse, thereby inhibiting angiogenesis [56]. In addition, recent reports revealed that PI(4,5)P2 acts as a second messenger and controls focal adhesion dynamics and actin cytoskeleton [57], both of which are critical for cell migration. In neuronal cells, inhibition of another isoform of PIP5K, PIPKI661, is required for Sema3A-induced axonal repulsion and growth cone collapse [42]. These data suggest that semaphorins may commonly utilize phospholipid-regulated signaling Figure 2 Anti-angiogenic signaling by Sema3E-plexin D1 in endothelial cells. The activation of plexin D1 by Sema3E induces the association of the Ras GAP domain of plexin D1 with R-Ras. This inactivates integrins and enables their subsequent internalization by the plexin D1-mediated activation of Arf6, thus inhibiting endothelial cell adhesion to the extracellular matrix (ECM) by disrupting integrin-mediated adhesive structures, and causing filopodial retraction in endothelial tip cells. The pathway by which Sema3E stimulation of plexin D1 leads to Arf6 activation involves phosphatidylinositol-4-phosphate-5-kinase activation, which generates PI(4,5)P2 locally. PI(4,5)P2 then binds to the PH domain of an Arf6 GEF, guanine nucleotide exchange protein 100, resulting in Arf6 activation. Sema3E-induced Arf6 activation induces rapid focal adhesion (FA) disassembly, integrin internalization, and endothelial cell collapse, thereby inhibiting angiogenesis. Cell Research | Vol 22 No 1 | January 2012 Atsuko Sakurai et al. npg 27 pathways to regulate cell adhesion and migration, by regulating lipid kinases such as PIP5K. Sema3F Sema3F decreases tumor growth in a number of in vivo tumor models, and although Sema3F is capable of interacting with Nrp1, its higher-affinity interaction with Nrp2 appears to be required for its tumor-suppressive activity in many models [58-60]. Sema3F exerts a repulsive effect on breast cancer cells [61] and reduces the growth and metastatic activity of colorectal carcinoma cells by modifying integrin v3 [62], suggesting that Sema3F affects tumor cells directly by controlling cell adhesion and migration. Similar to other Sema3s, the Sema3Fmediated suppression of tumor growth was associated with an anti-angiogenic phenotype, where Sema3F expression significantly decreased the vascularity of tumors [25, 58]. Consistently, Sema3F can induce endothelial cell collapse [63], repel endothelial cells and inhibit their survival [59], and this anti-angiogenic effect is synergistically enhanced by the addition of Sema3A [34]. At the molecular level, it has been shown that Sema3F inhibits multiple signaling pathways in cancer cells [63, 64]; however, whether these pathways are also affected in endothelial cells remains unclear. While it is clear that Sema3F can function as a potent tumor suppressor in vivo, further studies are required to understand the molecular mechanism behind this function and the role that Sema3F expression plays in cancer development and progression. Sema3G Far less is known about the role of Sema3G in cancer and signaling. Sema3G has been identified as a significant prognostic marker in glioma [65]. Sema3G appears to bind to Nrp2, but not Nrp1 [66], and is able to suppress tumor growth and inhibit soft-agar colony formation only in cells expressing high levels of this receptor [25]. However, further work is necessary to determine whether Sema3G has anti-angiogenic effects in vivo and the mechanism by which it achieves its Nrp2-dependent tumor growth-suppressive activity. Pro-angiogenic semaphorins Some plexins are highly expressed in endothelial cells. Among them, plexin-B1 is a receptor for Sema4D, and it has been reported that Sema4D-Plexin-B1 signaling promotes endothelial cell migration and tube formation [67, 68]. Interestingly, Sema4D expression is upregulated in head and neck squamous cell carcinoma as well as in some other solid tumors [69, 70]. Sema4D is processed and released from the membrane by membrane type www.cell-research.com | Cell Research 1-matrix metalloproteinase, which is frequently overexpressed in malignant tumors, and activates plexin-B1 to elicit pro-angiogenic signaling in endothelial cells [71]. Similarly, Sema4D can be released from platelets by the action of metalloprotease ADAM17 (TACE), thereby acting on endothelial cells as well as platelets that also express Sema4D receptors [72]. The pro-angiogenic effect of Sema4D is mediated by the activation of a small GTPase RhoA through Rho-specific GEFs, leukemiaassociated RhoGEF, and PDZ-RhoGEF, which bind to the C-terminal PDZ-binding motif of plexin-B1 [67, 73-76]. Upon ligation of Sema4D, PDZ-RhoGEF and leukemia-associated RhoGEF are recruited to plexin-B1, thus stimulating RhoA and its downstream effector Rho kinase, and Rho kinase activation promotes angiogenic response through a chain of events involving myosin light chain phosphorylation, stress fiber contaction, nonreceptor tyrosine kinase activation, and activation of the Akt and Erk pathways [77] (Figure 3). RhoA/Rho kinase signaling also activates PIP5K and leads to the generation of the lipid second messenger PI(4,5)P2, further supporting the idea that phosphoinositides may represent a common mediator for semaphorin signaling. In the case of Sema4D, PI(4,5)P2 serves as a substrate for PLC and increases intracellular calcium level, which is required for Sema4D-induced tube formation in vitro [78]. In addition to the direct effect of Sema4D on plexin-B1, this semaphorin can also promote endothelial cell migration through the activation of HGF receptor Met [68], while in other cell types, plexin-B1 activates members of the EGF receptor family [79] and inhibits RhoA through p190 RhoGAP [80], resulting in changes in the actin cytoskeleton and cell migration. Lymphangiogenesis and cancer Like angiogenesis, lymphangiogenesis is both a physiological and pathological process required for the formation of new vasculature, and the regulation of this process is mediated by a balance of pro- and anti-lymphangiogenic factors. The lymphatic system is critical for the removal of waste products and transport of cells and proteins, and contributes to the immune response and cancer progression. During embryogenesis, expression of the transcription factor PROX1 leads to the differentiation of venous endothelial cells into lymphatic endothelial cells [81-84]. This differentiation is followed by sprouting and proliferation of the lymphatic vasculature, which is primarily attributed to signaling by VEGF-C through its receptor VEGFR3 [85-87]. VEGF-C binds to receptors VEGFR3 and Nrp2, and loss of these molecules results in a variety of lymphangiogenic defects. Vegfc/ mice npg Semaphorin signaling in angiogenesis 28 Figure 3 Pro-angiogenic signaling by semaphorins in endothelial cells. Membrane-bound Sema4D expressed by cancer cells or by tumor-associated macrophages and platelets is cleaved by matrix metalloproteinases, and soluble Sema4D can then bind and activate plexin-B members expressed on endothelial cells. After stimulation by Sema4D, plexin-B can activate certain receptor tyrosine kinases, such as Met, EGFR, and Her2, in some cellular contexts, hence activating their regulated signaling pathways indirectly. Sema4D binding to plexin-B causes the recruitment and activation of PDZ-RhoGEF and leukemiaassociated RhoGEF through association mediated by their PDZ domains. These GEFs then activate Rho and its downstream effector Rho kinase (ROCK). In turn, ROCK signaling promotes the phosphorylation of myosin light chain. This induces polymerization and contraction of actin/myosin stress fibers. The tension generated by contracting stress fibers promotes the assembly of mature focal adhesion complexes at the sites of cell contact with the extracellular matrix via integrins, thereby activating non-receptor tyrosine kinases such as Pyk2 and Src. Pyk2 activation results in the phosphorylation of phosphatidylinositol 3-kinase and the activation of the Akt and Erk1/2 pathways, leading to increased endothelial cell migration and proliferation, which contribute to promoting an angiogenic response. are able to commit to the lymphatic lineage, but cannot undergo lymphangiogenesis and die in utero [88]. VEGFR3-knockout mice display a more severe phenotype, where they lack lymphatic vasculature entirely and fail to undergo lymphangiogenesis [89]. In contrast, Nrp2 is not required for lymphangiogenesis but appears to be a key regulator of the process. Mice deficient in Nrp2 show a variety of lymphatic defects, including reduction of small lymphatic vessels and capillaries [24]. Further, Nrp2 appears to be selectively expressed in tumor-associated lymphatic vessels, and inhibition of Nrp2 function blocked VEGF-C/VEGFR3-mediated lymphangiogenesis and reduced metastasis in vivo [90, 91]. By exten- sion, this suggests that anti-angiogenic semaphorins like Sema3F can function as anti-lymphangiogenic signaling molecules in vivo through their interaction with Nrp2. Sema3F overexpression has been shown to have a direct chemorepulsive effect on lymphatic endothelial cells [58]. Sema3F appears to compete for Nrp2 binding with VEGF-C and decreases endothelial cell survival and migration [92]. Nevertheless, direct evidence for an antilymphangiogenic role of Sema3F is limited. Indirectly, tumor-induced lymphangiogenesis is correlated with metastasis in a number of cancer models, including colorectal, breast, prostate, head and neck cancer, and melanoma [93-99], and expression of Semaphorin Cell Research | Vol 22 No 1 | January 2012 Atsuko Sakurai et al. npg 29 molecules decreases the incidence of metastasis [58, 62, 100]. Inhibition of tumoral lymphangiogenesis through neutralization of VEGFR3 or knockdown of VEGF-C expression blocks metastasis in vivo [101-103], indicating that this process is essential for metastasis in a variety of cancer models. Interestingly, this inhibition did not affect the normal lymphatic vasculature in adult mice, suggesting that alternative signaling mechanisms are employed after development, and targeting the VEGF-C/VEGFR3 signaling axis in adults may represent a potent means to target the tumor-associated lymphatics [104]. The precise molecular events that signal for lymphangiogenesis are poorly understood. Inhibition of the mTOR pathway decreases lymphangiogenesis and metastasis, and prolongs survival in a head and neck cancer animal model [99]. In support of this, a separate study found that inhibition of phosphatidylinositol 3-kinase/Akt similarly blocked lymphangiogenesis [105]. However, the specific signaling events required to initiate and sustain lymphangiogenesis in the context of tumor progression and metastasis are poorly defined. Given that tumoral lymphangiogenesis is likely a critical event in the progression of metastatic disease, the potential role of semaphorin-mediated regulation in this process warrants further investigation. Concluding remarks Recent studies have revealed the importance of semaphorins, plexins, and Nrps in tumor progression. These molecules directly control the behavior of tumor cells and also affect the other cell types which constitute the tumor microenviroment. In this review, we focused on vascular and lymphatic endothelial cells, both of which contribute to providing a suitable environment for tumor cells to grow and metastasize by inducing angiogenesis and lymphangiogenesis. A large fraction of the current information regarding semaphorin signaling was initially obtained from extensive studies in neuronal and cancer cells. It will be important to analyze whether the pathways deployed by the semaphorin-plexin signaling systems are shared by all cell types, with emphasis on those utilized in vascular and lymphatic endothelial cells to inhibit or promote angiogenesis and lymphangiogenesis. From the studies on Sema3B and Sema3F, loss of semaphorin function may represent an early event in cancer development, thus contributing to the acquisition of the angiogenic phenotype that characterizes most solid tumors. However, a number of questions remain: Which semaphorins are cleaved by furin-like proteases, and how does the proteolytic cleavage of semaphorins affect their function? Can the differential expression of these enzymatic factors account for the discrepancies in signaling www.cell-research.com | Cell Research and biological effects observed among different studies? Does differential expression of semaphorin receptors on tumor and endothelial cells affect their signaling potential? How does expression of competitive factors, such as VEGF, regulate the signaling capabilities of the semaphorins? Further, can their tumor-suppressive and anti-angiogenic roles be strengthened by multi-targeted approaches that take into account both the semaphorins and the regulators of their expression and function? What are the molecular differences in the signaling pathways activated by semaphorin binding, as opposed to the withdrawal of VEGF signaling? 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Cancer Res 2011; 71:7061-7070. Cell Research | Vol 22 No 1 | January 2012  ...

... Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Cancer Research Tumor and Stem Cell Biology Genetic Identication of SEMA3F as an Antilymphangiogenic Metastasis Suppressor Gene in Head and Neck Squamous Carcinoma Colleen L. Doci1, Constantinos M. Mikelis1, Michail S. Lionakis2, Alfredo A. Molinolo1, and J. Silvio Gutkind1 Abstract Head and neck squamous cell carcinomas (HNSCC) often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. HNSCCs are remarkably lymphangiogenic and represent a clear example of a cancer that utilizes the lymphatic vasculature for malignant dissemination; however, the molecular mechanisms underlying lymphangiogenesis in HNSCC is still poorly understood. Of interest, we found that an axon guidance molecule, Semaphorin 3F (SEMA3F), is among the top 1% underexpressed genes in HNSCC, and that genomic loss of SEMA3F correlates with increased metastasis and decreased survival. SEMA3F acts on its coreceptors, plexins and neuropilins, among which neuropilin-2 (NRP2) is highly expressed in lymphatic endothelial cells (LEC) but not in oral epithelium and most HNSCCs. We show that recombinant SEMA3F promotes LEC collapse and potently inhibits lymphangiogenesis in vivo. By reconstituting all possible plexin and neuropilin combinations, we found that SEMA3F acts through multiple receptors, but predominantly requires NRP2 to signal in LECs. Using orthotopic HNSCC metastasis mouse models, we provide direct evidence that SEMA3F re-expression diminishes lymphangiogenesis and lymph node metastasis. Furthermore, analysis of a large tissue collection revealed that SEMA3F is progressively lost during HNSCC progression, concomitant with increased tumor lymphangiogenesis. SEMA3F is localized to 3p21, an early and frequently deleted locus in HNSCC and many other prevalent human malignancies. Thus, SEMA3F may represent an antilymphangiogenic metastasis suppressor gene widely lost during cancer progression, hence serving as a prognostic biomarker and an attractive target for therapeutic intervention to halt metastasis. Cancer Res; 75(14); 293748.
2015 AACR. Introduction demonstrate intratumoral lymphangiogenesis that is associated with invasion, metastasis, and decreased survival (68). However, the relative contribution of angiogenesis and lymphangiogenesis to cancer progression and metastasis is still not fully understood. HNSCC is one of the ten most common cancers globally and less than half of patients diagnosed with nonlocalized disease will survive beyond 5 years (9, 10). This is partially attributed to the propensity of HNSCC to metastasize to locoregional lymph nodes, which is the single most signicant prognostic indicator and decreases the overall survival rate by more than 50% (1113). Furthermore, approximately 40% of the lymph nodes in the human body are located within the head and neck region (14), and HNSCC demonstrates signicant intratumoral lymphangiogenesis compared with other solid cancers, suggesting that lymphangiogenesis may play a pivotal role in HNSCC metastasis and survival. Therefore, HNSCC may represent a clinically relevant condition to begin to dissect the specic role of lymphangiogenesis in metastasis. The emergence of deep-sequencing approaches for human disease has led to the identication of a multitude of aberrant molecules in cancer that may contribute to its pathogenesis. While conducting analyses on altered molecules in the HNSCC genome, we observed that SEMA3F is among the top 1% underexpressed genes (15). SEMA3F is a member of the class 3 semaphorin family originally characterized in axonal guidance (16). In addition, semaphorins have been shown to play multiple roles in normal and pathologic angiogenesis by acting on their receptors, plexins and neuropilins (1720). Interestingly, SEMA3F can bind to One of the dening hallmarks of cancer is ability to form new vasculature to facilitate the growth and metastatic spread of cancer cells (1). Metastasis is the leading cause of morbidity in patients with a variety of solid tumors, where cancer cells often disseminate through blood and lymphatic vessels (1). Thus, the presence of intra- and peritumoral vasculature is a critical diagnostic and prognostic biomarker (2). Angiogenesis is required for tumors to grow beyond a critical limit and tumor-associated blood vessels have been suggested to participate in metastasis (3). Signicantly less is known about the role of lymphangiogenesis in cancer, although lymphatic invasion is one of the most relevant diagnostic parameters for solid tumors (2, 4, 5). Specically, melanoma and head and neck squamous cell carcinomas (HNSCC) 1 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland. 2Fungal Pathogenesis Unit, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: J. Silvio Gutkind, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive, Bldg 30, Rm 320, Bethesda, MD 20892. Phone: 301-496-6259; Fax: 301-4020823; E-mail: sg39v@nih.gov doi: 10.1158/0008-5472.CAN-14-3121
2015 American Association for Cancer Research. www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2937 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. neuropilin 2 (NRP2), and early studies indicated that SEMA3F expression prevents the growth of metastatic melanoma cells that express high levels of NRP2 (21). However, the relevance of SEMA3F expression in cancers lacking NRP2 has not been investigated. Furthermore, NRP2 is a coreceptor highly expressed on lymphatic endothelial cells (LEC). Therefore, these observations prompted us to explore whether SEMA3F loss may contribute to HNSCC lymphangiogenesis, and hence impact on cancer progression and metastasis. Materials and Methods The following represent a brief summary of the procedures. Please see the Supplementary Material for additional detailed methods. Cell culture 293T-17, HaCat, COS-7, UMSCC2, and UMSCC17B cells were cultured in DMEM 10% FBS. LECs and HMVECs were cultured in EGM2-MV and human umbilical vein endothelial cells (HUVEC) were cultured in EGM-2 (Lonza). All cells were cultured at 37 C in 5% CO2. UMSCC2 and UMSCC17B stable cell lines were achieved by selection with 1 mg/mL blasticidin. Transfection of plasmids and siRNAs can be found in the Supplementary Methods. All cell lines underwent DNA authentication (Genetica DNA Laboratories, Inc.) before the described experiments to ensure consistency in cell identity. The Cancer Genome Atlas analysis Data regarding the copy number of PIK3CA and SEMA3F in head and neck cancer was downloaded from the cBio Portal for Cancer Genomics (http://www.cbioportal.org/public-portal/ accessed February 5, 2014). Immunohistochemistry Tissue arrays containing normal and oral cancer tissues were purchased from US BioMax Inc. Histopathology of tongue sections was performed as previously described (22). Formalin-xed parafn-embedded (FFPE) slides were stained and for tissue arrays were classied on the basis of the intensity and the percentage of positive cells quantied as described (23). Correlations were determined using Pearson coefcient. SEMA3F purication Serum-free conditioned medium (CM) from 293T-17 cells expressing NTAP-SEMA3F construct was collected, dialyzed, then isolated using HisTALON cobalt beads (Clontech). DFLAG control was generated by incubating puried SEMA3F with anti-FLAG conjugated beads (Sigma-Aldrich) and collecting the unbound supernatant. Immunoblotting Cells were lysed in radioimmunoprecipitation assay buffer and concentration was determined using Bio-Rad DC protein assay. Twenty micrograms total protein was separated by SDS-PAGE and transferred to polyvinylidene diuoride membrane overnight at 4 C. Membranes were blocked for 1 hour at room temperature in 5% milk in TBST and then probed with primary antibodies overnight at 4 C. Membranes were washed four times in Trisbuffered saline 1% Tween-20, probed with horseradish peroxidaseconjugated secondary antibodies for 1 hour at room tem- 2938 Cancer Res; 75(14) July 15, 2015 perature in 5% milk, washed four times in TBST, and detected using chemiluminescent substrate (Millipore). Immunouorescence For LECs and normal oral keratinocytes (NOK), cells were xed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. LECs were stained with phalloidin-GFP (Invitrogen) and counterstained with Hoescht 33342 (Invitrogen). NOKs were stained with SEMA3F (Sigma-Aldrich) or 58K Golgi Protein (Abcam), imaged with anti-rabbit AlexaFluor 488 (Invitrogen) or anti-goat AlexaFluor 546 (Invitrogen), and counterstained with Hoescht 33342 (Invitrogen). For Matrigel and orthotopic tumor sections, FFPE slides were prepared and stained using the immunohistochemistry protocol described, and then counterstained Hoescht 33342 (Invitrogen). The images were taken using an Axio Imager Z1 microscope equipped with an ApoTome system. Cell adhesion and collapse assays For adhesion assays, LEC were treated and plated on collagencoated plates. Nonadherent cells were removed by washing and adherent cells xed and stained. For collapse, LECs were transfected with LifeAct GFP and treated, or treated, xed, and stained with uorescent phalloidin and nuclear counterstain. Cell area and perimeter were assessed using ImageJ. For heterologous assays, transfected COS-7 cells were treated as indicated. For all assays, quantication was performed using ImageJ from three independent experiments. Statistical signicance was determined using one-way ANOVA. For movies, cells were imaged on an Olympus IX-81 inverted confocal microscope with images obtained every 30 seconds for a total of 3 hours, and analyzed using ZEN software (Carl Zeiss). In vivo lymphangiogenesis assay and FACS Basement membrane extract (Trevigen) plugs with growth factors and inhibitors, as indicated, were injected subcutaneously into the ank of nude mice. Single-cell suspensions from plugs were prepared as described (24) and cell populations determined by FACS. Statistics were determined using ANOVA from three independent experiments. Cells were resuspended in PBS, stained with a LIVE/DEAD uorescent dye (L-23105; Invitrogen), and incubated with CD16/32 (2.4G2; BD Biosciences) to block Fc receptors. For staining, cells were incubated with Alexa Fluor 488-conjugated LYVE-1, PE-Cy7-conjugated CD31, allophycocyanin (APC)-conjugated TER-119, APC-Cy7conjugated CD45 and eFluor 450-conjugated CD102 (eBioscience or BD Biosciences) washed, and analyzed on a 5-laser LSRFortessa (BD Biosciences). Data were analyzed using FACS Diva (BD Biosciences) and FlowJo software (Treestar). Quantication of cell types was performed using PE-conjugated uorescent counting beads (Spherotech). Migration assay Cells were treated as indicated for overnight Boyden migration. Membranes were xed with methanol, counterstained with hematoxylin, and imaged on an Axiovert microscope. Calculations were based on 18 imaging elds each from three independent experiments. Statistical signicance was determined using ANOVA. Proliferation assay Cancer cell lines were plated to 60% conuence and transferred to serum-free media with doxycycline for 20 hours. Cells were Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor incubated an additional 4 hours with 1 mCi [methyl-3H]-thymidine (PerkinElmer). Proliferation was determined by liquid scintillation counting. Statistical signicance was determined using ANOVA. Orthotopic tumor xenografts in SCID/NOD mice All animal studies were carried out according to NIHapproved protocols (ASP# 10-569), in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Female SCID/NOD mice (NCI) were housed in appropriate sterile lter-capped cages, and fed and watered ad libitum. Each animal was injected with 105 cells in 50 mL of serum-free media in the tongue. Animals in the control group were fed regular chow, while animals in the prevention group were fed 6 g doxycycline/kg chow 1 week before injection and throughout the study. All animals underwent evaluation of the tongue for disease onset every 3 days, and the observed lesions were assessed for length and width, and tumor volume was determined as described previously (22). Animals were euthanized at the indicated time points and the cervical lymph nodes assessed for evidence of metastases by H&E staining. Results SEMA3F expression is lost during HNSCC progression While analyzing gene expression alterations in HNSCC in available datasets, we observed that SEMA3F is among the 1% most downregulated genes [2.09-fold decrease, P 3.6E20; ref. 15] and is localized to 3p21, one of the most commonly deleted loci in HNSCC (25). To investigate whether reduced SEMA3F expression is caused by genomic alterations, we interrogated the The Cancer Genome Atlas (TCGA) Head and Neck Cancer databases, using PIK3CA, one the most frequently amplied genes in HNSCC that is localized to the long arm of the same chromosome (26) as a control (Fig. 1A). Nearly 75% of HNSCC patients showed heterozygous loss of SEMA3F, suggesting that this semaphorin may represent a potential tumor suppressor (Fig. 1A). We assessed the KaplanMeier univariate survival of patients with heterozygous loss of SEMA3F (Fig. 1B) and the Cox proportional hazard multivariate survival, taking into consideration lymph node metastasis (Fig. 1C). In both analyses, SEMA3F was a strong negative indicator of survival and a statistically signicant independent biomarker [HR 2.1, P 0.01 and exp(b) 1.95, P 0.03, respectively]. The median survival of patients with heterozygous loss was nearly half of patients expressing normal levels of SEMA3F (Fig. 1D). In addition, patients with SEMA3F heterozygous loss showed signicantly increased percentage of metastatic lymph nodes and lymphovascular invasion (Fig. 1E and F). SEMA3F loss was reected at the protein level, as immunohistochemical evaluation of SEMA3F using a SEMA3F-specic antibody (Supplementary Fig. S1) revealed progressive loss of expression with advancing cancer severity. While normal oral epithelium demonstrated high level of SEMA3F expression, this was almost completely abolished in advanced cancers (Fig. 1G). Quantication of a large collection of HNSCC tissues revealed that nearly 70% of the proliferating basal cells in normal oral epithelium expressed high levels of SEMA3F, while over half of HNSCC samples express little to no SEMA3F (Fig. 1H). Interestingly, this correlated with enhanced tumor vascularity, with markers for blood and lymphatic endothelium www.aacrjournals.org signicantly increased in the absence of SEMA3F (Fig. 1I and Supplementary Fig. S2). These data support that decreased expression of SEMA3F correlates with poor clinical outcome, increased tumor vascularity, invasion, and metastasis, thus suggesting that SEMA3F may function as a tumor and metastasis suppressor in HNSCC. SEMA3F is a chemorepulsant for LECs As the SEMA3F coreceptor NRP2 is highly expressed on LECs (27), we asked whether SEMA3F has a functional impact on these endothelial cells. We engineered a SEMA3F construct that would preserve its extracellular secretion and posttranslational processing while allowing for coexpression of N-terminal tandem afnity purication (NTAP) tags (Fig. 2A). SEMA3F was expressed, fully processed, and the secreted form of the protein was capable of purication using histidine afnity resin (Fig. 2A). Class 3 semaphorins function through a mechanism that involves negative regulation of integrin adhesion to the extracellular matrix, thereby modulating the attachment of endothelial cells to extracellular matrices through cytoskeletal remodeling (28). Secreted SEMA3F was functionally active in HUVEC adhesion assays, and this effect is specic as FLAG-depleted SEMA3F CM but not IgGdepeleted CM was no longer able to inhibit attachment (Fig. 2B). Furthermore, SEMA3F caused a dose-dependent decrease of LEC attachment, which was abolished by FLAG-mediated depletion (Fig. 2C). SEMA3F also induced a nearly 80% collapse of the LEC actin cytoskeleton, while decreasing cell perimeter by less than 3% (Fig. 2D). Using either puried SEMA3F or FLAG-depleted control, we documented the cellular collapse using live-cell imaging in LECs expressing uorescent actin (Supplementary Movies S1 and S2). SEMA3F induced signicant retraction of the LEC cytoskeleton, while little change in actin was observed in the FLAGdepleted control (Fig. 2E). Together, this indicates that SEMA3F can be puried in a biologically active form that is capable of negatively regulating the function of LECs. SEMA3F inhibits LEC function in vivo We next asked whether SEMA3F can function as antilymphangiogenic factor in vivo. Matrigel combined with different growth factors were implanted in anks of nude mice and invaded cells were identied and quantied by FACS. As proangiogenic growth factors such as VEGF-A and -C use neuropilins as coreceptors (16), sphingosine-1-phosphate (S1P), and basic broblast growth factor (bFGF) were substituted to circumvent any potential competition with SEMA3F. A stepwise gating strategy was employed to identify specic types of vascular-associated cells and endothelial cells while excluding immune-reactive cells like macrophages (Supplementary Fig. S3). Using Matrigel alone as a negative control and S1P and bFGF as a positive control, we tested the ability of puried SEMA3F to block chemoattractant-mediated endothelial cell recruitment in a dose-dependent, quantitative in vivo setting (Fig. 3). Gross evaluation and H&E-staining Matrigel plugs demonstrated strong inltration of vasculature in the positive control that was attenuated by SEMA3F addition (Fig. 3A, top and middle). Immunouorescence against vascular (CD31) and lymphatic (LYVE-1) vessels demonstrate a SEMA3F dose-dependent decrease in S1P- and bFGF-induced vessel recruitment (Fig. 3A, bottom). SEMA3F caused approximately 70% decrease in the abundance of red blood cells, long-regarded as the standard for angiogenesis implantation assays (Fig. 3B; ref. 29). Similarly, Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2939 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 1. SEMA3F expression is lost during head and neck cancer progression. A, SEMA3F (3p21.13p21.2) and PIK3CA (3q26.22q26.3) are located on chromosome 3 and their genomic copy number in head and neck cancer patients according to TCGA was calculated. Clinical outcome for patients with normal (CN 2) or SEMA3F heterozygous loss (CN 1) were interrogated and KaplanMeier (B) and Cox proportional hazard survival curves (C), median survival (D), evidence of metastatic disease (E), and lymphovascular invasion (F) were determined. G, immunohistochemistry of SEMA3F in primary tumor sections of normal oral epithelium and cancer. H, quantication of SEMA3F staining in a tissue array including normal oral epithelium, stage I, II, III, and IV cancers cases. Tissues were evaluated for intensity and percentage of positive cells, with 0 being absent staining and 4 being intense staining in all cells. I, the same array was also stained for podoplanin (lymphatic vessels) and CD31 (blood vessels), and correlation to SEMA3F expression was calculated using Pearson coefcient. Statistical signicance compared to normal was determined by Student t test,  , P < 0.01;    , P < 0.001. 2940 Cancer Res; 75(14) July 15, 2015 Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor Figure 2. SEMA3F is a chemorepulsant that negatively impacts the function of lymphatic endothelial cells. A, Coomassie staining and Western blot analysis of serum-free CM from cells transfected with NTAP-SEMA3F. B, attachment of HUVEC cells CM from NTAP-SEMA3F cell supernatants alone or after FLAG or IgG immunoprecipitation depletion. C, attachment of LECs in the presence of increasing amounts of puried SEMA3F or supernatant after FLAG depletion. D, immunouorescent staining of actin (green) and nuclei (blue) in LEC treated with 100 ng/mL SEMA3F for 6 hours. Quantication of cell area and perimeter were determined using ImageJ using 25 independent elds. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. E, still images captured during live-cell imaging of LEC transfected with LifeAct GFP and treated with 1 mg/mL SEMA3F or DFLAG. we saw 60% to 70% decreased abundance of total CD31 CD102 endothelial cells (Fig. 3C) and vascular endothelial cells expressing little to no LYVE-1 (LYVE-1low), consistent with an antiangiogenic function previously reported for SEMA3F (Fig. 3D; refs. 21, 30, 31). Remarkably, SEMA3F prevented the recruitment of LECs (dened as LYVE-1hi), with a 70% to 85% reduction in the abundance of LECs with respect to control plugs (Fig. 3E). Thus, SEMA3F has potent antiangiogenic activity and an even more robust antilymphangiogenic function. NRP and plexin A coreceptor complexes are sufcient for SEMA3F signaling NRP2 and plexin A family members are capable of binding to SEMA3F (32, 33), but the relative contribution of each member of this family to SEMA3F-mediated signaling is unknown. Primary HUVEC, human microvascular endothelial cells (HMVEC), and LEC express NRP1, NRP2, and plexin A family members to varying degrees, while COS-7 cells do not express any of these receptors endogenously, thus serving as a heterologous system to investigate SEMA3F signaling (Fig. 4A). Each of the NRP and plexin A family members were expressed in COS-7 cells to test their relative www.aacrjournals.org contribution to SEMA3F signaling alone and in coreceptor complexes (Fig. 4B). As a control, we also tested the effect of SEMA3A, as this semaphorin signals primarily through NRP1-Plexin A1 and NRP1-Plexin A3 (34). Neither NRPs nor plexins alone were sufcient for SEMA3F or SEMA3A signaling (Fig. 4C and D). SEMA3A was most effective in cells expressing NRP1 and plexin A1, as predicted. SEMA3F potently induced the collapse of COS-7 cells that expressed NRP2 and plexin A3, although cells expressing NRP2 and plexin A1 also collapsed when compared with controls. Interestingly, a modest collapse was observed in SEMA3F-treated cells that expressed NRP1 and plexin A3, and to a lesser degree in those that expressed NRP1 and plexin A1. Some broader effect of SEMA3A was also observed, as NRP2-Plexin A1 complexes and NRP1-Plexin A4 complexes showed collapse. Plexin D1, a receptor for SEMA3E, did not appear to play a role in SEMA3F function (Supplementary Fig. S4). On the basis of these results, we summarized the relative contribution of different receptor combinations to SEMA3Finduced cytoskeletal collapse (Fig. 4E). Finally, we performed live cell imaging of the collapse in COS-7 cells transfected with NRP2 control or NRP2 and plexin A3 treated with SEMA3F Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2941 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 3. SEMA3F inhibits in vivo lymphangiogenesis. A, Matrigel plugs containing the indicated factors were examined for gross histology (top row), H&E (middle row), and immunouorescence for CD31 (red) and LYVE1 (green; bottom row). FACS analysis of the number of total red blood cells (B), total CD102 CD31 endothelial cells low (C), LYVE-1 vascular endothelial cells (D), hi and LYVE-1 lymphatic endothelial cells (E) are shown. Values were expressed as a percentage of the stimulated vehicle control from three independent experiments. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. (Supplementary Movies S3S4). Control cells demonstrated no changes in the actin cytoskeleton, while expression of NRP2 and plexin A3 was sufcient to induce a rapid collapse phenotype with signicant alterations in the actin cytoskeleton (Fig. 4F). NRP2 is predominantly required for SEMA3F signaling To determine whether both endogenous NRP1 and NRP2 are necessary for SEMA3F signaling, we abolished expression of these receptors using siRNAs in LECs. Little to no NRP expression remained in siRNA-targeted cells, with no apparent cross-specicity between the knockdown sequences (Fig. 5A). Loss of NRP1 expression only modestly attenuated the collapse of the cells upon SEMA3F treatment; however, signicantly less collapse was observed in SEMA3F-treated LEC that do not express NRP2 (Fig. 5B). In a doseresponse collapse (Fig. 5C) and attachment (Fig. 5D) assay, loss of NRP1 slightly inhibited the effectiveness of low doses of SEMA3F. In both assays, however, NRP2 knockdown had a pronounced effect, suggesting that NRP2 is the dominant 2942 Cancer Res; 75(14) July 15, 2015 SEMA3F receptor in LECs. This was further documented by logdose response analysis (Fig. 5E). While loss of NRP1 increased the IC50 of SEMA3F slightly compared with control, NRP2-decient cells required nearly ten times more SEMA3F to achieve the same effect. Taken together, these ndings suggest that while NRP1 may play a role in transmitting SEMA3F signal in the absence of NRP2, NRP2 is predominately required for SEMA3F biologic responses in LECs. A subset of HNSCC cells express NRP2 and respond to the antitumor activity of SEMA3F NOKs and most HNSCC cancer cells do not express NRP1 or NRP2. However, a subset of HNSCC cell lines acquired expression of NRP2 (Fig. 6A). Aligned with this observation, increased NRP2 mRNA expression correlates with loss of SEMA3F (P 0.003), poor prognosis, and decreased survival (Supplementary Fig. S5). To test the effect of SEMA3F in HNSCC cells with or without NRP2, we chose two representative highly tumorigenic and Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor Figure 4. NRP and plexin A coreceptors coordinate SEMA3F function. A, Western blot analysis demonstrating expression of the NRP, plexin, and VEGFR family members in a panel of immortalized and primary epithelial and endothelial cells. B, Western blot analysis demonstrated specic expression of each NRP and plexin A family member in COS-7 cells. C, COS-7 cells stably expressing GFP were transfected and treated as indicated. Collapse was quantied using ImageJ relative to vehicle control based on 25 imaging elds, each from quadruplicate wells in three independent experiments. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. D, representative images of cells transfected with different receptor combinations and treated with vehicle, SEMA3F, or SEMA3A. E, table summarizing the relative contributions of the different receptor combinations tested. F, confocal images of COS-7 cells transfected with LifeAct-GFP, NRP2, and/or plexin A3 and treated with SEMA3F or DFLAG. Images were taken 1 and 3 hours after treatment with SEMA3F. metastatic HNSCC cell lines (22), UMSCC17B, which do not express NRPs, and UMSCC2, with high level expression of NRP2. As an experimental approach, we used a doxycyclineinducible system and conrmed that doxycycline addition induced SEMA3F expression without changing the expression of NRP1 or NRP2 in these cells (Fig. 6B). In both cell types, expression of the rtTA3 tetracycline-dependent transactivating complex alone had no effect on proliferation (Fig. 6C and D). SEMA3F expression inhibited proliferation of UMSCC2 cells signicantly; however, this effect was abrogated upon NRP2 knockdown (Fig. 6C). Conversely, in UMSCC17B cells induction of SEMA3F did not alter proliferation (Fig. 6D). As a gainof-function strategy, transient expression of NRP2 in UMSCC17B rendered them sensitive to the growth inhibitory activity of SEMA3F (Fig. 6D). Induction of SEMA3F in UMSCC2 cells also blocked endogenous and directed migration (Fig. 6E). Interestingly, siRNA-mediated loss of NRP2 decreased the endogenous migration of these cells compared with control siRNA, although there was no difference between vehicle- and doxycyline-treated groups (Fig. 6E). www.aacrjournals.org Ectopic expression of NRP2 in UMSCC17B cells was sufcient to enhance cell migration over vector control, and induction of SEMA3F did not alter endogenous UMSCC17B migration unless NRP2 was introduced to these cells (Fig. 6F). Taken together, this supports that HNSCC cells expressing NRP2 may exhibit increased migratory capacity, but that loss of SEMA3F may need to precede gain of NRP2 expression. SEMA3F functions as a potent HNSCC metastasis suppressor SEMA3F inhibited the biologic activity of LECs and intratumoral lymphangiogenesis is highly prevalent in HNSCC, suggesting that SEMA3F may suppress metastasis in vivo through paracrine mechanisms. To test this hypothesis in the context of NRP2 expression or absence, we injected UMSCC2-rtTA3-SEMA3F and UMSCC17B-rtTA3-SEMA3F cells into the tongues of mice and assessed tumor growth, metastasis, and intratumoral vascularity. Consistent with our observations in vitro, doxycycline induction of rtTA3 alone had no effect on tumor growth (Supplementary Fig. S6). SEMA3F induction inhibited tumor growth in the UMSCC2-injected animals (Fig. 7A) and this correlated with a Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2943 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 5. NRP2 is predominantly required for SEMA3F function in LEC. A, Western blot analysis of endogenous NRP1 and NRP2 in LEC in the presence of control (siCtrl), NRP1, or NRP2 siRNA. B, uorescent imaging of LEC transfected with the indicated siRNA and then treated with vehicle or 100 ng/mL SEMA3F. Parental and siCtrl-transfected LECs treated with increasing doses of SEMA3F and evaluated for collapse (C) or attachment (D). For both collapse and attachment assays, values are reported relative to vehicle control and quantication was done by ImageJ, based on 25 imaging elds, each from quadruplicate wells in three independent experiments. E, IC50 of SEMA3F was calculated from the dose curve generated in C and D. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;  , P < 0.01;    , P < 0.001. statistically signicant decrease in cervical lymph node metastasis (Fig. 7C). Interestingly, SEMA3F induction in UMSCC17B tumors, which do not express NRP2, had no effect on tumor size (Fig. 7B). However, a similar decrease in lymph node metastasis was observed (Fig. 7D). This suggested that the paracrine signaling of SEMA3F to the tumor stroma may be sufcient for suppression of metastasis, while exerting an additional autoinhibitory effect on tumor growth in HNSCC cells that acquired expression of NRP2. Tongue sections were evaluated by immunohistochemistry for CD31 and LYVE1 and the intratumoral microvessel density (MVD) was quantied. In both the UMSCC2 and UMSCC17B tumors, induction of SEMA3F signicantly decreased intratumoral lymphangiogenesis and modestly decreased CD31 vasculature, albeit not to a statistically signicant degree (Fig. 7E and F). As a control, MVD outside the tumor was quantied and was not affected for either CD31 or LYVE1 (Fig. 7E and F). Immunouorescence staining revealed a high rate of cancer cells inltrating into lymphatic vessels in control animals that were not observed in SEMA3F-induced cohorts (Fig. 7G and H, white arrows). These results demonstrate that SEMA3F exerts a potent antilymphangiogenic and metastasis suppressor function in HNSCC, and that 2944 Cancer Res; 75(14) July 15, 2015 SEMA3F may also inhibit growth of a subset of HNSCC expressing NRP2. Discussion The tumor microenvironment is regulated through a complex autocrine and paracrine signaling network that is often hijacked and exploited by cancer cells to facilitate their survival, growth, and dissemination. In HNSCC, the interplay of these factors is shifted to promote intratumoral lymphangiogenesis and metastasis, which contribute to poor prognosis and outcome (6, 8). Our ndings suggest that SEMA3F is a key antilymphangiogenic molecule that may function at the core of these cell regulatory networks during HNSCC progression. Indeed, loss of SEMA3F is a frequent event in HNSCC, which correlates with increased tumor vascularity and metastasis. We show that SEMA3F can directly affect LEC function in vitro and in vivo through specic receptor complexes. In COS-7 reconstitution assays, NRP2 and plexin A3 were sufcient to mediate SEMA3F biologic responses, while NRP2-Plexin A1, NRP1-Plexin A3, and NPR1-Plexin A1 complexes can also transduce SEMA3F signals, albeit with decreasing efciency. NRP2 is the major coreceptor for SEMA3F in LECs, Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor Figure 6. NRP2 expression varies in HNSCC and mediates tumor cell responsiveness to SEMA3F. A, Western blot analysis for the expression of receptors in normal oral keratinocytes (NOKSI) and panel of HNSCC cell lines. B, Western blot analysis showing expression of inducible SEMA3F and knockdown or reintroduction of NRP2 expression in UMSCC2 and UMSCC17B cells, respectively. Proliferation assay of UMSCC2-rtTA3-SEMA3F (C) or UMSCC17B-rtTA3-SEMA3F (D) after SEMA3F induction. Cells were treated with control siRNA or NRP2 siRNA (C) or empty vector or NRP2 (D). Migration assay of UMSCC2-rtTA3SEMA3F (E) or UMSCC17B-rtTA3SEMA3F (F) cells with no stimulation (vehicle) or towards 20% FBS. Cells were treated with control siRNA or NRP2 siRNA (E) or empty vector or NRP2 (F). Proliferation and migration were reported as a percentage of control. Statistical signicance was determined using one-way ANOVA,  , P < 0.05;   , P < 0.01;    , P < 0.001. although NRP1 can transmit SEMA3F signals to a lesser extent. Overall, our in vitro and in vivo studies indicate that SEMA3F is potent antilymphangiogenic molecule. Thus, SEMA3F loss may represent an early event in HNSCC, enabling intratumoral lymphangiogenesis that may contribute to the high prevalence of HNSCC cases presenting with locoregional lymph node invasion, heralding a poor clinical outcome. NRP2 is a multiligand coreceptor that can both promote and inhibit the development of venous and lymphatic vasculature, in the stroma and on some tumor cells themselves (3537). In endothelial cells, NRP2 associates with plexin A family members and VEGFR2 and VEGFR3, and these complexes have been implicated in their proangiogenic and -lymphangiogenic signaling (38, 39). Certain malignant epithelial cells express NRP2 without concomitant expression of VEGF receptors (40). The observation that NRP2 but not VEGF receptors are expressed in a subset of HNSCC cells raises the possibility that in these cells NRP2 may act as a coreceptor for plexins or other partners, acting as a gain-of-function alteration during cancer development and www.aacrjournals.org progression. Indeed, exogenous NRP2 expression in HNSCC cells enhanced their endogenous migration while loss of upregulated NRP2 in HNSCC cells decreased their endogenous migration. This allows us to hypothesize that cancer cells may be selected for their NRP2 expression due to an increased growth or migratory potential, but that SEMA3F loss may need to precede NRP2 gain in HNSCC (Fig. 7I). Lymphatic metastasis is mainly attributed to increased expression and signaling of proangiogenic factors (2, 5, 4143). However, expression and secretion of these growth factors alone may not be sufcient for tumor-associated lymphangiogenesis and cancer dissemination. For example, in some cancers VEGF-C or VEGF-D expression levels do not correlate with lymph node metastasis (44, 45), suggesting that other factors may counteract the high level expression of prolymphangiogenic cytokines and attenuate their signaling capacity and prognostic value in vivo. On the basis of our current results, one possible explanation is that SEMA3F could repel LECs and thus dominate the activity of VEGFA and VEGF-C, rendering their expression alone insufcient for Cancer Res; 75(14) July 15, 2015 Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. 2945 Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Doci et al. Figure 7. SEMA3F functions as a tumor and metastasis suppressor in vivo. Tumor growth of UMSCC2-rtTA3-SEMA3F (A) and UMSCC17B-rtTA3-SEMA3F (B) after SEMA3F induction via doxycycline chow. Cervical lymph node metastasis was evaluated as the percentage of metastatic lymph nodes in control and doxycycline-fed animals for UMSCC2-rtTA3-SEMA3F (C) and UMSCC17BrtTA3-SEMA3F (D). MVD for lymphatic (LYVE-1) and blood (CD31) vessels was evaluated in the tumor and muscle of tongues for UMSCC2-rtTA3-SEMA3F (E) and UMSCC17B-rtTA3-SEMA3F (F). MVD was reported relative to the average density in vessels/mm. Statistical signicance was determined using Student t test,  , P < 0.05;   , P < 0.01;    , P < 0.001. Immunouorescent staining of tumors from control and doxycycline-fed animals for UMSCC2-rtTA3-SEMA3F (G) and UMSCC17B-rtTA3-SEMA3F (H) revealed a higher density and size of vessels in control animals. Lymphovascular invasion by cancer cells is indicated by white arrowheads. I, proposed mechanism for the role of SEMA3F loss in HNSCC. See text for details. 2946 Cancer Res; 75(14) July 15, 2015 Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 SEMA3F as an HNSCC Antilymphangiogenic Metastasis Suppressor lymphangiogenesis and metastasis. This hypothesis is supported by our ndings that induction of SEMA3F alone in orthotopic HNSCC cells signicantly inhibits intratumoral lymphangiogenesis and metastasis, even in HNSCC cells that do not express NRP2. Thus, loss of SEMA3F expression in HNSCC may simultaneously enhance VEGF-mediated signaling on endothelial cells and alleviate repressive semaphorin functions, resulting in increased lymphangiogenesis. Our observation may have a broad impact in multiple highly prevalent human malignancies. In an integrative multiplatform analysis, 3p deletion was identied as a key genomic signature shared by a squamous-like subtype of solid cancers (46). Furthermore, chromosomal loss at 3p21, which is expected to result in the genomic deletion of SEMA3F, has been observed for lung, breast, and kidney cancers in addition to HNSCC (4750). Immunohistochemistry on HNSCC tumors reveals a complete absence of SEMA3F expression in most advanced cases, suggesting that epigenetic regulation or transcriptional dowregulation may contribute to reduced SEMA3F expression of the remaining SEMA3F allele in cancers with SEMA3F heterozygous loss. We can conclude that SEMA3F is a potent chemorepellent molecule for lymphatic and vascular endothelial cells, which acts through NRP2-Plexin A and to a lesser extent NRP1-Plexin A signaling complexes. Loss of SEMA3F is an early event in HNSCC and likely many other highly prevalent human malignancies. This information can in turn be exploited for therapeutic purposes, as most HNSCC lesions may retain one intact SEMA3F allele. This suggests that reactivation of SEMA3F expression or ectopic SEMA3F delivery may offer the opportunity to halt intratumoral lymphangiogenesis and suppress metastasis regardless of the NRP2 expression status of the HNSCC lesion. We can conclude that SEMA3F-NRP2 represents a novel regulatory axis during HNSCC development, progression, and metastasis, thus providing new prognostic markers and therapeutic options in this highly aggressive disease. Disclosure of Potential Conicts of Interest No potential conicts of interest were disclosed. Authors' Contributions Conception and design: C.L. Doci, J.S. Gutkind Development of methodology: C.L. Doci, C.M. Mikelis, A.A. Molinolo, J.S. Gutkind Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.L. Doci, C.M. Mikelis, M.S. Lionakis, A.A. Molinolo Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.L. Doci, M.S. Lionakis, A.A. Molinolo Writing, review, and/or revision of the manuscript: C.L. Doci, J.S. Gutkind Study supervision: J.S. Gutkind Acknowledgments The authors thank Dr. Roberto Weigert for providing the LifeAct plasmid. They also thank Dr. Gera Neufeld for providing plexin A1, A3, and A4 receptor plasmids and puried Sema3E. Grant Support This research was supported by the Intramural Research Program of the National Institute of Dental and Craniofacial Research, and in part by the National Institute of Allergy & Infectious Diseases, NIH (Z01DE00558-23 and Z01DE00551-23). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 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Sekido Y, Ahmadian M, Wistuba II, Latif F, Bader S, Wei MH, et al. Cloning of a breast cancer homozygous deletion junction narrows the region of search for a 3p21.3 tumor suppressor gene. Oncogene 1998;16:31517. 49. Roche J, Boldog F, Robinson M, Robinson L, Varella-Garcia M, Swanton M, et al. Distinct 3p21.3 deletions in lung cancer and identication of a new human semaphorin. Oncogene 1996;12:128997. 50. Hesson LB, Cooper WN, Latif F. Evaluation of the 3p21.3 tumour-suppressor gene cluster. Oncogene 2007;26:7283301. Cancer Research Downloaded from cancerres.aacrjournals.org on March 8, 2021. 2015 American Association for Cancer Research. Published OnlineFirst May 7, 2015; DOI: 10.1158/0008-5472.CAN-14-3121 Genetic Identification of SEMA3F as an Antilymphangiogenic Metastasis Suppressor Gene in Head and Neck Squamous Carcinoma Colleen L. Doi, Constantinos M. Mikelis, Michail S. Lionakis, et al. Cancer Res 2015;75:2937-2948. Published OnlineFirst May 7, 2015. 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... Oncogene (2013) 32, 43774386 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE The novel tumor suppressor NOL7 post-transcriptionally regulates thrombospondin-1 expression CL Doci, G Zhou and MW Lingen Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis whose expression suppresses tumor growth in vivo. Like many angiogenesis-related genes, TSP-1 expression is tightly controlled by various mechanisms, but there is little data regarding the contribution of post-transcriptional processing to this regulation. NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype and suppresses tumor growth, in part through upregulation of TSP-1. Here we demonstrate that NOL7 is an mRNA-binding protein that must localize to the nucleoplasm to exert its antiangiogenic and tumor suppressive effects. There, it associates with the RNA-processing machinery and specically interacts with TSP-1 mRNA through its 30 UTR. Reintroduction of NOL7 into SiHa cells increases luciferase expression through interaction with the TSP-1 30 UTR at both the mRNA and protein levels. NOL7 also increases endogenous TSP-1 mRNA half-life. Further, NOL7 post-transcriptional stabilization is observed in a subset of angiogenesis-related mRNAs, suggesting that the stabilization of TSP-1 may be part of a larger novel mechanism. These data demonstrate that NOL7 signicantly alters TSP-1 expression and may be a master regulator that coordinates the posttranscriptional expression of key signaling factors critical for the regulation of the angiogenic phenotype. Oncogene (2013) 32, 43774386; doi:10.1038/onc.2012.464; published online 22 October 2012 Keywords: NOL7; TSP-1; angiogenesis; post-transcriptional INTRODUCTION Angiogenesis is one of the hallmarks of cancer and is required for tumors to grow beyond a diffusion gradient.1,2 It is regulated by a balance of pro-and antiangiogenic factors, and, in many cancers, the differential regulation of these factors is a precursor to the angiogenic switch and subsequent malignant transformation.35 Current therapies aimed at disrupting this process have been hindered by off-target effects owing to the diversity of angiogenic molecules and their downstream signaling pathways.69 This diversity is mediated in part by the post-transcriptional processing of many of these angiogenesis-related transcripts, where alternative splicing, polyadenylation, stability, and translational control contributes to their expression level and functionality. In some cases, as in the alternative splicing of vascular endothelial growth factor (VEGF) and its receptor, VEGF receptor 1, these processes can change the function of the molecule itself.1013 In other cases, these processes can inuence the bioavailability through modulation of secretory domains or mRNA stability.1418 The post-transcriptional regulation of mRNA stability is particularly critical for the rapid cellular response to internal and external stimuli and is therefore a crucial mechanism in the control of growth factors, cytokines, and angiogenic molecules.19 While advances in our understanding of the post-transcriptional regulation of proangiogenic factors have emerged, considerably less is known about the post-transcriptional processing of antiangiogenic molecules. The rst endogenous antiangiogenic molecule reported was thrombospondin-1 (TSP-1).20,21 TSP-1 suppresses angiogenesis by inhibiting cell migration, inducing apoptosis, and modulating the signaling of growth factors.2224 TSP-1 expression is downregulated in a number of cancers, and reintroduction of TSP-1 has been shown to signicantly suppress tumor growth.2528 Owing to its central role in physiological and pathological angiogenesis, expression of TSP-1 mRNA is tightly controlled, with no known alternative splicing or polyadenylation isoforms. TSP-1 mRNA is post-transcriptionally regulated by TGF-b stimulation, hypoxia, or heat shock, but the underlying mechanism is poorly understood.2934 Post-transcriptional regulation is achieved in part through interaction with RNA-binding proteins (RBPs) such as HuR. HuR regulates a number of angiogenesis-related transcripts, including VEGF, COX-2, and HIF-1a3538 and recently has been shown to bind to the 30 UTR of TSP-1.39 While many of the well-characterized mRNA-stabilizing RBPs such as HuR function in the cytoplasm, the existence of signicant parallel mechanisms for nuclear RNA turnover argues for similar stabilizing roles in this compartment. Many of the decay pathways active in the nucleus are tied to processing events such that degradation of aberrant transcripts and further processing of mRNAs occur co-transcriptionally and are tied to the RNA polymerase II machinery in ribonucleoprotein (RNP) complexes.4043 These RNPs, including nuclear-processing factors such as XRN2, EXOSC10, TRAMP, and others are critical for appropriate expression tied to transcriptional termination, 30 end processing, and mRNA quality control.4455 Within the nucleus, RBPs have a central role in coordinating mRNA quality control through alternative decay, stabilization, and export.5659 NOL7 is a novel tumor suppressor that induces an antiangiogenic phenotype through differential regulation of VEGF and TSP-1.60 However, the mechanism underlying this function is unknown. In this study, we demonstrate that NOL7 must reside in the nucleoplasm to upregulate TSP-1 and induce an antiangiogenic and tumor suppressive phenotype. There, it functions as an RBP that associates with the RNA-processing machinery. NOL7 interacts with polyadenylated transcripts, specically with TSP-1 mRNA through its 30 UTR. Reintroduction of NOL7 increases Departments of Pathology, Medicine and Radiation, and Cellular Oncology, University of Chicago, Chicago, IL, USA. Correspondence: Dr MW Lingen, Department of Pathology, Medicine and Radiation, and Cellular Oncology, University of Chicago, 5841S. Maryland Avenue MC6101, Chicago, IL 60637, USA. E-mail: mark.lingen@uchospitals.edu Received 18 April 2012; revised 22 August 2012; accepted 24 August 2012; published online 22 October 2012 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4378 reporter gene expression through this interaction at both the mRNA and protein levels. This is also observed in endogenous TSP-1, where reintroduction of NOL7 signicantly increases the TSP-1 transcript half-life. NOL7 post-transcriptional stabilization is limited to a distinct subset of mRNAs through a novel mechanism. Together, these data demonstrate that NOL7 is a critical regulator of TSP-1 expression and may participate in the coordination of cellular signaling pathways through post-transcriptional mRNA regulation to control the angiogenic process. RESULTS NOL7 must reside in the nucleoplasm to induce an antiangiogenic phenotype and suppress tumor growth NOL7 is a highly basic protein that suppresses tumor growth and induces an antiangiogenic phenotype in part through upregulation of TSP-1 expression.60 NOL7 is localized exclusively to the nucleus and nucleolus of cells and completely absent from the cytoplasm (Supplementary Figure S1), but it is not known if NOL7 is sequestered or functional within these compartments. To determine the compartment in which NOL7 must reside for its function, we made a series of mutational constructs that targeted NOL7 to the nucleolus (NOL7 wild-type), nucleoplasm (N23(  )) or cytoplasm (N123(  )) (Zhou et al 61; Figure 1a). In SiHa cells, which essentially lack endogenous NOL7,60,62 reintroduction of wild-type nucleolar NOL7 was able to signicantly upregulate endogenous TSP-1 expression compared with GFP controls, while cytoplasmic NOL7 had no effect on TSP-1 levels (Figure 1b). However, nucleoplasmic NOL7 was still able to upregulate endogenous TSP-1 to the same levels as wild-type NOL7 (Figure 1b). In the same fashion, there was no difference in endothelial cell migration between cells treated with conditioned media from GFP control or cytoplasmic NOL7 (Figure 1c). However, conditioned media from wild-type NOL7 and nucleoplasmic NOL7 were able to suppress Figure 1. NOL7 must reside in the nucleus to modulate TSP-1 expression, inhibit endothelial cell migration and suppress tumor growth. (a) Cells were transfected with GFP-tagged wild-type NOL7 or mutants that target NOL7 to the nucleoplasm (N23(  ), clones 1 and 2) or the cytoplasm (N123(  ), clones 1 and 2). Cell nuclei were counterstained with DAPI and imaged by fluorescence microscopy. (b) Conditioned media from transfected cells was analyzed by enzyme-linked immunosorbent assay and concentration calculated from a standard curve. Data are represented as means.e.m. Significance was calculated using Students t-test from three independent experiments. *Po0.005; **Po0.002; ***Po0.001; n.s., not significant. (c) Serum-free conditioned media from parental SiHa, GFP and NOL7 wt- and mutant-transfected clones were functionally tested for their ability to stimulate endothelial cell migration. Data are represented as means.e.m. Significance was calculated using Students t-test from four independent experiments. *Po1  10  5; **Po1  10  8; n.s., not significant. (d) SiHa parental, GFP and NOL7 wtand mutant-transfected cells were subcutaneously injected into nude mice and monitored over a period of 30 (n 6 animals per group). Data are represented as means.e.m. Significance was calculated using two-way analysis of variance. *Po0.001; **Po0.0001; n.s., not significant. (e) Tumor angiogenesis was assessed by CD31 staining, followed by microvessel density quantification. Data are represented as means.e.m. Significance was calculated using Students t-test (n 6 animals per group). *Po2  10  6; ** Po1  10  10; n.s., not significant. Oncogene (2013) 4377 4386 & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4379 Figure 2. NOL7 interacts with a large RNP complex in an RNA-dependent manner. NOL7-transfected lysate was separated on a 1030% gradient. Individual fractions were collected and plotted on the basis of their absorbance at 260 nm. Individual fractions were also subjected to western blotting for NOL7. The co-migration of NOL7 with a large RNP complex is marked with a box. (a) Total lysate was evaluated for association of NOL7 into RNP complexes. (b) Lysate was either mock-treated or digested with RNase A before separation. endothelial cell migration to the same degree (Figure 1c). This correlated with a signicant suppression in tumor growth, where both nucleoplasmic and nucleolar NOL7 demonstrated 90% less tumor volume than GFP control or cytoplasmic NOL7 (Figure 1d). Tumor angiogenesis, as assessed by CD31 microvessel density, revealed that wild-type and nucleoplasmic NOL7 suppressed tumor angiogenesis, whereas GFP control and the cytoplasmic NOL7 mutant displayed robust angiogenesis in these tumors (Figure 1e). Taken together, this suggests that NOL7 must reside in the nucleoplasm to induce an antiangiogenic phenotype and suppress tumor growth. NOL7 interacts with a large ribonucleoprotein complex in an RNAdependent manner To determine the potential mechanism by which NOL7 suppresses tumor growth and induces an antiangiogenic phenotype in the nucleoplasm, we hypothesized that NOL7 may be part of a nucleic-acid-interacting complex. To test this, we performed sucrose gradient ultracentrifugation of NOL7-expressing lysate (Figure 2a). NOL7 co-migrated with a large, B70S complex (Figure 2a, box). To determine if the association of NOL7 was dependent upon RNA, lysate was either mock-treated or treated with RNase, and separated as before (Figure 2b). Treatment with RNase caused NOL7 to revert completely to the soluble fraction & 2013 Macmillan Publishers Limited (Figure 2b, arrow). This demonstrates that NOL7 interacts with a large RNP complex in an RNA-dependent manner. NOL7 interacts with mRNA-processing factors To identify the RNP complex and protein cofactors of NOL7, mass spectroscopic (MS) analysis was performed on comigrating NOL7 immunoprecipitates (Figure 3a). This sample was highly enriched for RNA-processing factors, particularly those involved in mRNA maturation. To conrm the putative cofactors identied by mass spectroscopy, coimmunoprecipitation of NOL7 or controls was performed and analyzed by western blot against the endogenous MS-identied proteins. To determine if these putative cofactors interact in an RNA-dependent or -independent manner, lysates were mock-treated or digested with RNase A before immunoprecipitation (IP). SR proteins SF2-ASF and SRp40, RNAprocessing factor NCL, and the 30 -processing and decay factors XRN2, CNOT3, and CPSF2 were found to specically associate with NOL7 in either the presence of absence of RNA (Figure 3b). In addition, the multifunctional RBPs HuR, NPM, and EXOSC10 associated with NOL7 only in the presence of RNA. These data demonstrate that NOL7 interacts with mRNA-processing complexes and specically associates with proteins involved in mRNA maturation. Oncogene (2013) 4377 4386 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4380 Figure 3. NOL7 interacts with 30 end-processing proteins. (a) Lysate from SiHa cells stably expressing GFP-V5 or NOL7-V5 was separated by gradient ultracentrifugation and the large 70S fractions were pooled, immunoprecipitated and separated by SDSpolyacrylamide gel electrophoresis and stained with Coomassie before analysis by mass spectroscopy. Data was curated from the mass spectroscopy results to identify putative functional cofactors of NOL7. (b) GFP-V5 or NOL7-V5 lysate was mock-treated (  ) or digested with RNase ( ). Lysates were immunoprecipitated using a-V5-conjugated beads and coimmunoprecipitating proteins were analyzed by western blot. As a control for RNase digestion, RNA was extracted from the lysates after treatment, reverse transcribed and RTPCR against the 18S rRNA was performed. NOL7 interacts specically with mRNA The protein cofactors, identied suggested that NOL7 may be involved in mRNA processing. To determine if NOL7 interacts with mRNA, total lysate overexpressing GFP control or NOL7 was incubated with oligo(dT) beads to precipitate polyadenylated transcripts and coprecipitated proteins were visualized by western blotting. NOL7, but not GFP, interacted with the polyA transcripts, demonstrating that this was not an artifact of overexpression (Figure 4, lanes 3 and 4). This was not due to nonspecic binding to the beads, as treatment of the lysate with RNase before incubation completely abolished NOL7 binding (Figure 4, lane 5). Finally, to demonstrate that this interaction was mRNA-specic and not a random, charge-based interaction, the competition of the bound proteins with either polyA or polyC at ve or ten times the input RNA was performed. NOL7 binding was lost in the polyA treatments (Figure 4, lanes 6 and 8) but unaffected by polyC competition (Figure 4, lanes 7 and 9), indicating that NOL7 interacts specically with mRNA. NOL7 interacts with TSP-1 mRNA through its 30 UTR NOL7 induces an antiangiogenic phenotype in the nucleus through upregulation of TSP-1 (Figure 1). To determine if NOL7 interacts with TSP-1 mRNA, total lysate overexpressing GFP, NOL7, or the TSP-1 mRNA-binding protein HuR was immunoprecipitated and the associated mRNAs were analyzed by northern blotting (Figure 5a). TSP-1 mRNA was signicantly associated with both NOL7 and HuR, and absent from GFP (Figure 5a). This demonstrates that NOL7 is capable of specically interacting with TSP-1 mRNA. The association of NOL7 with polyadenylated transcripts and 30 -processing factors suggested that NOL7 might interact with TSP-1 through its 30 UTR. To determine if NOL7 might bind in this region, the 30 UTR of TSP-1 or R01, a random, nongenic sequence of the same length were in vitro transcribed and Oncogene (2013) 4377 4386 biotinylated. Total lysate from cells overexpressing GFP, NOL7, or HuR were incubated with increasing amounts of biotinylated transcript, precipitated using streptavidin beads, and analyzed by western blotting (Figure 5b). GFP did not bind to either transcript at any of the concentrations assayed. Both NOL7 and HuR bound to TSP-1 30 UTR in a dose-dependent manner, but was not detected in the negative-control lanes (Figure 5b). Together, this demonstrates that NOL7 interacts specically with TSP-1 mRNA through its 30 UTR. The 30 UTR of TSP-1 is sufcient for NOL7-mediated posttranscriptional upregulation Interaction of NOL7 with the 30 UTR of TSP-1 suggested it may have a role in its post-transcriptional regulation. To determine if this interaction can affect downstream expression levels, luciferase reporters were cloned in-frame with the 30 UTR of TSP-1, positivecontrol SV40 late polyA signal (EMP), or a negative-control nongenic (R01) sequence. SiHa cells express extremely low levels of endogenous NOL7, such that reintroduction in stable cell lines restored NOL7 to near-endogenous levels observed in 293T cells, and expression of exogenous NOL7 or HuR did not affect endogenous levels of either gene (Supplementary Figure S2). Luciferase levels for clones bearing the TSP-1 30 UTR were signicantly increased in NOL7- and HuR-expressing cells (Figure 6). No difference was observed between GFP, NOL7, or HuR in EMP or R01 constructs, indicating that the increase in luciferase was due specically to regulation through the TSP-1 30 UTR. To determine if this upregulation was due to an increase in mRNA levels or through an increase in translational rate or protein stability, luciferase levels were analyzed by real-time PCR. The upregulation of luciferase was observed at the mRNA as well as protein levels, suggesting that the NOL7 regulates expression at the level of mRNA abundance. & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4381 Figure 4. NOL7 specifically interacts with mRNA. HEK293T cells were transfected with GFP control or NOL7. Proteins associated with poly(A) mRNA were pulled down using oligo(dT) cellulose. Input lanes represent 10% of total input. As controls, lysate was digested with RNase A before incubation or bound proteins were competed using five or ten times input RNA of polyA or polyC. Coprecipitation of GFP and NOL7 was evaluated by western blot. Data are represented as means.e.m. Significance was calculated using Students t-test relative to NOL7 input (a, b) and relative to the NOL7-bound (c, d) fractions. aPo0.002; bPo4  10  4; cPo0.001; d Po0.0001. Statistical significance between groups was also calculated (bars). *Po0.001, **Po1  10  4. Data are represented as means.e.m. from three independent assays. NOL7 post-transcriptionally regulates TSP-1 expression To determine if NOL7 could post-transcriptionally upregulate endogenous TSP-1, two clones each from SiHa cells stably expressing GFP, NOL7 or HuR were treated with a-amanitin to block RNA Pol II-mediated mRNA transcription. TSP-1 expression levels were measured by real-time PCR for increasing durations of a-amanitin treatment. In cells expressing GFP, TSP-1 expression fell to almost half its original level within 1 hour. However, cells expressing NOL7 or HuR decreased only slightly throughout the timecourse (Figure 7a). Reintroduction of NOL7 doubled the halflife of endogenous TSP-1 mRNA (Figure 7b), suggesting that NOL7 regulates TSP-1 expression by enhancing the post-transcriptional stability of its mRNA. NOL7 may stabilize a specic subset of angiogenesis-related mRNAs To determine if NOL7 was capable of post-transcriptionally stabilizing other transcripts in addition to TSP-1, we assessed steady-state and post-transcriptional mRNA levels in a panel of angiogenesis-related mRNAs. SiHa cells stably expressing GFP or NOL7 were left untreated or transcriptionally inhibited with a-amanitin for 4 hours, when the maximal effect of NOL7 posttranscriptional stabilization was observed for TSP-1 (Figure 7). Using the TaqMan 384-well Human Angiogenesis Array, we found approximately one-third of the mRNAs analyzed were differentially expressed at a statistically signicant level between the untreated samples, suggesting that NOL7 can modulate the steady-state expression of angiogenesis-related genes at the mRNA level (Figure 8a). Within these genes, a specic subset of mRNAs was post-transcriptionally stabilized upon reintroduction of NOL7 (Figure 8b). Further, over half of the post-transcriptionally stabilized genes were also upregulated and functionally associated with antiangiogenic and antitumorigenic cell phenotypes & 2013 Macmillan Publishers Limited Figure 5. NOL7 coimmunoprecipitates TSP-1 mRNA. (a) Lysate from cells expressing GFP, NOL7 or HuR were immunoprecipitated with a-V5 beads and RNA was extracted. RNA was then subjected to northern blotting using a probe against the 30 UTR of TSP-1. Western blotting to confirm equivalent protein IP was performed. Densitometry scanning was performed on northern bands and normalized to protein IP lanes. Data are represented as means.e.m. from three independent experiments. *Po0.01. (b) The 30 UTR of TSP-1 and a negative-control sequence R01 were in vitro transcribed and biotinylated. Increasing amounts of transcript were incubated with lysate expressing GFP, NOL7 or HuR. Transcripts were precipitated using streptavidin beads and coprecipitating proteins were analyzed by western blot. (Figure 8c). Together, this indicates that NOL7 is capable of modulating the expression of a specic subset of mRNAs posttranscriptionally, while others may be differentially regulated as a consequence of NOL7 modulation of upstream targets in linked signaling pathways. Finally, it indicates that for some genes, such as TSP-1, this post-transcriptional regulation may signicantly alter the available mRNA pool and inuence downstream function and phenotype. DISCUSSION NOL7 is a novel tumor suppressor that signicantly suppresses in vivo tumor growth and induces an antiangiogenic phenotype in part through upregulation of TSP-1. In this work, we characterize NOL7 as a novel RBP that increases TSP-1 expression through post-transcriptional mRNA stabilization. NOL7 must reside in the nucleoplasm to exert its anticancer phenotype. Within the nucleus, NOL7 is component of a large, RNA-dependent RNP comprised of mRNA-processing factors. NOL7 specically interacted with a number of these putative cofactors as well as polyadenylated transcripts. NOL7 was shown to interact specically with the TSP-1 transcript through binding to its 30 UTR. Reporter constructs bearing the TSP-1 30 UTR were signicantly upregulated at both the mRNA and protein level, and endogenous TSP-1 mRNA was stabilized in cells re-expressing NOL7. Finally, this post-transcriptional regulation was demonstrated to be specic to a subset of angiogenesis-related mRNAs. Taken together, this demonstrates that NOL7 is a novel RBP that post-transcriptionally upregulates TSP-1 through an increase in mRNA stability. Further, this suggests that NOL7 may regulate the antiangiogenic phenotype and suppress tumor growth through post-transcriptional modulation of gene expression. Interaction of NOL7 with components of the RNA-processing machinery in a large RNP suggests that NOL7 may contribute to Oncogene (2013) 4377 4386 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4382 Figure 6. The 30 UTR of TSP-1 is sufficient for NOL7-mediated post-transcriptional upregulation. Two clones each from SiHa cells stably co-expressing GFP, NOL7 or HuR and luciferase-EMP, -R01 or TSP-1 were assayed for luciferase expression at the mRNA (dark-gray bar) or protein (light-gray bar). To control for vector expression artifacts, values were normalized to luciferase-EMP and reported as a percentage of GFP expression for each set of constructs. Data are represented as means.e.m. from four independent experiments. *Po3  10  5; **Po9  10  5; ***Po2  10  7. Figure 7. NOL7 post-transcriptionally stabilizes TSP-1 mRNA. SiHa cells stably expressing GFP, NOL7 or HuR were treated for 0, 2, 4 or 6 with 5 mg/ml a-amanitin. Endogenous TSP-1 levels were assayed by real-time PCR and calculated via DDCt method. Half-life calculations were calculated from the nonlinear regression of the exponential decay curve N0 N(t)e  lt, where the TSP-1 mRNA half-life t1/2  ln(2)/l. Data are represented as means.e.m. from four independent experiments. *Po0.03; **Po0.001; ***Po0.0001. the co-transcriptional processing of mRNA at multiple levels. These data characterize the role of NOL7 during the 30 end processing and maturation of TSP-1 mRNA, but do not exclude the possibility that NOL7 may contribute to other aspects of mRNA metabolism. It has been demonstrated that the efcacy of downstream processing steps can form feedbacks that inuence upstream transcriptional initiation and elongation.63,64 In addition, some nuclear mRNA complexes are active both in late-maturation and early-initiation steps of processing. One such complex, the CCR4-Not complex, has roles in mRNA transcription and degradation and its activity can inuence cellular signaling pathways.40,41 NOL7 was recently identied in a proteomic study of the CCR4-Not complex as a factor that interacts specically with multiple core subunits of the complex,65 validated in this work by evidence that NOL7 specially interacts with the subunit CNOT3 (Figure 3). Similarly, NOL7 also interacts specically with the 50 -30 endonuclease complex and nuclear exosome via its binding with XRN2 and EXOSC10. While it is unclear if NOL7 functions as a part of these complexes, it does suggest that NOL7 could be playing a role as a master regulator of gene expression through regulation of mRNA maturation and degradation, and subsequent control of critical signaling molecules on multiple levels. This may be particularly signicant in its regulation of the angiogenic phenotype, as feedback mechanisms and integrated signaling can have a major role in driving the angiogenic switch. Oncogene (2013) 4377 4386 Angiogenesis is critical in cancer development and represents a promising target for therapy. However, the diversity and redundancy of many angiogenic molecules, coupled with the complexity of angiogenic signaling, have hindered progress in the eld. Particularly, the ability of post-transcriptional regulation to rapidly and signicantly alter the signaling capability of many of these factors, and in some cases change the functionality of these molecules entirely has been overlooked.12,6670 Proangiogenic molecules such as broblast growth factor 2 (FGF-2), VEGF and COX-2 all undergo alternative post-transcriptional processing that results in modulation of their half-life and downstream functionality.17,18,7175 Importantly, some major angiogenic signaling factors such as TGF-b have been found to modulate gene expression through a dual regulation of transcriptional activity and mRNA turnover.68 Coupled with constitutive low-level expression of many of these mRNAs, rapid changes in mRNA stability can signicantly alter the steadystate pool of a given mRNA in response to stimulus19 and represent a key target for modulating the angiogenic phenotype in cancer. While post-transcriptional regulation of proangiogenic factors has been described in the literature, evidence regarding alternative processing and stability and angiogenic inhibitors is lacking. Despite decades of research focused on TSP-1, few reports address the issue of its post-transcriptional regulation. The evidence available suggests that post-transcriptional regulation is tied to conditions that promote or suppress tumorigenesis in vivo, such as & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4383 Figure 8. NOL7 specifically regulates distinct subset of angiogenic transcripts. Total complementary DNA from GFP- or NOL7-expressing cells untreated or transcriptionally inhibited with a-amanitin were analyzed by real-time PCR on TaqMan angiogeniesis array cards. Data are represented as means.e.m. from three independent experiments. (a) Steady-state expression (untreated NOL7 normalized to untreated GFP). Dashed line indicates the GFP control normalization. aPo0.05; bPo0.01; cPo0.001; dPo0.0001. (b) Post-transcriptional expression (a-amanitin-treated GFP/NOL7 normalized to untreated controls). *Po0.05, **Po0.01. (c) Genes significantly upregulated (yellow), unchanged (white), or downregulated (blue) are represented schematically based on their functional annotations, ligand-receptor interactions, and signaling. Those genes that were post-transcriptionally stabilized by NOL7 are outlined in red. hypoxia or oncogene expression. For example, TSP-1 mRNA is destabilized by overexpression of the oncogene Myc or hypoxic conditions, while its half-life is increased by heat shock or TGF-b stimulation.2934 Interestingly, TGF-b is activated by TSP-1, indicating that the post-transcriptional stabilization may be part of a feedback loop to suppress tumor growth. To the best of our knowledge, no studies have investigated any tumor-associated somatic mutations within the TSP-1 30 UTR. The contribution of 30 UTR cis-elements to the overall regulation of TSP-1 expression and the trans-factors that bind to them, such as AUF1 and HuR, are also not well characterized. Here, we demonstrate that NOL7 is a novel TSP-1 30 UTR-interacting protein that stabilizes the TSP-1 transcript. NOL7 may represent a novel type of RBP in cancer, because of its unique domain structure and role as a tumor suppressor in vivo. & 2013 Macmillan Publishers Limited NOL7 lacks any sequence similarity to known proteins or domains, suggesting NOL7 may employ a novel method of interaction with its targets. While the data here are insufcient to identify a NOL7interacting cis-element, it does rule out classic binding elements such as AREs, as VEGF, FGF-2, and interleukin 8 are not posttranscriptionally regulated by NOL7. Nonetheless, these data suggest that NOL7 binds and regulates a specic subset of mRNAs, and for some of these transcripts, that stabilization may signicantly alter the steady-state expression level. In addition, some genes were differentially regulated only at the steady-state, suggesting that for some genes the effect of NOL7 expression may be propagated through signaling pathways or be secondary effects of NOL7s function on upstream targets. Assessment of mRNA levels over a transcriptional inhibition timecourse will be necessary to conrm the modulation of mRNA half-life and Oncogene (2013) 4377 4386 NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4384 expansion of these results to include more genes and pathways is necessary. For example, a major angiogenic transcription factor, HIF-1a, was not proled on this array, and could contribute signicantly to the differential expression of NOL7 targets, including downstream genes affected only at the steady state. The differentially regulated genes further suggest that NOL7 may be having a role in mediating focal adhesion, remodeling of the extracellular matrix and epithelial to mesenchymal (EMT) transition through its post-transcriptional mechanisms, pathways that are critical for tumor growth suppression, metastasis and antiangiogenic therapy. Acquisition of an EMT phenotype is associated with acquired resistance to angiogenic therapies, metastasis, and has recently been tied to global enrichment of ARE-containing mRNAs.7678 Therefore, the role of NOL7 in posttranscriptional regulation of these phenotypes may be critical in avoiding acquired resistance and increasing the efcacy of current angiogenic therapies. In conclusion, NOL7 is a novel tumor suppressor that must reside in the nucleoplasm to suppress in vivo tumor growth. Reintroduction of NOL7 induces an antiangiogenic phenotype drive in part by the post-transcriptional stabilization of TSP-1. This is achieved through specic interaction with the TSP-1 30 UTR, which is sufcient to post-transcriptionally upregulate gene expression at the mRNA and protein levels. Finally, this posttranscriptional regulation was demonstrated to be specic to a small subset of mRNAs. Taken together, this demonstrates that NOL7 is a novel RBP that post-transcriptionally upregulates TSP-1 through an increase in mRNA stability. Further characterization of the mechanism underlying this function and the phenotypic consequences will illustrate the potential role of NOL7 as a master regulator of the angiogenic phenotype through post-transcriptional modulation of gene expression. 2% sucrose, protease inhibitors). RNA digestion was performed by incubation with 500 mg/ml RNase A and 50 mg/ml EDTA at 37 1C for 1 h. All gradients were separated at 27 500 rpm at 4 1C on Beckman LM-80 Centrifuge (Beckman-Coulter, Inc., Brea, CA, USA) for 8 h. Equivalent fractions were collected manually measured for absorbance at 260 nm. Immunoprecipitation and pulldowns Cells stably expressing GFP-V5 or NOL7-V5 were resuspended in sucrose lysis buffer and lysed by freezethaw. RNase digestion was performed, as described above. For IP, 250 mg total protein was mock-treated or RNase digested and incubated with 25 ml a-V5 agarose beads (SigmaAldrich, St Louis, MO, USA). Puried complementary DNA was reverse transcribed using the Superscript III one-step RT-PCR kit and amplied with 18S specic primers (Invitrogen, Carlsbad, CA, USA). For oligo(dT) pulldowns, 500 A260 units were bound to 10 mg oligo(dT) cellulose beads (Ambion) and incubated with buffer alone or buffer containing 2500 or 5000 A260 units of polyadenylic or polycytidylic acid (SigmaAldrich). For 30 UTR pulldowns, the 30 UTR of TSP-1 or R01 was in vitro transcribed using the MEGAscript kit from Ambion (Ambion/Applied Biosystems, Austin, TX, USA). RNA was incubated with 100 mg lysate and bound to streptavidin Dynabeads (Invitrogen). In all cases, beads were washed thoroughly and bound proteins were eluted by boiling in SDS sample buffer. Results were quantied using Bio-Rad QuantityOne software (Bio-Rad) and normalized to input. Statistical signicance was determined from three independent assays using Students t-test. 30 UTR luciferase assays Two clones each of SiHa cells stably co-expressing GFP, NOL7, or HuR and EMP, R01, or TSP-1 30 UTR luciferase reporter constructs were measured using the Steady-Glo luciferase assay system (Promega, Madison, WI, USA), according to manufacturers instructions. Values for GFP, NOL7 and HuR clones were averaged, normalized to luciferase control, and reported as a percentage of GFP. Luciferase mRNA levels were measured by quantitative PCR. Statistical signicance was calculated using Students t-test from four independent assays against log values to control for normalization bias. MATERIALS AND METHODS Further details and additional methods can be found in the Supplementary section. Cell culture and uorescence microscopy HEK293T and SiHa cells were obtained from the ATCC (Manassas, VA, USA) and cultured, as previously described.60,61 Fluorescence microcopy was performed as described.61 Quantitative real-time PCR RNA levels were measured by real-time quantitative PCR using the Ag-Path One Step RTPCR kit (Ambion/Applied Biosystems). For each, 30 ng total RNA was amplied on the CFX-1000 (Bio-Rad) and detected using TaqMan probes against target transcripts (Applied Biosystems). Relative expression levels were calculated using the DDCt method relative to 18S. Statistical differences were calculated as indicated. In vivo tumor studies, ELISA and migration assay Conditioned media from the SiHa parental, GFP control, NOL7 wild-type or NOL7 mutant cells were assayed for TSP-1 and VEGF by enzyme-linked immunosorbent assay (ELISA), as described by the manufacturer (R&D Systems, Minneapolis, MN, USA). The migration assay was performed, as previously described.79 For tumor studies, 107 cells in PBS were injected subcutaneously into 68-week-old female nu/nu mice (Charles River Laboratories, Wilmington, MA, USA) (n 6 per group). Tumor growth was monitored with a caliper. Statistics were calculated using two-way analysis of variance. Microvessel density was calculated from CD31 staining, as previously described.60 Measurement of post-transcriptional mRNA abundance Two clones each of SiHa cells stably co-expressing GFP, NOL7 or HuR were plated in six-well plates, washed with PBS and transcriptionally inhibited in complete media containing 5 mg/ml a-amanitin for 0, 2, 4 or 6 hours. After treatment, RNA was collected from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Post-transcriptional TSP-1 abundance was measured by real-time quantitative PCR. TSP-1 mRNA levels were normalized to time zero and plotted as a function of a-amanitin treatment duration such that N0 N(t)e  lt. Half-life was calculated as t1/2  ln(2)/l. Statistical signicance was calculated using two-way analysis of variance and Students t-test from four independent assays. Northern and western blotting Northern blotting was performed using the NorthernMax kit (Ambion, Austin, TX, USA). The TSP-1-specic RNA probe was in vitro transcribed and labeled with [a-32P]-UTP (PerkinElmer, Waltham, MA, USA) using the MAXIscript kit, according to the manufacturers instructions (Ambion). For westerns, proteins were separated on SDSpolyacrylamide gel electrophoresis gels, transferred to ImmunoBlot PVDF membrane (Bio-Rad, Hercules CA, USA), blocked in 5% milk-TBST and probed overnight. Secondary was probed at 10 ng/ml for 1 h at room temperature. Blots were visualized using Pierce SuperSignal West Dura substrate. Sucrose gradient ultracentrifugation Lysate was separated on 1030% continuous gradients prepared manually in sucrose gradient buffer (50 mM TrisHCl, 80 mM KCl, 5 mM Mg(C2H3O2)2, Oncogene (2013) 4377 4386 TaqMan Array SiHa cells stably expressing GFP or NOL7 were untreated or transcriptionally inhibited with 5 mg/ml a-amanitin for 4 h. After treatment, RNA was collected from cells using the RNeasy Mini Kit (Qiagen) and reverse transcribed with Superscript VILO (Invitrogen). 100 ng complementary DNA from treated and untreated GFP and NOL7 expressing cells was combined with TaqMan Gene Expression Master Mix, loaded onto the Human Angiogenesis Array, and analyzed on the 7900HT Fast real-time PCR system (Applied Biosystems). For steady state, expression was calculated between untreated samples relative to GFP. For post-transcriptional expression, mRNA levels were normalized to untreated samples and compared directly. Statistical signicance was calculated using Students t-test from an average of three independent assays. & 2013 Macmillan Publishers Limited NOL7 post-transcriptionally upregulates TSP-1 CL Doci et al 4385 CONFLICT OF INTEREST The authors declare no conict of interest. ACKNOWLEDGEMENTS We wish to thank Drs Ira Wool and Yuen-Ling Chan for their assistance in sucrose gradient ultracentrifugation. This work was supported in part by Illinois Department of Public Health Penny Severns Cancer Research Fund (MWL). REFERENCES 1 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 5770. 2 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell (Research Support, NIH, Extramural Review) 2011; 144: 646674. 3 Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407: 249257. 4 Folkman J. Role of angiogenesis in tumor growth and metastasis. 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... Oral Oncology 49 (2013) 93101 Contents lists available at SciVerse ScienceDirect Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology DSG3 as a biomarker for the ultrasensitive detection of occult lymph node metastasis in oral cancer using nanostructured immunoarrays Vyomesh Patel a, Daniel Martin a, Ruchika Malhotra b, Christina A. Marsh a, Colleen L. Doi a, Timothy D. Veenstra f, Cherie-Ann O. Nathan c, Uttam K. Sinha d, Bhuvanesh Singh e, Alfredo A. Molinolo a, James F. Rusling b, J. Silvio Gutkind a, a Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4330, United States Departments of Chemistry and Cell Biology, University of Connecticut, Storrs, CT, United States Department of Otolaryngology, Head and Neck Surgery, Louisiana State University Health Sciences Center, Shreveport, LA, United States d Department of Otolaryngology, Head and Neck Surgery, University of Southern California, Keck School of Medicine, Los Angeles, CA, United States e Laboratory of Epithelial Cancer Biology, Head and Neck Service, Memorial Sloan-Kettering Cancer Center, New York, NY, United States f Laboratory of Proteomics and Analytical Technologies, Science Applications International Corporation-Frederick, Inc., National Cancer Institute, Frederick, MD, United States b c a r t i c l e i n f o Article history: Received 26 April 2012 Received in revised form 19 July 2012 Accepted 1 August 2012 Available online 23 September 2012 Keywords: DSG3 Head and neck cancer Desmosomes Biomarker Sentinel lymph nodes Nanosensors Immunoarray Lymph nodes metastasis Proteomics SCC s u m m a r y Objectives: The diagnosis of cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) patients constitutes an essential requirement for clinical staging and treatment selection. However, clinical assessment by physical examination and different imaging modalities, as well as by histological examination of routine lymph node cryosections can miss micrometastases, while false positives may lead to unnecessary elective lymph node neck resections. Here, we explored the feasibility of developing a sensitive assay system for desmoglein 3 (DSG3) as a predictive biomarker for lymph node metastasis in HNSCC. Materials and methods: DSG3 expression was determined in multiple general cancer- and HNSCC-tissue microarrays (TMAs), in negative and positive HNSCC metastatic cervical lymph nodes, and in a variety of HNSCC and control cell lines. A nanostructured immunoarray system was developed for the ultrasensitive detection of DSG3 in lymph node tissue lysates. Results: We demonstrate that DSG3 is highly expressed in all HNSCC lesions and their metastatic cervical lymph nodes, but absent in non-invaded lymph nodes. We show that DSG3 can be rapidly detected with high sensitivity using a simple microuidic immunoarray platform, even in human tissue sections including very few HNSCC invading cells, hence distinguishing between positive and negative lymph nodes. Conclusion: We provide a proof of principle supporting that ultrasensitive nanostructured assay systems for DSG3 can be exploited to detect micrometastatic HNSCC lesions in lymph nodes, which can improve the diagnosis and guide in the selection of appropriate therapeutic intervention modalities for HNSCC patients. Published by Elsevier Ltd. Introduction With more than 500,000 new cases annually, squamous cell carcinomas of the head and neck (HNSCC) represent one of the ten most common cancers globally,1 and result in more than 11,000 deaths each year in the US alone.2 The 5 year survival of newly diagnosed HNSCC patients is 50%, and despite new treatment approaches, it has improved only marginally over the past decades.3 HNSCC has a high propensity to metastasize to loco Corresponding author. Address: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, 30 Convent Drive, Building 30, Room 211, Bethesda, MD 20892-4330, United States. Tel.: +1 301 496 6259; fax: +1 301 402 0823. E-mail address: sg39v@nih.gov (J.S. Gutkind). regional lymph nodes due to the presence of a rich lymphatic network and the overall high number of lymph nodes in the neck region.38 Even in patients without clinical evidence of lymph node involvement (N0), there is a high incidence of occult lymph node metastasis, ranging from 10% to 50%.4,5,7 The diagnosis of cervical lymph node metastasis an essential requirement for clinical staging and treatment,9 and is now widely accepted as the most important factor in HNSCC prognosis.3,5,6,10 However, due to limitations in the accurate diagnosis of lymph node metastasis, patients with clinically negative nodes often undergo elective neck resection or radiation,11,12 with the consequent associated morbidity and adverse impact in the quality of life.12 Clinical assessments of lymph node metastases include physical examination, imaging modalities such as computed tomography (CT), magnetic resonance imaging (MRI), ultrasonography, and 1368-8375/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.oraloncology.2012.08.001 Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 94 V. Patel et al. / Oral Oncology 49 (2013) 93101 [18F]-2-uorodeoxyglucose positron emission tomography scans (PET).13,14 However, poor spatial resolution, false positive detection of reactive lymph nodes, and limited sensitivity under 5 mm size,1518 can add to potential false negative results. Histopathological, immunohistochemical (IHC), and molecular approaches to evaluate sentinel lymph node biopsies have improved the detection rate of metastatic disease in some cancers,19,20 but histopathology-based methods can often miss micrometastases, while more sensitive techniques such as IHC and real-time PCR for validated cancer markers are time consuming and require stringent handling procedures and technical expertise. A recent proteomic analysis of parafn embedded normal oral mucosa and HNSCC lesions revealed a very high abundance of Desmoglein 3 (DSG3) in both non-neoplastic epithelium and cancer lesions.21 DSG3 is a transmembrane glycoprotein involved in cell-to-cell adhesion that is exclusively expressed in stratied epithelium.22 These observations prompted us to explore whether the assessment of DSG3 protein levels could be used to investigate the presence of malignant squamous epithelial cells in cervical lymph nodes, and hence serve as a predictive biomarker for metastasis. In this regard, high sensitivity electrochemical immunoassays have recently gained acceptance in biomedicine.23 For example, we have developed immunosensors based on nanostructured electrodes coupled to microuidics and multilabel strategies to achieve highly sensitive detection of protein cancer biomarkers in serum.24,25 We have combined these strategies into a simple microuidic immunoarray26,27 and here explore the suitability of this platform for the rapid and sensitive detection of DSG3 protein. We show that this system can be used to rapidly detect and quantify DSG3 in frozen human tissue sections, distinguishing between clinically positive and negative cervical lymph nodes. Overall, these studies may help develop point-of-care procedures aiding in the diagnosis of invaded lymph nodes in HNSCC patients, thereby facilitating educated decisions regarding appropriate therapeutic intervention modalities, and decreasing the morbidity often associated with HNSCC. Materials and methods Reagents, antibodies, and cell culture All chemicals and reagents were from SigmaAldrich (St. Louis, MO), unless indicated. The following antibodies: goat-anti-human DSG3 [AF1720]; mouse-anti-human DSG3 [MAP1720], biotin labeled goat-anti-human DSG3 [BAF1720], recombinant human DSG3 Fc Chimera protein [1720-DM], were from R&D Systems (MN, USA). The mouse anti-human DSG3 antibody [32-6300] from Invitrogen (MA, USA), and rabbit-anti-cytokeratin Wide Spectrum Screening [N1512] from Dako (CA), were used for immunohistochemistry (IHC). The a-tubulin antibody [11H10] was from Cell Signaling Technology (MA, USA). Biotinylated peroxidase and streptavidin coated magnetic beads were from Invitrogen. Antirabbit and anti-mouse biotinylated secondary antibodies were from Vector, Burlingame, CA, US. HN12, HN13 and HN30 cells were described previously.28 Cal27 and Jurkat cells were from ATCC (VA); and primary human cells from Lonza (MD). See Supplemental information for additional information. Human clinical tissues and tissue microarrays (TMAs), immunohistochemistry and immunouorescence Formalin xed, parafn-embedded, and freshly frozen HNSCC and lymph node samples were obtained anonymized with Institutional Review Board approval. Five lm sections from all tissues underwent standard H&E staining for histopathological evaluation and immunostaining. Tissue microarrays used include TMA MC2081 US (Biomax, MD) with 208 representative cases of colorectal, breast, prostate and lung cancers, and normal tissue; TMA LC810 (Biomax, MD), consisting of 40 cases of different types of lung cancers with their matched metastatic lymph nodes (total 80 tissue cores); and the Head and Neck Tissue Microarray Initiative, including 317 HNSCC cases.29 Tissue processing and analysis are described in detail in Supplemental information. All slides were scanned at 400 magnication using an Aperio CS Scanscope (Aperio, CA) and quantied using the available Aperio algorithms. Immunodetection of DSG3 was quantied according to percent of tumor cells stained (125%, 2650%, 5175%, or 76100%).29 For immunouorescence, 10 lm cryosections were immunostained with goat-anti-human DSG3 (AF1720), mouse-anti-vimentin and DAPI containing. See Supplemental information for additional details. Western blot analysis of cell and tissue extract, and microuidic immunoarrays systems for DSG3 A detailed description of the procedures used for tissue lysate preparation, SDSPAGE gel analysis and Western blotting, and the fabrication of the microuidic immunoarrays made of gold nanoparticles layered with glutathione are described in detail in the Supplemental information. Briey, the immunoarrays consisting of eight sensor elements, made of gold nanoparticles layered with glutathione, were rst coated with the capture antibody and transferred to a microuidic chamber. In parallel, biotinylated horseradish peroxidase and a biotinylated secondary antibody were attached to streptavidin-coupled magnetic beads and collected with a magnet. Next, 5 ll of 5750 fg/mL of recombinant DSG3 protein standards or 4 ll tissue extract were diluted 1:6000 in RIPA buffer and added to the bioconjugates. The bioconjugates with captured proteins were then magnetically separated, washed, resuspended in a nal volume of 110 lL, and immediately injected into the microuidic channel housing the immunoarrays. At this step, the ow was stopped, incubated for 20 min, washed, and hydroquinone solution was passed through the channel. The amperometric signal was developed by injecting 50 lL of 100 lM H2O2. Tissue lysates used for Western blot analysis and microuidic immunoarrays were made from primary tumors (n = 4), lymph node ( ) (n = 3), and lymph node (+) (n = 3). Results In a previous proteome-wide analysis of HNSCC progression, we noted that DSG3 was highly expressed in normal oral mucosa and HNSCC lesions.21 To further investigate a possible use for DSG3 as a predictive biomarker, we rst assessed DSG3 expression by immunohistochemistry in an independent cohort of human normal and malignant oral squamous tissues. By H&E histological evaluation, normal squamous epithelium shows a dened basement membrane with layers of differentiating keratinocytes, whereas in the malignant counterpart, this organized pattern is lost (Fig. 1A). Normal tissues sections stained for DSG3 show that it is predominantly expressed in the basal and suprabasal layers of the normal squamous epithelium, while in SCC DSG3 expression is restricted to cancer cells. Stromal cells were negative. We next evaluated DSG3 expression in a HNSCC tissue microarray (TMA) containing 317 evaluable cores.29 DSG3 was readily detected in all HNSCC cores and localized to tumors cells (Fig. 1B). Within these cases, well-differentiated carcinomas (n = 120) had the highest percentage of DSG3-positive cells (90%). The moderate- (n = 119), and poorlydifferentiated (n = 66) cores showed slightly lower proportion of DSG-reactive cells (80% and 70%, respectively), the remaining 12 cores consisting of corresponding to non-squamous tissues were Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 95 V. Patel et al. / Oral Oncology 49 (2013) 93101 Normal HNSCC H&E A 100 100 100 DSG3 100 B DSG3 immunostaining WD % positive cancer cells C 100 100 n: 120 119 66 76-100 51-75 26-50 1-25 80 60 40 20 0 PD 30 10 1 3 0.3 Recombinant DSG3 (ng) 100 Tubulin PD DSG3 100 HN13 HN30 CAL27 HaCaT Total cell lysates JURKAT HMVEC LEC HUVEC HN12 MD D MD 0.1 WD Figure 1 Validation of DSG3 expression in normal and malignant HNSCC. Normal oral mucosa biopsies and HNSCC were evaluated for DSG3 by IHC. (A) DSG3 is expressed throughout the normal epithelium, but is stronger in the basal and parabasal layers. Diffuse expression was seen in the epithelial component of all HNSCC. (B) Representative well (WD), moderate (MD) and poorly (PD) differentiated HSCC cases are shown. (C) DSG3 was expressed in all tumors regardless of differentiation, with increased expression in WD cases. Numbers of cases analyzed is depicted. (D) Total cell extracts from non-squamous (Jurkat, HMVEC, LEC, HUVEC) and oral-squamous (HN12, HN13, HN30, Cal27), and epidermal-squamous (HaCaT) were processed for Western blot analysis. Native DSG3 and its glycosylated forms were readily detected in squamous cells extracts, while absent from the non-squamous counterparts. These levels were compared with human recombinant DSG3 that was processed in a background of Jurkat cell lysate. Tubulin staining indicates equivalent loading and protein integrity. negative for DSG3 (Fig. 1C). The data demonstrates that DSG3 is highly expressed in human oral squamous epithelium and HNSCC. We next sought to assess in vitro the specicity of the epithelial expression of DSG3 in a panel of squamous and non-squamous model cells. The latter included Jurkat cells (immortalized T lymphocyte cells), HMVEC (skin human microvascular endothelial cells), LEC (lymphatic endothelial cells), and HUVEC (human umbilical vein endothelial cells). HaCaT cells are squamous, nonoral immortalized epidermal keratinocytes, while the oral squamous cell carcinoma lines included HN12, HN13, HN30 and Cal27.28 DSG3 was readily detected in all squamous oral cancer cell lines and HaCaT cells, with lower levels in HN12 and higher in Cal27 (Fig. 1D). No expression for DSG3 was observed in any of four non-squamous lines, while levels of a-tubulin indicated equal loading as well as protein integrity. The data seem to indicate that DSG3 is exclusively expressed in squamous epithelial-derived cells. To further validate the specicity of DSG3 expression, we evaluated TMAs containing cores representing the four most common cancers (breast, lung, prostate, and colon cancer) for DSG3 levels, and scored cases based on the presence or absence of DSG3 expression. As seen in Fig. 2A), DSG3 is poorly expressed in breast and prostate cancers, as well in adenocarcinoma of the lung (ADC), which likely reects the glandular epithelial cell origin of these human malignancies. In colon carcinoma, the expression was variable, and in all cases the pattern of expression was diffused and not the characteristic membrane lace-like pattern. In contrast, Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 96 V. Patel et al. / Oral Oncology 49 (2013) 93101 A TMA major cancer types Lung ADC Breast n: 42 47 16 45 37 13 298 100 + - Lung 100 m Colon % Cases 80 100 m 60 40 20 100 m OSCC Lung SCC Lung ADC Prostate Breast Prostate OSCC Rectum 0 100 m Colon 100 m 100 m Lung cancer B TMA lung cancer 100 SCC Met 100 m 100 m SCLC ADC n=30 % positive tumors SCC 80 60 40 20 100 m n=42 100 m n=6 0 SCC ADC SCLC Figure 2 Immunoreactivity of DSG3 in common tumor types. (A) Multi-tumor TMAs (lung, breast, colon, prostate), and an oral specic TMA were assessed for DSG3 expression by IHC, and the staining scored for the presence of specic staining as (+) or ( ). Most squamous cell lung cancers stained positive for DSG3, but very few of the adenocarcinomas gave positive reaction. All cores from the OSCC TMA scored positive. (B) In lung cancer, DSG3 expression was positive in most squamous cell carcinomas (SCC) including lymph node metastasis (SCC Met), while few cases of adenocarcinomas (ADC) gave positive reaction, and all small cell lung carcinoma samples (SCLC), were negative. DSG3 is highly expressed in tumors derived from cells of squamous epithelial origin, such as lung squamous carcinoma (SCC) and additional oral squamous carcinoma (OSCC) that were included in these arrays, showing a membrane localized staining. All stainings were scored blindly and tabulated (Fig. 2A). All cancers of squamous origin (oral and lung SCC) were strongly positive for DSG3 expression. As lung cancers include SCC, ADC, and small cell lung carcinomas (SCLCs), we examined further the specicity of DSG3 expression in these distinct lung cancer lesions. All lung SCC show strong membrane localized staining in both the primary tumor and metastasis, while ADC and SCLC show marginal to no DSG3 expression, as reected by scoring their corresponding tissue cores (Fig. 2B). Collectively, our results indicate the high specicity of DSG3 expression in oral and lung SCC lesions. Based on the observation that DSG3 is highly expressed in HNSCC, we wanted to determine if the presence of this protein in cervical lymph nodes of the neck region could be used as a predictive biomarker of HNSCC invasion. To this end, we evaluated formalin-xed, parafn-embedded and anonymized tissue sections of non-metastatic (N0) or metastatic (N+) human cervical lymph node biopsies from patients diagnosed with HNSCC for expression of DSG3 and cytokeratin, a squamous-specic protein marker. Negative lymph nodes were negative (Fig. 3A) whereas clusters of tumor cells stained positive for membrane-localized DSG3 (inset, top right), can be seen throughout the invaded lymph node (N+), indicating the metastatic spread of squamous tumor cells of the primary tumor lesions from the oral cavity (Fig. 3A). Noteworthy, small clusters of 23 isolated tumor cells, constituting micrometastases were readily detected by the presence of DSG3 protein, and this size tumor island could potentially be missed by histopathological evaluation (Inset). Next, we screened a larger cohort of metastatic and non-metastatic human cervical lymph nodes for cytokeratin and DSG3 expression (n = 35). All metastatic (n = 30), but not non-metastatic cases (n = 5) were positive for DSG3. Serial sections stained for H&E and cytokeratin conrmed the epithelial nature of the malignant DSG3-positive cells. (Fig. 3B, and low magnication of a whole lymph node in Suppl. Fig. 1). This indicates that DSG3 expression can help identify small numbers of malignant squamous tumor cells in lymph nodes, and hence the metastatic nature of the primary lesion. The sensitivity and specicity of this detection suggests that DSG3 may hold promise for accurately detecting micrometastasis in cervical lymph nodes in newly diagnosed HNSCC patients. Our previous study adapted amperometric sandwich immunoassays to a microuidic system for the ultrasensitive, multiplexed detection of secreted biomarker proteins.26 Here, we used a similar strategy for detecting DSG3 in complex tissue extracts using the microuidic immunoassay system. As shown in Fig. 4A, DSG3 Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 97 V. Patel et al. / Oral Oncology 49 (2013) 93101 A N- 100m B N+ 100m N+ 100m DSG3 N+ 50m 100m H&E N+ 50m DSG3 50m 100m N- 100m DSG3 50m CK 50m DSG3 50m Figure 3 Specic detection of DSG3 in human cervical lymph nodes. (A) Formalin xed and parafn embedded tissue sections of non-metastatic (N ) and metastatic lymph nodes (N+) show DGS3 expression only in N+, with the staining localized to the malignant squamous cells (n = 30). All N cases were negative (n = 5). B. The epithelial specicity of DSG3 immunoreactivity was further conrmed using simultaneous cytokeratin (CK) staining. A representative example is shown, whereby the H&E stained tumor island is matched with CK and DSG3 expression, with no non-specic staining. An example of a N case stained for DSG3 is shown. capture antibodies are attached to up 8 sensor elements, and streptavidin-coated paramagnetic beads (MB) loaded with 400,000 biotin-HRPs and thousands of secondary biotin-labeled antibodies to DSG3 (Ab2) are used to capture the protein off-line. After washing and magnetic separation, the MBs that have bound DSG3 (DSG3MB) are injected into the microuidic channel. Incubation at stopped ow allows the sensor antibodies to capture DSG3-MBs, and amperometric signals are developed by injecting hydrogen peroxide to activate HRP and hydroquinone to mediate the amperometic oxidation, resulting in peak currents proportional to DSG3 concentration (Fig. 4B). Noteworthy, the entire assay from incubation of sample with Ab2-MB-HRP to measurement takes 50 min. Remarkably, using this approach we were able to accurately and reproducibly detect DSG3 protein at levels down to 5 fg mL 1 in complex tissue extracts, with minimal non-specic binding. We next used these protocols for capturing DSG3 from clinical samples of human HNSCC. Desmosomes are notoriously insoluble, and multiple buffers tested, RIPA buffer afforded excellent solubility and retaining antigenicity of DSG3 extracted directly from cryosections of HNSCC and lymph node tissue. Protein extracts were made from a single 10 lm cryosection from each sample and used as input. Picogram levels of DSG3 protein were detected in all tumor samples (T14), and this was conrmed by Western blot analysis, where high levels of the protein were also detected in total cell extracts that were used for these analyses (Fig. 4C and D). The specicity of DSG3 identication was further conrmed by simultaneous uorescence microscopy of DSG3 and vimentin, as a stromal marker, in frozen sections of a series of metastatic and non-metastatic lymph nodes. As seen in Fig. 5A, only vimentin (red) was identied in non-metastatic lymph nodes (in blue, nuclear DAPI staining), whereas all metastatic lymph nodes showed pockets of very strong staining for DSG3 (green). To further explore the sensitivity of the method, we decided to analyze metastatic lymph node tissues in which the number of malignant epithelial cells was known. For this, H&E stained slides of each case was scanned, analyzed histologically, the malignant areas identied, and the number of cells quantied using the Aperio nuclear algorithm (Aperio, Vista, CA). The number of cancer cells per lymph node section is indicated in Table 1. No tumor cells were be present in the negative lymph nodes, while all three metastatic lymph nodes (13) evaluated had differing number of tumor cells. Noteworthy, positive lymph node 1 had less than Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 98 V. Patel et al. / Oral Oncology 49 (2013) 93101 A B Ab1 DSG3 (fg mL-1) 40 750 500 250 30 DSG3 PDDA HRP Ab2 I, nA 100 AuNPs 50 20 25 5 10 10 0 0 1 m streptavidin coated magnetic bead -10 0 400 800 1200 1600 t, s Ab2 -MB-HRP 40 35 y = -7.4659 + 14.987log(x) R = 0.99315 30 I, nA 25 20 15 10 5 Voltage + H2O2 HQ amperometric signal 0 100 10 C 1000 [DSG3], pg mL-1 1000 [DSG3], fg mL-1 D Primary tumor T1 T2 T3 T4 100 10 DSG3 1 0.1 0.01 Tubulin Figure 4 Rapid and ultrasensitive detection of DSG3 in human HNSCC samples using nanosensors. (A) Scheme used for the ultrasensitive detection by the microuidic immunoarray showing a single sensor in the array with capture DSG3 antibodies attached. Proteins are captured off-line on Ab2-magnetic bead (MB)-HRP bioconjugates , and after magnetic separation and washes, the MBs are injected into the immunoarray containing 8 sensors. A single immunoarray sensor is depicted. Following incubation, amperometric signals are generated by applying 0.3 V versus Ag/AgCl to the sensors by injecting a mixture of HRP-activator H2O2 and mediator hydroquinone (HQ). (B) Varying recombinant DSG3 protein concentrations were used to generate a calibration plot. The sensitivity of DSG3 sensor using recombinant protein was 5646 nA mL [fg protein] 1 cm 2. (C) Protein extracts of primary human oral squamous carcinomas (T14) made with RIPA buffer were processed for detection of DSG3. High DSG3 levels were found to be present in all the samples, and this was conrmed by Western blot analysis of the same extracts for DSG3 (D). Tubulin was used as loading control. 1000 tumor cells, while the remaining had between 13,000 and 16,000 cells (Table 1). Using the nanosensors, we evaluated total protein extracts from lymph nodes for levels of DSG3. As seen in Fig. 5A, DSG3 was essentially not detected in the normal lymph nodes, with marginal values likely a reection of very limited residual non-specic binding of Ab2-MB-HRP complex, giving rise to minimal amperometric signal. In contrast, all of the positive, metastatic lymph nodes showed high levels of DSG3 protein expression. To validate these measurements, the same protein extracts were also analyzed by Western blotting (Fig. 5B). No DSG3 was detected in the negative lymph nodes, while in all the positive lymph nodes, bands for DSG3 and its multiple glycosylated forms were readily seen, and the intensity of the corresponding bands correlated with DSG3 levels quantied by the nanosensors. When total DSG3 was normalized by the number of tumor cells in each metastatic lesion, positive lymph nodes expressed approximately 150 fg DSG3 per tumor cell (Table 1), well above the threshold for DSG3 detection. Together, this indicates that the nanosensor-based detection of DSG3 could be a highly sensitive and specic method for the identication of squamous carcinoma metastases in clinical practice. This rapid method was capable of measuring DSG3 levels even from as little as a single cell, suggesting that this technique may represent a potent tool for the ultrasensitive detection of the presence or absence of lymph node invasion in human oral cancer patients. Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 99 V. Patel et al. / Oral Oncology 49 (2013) 93101 Lymphnodes(+) 1 2 2 H&E Lymphnodes(-) 1 50m 100m 100m 50m 100m 50m 100m 50m 100m 50m 100m 50m 100m 50m IF 100m 50m DAPI/Vimentin/DSG3 Lymph node (-) Lymph node (+) 1000 [DSG3], pg mL-1 1 2 3 1 2 3 100 10 DSG3 1 0.1 0.01 1 2 3 1 2 3 Tubulin Lymph node - Lymph node + Figure 5 Detection of DSG3 in metastatic human cervical lymph nodes. H&E stained cryosections of representative non-metastatic ( ) and metastatic (+) human cervical lymph nodes were scanned and the total number of tumor cells per section was quantied (Table 1). Serial sections of these lymph nodes were evaluated by immunouorescence for DSG3 and detected only in metastatic lymph nodes (green). Vimentin (red) was used to identify stromal tissue, and nuclei of all cells were stained blue with DAPI (Fig. 5A). Protein extracts made from single cryosections of lymph nodes were used for the detection of DSG3 by Western blot analysis and DSG3 quantication using nanosensors. DSG3 levels were similar to background for all non-metastatic samples, while DSG3 levels in all metastatic cases were proportional to the number of invading HNSCC cells (Fig. 5B). Table 1 DSG3 detection in metastastic lymph nodes. Cryosections of non-metastatic (N ) (n = 3) and metastatic (N+) (n = 3) human cervical lymph nodes were collected and analyzed by nanosensor detection. The total number of tumor cells per cryosection was evaluated, and used to estimate the total amount of DSG3 per tumor cell. Samples Detected DSG3 (pg/mL) Tumor cells per section Detected DSG3 (fg/tumor cell) N 1 N 2 N 3 N+1 N+2 N+3 0.01 0.03 0.02 427 6274 4512 697 13,843 16,576 202 150 90 Discussion The spread of primary HNSCC lesions to locoregional lymph nodes has often already occurred at the time of diagnosis, thus compromising the prognosis and long-term survival of HNSCC patients.3 Accurate diagnosis of lymph node metastases remains difcult, and many patients that do not present cancer dissemination to the lymph nodes (N0) may be subjected to unnecessary elective surgery. On the other hand, small lesions may be difcult to identify within the lymph nodes in cryosections when histophatologic evaluation is performed while the patient is in the operating room. Hence, some patients may miss a therapeutic opportunity due to false negative diagnosis of lymph node metastasis. Here, we demonstrate that DSG3 is expressed in normal oral squamous mucosa, and in all HNSCC lesions and their metastatic cervical lymph nodes. Indeed, the presence of DSG3 in lymph nodes can be exploited to detect Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. 100 V. Patel et al. / Oral Oncology 49 (2013) 93101 micrometastatic lesions, which can serve as a sensitive marker of HNSCC progression. We also show the feasibility of using a rapid, low-cost nanostructured immunoarray device for the detection of DSG3 protein in metastatic lymph nodes in newly diagnosed HNSCC patients, which can improve diagnosis and guide the most effective therapeutic options. Most current technologies for cancer detection and diagnostics are not suitable for the differentiation of normal versus metastatic lymph nodes at early stages of cancer progression, and efforts to address this gap have been met with mixed results. Currently, the gold standard for identication of metastasis is the serial sectioning and histopathological analysis of tissue specimens by H&E staining and immunohistochemistry.30 This provides key information needed for TNM (tumor-node-metastasis criteria) classication of HNSCC patients. However, a risk remains that micrometastases may go undetected in otherwise negative lymph nodes. IHC performed on serial sections for cytokeratin may help in detecting metastases, but unfortunately this low-throughput method requires signicant investment of time and expense, and it is often performed after surgery. Considering the false-negative rate and the sampling error that are encountered by H&E examination alone, a reliable and rapid predictive test to determine lymph node metastases is needed.31 Application of new technologies such as real-time quantitative PCR (qPCR), to look at mRNA levels of molecules expressed by oral squamous tissues have shown encouraging results.32 While this improves upon some of the limitations of IHC detection of cytokeratins, independent studies have found some inconsistencies in the precise cytokeratin to be analyzed. For example, from a three-marker analysis, only cytokeratin 14 was reliably detected by qPCR in several oral cancer cell lines and tissues, and sensitive enough to detect down to a single cancer cell in a background of Jurkat cells, essentially representing a model of lymph node metastasis.33 In another study, cytokeratin 17 was demonstrated to be far superior at discriminating positive lymph nodes while cytokeratin 14 was less informative, although in parallel histological analysis, this was only achieved if metastasis had exceeded 450 lm, leaving a high probability of micrometastasis going undetected.34 While the sensitivity of qPCR for detecting cytokeratins is unquestionable, its ability to reliably and consistently detect these molecules in a single tumor cell embedded within normal lymphatic tissues still remains a challenge. The met-receptor is over-expressed in several metastatic carcinomas including HNSCC,35,36 and with minimal to none in lymphatic cells, its presence in lymph nodes may be exploited for predicting metastasis. Indeed, qPCR analysis detected met expression in 40% of invaded lymph nodes, and interestingly exceeding the sensitivity of cytokeratins, which were tested in the same sample cohort.37 Use of multiple markers may improve detection of metastatic lymph nodes, and in this regard, mRNA for DSG3 (referred to as pemphigus vulgaris antigen, PVA) and TACSTD1 (tumor-associated calcium signal transducer 1), have been previously reported to be highly expressed in HNSCC, and successfully integrated into a multiplex qRT-PCR assay for metastatic prediction, achieving a remarkable accuracy.38,39 DSG3 mRNA levels in lymph nodes have been also touted as potential predictors of HNSCC progression.38,40 Generally, the use of qPCR greatly improves the sensitivity of detection of target genes, but the need of high quality RNA extracted from tissues remains a signicant technical hurdle, such as the presence of contaminants and RNA degradation that can severely interfere with data interpretation. It is noteworthy that mRNA levels may not accurately reect protein expression, as many post translational regulatory processes may allow or prevent the accumulation of translated products, and for predictive biomarkers, the presence of the target protein may be better suited. In this regard, our proteomics analysis of HNSCC and normal oral epithelial tissues suggested that DSG3 is preferentially expressed in squamous tissues.21 By examining hundreds of cancer lesions representing some of the most prevalent human malignancies we now show that DSG3 is highly expressed in all tumors derived from cells of squamous epithelial origin, such as lung SCC and HNSCC, with more variable expression in adenocarcinomas of the colon, prostate, breast and lung, likely reecting their glandular epithelial cell of origin. For HNSCC, we noticed a lower expression of DSG3 in poorly differentiated lesions, aligned with prior reports.41 However, all HNSCC cases analyzed expressed DSG3, albeit in some lesions not all tumor cells expressed this marker. Thus, although the possibility exists that in some invaded lymph nodes the level of DSG3 may be below our detection limit, our collective ndings indicate that DSG3 is highly expressed in all human SCCs but not expressed in normal lymph nodes, and that DSG3 can be detected even in small clusters of malignant cells invading the lymph nodes, thus serving as a marker for the metastatic spread of the cancerous lesions. We now exploited this information, the availability of highly specic monoclonal antibodies detecting different epitopes in DSG3, and our recently established biomarker detection platform using microuidic immunoarray devices featuring nanostructured electrodes,26 to develop an assay system enabling the rapid and ultrasensitive detection of DSG3 protein in complex tissue extracts, with minimal non-specic binding. The method was sensitive enough to detect isolated tumor cells, and certainly small groups of cells in a single cryosection of a positive lymph node. Taken together, we can conclude that the ability to quantitate femtogram levels of DSG3 can be used for the intraoperational detection of the presence of even few invasive human squamous epithelial cells in cryosections of lymph nodes of HNSCC patients, hence aiding the pathologists and surgeons to make informed decisions about appropriate treatment options. We expect that similar approaches can be used to optimize the detection of additional cancer biomarkers in lymph node sections, thus increasing the basis for successful clinical prediction. Collectively, combined with a simple work-ow and a short assay time, these features described in this study, hold promise for the development of point-of-care clinical screening techniques to identify HNSCC patients with metastatic disease. Indeed, the encouraging results described in this proof of principle study may provide the rationale for future validation of this diagnostic strategy in larger multicenter studies. Conict of interest statement None declared. Acknowledgements This work was supported by the Intramural Program of the National Institute of Dental and Craniofacial Research, National Institutes of Health, and through grant R01EB014586 from the National Institute of Biomedical Imaging and Bioengineering (JRF). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.oraloncology. 2012.08.001. References 1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127(12):2893917. 2. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62(1):1029. Downloaded for Anonymous User (n/a) at Marian University from ClinicalKey.com by Elsevier on March 08, 2021. For personal use only. No other uses without permission. Copyright 2021. Elsevier Inc. All rights reserved. V. Patel et al. / Oral Oncology 49 (2013) 93101 3. Forastiere A, Koch W, Trotti A, Sidransky D. Head and neck cancer. N Engl J Med 2001;345(26):1890900. 4. Clark JR, Naranjo N, Franklin JH, de Almeida J, Gullane PJ. Established prognostic variables in N0 oral carcinoma. Otolaryngol Head Neck Surg 2006;135(5):74853. 5. Kuriakose MA, Trivedi NP. 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