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• W2090395004 abstract "Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo. Tumor cell–specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability, migration of tumor cells, and invasion of tumor cells. Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13. Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer. Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo. Tumor cell–specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability, migration of tumor cells, and invasion of tumor cells. Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13. Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer. actinic keratosis cutaneous squamous cell carcinoma cSCC in situ erythropoietin-producing hepatocellular immunohistochemistry matrix metalloproteinase normal human epidermal keratinocyte quantitative real-time reverse-transcriptase–PCR receptor tyrosine kinase severe combined immunodeficient small interfering RNA Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic malignant tumor of skin (Madan et al., 2010Madan V. Lear J.T. Szeimies R.M. Non-melanoma skin cancer.Lancet. 2010; 375: 673-685Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). The incidence of cSCC is increasing worldwide, making it the most common form of metastatic skin cancer (Rogers et al., 2010Rogers H.W. Weinstock M.A. Harris A.R. et al.Incidence estimate of nonmelanoma skin cancer in the United States, 2006.Arch Dermatol. 2010; 146: 283-287Crossref PubMed Scopus (974) Google Scholar). Important risk factors for cSCC include solar UV radiation, chronic ulcers, and immunosuppression (Alam and Ratner, 2001Alam M. Ratner D. Cutaneous squamous-cell carcinoma.N Engl J Med. 2001; 344: 975-983Crossref PubMed Scopus (977) Google Scholar). Primary cSCCs have a tendency for recurrence and metastasis, and treatment of metastatic cSCCs is challenging in the absence of targeted therapies (Rowe et al., 1992Rowe D.E. Carroll R.J. Day Jr., C.L. Prognostic factors for local recurrence, metastasis, and survival rates in squamous cell carcinoma of the skin, ear, and lip. Implications for treatment modality selection.J Am Acad Dermatol. 1992; 26: 976-990Abstract Full Text PDF PubMed Scopus (1161) Google Scholar; Czarnecki et al., 2002Czarnecki D. Sutton T. Czarnecki C. et al.A 10-year prospective study of patients with skin cancer.J Cutan Med Surg. 2002; 6: 427-429Crossref PubMed Scopus (19) Google Scholar). UV-induced inactivation of both alleles for tumor protein 53 (TP53) gene is an early event in keratinocyte carcinogenesis resulting in marked expansion in simple mutations, making cSCCs one of the human cancers with highest mutation rate (Durinck et al., 2011Durinck S. Ho C. Wang N.J. et al.Temporal dissection of tumorigenesis in primary cancers.Cancer Discov. 2011; 1: 137-143Crossref PubMed Scopus (201) Google Scholar; Ratushny et al., 2012Ratushny V. Gober M.D. Hick R. et al.From keratinocyte to cancer: the pathogenesis and modeling of cutaneous squamous cell carcinoma.J Clin Invest. 2012; 122: 464-472Crossref PubMed Scopus (390) Google Scholar). Another early event in the progression of cSCC is loss-of-function mutation of NOTCH1 (Wang et al., 2011Wang N.J. Sanborn Z. Arnett K.L. et al.Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma.Proc Natl Acad Sci USA. 2011; 108: 17761-17766Crossref PubMed Scopus (331) Google Scholar; South et al., 2014South A.P. Purdie K.J. Watt S.A. et al.NOTCH1 mutations occur early during cutaneous squamous cell carcinogenesis.J Invest Dermatol. 2014; 134: 2630-2638Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). However, at present, the knowledge on the molecular basis of cSCC progression from premalignant lesion (actinic keratosis (AK)) to cSCC in situ (cSCCIS) and eventually to invasive cSCC is incomplete (Ratushny et al., 2012Ratushny V. Gober M.D. Hick R. et al.From keratinocyte to cancer: the pathogenesis and modeling of cutaneous squamous cell carcinoma.J Clin Invest. 2012; 122: 464-472Crossref PubMed Scopus (390) Google Scholar). Therefore, molecular markers for rapid progression and metastatic capacity of cSCCs, as well as new therapeutic targets for these tumors, are in need (Kivisaari and Kähäri, 2013Kivisaari A. Kähäri V.M. Squamous cell carcinoma of the skin: Emerging need for novel biomarkers.World J Clin Oncol. 2013; 4: 85-90Crossref PubMed Google Scholar). Erythropoietin-producing hepatocellular (Eph) receptors represent the largest family of receptor tyrosine kinases (RTKs) (Pasquale, 2010Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat Rev Cancer. 2010; 10: 165-180Crossref PubMed Scopus (905) Google Scholar). According to their structures and ligand-binding affinities, the human Eph RTKs and their ligands, ephrins, are divided into subclasses A and B (Pasquale, 2010Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat Rev Cancer. 2010; 10: 165-180Crossref PubMed Scopus (905) Google Scholar; Nikolov et al., 2013Nikolov D.B. Xu K. Himanen J.P. Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation.Biochim Biophys Acta. 2013; 1834: 2160-2165Crossref PubMed Scopus (73) Google Scholar). Glycosylphosphatidylinositol-linked ephrin-A ligands (5 members) promiscuously bind to the EphA RTKs (9 members) and transmembrane ephrin-B ligands (3 members) bind to EphB RTKs (5 members). As both receptor and ligand are located on the cell surface, cell–cell contact is usually needed for their interaction. Upon binding, both receptors and ligands can activate cellular signaling resulting in bidirectional reverse and forward signaling in adjacent cells (Pasquale, 2010Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat Rev Cancer. 2010; 10: 165-180Crossref PubMed Scopus (905) Google Scholar; Nikolov et al., 2013Nikolov D.B. Xu K. Himanen J.P. Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation.Biochim Biophys Acta. 2013; 1834: 2160-2165Crossref PubMed Scopus (73) Google Scholar). In addition, there is evidence for ligand-independent clustering and activation of Eph RTKs on cell surface (Nikolov et al., 2013Nikolov D.B. Xu K. Himanen J.P. Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation.Biochim Biophys Acta. 2013; 1834: 2160-2165Crossref PubMed Scopus (73) Google Scholar). The role of Eph/ephrin signaling in numerous biological processes has been established (Lin et al., 2010Lin S. Gordon K. Kaplan N. et al.Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1.Mol Biol Cell. 2010; 21: 3902-3914Crossref PubMed Scopus (38) Google Scholar, Lin et al., 2012Lin S. Wang B. Getsios S. Eph/ephrin signaling in epidermal differentiation and disease.Semin Cell Dev Biol. 2012; 23: 92-101Crossref PubMed Scopus (33) Google Scholar; Nievergall et al., 2012Nievergall E. Lackmann M. Janes P.W. Eph-dependent cell-cell adhesion and segregation in development and cancer.Cell Mol Life Sci. 2012; 69: 1813-1842Crossref PubMed Scopus (75) Google Scholar; Gordon et al., 2013Gordon K. Kochkodan J.J. Blatt H. et al.Alteration of the EphA2/Ephrin-A signaling axis in psoriatic epidermis.J Invest Dermatol. 2013; 133: 712-722Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and their role in skin homeostasis has been well documented (Egawa et al., 2009Egawa G. Osawa M. Uemura A. et al.Transient expression of ephrin b2 in perinatal skin is required for maintenance of keratinocyte homeostasis.J Invest Dermatol. 2009; 129: 2386-2395Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar; Lin et al., 2010Lin S. Gordon K. Kaplan N. et al.Ligand targeting of EphA2 enhances keratinocyte adhesion and differentiation via desmoglein 1.Mol Biol Cell. 2010; 21: 3902-3914Crossref PubMed Scopus (38) Google Scholar). In human epidermal keratinocytes, EphB2 acting as a ligand triggers reverse signaling, and consequently promotes epidermal differentiation (Walsh and Blumenberg, 2012Walsh R. Blumenberg M. Eph-2B, acting as an extracellular ligand, induces differentiation markers in epidermal keratinocytes.J Cell Physiol. 2012; 227: 2330-2340Crossref PubMed Scopus (11) Google Scholar). The role of Eph/ephrin signaling in tumor progression in general appears complex (Pasquale, 2010Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat Rev Cancer. 2010; 10: 165-180Crossref PubMed Scopus (905) Google Scholar; Nievergall et al., 2012Nievergall E. Lackmann M. Janes P.W. Eph-dependent cell-cell adhesion and segregation in development and cancer.Cell Mol Life Sci. 2012; 69: 1813-1842Crossref PubMed Scopus (75) Google Scholar). Knowledge available on their role in the progression of skin cancer indicates that members of EphA subfamily serve as tumor suppressors in epidermal keratinocytes, and that loss of EphA2 enhances formation of chemically induced skin cancer in mouse model (Guo et al., 2006Guo H. Miao H. Gerber L. et al.Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin.Cancer Res. 2006; 66: 7050-7058Crossref PubMed Scopus (113) Google Scholar). Furthermore, downregulation of EphA1 expression has been documented in human nonmelanoma skin cancer (Hafner et al., 2006Hafner C. Becker B. Landthaler M. et al.Expression profile of Eph receptors and ephrin ligands in human skin and downregulation of EphA1 in nonmelanoma skin cancer.Mod Pathol. 2006; 19: 1369-1377Crossref PubMed Scopus (70) Google Scholar). At present, the role of other members of Eph family in skin cancer is unclear. Here, we provide evidence that EphB2 plays a role in the progression of cSCC. EphB2 is specifically overexpressed by tumor cells in cSCCs and EphB2 promotes proliferation, migration, and invasion of cSCC cells and growth of cSCC in vivo by regulating the expression of genes associated with cell migration and invasion. These results provide molecular background for the role of EphB2 in cSCC progression and identify EphB2 as a potential therapeutic target in this invasive cancer of skin. Expression of the entire EPH receptor and EFN ligand families in primary (n=5) and metastatic (n=3) cSCC cell lines and normal human epidermal keratinocytes (NHEKs) from 5 individuals was analyzed using microarray technique. Significant upregulation of EPHA4 and EPHB2 mRNA was noted in cSCC cells as compared with NHEKs (Figure 1a). Moreover, EFNB1 mRNA levels were significantly lower in cSCC cell lines than in NHEKs. Expression levels of EPH and EFN mRNAs in cSCC cell lines and NHEKs were also determined by next-generation sequencing. This analysis confirmed specific upregulation of EPHB2 mRNA and downregulation of EFNB1 mRNA in cSCC cells as compared with NHEKs (Figure 1b). The expression profiling analyses with Affymetrix and RNA sequencing identified EPHB2 as the only Eph receptor significantly overexpressed in cSCC cells with negligible expression in NHEKs (Figure 1a and b). In addition, high expression of EFNB2, which codes for ephrin-B2, a high-affinity ligand for EphB2, was detected in cSCC cells (Figure 1a and b). Therefore, EPHB2 and EFNB2 mRNA levels in cSCC cell lines and NHEKs were determined with quantitative real-time reverse-transcriptase–PCR (qRT-PCR). The results showed significant upregulation of EPHB2 mRNA in cSCC cells, whereas the expression was very low in NHEKs (Figure 1c). In contrast, abundant EFNB2 mRNA expression was detected in both cSCC cells and NHEKs (Figure 1c). Next, EPHB2 mRNA levels in cSCC tumors (n=6) and normal skin samples (n=7) in vivo were determined with qRT-PCR. The mean level of EPHB2 mRNA expression was significantly higher in cSCC tumors compared with normal skin (Figure 1d). Abundant expression of EFNB2 mRNA was detected in both cSCCs and normal skin (Figure 1d). Level of EphB2 production by NHEK and cSCC cell lines was determined by western blot analysis of the total cell lysate. Specific bands corresponding to EphB2 were detected in all cSCC cell lines, whereas EphB2 protein level was very low in NHEKs (Figure 1e). The levels of EphB2 on the surface of cSCC cells and NHEKs were determined by western blot analysis of the biotinylated cell surface proteins pulled down with avidin. Specific EphB2 bands were detected on cell surface in all cSCC cell lines examined, but not in NHEKs (Figure 1f). Cell surface EphB2 and ephrin-B2 on cSCC cells and NHEKs was also analyzed with immunofluorescence staining. Clustering of EphB2 was observed on the surface of individual cSCC cells and in cell–cell contact sites between adjacent cSCC cells (Figure 1g). Prominent staining for ephrin-B2 was noted on the surface of cSCC cells colocalizing with EphB2 clusters. In contrast, no labeling for EphB2 was detected on the surface of NHEKs and staining for ephrin-B2 on NHEKs was not clustered (Figure 1g). To study the role of EphB2 in the progression of cSCC in vivo, tissue microarrays consisting of normal skin (n=12), UV-induced premalignant lesions (AK; n=69), cSCCIS (n=56), and cSCCs (n=68) were analyzed by immunohistochemistry (IHC) using anti-EphB2 antibody. Positive EphB2 staining was observed on cell surface (Figure 2a) or in cytoplasm (Figure 2b and c) of tumor cells in invasive cSCCs and cSCCIS (Figure 2f) and the intensity of EphB2 labeling was in general stronger in cSCCs and cSCCIS than in premalignant lesions (Figure 2e) and normal skin (Figure 2d). Semiquantitative analysis of EphB2 stainings (Figure 2g) revealed that the epidermal layer in normal skin (Figure 2d) was negative in 58% and weakly positive (+) in 42% of samples. In AK lesions (Figure 2e), EphB2 staining was mainly negative (48%) or weakly positive (+) (49%). Stronger EphB2 labeling was observed in cSCCIS tissue samples, as moderate (++) staining was observed in 19% of cSCCIS cases (Figure 2f). In invasive cSCCs, strong (+++) (Figure 2c) and moderate (++) (Figure 2b) tumor cell–associated EphB2 staining was noted in 24% of the tumors. In general, EphB2 staining was significantly stronger in cSCCIS and cSCCs than in normal skin and AK compared as groups (Figure 2g). To obtain further evidence for the role of EphB2 in cSCC progression in vivo, we employed the well-characterized model of chemically induced mouse skin carcinogenesis (Abel et al., 2009Abel E.L. Angel J.M. Kiguchi K. et al.Multi-stage chemical carcinogenesis in mouse skin: fundamentals and applications.Nat Protoc. 2009; 4: 1350-1362Crossref PubMed Scopus (383) Google Scholar). Tissue samples from untreated normal mouse skin (n=5), vehicle-treated skin (n=2), hyperplastic mouse skin (n=6), and mouse cSCCs (n=19) were stained for EphB2 by IHC and semiquantitative analysis was performed based on staining intensity. Strong (+++) (Figure 3b and c) or moderate (++) (Figure 3a) EphB2 staining was noted in tumor cells in nearly all mouse cSCCs studied (95% of cases) and none of the tumors were negative. The majority of untreated (Figure 3d) and vehicle-treated (Figure 3e) mouse skin tissues were negative for EphB2 (86%) and weak (+) staining was noted in the epidermal layer of only 14% of samples. In hyperplastic mouse epidermal layer induced by 12-O-tetradecanoyl phorbol-13-acetate treatment, weak (+) staining for EphB2 (Figure 3f) was noted in 83% of cases and 17% were negative. Significantly stronger EphB2 staining was detected in cSCC tumors compared with all nonmalignant mouse skin tissues as a group (Figure 3g). To elucidate the molecular basis of the role of EphB2 in cSCC progression, microarray-based global gene expression profiling and pathway analysis were performed after small interfering RNA (siRNA) knockdown of EphB2 expression in cSCC cell lines (n=3; Figure 4a). Analysis of the genes significantly regulated after EphB2 knockdown revealed that the differentially expressed genes were significantly associated with the biofunction categories cell death, cellular movement, cell-to-cell signaling and interactions, and cellular growth and proliferation (Figure 4b). Interestingly, based on the regulation z-score, cell viability, invasion of tumor cells, migration of tumor cells, migration of cells, and cell movement were the top biofunctions significantly downregulated following EphB2 knockdown (Figure 4b). Analysis of the molecular network in the top biofunction invasion of tumor cells revealed downregulation of mRNAs for several matrix metalloproteinases (MMPs), especially MMP1 (collagenase-1) and MMP13 (collagenase-3) (Supplementary Figure S1 online). Interestingly, out of 2,460 differentially regulated probe sets, MMP1 and MMP13 were among the most downregulated genes after EphB2 knockdown (Figure 4c). In addition, comparison of the expression of the entire MMP family revealed that the expression of MMP1 and MMP13 was significantly downregulated following EphB2 knockdown (Figure 4d). MMP1 and MMP13 were also involved in other significantly downregulated biofunctions migration of tumor cells (Supplementary Figure S2 online), migration of cells, cell movement, and cell viability (data not shown). Download .pdf (.8 MB) Help with pdf files Supplementary Material To explore the functional role of EphB2 in cSCC cells (n=3), cell viability, cell migration, and invasion assays were performed following EphB2 knockdown. A significant reduction in the number of viable cSCC cells was noted 24, 48, and 72 hours after EphB2 knockdown (Figure 5a). Cell migration assay was performed 48 hours after transfection of the cSCC cells with control or EphB2 siRNA. Whereas the scratch wounds healed completely within 24 hours in the cSCC cell cultures transfected with control siRNA, knockdown of EphB2 expression clearly inhibited the motility of the cSCC cells (Figure 5b and c). Analysis of cSCC cell migration with time-lapse microscopy revealed effective and rapid migration of cSCC cells transfected with control siRNA (Supplementary Video S1 online). In contrast, a severe defect in the directional migration of cSCC cells was noted following EphB2 knockdown (Supplementary Video S2 online).eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyYzc2NTJhMTNlMjgzOGMzZGIwOTIzMjE1NzJmY2I2MyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NjE4NTM2fQ.sKC7G4-oQlwd7QPzGbLZfCllN7fAVVnADkjP1yoYVhUisHUB9Bb24TzdqYyEHISDApf9AZ1F_b-EqkXhYp4IxaRULWfLGUu2ZYh5gi5aFCyGvj5EW5YnkeppcLoriem14Rz_HuKj0FsLBt7JpJxZLUvlhvI4DNWssyXOuyH2DTjWgBb-4KvSrlSE_KkiObkuqj53vZF_OGYfZzDvIFPxIAPUV9nrQP__W0Ix52aEZxmeUKQa-JINOS8w-Wv2nYNA2TYb6ATdjZKYTHBFW4MeL5m6-QU2Q_3fl1Z0ZSlWODZfSDzTFpbvUEQIxRRUW2kSnDXnvFm341Wnyw_U7Ca6Fg Download .mp4 (3.96 MB) Help with .mp4 files Supplementary Movie 1eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI5YjVmY2VkZTkwZTg3NjAxZjU5YmE4NWUyMmFjODM0YiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NjE4NTM2fQ.f3kyv2JvrRGHB_Nkbn6Ru_s7TD5ZpBmoePFJohW37kdKRKppPRaqupl4hJh6lndaC-zLVwXpb6wWa2IAXcd3d_tjozR9I2psuD86Koo47bygcDb6NaWS0Ms8iYCIJgrcdNuOvkKpBIj0bW31BWafawy9ckUgWjVaOGhwisLhkvT2jwPz_3SL3YK_KIN5YvWIEQF5HS6_bFg7utNK7KVAAaeAlc58X0dmY-lOaNCj5dfGkx8G58iunmPDuKhQyQwI2VrYVEI_0kdjDws5eNz74nrqxFmbZSNWnTw4JpqxhEqjoCnVzaS9IN-sRCNeQ9qwaWoXMn5JJo7yJ_zpKCry_Q Download .mp4 (3.9 MB) Help with .mp4 files Supplementary Movie 1 In addition, cSCC cells displayed significantly reduced invasion through collagen following knockdown of EphB2 as compared with the control siRNA-transfected cSCC cells (Figure 5d). EphB2 knockdown potently inhibited production of MMP1 and MMP13 in cSCC cell line with high basal expression level of MMP1 and MMP13 (Figure 5e). Furthermore, activation of EphB2 signaling by soluble EphB2 ligand, ephrin-B2-Fc (Figure 5f), enhanced invasion of cSCC cells through collagen (Figure 5g) and stimulated production of MMP13 (Figure 5h) and MMP1 (Supplementary Figure S3 online). The role of EphB2 in cSCC growth in vivo was examined in xenograft model established with cSCC cells implanted subcutaneously in the back of severe combined immunodeficient (SCID) mice 72 hours after knockdown of EphB2 by transfection with siRNA. A significant delay in the growth of tumors established with EphB2 siRNA-transfected cells was noted already 4 days after implantation of the tumor cells as compared with the control siRNA group, and the difference increased throughout the observation period of 28 days (Figure 6a). Extended incubation of cSCC cells transfected with siRNA showed persistence of EphB2 knockdown up to 8 days (data not shown). Histological analysis of the xenografts harvested at day 28 revealed that the tumors established with EphB2 siRNA-transfected cSCC cells contained more inert material and were less cellular as compared with the xenografts in the control siRNA group (Figure 6b, upper panels). Accordingly, the relative number of proliferating (Ki-67-positive) tumor cells was significantly lower in the xenografts established with cSCC cells transfected with EphB2 siRNA (17%) compared with control siRNA tumors (39%) (Figure 6b, middle panels, and Figure 6c). In addition, IHC analysis of the xenografts for vascular marker CD34 revealed significantly reduced density of CD34-positive blood vessels in EphB2 knockdown tumors compared with control siRNA xenografts (Figure 6b, lower panels, and Figure 6d). The role of Eph/ephrin signaling in normal biological functions such as axon guidance, neural development, and cell migration and adhesion is well established, but their role in cancer progression is complex (Merlos-Suarez and Batlle, 2008Merlos-Suarez A. Batlle E. Eph-ephrin signalling in adult tissues and cancer.Curr Opin Cell Biol. 2008; 20: 194-200Crossref PubMed Scopus (111) Google Scholar; Coulthard et al., 2012Coulthard M.G. Morgan M. Woodruff T.M. et al.Eph/Ephrin signaling in injury and inflammation.Am J Pathol. 2012; 181: 1493-1503Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Distinct members of Eph/ephrin families have emerged both as tumor suppressors and as promoters of cancer progression (Merlos-Suarez and Batlle, 2008Merlos-Suarez A. Batlle E. Eph-ephrin signalling in adult tissues and cancer.Curr Opin Cell Biol. 2008; 20: 194-200Crossref PubMed Scopus (111) Google Scholar; Pasquale, 2010Pasquale E.B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond.Nat Rev Cancer. 2010; 10: 165-180Crossref PubMed Scopus (905) Google Scholar; Nievergall et al., 2012Nievergall E. Lackmann M. Janes P.W. Eph-dependent cell-cell adhesion and segregation in development and cancer.Cell Mol Life Sci. 2012; 69: 1813-1842Crossref PubMed Scopus (75) Google Scholar). Upregulation of EphA2 expression has been documented in prostate cancer (Walker-Daniels et al., 1999Walker-Daniels J. Coffman K. Azimi M. et al.Overexpression of the EphA2 tyrosine kinase in prostate cancer.Prostate. 1999; 41: 275-280Crossref PubMed Scopus (227) Google Scholar), breast cancer (Zelinski et al., 2001Zelinski D.P. Zantek N.D. Stewart J.C. et al.EphA2 overexpression causes tumorigenesis of mammary epithelial cells.Cancer Res. 2001; 61: 2301-2306PubMed Google Scholar), melanoma (Udayakumar et al., 2011Udayakumar D. Zhang G. Ji Z. et al.EphA2 is a critical oncogene in melanoma.Oncogene. 2011; 30: 4921-4929Crossref PubMed Scopus (59) Google Scholar), and glioblastoma (Wykosky and Debinski, 2008Wykosky J. Debinski W. The EphA2 receptor and ephrinA1 ligand in solid tumors: function and therapeutic targeting.Mol Cancer Res. 2008; 6: 1795-1806Crossref PubMed Scopus (228) Google Scholar), providing evidence for its role in tumor progression. Furthermore, EphB2 displays both tumor-suppressing and tumor-promoting effects. EphB receptor activation suppresses growth of colorectal cancer (Batlle et al., 2005Batlle E. Bacani J. Begthel H. et al.EphB receptor activity suppresses colorectal cancer progression.Nature. 2005; 435: 1126-1130Crossref PubMed Scopus (340) Google Scholar). Loss-of-function mutations or deletion of EPHB2 have been reported in prostate cancer (Huusko et al., 2004Huusko P. Ponciano-Jackson D. Wolf M. et al.Nonsense-mediated decay microarray analysis identifies mutations of EPHB2 in human prostate cancer.Nat Genet. 2004; 36: 979-983Crossref PubMed Scopus (161) Google Scholar), colorectal cancer (Alazzouzi et al., 2005Alazzouzi H. Davalos V. Kokko A. et al.Mechanisms of inactivation of the receptor tyrosine kinase EPHB2 in colorectal tumors.Cancer Res. 2005; 65: 10170-10173Crossref PubMed Scopus (78) Google Scholar), and gastric cancer (Davalos et al., 2007Davalos V. Dopeso H. Velho S. et al.High EPHB2 mutation rate in gastric but not endometrial tumors with microsatellite instability.Oncogene. 2007; 26: 308-311Crossref PubMed Scopus (36) Google Scholar). Reduction in the expression of EphB2 is associated with invasion, metastasis, and poor prognosis in colorectal tumors (Lugli et al., 2005Lugli A. Spichtin H. Maurer R. et al.EphB2 expression across 138 human tumor types in a tissue microarray: high levels of expression in gastrointestinal cancers.Clin Cancer Res. 2005; 11: 6450-6458Crossref PubMed Scopus (83) Google Scholar; Guo et al., 2006Guo D.L. Zhang J. Yuen S.T. et al.Reduced expression of EphB2 that parallels invasion and metastasis in colorectal tumours.Carcinogenesis. 2006; 27: 454-464Crossref PubMed Scopus (97) Google Scholar), and high level of EphB2 expression in colorectal cancer is associated with longer survival (Jubb et al., 2005Jubb A.M. Zhong F. Bheddah S. et al.EphB2 is a prognostic factor in colorectal cancer.Clin Cancer Res. 2005; 11: 5181-5187Crossref PubMed Scopus (90) Google Scholar). In contrast, activation of EphB2 has been shown to promote invasion of glioblastoma in vivo (Wang et al., 2012Wang S.D. Rath P. Lal B. et al.EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase.Oncogene. 2012; 31: 5132-5143Crossref PubMed Scopus (69) Google Scholar). In transitional cell carcinoma of the bladder, loss of EphB2 and gain of EphB4 expression have been reported to be associated with the tumor progression, and EphB4 inhibitors have been shown to have antitumor properties in xenograft model of this tumor (Li et al., 2014Li X. Choi W.W. Yan R. et al.The differential expression of EphB2 and EphB4 receptor kinases in normal bladder and in transitional cell carcinoma of the bladder.PLoS One. 2014; 9: e105326Crossref PubMed Scopus (21) Google Scholar). Previous studies on the role of Eph/ephrin signaling in the progression of skin cancer have identified members of EphA subfamily, especially EphA2, as tumor suppressor in epidermal keratinocytes (Guo et al., 2006Guo H. Miao H. Gerber L. et al.Disruption of EphA2 receptor tyrosine kinase leads to increased susceptibility to carcinogenesis in mouse skin.Cancer Res. 2006; 66: 7050-7058Crossref PubMed Scopus (113) Google Scholar). Here, we have examined the role of EphB2 in the progression of cSCC, the most common metastatic skin cancer (Alam and Ratner, 2001Alam M. Ratner D. Cutaneous squamous-cell carcinoma.N Engl J Med. 2001; 344: 975-983Crossref PubMed Scopus (977) Google Scholar; Czarnecki et al., 2002Czarnecki D. Sutton T. Czarnecki C. et" @default.
• W2090395004 created "2016-06-24" @default.
• W2090395004 creator A5004214960 @default.
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• W2090395004 date "2015-07-01" @default.
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• W2090395004 title "EphB2 Promotes Progression of Cutaneous Squamous Cell Carcinoma" @default.
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• W2090395004 doi "https://doi.org/10.1038/jid.2015.104" @default.
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