ErbB-2 Receptor Cooperates with E6/E7 Oncoproteins of HPV Type 16 in Breast Tumorigenesis

ErbB-2 Receptor Cooperates with E6/E7 Oncoproteins of HPV Type 16 in Breast Tumorigenesis
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  [Cell Cycle 6:23, 2939-2943, 1 December 2007]; ©2007 Landes Bioscience Brief Report ErbB-2 Receptor Cooperates with E6/E7 Oncoproteins of HPV Type 16 in Breast Tumsrcenesis  Amber Yasmeen 1,2  Tarek A. Bismar 1-3 Hafedh Dekhil 1  Riccardo Ricciardi 1   Amal Kassab 1  Carlo Gambacorti-Passerini 1,4  Ala-Eddin Al Moustafa 1,2, * 1 Montreal Center for Experimental Therapeutics in Cancer; Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital; 2 Program in Cancer Genetics; Department of Oncology; 3 Department of Pathology 4 Department of Haematology; Jewish General Hospital; McGill University; Montreal, Quebec, Canada*Correspondence to: Ala-Eddin Al Moustafa; Montreal Center for Experimental Therapeutics in Cancer of the Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis-Jewish General Hospital; 3755 Ch. de la Cote Ste-Catherine; Montreal, Quebec H3T 1E2 Canada; Tel.: 514.340.8222 x4510; Fax: 514.340.8716; Email: or aalmoust@ldi.jgh.mcgill.caOriginal manuscript submitted: 06/19/07Revised manuscript submitted: 08/28/07Manuscript accepted: 08/30/07Previously published online as a Cell Cycle   E-publication: KEY WORDS Breast tumsrcenesis, transgenic mice, ErbB-2 and E6/E7 of high-risk HPV  ACKNOWLEDGEMENTS  We are grateful to Drs G. Batist, M. Alaoui- Jamali and Foulkes for their support of this  work. We are thankful to Dr. A. Darnel for his critical reading of the manuscript. We also thank Dr. T. Li-Qun and G. Mousavi for their technical assistance. This work is supported by the Canadian Institutes for Health Research (Ala-Eddin Al Moustafa) and the National Colorectal Cancer Campaign (Ala-Eddin Al Moustafa and Tarek A. Bismar).  ABSTRACT The ErbB-2 receptor is over-expressed in roughly 30% of human breast cancers. Moreover, approximately 50% of breast cancers are positive for high-risk human papillomaviruses (HPVs). Recently, we reported that ErbB-2 cooperates with E6/E7 oncoproteins of HPV type 16 to induce neoplastic transformation of human normal oral epithelial cells. We also demonstrated that E6/E7 of HPV type 16 converts non-invasive breast cancer cells to an invasive form. In order to investigate the effect of ErbB-2/E6/E7 cooperation in breast carcinogenesis, we generated double transgenic mice carrying ErbB-2 and E6/E7 of HPV type 16 under mouse mammary tumor virus (MMTV) and human keratin 14 promoters, respectively. Within six months, these double transgenic mice developed large and extensive invasive breast cancer in comparison to ErbB-2 or E6/E7 singly transgenic mice. Histological analysis of ErbB-2/ E6/E7 transgenic mice tumors showed the presence of invasive breast carcinomas. However, the breast tissues from ErbB-2 and E6/E7 transgenic mice showed only in-situ cancer and normal mammary phenotype, respectively. In parallel, we examined the cooperation effect of ErbB-2 and E6/E7 in the human breast cancer cell line, BT20; in comparison to ErbB-2 and E6/E7 alone as well as wild type cells, we found that ErbB 2/E6/E7 together stimulate colony formation and cell migration in the BT20 cell line. Furthermore, we found that b -catenin is constitutively phosphorylated by c-Src and conse-quently trans-located to the nucleus in ErbB-2/E6/E7-breast cancer cells. These findings provide evidence that the ErbB-2 receptor cooperates with high-risk HPVs in breast tumori-genesis via b -catenin activation. INTRODUCTION Breast cancer is the most common malignancy in women and causes over 400,000 deaths per year. 1  About 30% of all breast cancers carry amplifications of the ErbB-2 locus, which encodes the receptor tyrosine kinase ErbB-2. 2  These tumors have high rates of relapse and poor prognosis. 3  In agreement with the hypothesis that ErbB-2 signaling initiates mammary tumsrcenesis, the levels of ErbB-2 increase at the onset of ductal carci-noma in situ. 4  Additionally, transgenic mice expressing constitutively activated ErbB-2 ( Neu ) in their mammary glands develop ductal carcinomas within one year, 5  directly implicating ErbB-2 signaling in mammary oncogenesis. However, the mechanisms by  which ErbB-2 amplifications and its interaction with other oncogenes promote breast carcinoma progression in vivo remain poorly understood.Several studies demonstrated that high-risk HPVs are present in human breast cancer and normal mammary epithelial cells; 6-9  however, other studies reported that HPVs could not be detected in breast cancer and normal tissues. 10-12  Nevertheless, the high-risk HPVs E6 and E7 oncoproteins, which are constitutively expressed in these cancers, inactivate p53 and pRb tumor suppressors, respectively. 13  We recently reported that E6/E7 of HPV type 16 induce cell invasive and metastatic abilities of two non-invasive breast cancer cell lines, MCF7 and BT20, both in vitro and in vivo, respectively, in comparison with  wild type cells. Furthermore, we found that HPV type 16 is present in all invasive and metastatic breast cancers and less frequently in in-situ breast cancer in contrast to normal mammary tissue. 14  Recently, Woods Ignatoski et al. reported that E6/E7 of HPV type 16 and ErB-2 cooperate with endogenous epidermal growth factor-receptor (EGF-R/ErbB1) to fully transform human breast immortalized cell line, MCF-10A. 15  Our group has devel-oped a new in vitro model to study the effects of E6/E7 of HPV type 16 and ErbB-2 in human normal oral epithelial (NOE) cells. Using this model, we demonstrated that E6/E7 Cell Cycle 2939  E6/E7 of HPV/ErbB-2 Interaction in Breast Carcinogenesis cooperates with ErbB-2 receptor to induce cellular transformation of NOE via the conversion of b -catenin from a cell-adhesion molecule to a potential transcriptional regulator. 16 The objective of this project was to examine the interactive effect of ErbB-2 and high-risk HPVs infection in breast carcinogenesis.  We demonstrate that the ErbB-2 receptor cooperates with E6/E7 of HPV type 16 to stimulate breast tumsrcenesis, colony formation and cell migration both in vivo and in vitro, using double transgenic mice and human breast cancer cells, respectively. In addition, we report that the cooperation effect of ErbB-2 with E6/E7 occurs via b -catenin tyrosine phosphorylation by pp60 (c-Src) kinase activation in breast carcinogenesis. MATERIALS AND METHODS Generation of ErbB-2/E6/E7 double transgenic mice.  The ErbB-2/E6/E7 double-transgenic mice were generated by cross-breeding MMTV/Neu (ErbB-2) 5  with human keratin 14 (k14)-E6/E7 mice (which were generated in our laboratory from K14-E6 and K14-E7 mice). 17,18  We analyzed the first generation by PCR for the presence of the transgenes. The mice that were positive for both transgenes were included in further studies. The incidence of tumors  was evaluated once weekly. Histological and immunohistochemical analyses.  Hematoxylin and eosin (H&E) histology, and immunohistochemical analyses for ErbB-2 and b -catenin of tumors derived from the transgenic mice cells were performed as previously described in ref. 15 except that anti- b -catenin mAb (clone 14, Bio/Can Scientific) was used for the immunohistochemistry analysis. Reverse transcription (RT)-PCR analysis.  RT-PCR amplification  was performed using the following primer sets: E6, 5'-ATGCACC AAAAGAGAACTGCA-3' and 5'-TTACAGCTGGGTTTCTCTA CG-3'; E7, 5'-ATGCATGGAGATACACCTACAT-TGCAT-3' and 5'-GTTTCTGAGAACAG-ATGGGGCACAC-3'. Primers specific for GAPDH gene, 5'-GAAGGC-CATGCCAGTGAGCT-3' and 5'-CCGGGAAACTGTGGC-GTGAT-3', were used to control the amounts of cDNA generated from each sample. Synthesis of the first-strand cDNA was carried out using a cDNA kit for RT-PCR (MBI Fermentas). Synthesis of the first-strand cDNA was carried out using a cDNA kit for RT-PCR (MBI Fermentas). One fifth of the RT product was amplified for 30 cycles (1 minute at 95°C, 1 minute at 58°C, and 1 minute at 72°C) followed by an extension of 10 minutes at 72°C. RT-PCR amplification products were analyzed on a 1% agarose gel stained with ethidium bromide. Cell transduction assay.  The human breast cancer cell line, BT20,  was used in this study. This cell line was obtained from American Type Culture Collection (ATCC). Cells were grown in DMEM medium supplemented with 10% fetal bovine serum. The cells  were transduced with E6/E7 and/or ErbB-2 retro-vectors as previ-ously described by Al Moustafa et al., ref. 19. Briefly, BT20 cells transduced with PLXSN vector were also treated with G418. Over 95% of the E6/E7-infected cells were healthy following G418 treat-ment. BT20-E6/E7 cells were trypsinized and passaged twice while maintained on G418. Wild type cells died after approximately four days of treatment with G418, whereas PLXSN-transduced cells with empty vector were used as a control. The BT20-E6/E7 cells were subsequently maintained in long-term culture with DMEM medium supplemented with 10% fetal bovine serum. In parallel, BT20 and BT20-E6/E7 cells were transduced with AP2-ErbB-2-GFP retroviral particles. 20  Stably transduced cells were expanded and flow cytometric analysis was performed with an Epics XL/MCL Coulter analyzer to verify gene transfer efficiency as measured by GFP fluorescence. Cell treatment with SKI-606.  In all of our experiments, BT20 cell lines were treated with 15 m M of 4-anilino-3-quinolinecarbonitrile (SKI-606), an inhibitor of Src kinase activity, 0.001 m l of DMSO (as control) or without any treatment as described by Coluccia et al ref. 21. Soft agar growth assay.  2 x 10 3  cells from the breast cancer cell line BT20, as well as those expressing ErbB-2, E6/E7 and ErbB-2/E6/E7 together were placed in DMEM medium containing 0.4% agar and plated over a layer of DMEM medium containing 0.7% agar. The cultures were examined every one to two days for three weeks. Cell wounding assay.  The BT20 breast cancer cells were grown to confluence and serum-starved for 24 hours, and finally wounded  with a 2 ml pipette. Cells were examined by light microscopy 24 and 48 hours later for the ability to repopulate the wound as well as for cell migration.  Western blot and immunoprecipitation analyses.  These assays  were performed as previously described in refs. 16 and 22; however, anti- b -catenin (clone 14, Bio/Can Scientific) and anti-actin (clone C4, Roche Diagnostics) mAbs were used for the assays. For immu-noprecipitation, 200 m g of proteins were immunoprecipitated with anti- b -catenin mAb (clone 14, Bio/Can Scientific) or anti-src mAb (Ab-3, Oncogene Science). Immunoprecipitated samples were then blotted on a nitrocellulose membrane and detected with anti-phos-photyrosine (clone 4G10, Upstate Biotechnology), anti- b -catenin or anti-src (clone GD11, Upstate Biotechnology) mAbs. Immunofluorescence analysis.  BT20 cell lines were seeded and treated with/without SKI-606 on cover-slips for two days at a density of 50,000 cells/35 mm dish. The cells were rinsed with PBS and fixed  with 3% (w/v) formaldehyde in PBS for 5 minutes, followed by an incubation in pre-cooled methanol (-20°C) for 15 minutes. The cells were then incubated with the primary antibody anti- b -catenin mAb (Bio/Can Scientific) for 1 hr at room temperature. After three washes with 0.2% BSA in PBS, the cells were incubated with appropriate secondary antibody conjugated to Texas Red (Jackson Immunoresearch Laboratories) for 30 minutes. The cells were subse-quently treated with Hoechst nuclear stain for one minute. Finally, the coverslips were washed with PBS and mounted with Airvol (Air Products and Chemicals, Inc.), and viewed with a Zeiss Axiophot fluorescent microscope equipped with 63X plan apochromat objec-tives and photographed with a digital camera. RESULTS AND DISCUSSION In order to study the cooperation effect of ErbB-2 receptor with high-risk HPVs infection in breast tumsrcenesis, we generated double transgenic mice carrying activated Neu  (rat ErbB-2) and E6/E7 oncoproteins of high-risk HPV type 16 under MMTV and human keratin 14 (K14) promoters, respectively. First, we created E6/E7 transgenic mice by crossbreeding K14-E6 mice with K14-E7 mice; and then we crossed these mice with MMTV/activated Neu  (rat ErbB-2). 5  The MMTV/ErbB-2 female mice develop in-situ mammary cancers usually within one year, which could subsequently evolve into an invasive form. On the contrary, female K14-E6/E7 mice do not form any breast tumors within the same time period. 2940 Cell Cycle 2007; Vol. 6 Issue 23  E6/E7 of HPV/ErbB-2 Interaction in Breast Carcinogenesis Furthermore, the E6 or E7 of HPV type 16 individually and E6/E7 transgenic mice develop skin tumors, and cervical cancer following chronic estrogen administration. 23  However, we found that the ErbB-2/E6/E7 double transgenic mice develop large and extensive breast tumors within a six month period without any treatment (Fig. 1A). The histological analysis of ErbB-2/E6/E7 transgenic mouse tumors revealed that they are invasive high grade breast cancers, while the breast tissue from ErbB-2 or E6/E7 transgenic mice shows in-situ breast cancer and normal mammary phenotype, respectively (Fig. 1B). Furthermore, using immunohistochem-istry and RT-PCR analyses we confirmed that double transgenic breast tumors overexpress/express ErbB-2 and E6/E7, respectively, (Fig. 2A and B).To assess the outcome of ErbB-2/E6/E7 cooperation in human breast carcinogenesis, we examined the effect of ErbB-2 and E6/E7 of HPV type 16 on the BT20 breast cancer cell line. We transduced BT20 cells with E6/E7 using a recombinant retroviral system as described previously by Al Moustafa et al. in ref. 16. Next, we transduced the cell line and its wild type cells with a bicistronic retrovirus that co-expresses both ErbB-2 and enhanced green fluorescent protein (GFP-ErbB-2 and its control GFP-AP2). We confirmed that these cells overexpress/express ErbB-2 and E6/E7, in that order, when compared with their wild type counter-parts by Western blot and RT-PCR analyses (data not shown). Furthermore, we examined the colony formation and cell migration abilities of BT20 cells and those expressing ErbB-2, E6/E7 and ErbB-2/E6/E7 using soft agar and wound healing assays, respec-tively. We found that ErbB-2/E6/E7 cooperate in BT20 cell line to induce large colony formation and cell migration in comparison to ErbB-2, E6/E7 and  wild type cells (Fig. 3A and B). We recently reported that E6/E7 of HPV type 16 induce cell invasive ability of the BT20 cell line, both in vitro and in vivo, when compared with wild type cells. 14  In this study, we clearly demonstrate, for the first time, that ErbB-2 overexpression cooperates with E6/E7 oncoproteins of high-risk HPV type 16 in breast carcinogenesis both in vivo and in vitro. Earlier studies reported that cyclin D1 and c-myc are targets of nuclear b -catenin, via interaction with the T-cell factor/lymphoid enhancer factor (Tcf/Lef) family of transcription factors. 24,25  We recently reported that D-type cyclins are downstream targets of the nuclear b -catenin provoked by ErbB-2/E6/E7 coop-eration in NOE-transformed cells. 16,26  Moreover,  we observed that crossbreeding of ErbB-2/E6/E7 double transgenic mice with cyclin D1 knockout mice dramatically reduce breast tumor progres-sion (Al Moustafa et al, in preparation). Therefore,  we reasoned that the mechanism of ErbB2/E6/E7 cooperation in breast tumsrcenesis may be deduced from the analysis of b -catenin expression patterns; for that reason, we examined the ErbB-2/E6/E7 double transgenic tumors for b -catenin expression patterns using immunohistochemistry. We found that b -catenin is trans-located from the undercoat membrane to the nucleus in ErbB-2/E6/E7 mouse tumors and BT20-ErbB-2/E6/E7 cells in comparison with their respective controls (Fig. 4A and B). Regarding the mechanism of b -catenin trans-location, we hypothesized that ErbB-2/E6/E7 cooperation provokes a dissociation of E-cadherin/catenin complex by tyrosine phosphorylation of b -catenin through pp60(c-Src) kinase phos-phorylation. Subsequently, the free b -catenin enters to the nucleus and modulates cell transcription via its association with the Tcf/Lef transcription factors. Using SKI-606, a novel Src family kinase inhibitor, we recently demonstrated that b -catenin is physically associated to activated pp60(c-Src) kinase and constitutively phos-phorylated on the tyrosine residue in human colorectal cancer cells. 21  To assess this possibility in ErbB-2/E6/E7 cooperation, we examined the effect of SKI-606 on b -catenin regulation patterns in BT20 breast cancer cells and those expressing ErbB-2, E6/E7 and ErbB2/E6/E7. These cell lines were treated for 48 hours with vehicle alone (DMSO) or 15 m M/L of SKI-606 and then immunoprecipitated  with anti- b -catenin. Western blot analysis with anti-phosphotyrosine Figure 1. ErbB-2 receptor cooperates with E6/E7 in vivo. (A) Generation of double transgenic mice carrying E6/E7 of HPV type 16 and ErbB-2 receptor. We successfully gener-ated ErbB-2/E6/E7 double transgenic mice by crossbreeding ErbB-2 with E6/E7 transgenic mice. We note that the double transgenic mice develop large and extensive breast tumors with-in six months, when compared with E6/E7 (alone) and ErbB-2 (alone) mice. (B) Histological sections of breast tissues derived from ErbB-2/E6/E7, ErbB-2 and E6/E7 transgenic mice. Routine H&E stains show that ErbB-2/E6/E7 breast tumors are high-grade invasive carcino-mas in comparison with ErbB-2 tumors which are in-situ breast cancers; in contrast, the E6/E7 breast tissues are normal. Cell Cycle 2941  E6/E7 of HPV/ErbB-2 Interaction in Breast Carcinogenesis revealed that b -catenin is strongly phosphorylated in ErbB-2/E6/E7 cells in comparison with ErbB-2, E6/E7 and wild type cells (data not shown). In parallel, we found that SKI-606 inhibits b -catenin phosphorylation and consequently its trans-location to the nucleus (re-localize it to undercoat membrane) in these cells (Fig. 4C); thus, SKI-606 blocks cell migration through the conversion of b -catenin’s role from transcriptional regulator to a cell-cell adhesion function in ErbB-2/E6/E7-breast cancer cells (Fig. 4D). This data suggests that b -catenin phosphorylation and trans-location induced by ErbB-2/E6/E7 cooperation occurs via (c-Src) activation.In conclusion, using our in vitro and in vivo models we demon-strate that the ErbB-2 tyrosine kinase receptor cooperates with E6/E7 of high-risk HPVs in breast tumsrcenesis and metastasis. This discovery provides a new basis for understanding the mechanisms of ErbB-2/high-risk HPVs interaction in human breast carcinogenesis. Furthermore, our data will open a new therapeutic direction by co-targeting the E6/E7 of HPVs/ErbB-2 pathways in breast cancer. References  1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005; 55:74-108. 2. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987; 235:177-82. 3. Berger MS, Locher GW, Saurer S, Gullick WJ, Waterfield MD, Groner B, Hynes NE. Correlation of c-erbB-2   gene amplification and protein expression in human breast carci-noma with nodal status and nuclear grading. Cancer Res 1988; 48:1238-43. 4. van de Vijver MJ, Peterse JL, Mooi WJ, Wisman P, Lomans J, Dalesio O, Nusse R. Neu-protein overexpression in breast cancer: Association with comedo-type ductal carci-noma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 1988; 319:1239-45. 5. Guy CT, Webster MA, Schaller M, Parsons TJ, Cardiff RD, Muller WJ. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci USA 1992; 89:10578-82. 6. de Villiers EM, Sandstrom RE, zur Hausen H, Buck CE. Presence of papillomavirus sequences in condylomatous lesions of the mamillae and in invasive carcinoma of the breast. Breast Cancer Res 2005; 7:R1-11. 7. Kan CY, Iacopetta BJ, Lawson JS, Whitaker NJ. Identification of human papillomavirus DNA gene sequences in human breast cancer. Br J Cancer 2005; 93:946-8. 8. Liu Y, Klimberg VS, Andrews NR, Hicks CR, Peng H, Chiriva-Internati M, Henry-Tillman R, Hermonat PL. Human papillomavirus DNA is present in a subset of unselected breast cancers. J Hum Virol 2001; 4:329-34. 9. Yu Y, Morimoto T, Sasa M, Okazaki K, Harada Y, Fujiwara T, Irie Y, Takahashi E, Tanigami  A, Izumi K. Human papillomavirus type 33 DNA in breast cancer in Chinese. Breast Cancer 2000; 7:33-6. 10. Wrede D, Luqmani YA, Coombes RC, Vousden KH. Absence of HPV 16 and 18 DNA in breast cancer. Br J Cancer 1992; 65:891-4. 11. Gopalkrishna V, Singh UR, Sodhani P, Sharma JK, Hedau ST, Mandal AK, Das BC.  Absence of human papillomavirus DNA in breast cancer as revealed by polymerase chain reaction. Breast Cancer Res Treat 1996; 39:197-202. 12. Lindel K, Forster A, Altermatt HJ, Greiner R, Gruber G. Breast cancer and human papillo-mavirus (HPV) infection: No evidence of a viral etiology in a group of Swiss women. Breast 2007; 16:172-7. 13. Vousden KH. Regulation of the cell cycle by viral oncoproteins. Semin Cancer Biol 1995; 6:109-16. 14. Yasmeen A, Bismar T, Kandouz M, Desprez PY, Al Moustafa AE. E6/E7 of HPV type 16 promotes cell invasion and metastasis of human breast cancer cells. Cell Cycle 2007; 6:2038-42. 15. Woods Ignatoski KM, Dziubinski ML, Ammerman C, Ethier SP. Cooperative interactions of HER-2 and HPV-16 oncoproteins in the malignant transformation of human mammary epithelial cells. Neoplasia 2005; 7:788-98. 16. Al Moustafa AE, Foulkes WD, Benlimame N, Wong A, Yen L, Bergeron J, Batist G, Alpert L, Alaoui-Jamali MA. E6/E7 proteins of HPV type 16 and ErbB-2 cooperate to induce neo-plastic transformation of primary normal oral epithelial cells. Oncogene 2004; 23:350-8. Figure 2. Immunohistochemical and RT-PCR analyses of ErbB-2 and E6/E7 expression, respectively, in mammary tissues of ErbB-2/E6/E7, ErbB-2 and E6/E7 transgenic mice. (A) ErbB-2/E6/E7 and ErbB-2 breast tumors express high levels of ErbB-2 receptor; whereas, the normal breast tissues of E6/E7 mice do not express this receptor. (B) RT-PCR analysis confirms that E6/E7 oncoproteins are expressed in E6/E7 and ErbB-2/E6/E7 but not in ErbB-2 transgenic and wild type (WT; from normal mouse) mammary tissues.Figure 3. ErbB-2 cooperates with E6/E7 of HPV type 16 in BT20 human breast cancer cell line. (A) ErbB-2 cooperates with E6/E7 to induce large colonies in soft agar in comparison with ErbB-2, E6/E7 and wild type cells. Moreover, (B) ErbB-2/E6/E7 cooperation provokes cell motility of BT20 cells within 48 hours, in comparison with BT20-ErbB-2 and BT20-E6/E7 cells, as well as their wild type counterparts, (p < 0.001) using cell wounding assay. 2942 Cell Cycle 2007; Vol. 6 Issue 23  E6/E7 of HPV/ErbB-2 Interaction in Breast Carcinogenesis 17. Herber R, Liem A, Pitot H, Lambert PF. Squamous epithelial hyperplasia and carcinoma in mice transgenic for the human papillomavirus type 16 E7   oncogene. J Virol 1996; 70:1873-81. 18. Song S, Pitot HC, Lambert PF. The human papillomavirus type 16 E6   gene alone is suf-ficient to induce carcinomas in transgenic animals. J Virol 1999; 73:5887-93. 19. Al Moustafa AE, Foulkes WD, Wong A, Jallal H, Batist G, Yu Q, Herlyn M, Sicinski P,  Alaoui-Jamali MA. Cyclin D1 is essential for neoplastic transformation induced by both E6/E7 and E6/E7/ErbB-2 cooperation in normal cells. Oncogene 2004; 23:5252-6. 20. Yen L, Benlimame N, Nie ZR, Xiao D, Wang T, Al Moustafa AE, Esumi H, Milanini J, Hynes NE, Pages G, Alaoui-Jamali MA. Differential regulation of tumor angiogenesis by distinct ErbB homo- and heterodimers. Mol Biol Cell 2002; 13:4029-44. 21. Coluccia AM, Benati D, Dekhil H, De Filippo A, Lan C, Gambacorti-Passerini C. SKI-606 decreases growth and motility of colorectal cancer cells by preventing pp60(c-Src)-depen-dent tyrosine phosphorylation of beta-catenin and its nuclear signaling. Cancer Res 2006; 66:2279-86. 22. Al Moustafa AE, Yansouni C, Alaoui-Jamali MA, O’Connor-McCourt M. Up-regulation of E-cadherin by an anti-epidermal growth factor receptor monoclonal antibody in lung cancer cell lines. Clin Cancer Res 1999; 5:681-6. 23. Riley RR, Duensing S, Brake T, Munger K, Lambert PF, Arbeit JM. Dissection of human papillomavirus E6 and E7 function in transgenic mouse models of cervical carcinogenesis. Cancer Res 2003; 63:4862-71. 24. Lin SY, Xia W, Wang JC, Kwong KY, Spohn B, Wen Y, Pestell RG, Hung MC. Beta-catenin, a novel prognostic marker for breast cancer: Its roles in cyclin D1 expression and cancer progression. Proc Natl Acad Sci USA 2000; 97:4262-6. 25. Shtutman M, Zhurinsky J, Simcha I, Albanese C, D’Amico M, Pestell R, Ben-Ze’ev A. The cyclin D1  gene is a target of the beta-catenin/LEF-1 pathway. Proc Natl Acad Sci USA 1999; 96:5522-7. 26. Yasmeen A, Hossein A, Yu Q, Al Moustafa AE. Critical role for D-type cyclins in cellular transformation induced by E6/E7 of HPV type 16 and E6/E7/ErbB-2 cooperation. Cancer Science 2007; 98:973-7. Figure 4. Effect of ErbB-2/E6/E7 cooperation on b -catenin regulation patterns in breast cancer cells. (A) Immunohistochemical analysis of mammary tumor of ErbB-2 transgenic mice shows that b -catenin is located in the undercoat membrane; in contrast, it is trans-located into the nucleus in ErbB-2/E6/E7 transgenic mouse tumors. (B) Western bolt analysis of the nuclear b -catenin in BT20 wild type cells and those expressing E6/E7, ErbB-2 and ErbB-2/E6/E7. We note that ErbB-2/E6/E7 cooperation provokes the translocation of b -catenin to the nucleus in this human breast cancer cell line. (C) Double -staining with anti- b -catenin mAb and Hoechst 33258 in BT20-ErbB-2/E6/E7 cells with/without treatment of 15 m M SKI-606 for 48 hours. We observe that SKI-606 restores b -catenin in these cells in comparison with the control cells (treated with DMSO). Consequently, (D) SKI-606 inhibits cell migration induced by ErbB-2/E6/E7 cooperation in BT20 human breast cancer cells (p < 0.001). Cell Cycle 2943
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