The transcription factors Snail, Slug and Twist repress E-cadherin and induce epithelial-mesenchymal transition (EMT), a process exploited by invasive cancer cells. In this study, we evaluated the role of EMT in the tumorgenesis of neuroendocrine tumors of the pancreas (PNETs) in vitro, in vivo and human tumor specimen. Expression of EMT markers was analyzed using immunohistochemistry and real-time PCR. For in vitro studies, BON-1 cells were analyzed regarding expression of EMT markers before and after transfection with siRNA against Slug or Snail, and cell aggregation assays were performed. To asses in vivo effects, Rip1Tag2 mice were treated with vehicle or the snail-inhibitor polythlylenglykol from week 5-10 of age. The resected pancreata were evaluated by weight, tumor cell proliferation and apoptosis. Snail and Twist was expressed in 61 % and 64% of PNETs. This was associated with loss of E-cadherin. RT-PCR revealed conservation of the EMT markers Slug and Snail in BON-1 cells. Transfection with siRNA against Slug was associated with upregulation of E-cadherin, enhanced cell-cell adhesion and inhibition of cell proliferation. Snail-inhibition in vivo by PEG was associated with increased apoptosis, decreased tumor cell proliferation and dramatic reduced tumor volume in Rip1Tag2 mice. The presented data show that EMT plays a key role in tumorgenesis of PNETs. The activation of Snail in a considerable subset of human PNETs and the successful effect of Snail inhibition by PEG in islet cell tumors of transgenic mice provides first evidence of Snail as a drug target in PNETs.
References
[1]
？berg, K.; Eriksson, B. Endocrine tumors of the pancreas. Best Pract. Res. Clin. Gastroenterol. 2005, 19, 753–781, doi:10.1016/j.bpg.2005.06.002.
Zhou, B.P.; Hung, M.C. Wnt, hedgehog and snail: Sister pathways that control by GSK-3beta and beta-Trcp in the regulation of metastasis. Cell Cycle 2005, 4, 772–776, doi:10.4161/cc.4.6.1744.
Fendrich, V.; Waldmann, J.; Esni, F.; Ramaswamy, A.; Mullendore, M.; Buchholz, M.; Maitra, A.; Feldmann, G. Snail and Sonic Hedgehog activation in neuroendocrine tumors of the ileum. Endocr. Relat. Cancer 2007, 14, 865–874, doi:10.1677/ERC-07-0108.
[8]
Fendrich, V.; Waldmann, J.; Feldmann, G.; Schlosser, K.; K？nig, A.; Ramaswamy, A.; Bartsch, D.K.; Karakas, E. Unique expression pattern of the EMT markers Snail, Twist and E-cadherin in benign and malignant parathyroid neoplasia. Eur. J. Endocrinol. 2009, 160, 695–703. 19176646
[9]
Waldmann, J.; Feldmann, G.; Slater, E.P.; Langer, P.; Buchholz, M.; Ramaswamy, A.; Saeger, W.; Rothmund, M.; Fendrich, V. Expression of the zinc finger transcription factor Snail in Adrenocortical Carcinoma is associated with decreased survival. Br. J. Cancer 2008, 99, 1900–1907, doi:10.1038/sj.bjc.6604755.
[10]
Waldmann, J.; Slater, E.P.; Langer, P.; Buchholz, M.; Ramaswamy, A.; Walz, M.K.; Schmid, K.W.; Feldmann, G.; Bartsch, D.K.; Fendrich, V. Expression of the transcription factor snail and its target gene twist are associated with malignancy in pheochromocytomas. Ann. Surg. Oncol. 2009, 16, 1997–2005, doi:10.1245/s10434-009-0480-y.
[11]
Hanahan, D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogene. Nature 1985, 315, 115–122, doi:10.1038/315115a0.
Parangi, S.; O’Reilly, M.; Christofori, G.; Holmgren, L.; Grosfeld, J.; Folkman, J.; Hanahan, D. Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc. Natl. Acad. Sci. USA 1996, 93, 2002–2007, doi:10.1073/pnas.93.5.2002. 8700875
[14]
Feldmann, G.; Fendrich, V.; McGovern, K.; Bedja, D.; Bisht, S.; Alvarez, H.; Koorstra, J.B.; Habbe, N.; Karikari, C.; Mullendore, M.; et al. An orally bioavailable small-molecule inhibitor of Hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer. Mol. Cancer Ther. 2008, 7, 2725–2735, doi:10.1158/1535-7163.MCT-08-0573.
[15]
Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001, 25, 402–408, doi:10.1006/meth.2001.1262.
[16]
Vogelmann, R.; Nguyen-Tat, M.D.; Giehl, K.; Adler, G.; Wedlich, D.; Menke, A. TGFbeta-induced downregulation of E-cadherin-based cell-cell adhesion depends on PI3-kinase and PTEN. J. Cell Sci. 2005, 118, 4901–4912, doi:10.1242/jcs.02594. 16219695
[17]
Wali, R.K.; Kunte, D.P.; Koetsier, J.L.; Bissonnette, M.; Roy, H.K. Polyethylene glycol-mediated colorectal cancer chemoprevention: Roles of epidermal growth factor receptor and Snail. Mol. Cancer Ther. 2008, 7, 3103–3111, doi:10.1158/1535-7163.MCT-08-0434.
[18]
Gotzmann, J.; Mikula, M.; Eger, A.; Schulte-Hermann, R.; Foisner, R.; Beug, H.; Mikulits, W. Molecular aspects of epithelial cell plasticity: Implications for local tumor invasion and metastasis. Mutat. Res. 2004, 566, 9–20, doi:10.1016/S1383-5742(03)00033-4.
[19]
Jiao, W.; Miyazaki, K.; Kitajima, Y. Inverse correlation between E-cadherin and Snail expression in hepatocellular carcinoma cell lines in vitro and in vivo. Br. J. Cancer 2002, 86, 98–101, doi:10.1038/sj.bjc.6600017.
[20]
Rosivatz, E.; Becker, K.F.; Kremmer, E.; Schott, C.; Blechschmidt, K.; Hofler, H.; Sarbia, M. Expression and nuclear localization of Snail, an E-cadherin repressor, in adenocarcinomas of the upper gastrointestinal trac. Virchows Arch. 2006, 448, 277–287, doi:10.1007/s00428-005-0118-9.
[21]
Roy, H.K.; Smyrk, T.C.; Koetsier, J.; Victor, T.A.; Wali, R.K. The transcriptional repressor SNAIL is overexpressed in human colon cancer. Dig. Dis. Sci. 2005, 50, 42–46, doi:10.1007/s10620-005-1275-z.
[22]
Rosenau, J.; Bahr, M.J.; von Wasielewski, R.; Mengel, M.; Schmidt, H.H.; Nashan, B.; Lang, H.; Klempnauer, J.; Manns, M.P.; Boeker, K.H. Ki67, E-cadherin, and p53 as prognostic indicators of longterm outcome after liver transplantation for metastatic neuroendocrine tumour. Transplantation 2002, 73, 386–394, doi:10.1097/00007890-200202150-00012. 11884935
[23]
Fendrich, V.; Langer, P.; Celik, I.; Bartsch, D.K.; Zielke, A.; Ramaswamy, A.; Rothmund, M. An aggressive surgical approach leads to long- term survival in patients with pancreatic endocrine tumors. Ann. Surg. 2006, 244, 845–851, doi:10.1097/01.sla.0000246951.21252.60.
[24]
Folkman, J.; Watson, K.; Ingber, D.; Hanahan, D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989, 339, 58–61, doi:10.1038/339058a0. 2469964
[25]
Inoue, M.; Hager, J.H.; Ferrara, N.; Gerber, H.P.; Hanahan, D. VEGF-A has a critical, nonredundant role in angiogenic switching and pancreatic beta cell carcinogenesis. Cancer Cell 2002, 1, 193–202, doi:10.1016/S1535-6108(02)00031-4.
[26]
Bergmann, F.; Breinig, M.; H？pfner, M.; Rieker, R.J.; Fischer, L.; K？hler, C.; Esposito, I.; Kleeff, J.; Herpel, E.; Ehemann, V.; et al. Expression pattern and functional relevance of epidermal growth factor receptor and cyclooxygenase-2: Novel chemotherapeutic targets in pancreatic endocrine tumors? Am. J. Gastroenterol. 2009, 104, 171–181, doi:10.1038/ajg.2008.33. 19098866
