Abstract 3043: Mesenchymal phenoptype of metastasizing primary human colorectal TIC is maintained through epigenetic silencing of miR-200
Authors: Hoffmann Christopher M. , Giessler Klara M. , Ball Claudia R. , Dubash Taronish D. , Dieter Sebastian M. , Bergmann Sarah , Weichert Wilko , Kalle Christof Von , Schneider Martin , Baer Constance , Plass Christoph , Schmidt Manfred , Glimm Hanno 
Published: May 2015

Abstract
Metastasis formation is the most common cause of colorectal cancer (CRC) related death. How cancer cells disseminate from the primary tumor, gain access to blood and lymphatic vessels and seed to distant organs is not fully understood. To gain deeper mechanistic insights into metastasis formation, we developed a metastasis xenotransplantation model for primary human colorectal cancer cells. Patient (n=12) derived tumor-initiating cells (TIC) were enriched in spheroid cultures and subsequently transplanted into the kidney capsule of immunodeficient NOD/SCID-IL2RGnull (NSG) mice. Importantly, cultures derived from 3 distinct patients frequently metastasized into the murine liver and spleen, whereas spheroids derived from all other patients (n=9) never did. Compared to non-metastasizing spheroids, metastasizing spheroids strongly expressed mesenchymal markers (e.g. Vimentin and N-cadherin), whereas epithelial and colonic differentiation markers (e.g. E-cadherin, Trefoil factor 3, Defensin alpha 5, Keratin-8, -18 -20) were down-regulated suggesting that metastasizing primary patient derived CRC spheroids may have passed through an epithelial to mesenchymal transition (EMT). As the miR-200 family has been shown to support an epithelial phenotype and has been implicated in the process of EMT in tumor cells, we analyzed miRNA expression in metastasizing versus non-metastasizing spheroid cultures. Interestingly, metastasizing spheroids strongly down-regulated the miR-200 clusters miR-200a/-200b/-429 and miR-200c/-141 as well as miR-194 and miR-203. To further understand the mechanism of deregulated gene- and miRNA expression in metastasizing spheroids, we evaluated the DNA methylation status of miRNA promoters. In contrast to non-metastasizing spheroid cultures, metastasizing spheroids showed pronounced DNA methylation within the miR-200 promoter regions, indicating that miR-200 expression in primary metastasizing human colorectal cancer spheroids is suppressed through epigenetic mechanisms. Forced overexpression of both miR-200 clusters in metastasizing spheroids restored expression of epithelial genes like E-Cadherin and Keratin-8 and reduced expression of the mesenchymal markers Vimentin and N-cadherin. Moreover, incubation with low dose 5-aza-2′-deoxycytidine increased the expression of epithelial cell adhesion molecule (EpCAM) in metastasizing spheroids, indicating a partial reversion of the mesenchymal phenotype by demethylating agents. In summary, our findings indicate that metastasizing primary human CRC spheroids are epigenetically fixed in a mesenchymal state. This model allows further dissecting and understanding mechanisms of EMT and metastasis formation in human CRC thereby providing the basis for the development of future therapeutic intervention strategies against metastasizing human colorectal cancer.
Citation Format: Christopher M. Hoffmann, Klara M. Giessler, Claudia R. Ball, Taronish D. Dubash, Sebastian M. Dieter, Sarah Bergmann, Wilko Weichert, Christof Von Kalle, Martin Schneider, Constance Baer, Christoph Plass, Manfred Schmidt, Hanno Glimm. Mesenchymal phenoptype of metastasizing primary human colorectal TIC is maintained through epigenetic silencing of miR-200. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3043. doi:10.1158/1538-7445.AM2014-3043