Abstract 1012: MicroRNA-203 restricts the proliferation capacity of normal colon and colon cancer stem cells by regulating the expression of Tcf4

Abstract

Abstract The canonical Wnt/β-catenin pathway plays a crucial role in driving proliferation of normal colon epithelial cells and colon cancer cells. Tcf4, which is encoded by the Tcf7l2 gene, is a critical downstream molecule of the Wnt/β-catenin pathway. Although several downstream target genes of the Wnt/β-catenin pathway have been identified (e.g. CD44, Lgr5 and EphB2 receptor which mark both normal colon stem cells (NCSCs) and colon cancer stem cells (CoCSCs)), upstream regulators of this pathway remain poorly characterized. MicroRNAs (miRNAs) are endogenous small noncoding RNAs which suppress translation by binding to specific seed sequences in 3′untranslated region (3′UTR) of target mRNAs. In this study, we aimed to find miRNAs that are highly expressed during normal colonic epithelial differentiation and can also regulate the canonical Wnt/β-catenin pathway; in addition, their roles in the regulation of proliferation and differentiation in both normal colon and colon cancer are investigated. We isolated similar numbers of immature progenitor cells (EpCAMhigh CD44+; crypt base) and mature cells (EpCAMhigh CD44− CD66a+; top of the crypt) from both normal and colon cancer primary samples by FACS and performed real-time PCR. In normal human colon, we found that Tcf4 is highly expressed at the crypt base and is absent or low at the top of the crypt. In primary human colon cancer, Tcf4 expression is higher in tumorigenic cells (the fraction containing the CoCSCs) than the non-tumorigenic cells. Furthermore, we found several miRNAs that are differentially expressed in the top and bottom of the crypt of human and murine normal colon epithelium. One of the miRNAs, miR-203, is downregulated both in human NCSCs and CoCSCs. MiR-203 suppresses Tcf7l2 expression by specifically targeting its 3′UTR, as shown with a luciferase reporter. Western blotting showed that Tcf4 protein expression was decreased in colon cancer cell lines transfected with miR-203. Moreover, miR-203 suppresses Wnt/β-catenin signaling as assayed by TOP-flash. Overexpression of miR-203 inhibited the organoid-formation of murine normal colon and human xenograft CoCSCs in vitro. Furthermore, miR-203 suppressed in vivo tumorigenic capacity of human CoCSCs when injected subcutaneously in NOD/SCID interleukin-2 receptor γ chain null (Il2rg(-/-)) immunodeficient mice. In conclusion, miR-203 is downregulated in both in NCSCs and CoCSCs as compared to their more mature progeny, and regulates the canonical Wnt/β-catenin pathway by suppressing the expression of Tcf4. These findings suggest that normal stem/progenitor cells and CoCSCs share a similar molecular machinery to regulate their proliferation capacity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1012. doi:1538-7445.AM2012-1012