Abstract 3903: Mybl2, a link between proliferation and differentiation in maturing colon epithelial cells

Reprogramming of highly proliferative, multipotent colon epithelial stem/progenitor cells into 3 specialized cell types (enterocytic, goblet, and enteroendocrine cells) that migrate along the crypt axis toward the lumen and another (Paneth cells) that remains at the bottom of the crypt relies heavily on tightly coordinated changes in gene expression. This reprogramming is modeled in immortalized cell lines such as Caco-2 and HT29Cl.16E and Cl.19A that proliferate in an undifferentiated state in culture but acquire absorptive (Caco-2) or secretory (16E and 19A) phenotypes in response to contact inhibition of growth or treatment with the short chain fatty acid butyrate. Identification of genes that are critical in regulating this process, and elucidation of how these genes are themselves modulated, will reveal critical insights into mechanisms that drive normal colon cell differentiation and suppress tumorigenesis. We identified 14 genes that are commonly downregulated during lineage-specific maturation of Caco-2 and HT29Cl.16E and Cl.19A cells induced by contact inhibition of growth or by butyrate treatment. One of these, Mybl2 (Myb related protein B), is linked to the stem cell phenotype and is dramatically suppressed in maturing cells along the crypt-luminal axis in vivo. In spontaneously differentiating colonic epithelial tumor cells, the Mybl2 promoter is not significantly downregulated and pre-mRNA levels (indicating ongoing transcription) of Mybl2, unlike those of the Wnt target genes cyclin D2 and c-myc, do not decrease relative to levels in proliferating cells. We have identified several microRNA (miRNA) binding sites in the Mybl2 3′ untranslated region (UTR) and shown that one of these is functional in suppressing reporter gene activity in maturing colon epithelial cells. Several miRNAs are upregulated in differentiating colon epithelial cells, and overexpression of miR-365, predicted to bind a functional Mybl2 3′ UTR miRNA target, in proliferating colon epithelial cells suppresses Mybl2 protein expression while overexpression of miR-324-5p, predicted to bind a nonfunctional Mybl2 3′ UTR miRNA target, does not alter Mybl2 protein. In order to identify genes regulated by Mybl2, we silenced Mybl2 by 86% using siRNA in proliferating colon epithelial cells and assayed the expression of 48 genes we previously determined to be modulated during maturation of colon epithelial cells in vitro and in vivo. Mybl2 suppression induces expression of the cell cycle regulators cdk2, cdk6, cyclin D2, and c-myc and downregulates expression of cyclin B2 and cdc25B. Although Mybl2 suppression does not alter cell proliferation, it induces a 10% increase in G2/M with a complementary 10% reduction in G1. Therefore, Mybl2 may link cell cycle progression with generation of differentiation-specific signals and play a key role in commitment to colon epithelial cell maturation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3903.