Abstract 3959: Growth suppression by TGF-β in responsive colon carcinoma is opposed by miR-20a through mechanisms altering p21WAF1 up-regulation

Abstract

Abstract Loss of response to TGF-β occurs in many cancers and disruption of its regulatory circuitry appears as a central event in the genesis of colorectal cancer (CRC) malignancy. Lack of inhibitory response to TGF-β is common to most colon cancer cell lines. However, inactivating mutations at receptors and transducers occur in less than a half of neoplastic colon tissues, which underscore the significance of additional mechanisms diverting TGF-β growth suppression. In this context, abrogation of TGF-β response by some miRNAs has been recently reported. By searching for miRNAs in regions showing copy number changes and concordant gene expression in 36 sporadic CRCs compared to their normal counterpart, we identified the miR-17-92 cluster on the 13q31 locus, which is gained and highly expressed at early stages of CRC. MiR-20a from miR-17-92 cluster is a paralog of the miR-106b, which affects TGF-β sensitivity in gastric carcinoma. We hypothesized an involvement of miR-20a in the suppression of TGF-β response in CRCs and selected the TGF-β sensitive FET colon carcinoma, expressing low miR-20a levels, to investigate the relationship between enhanced expression of miR-20a and TGF-β sensitivity and address growth inhibition. We report that miR-20a affects regulation of p21WAF1 expression, has a negative and significant effect on the cytostatic response mediated by TGF-β – evaluated by BrdU incorporation, MTT assay, and cell-cycle analysis – but shows little effect on TGF-β untreated cells. Although p21WAF1 transcript and protein are significantly decreased in cells treated or not with the cytokine, we could observe that the p21 up-regulation driven by TGF-β is strongly subverted by miR-20a. One potential mechanism of p21 down-modulation by miR-20a can be the targeting of its 3’UTR, as occurring for miR-106a. Moreover, we could observe that miR-20a is also able to block the transactivation of the 2.3-kb p21 promoter upon TGF-β stimulation, as assessed by luciferase-based assay, but not the activation of the Smad3/4-reponsive reporter. Down-modulation of c-Myc by TGF-β, crucial to regulation of p21 transcription, is also subverted by miR-20a delivery into cells. Our experiments suggest for miR-20a an interference with the TGF-β homeostasis in colon addressing the up-regulation of p21 expression, through mechanisms involving more effectors of the TGF-β cascade. Overall, miR-20a seems to participate in the abrogation of this key regulatory response in colonic epithelium. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3959. doi:10.1158/1538-7445.AM2011-3959