Mir-194 Counterbalances Transcriptional Activation Of The Anti-Angiogenic Factor Thrombospondin-1 By P53

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Thrombospondin-1 (TSP-1) is a key endogenous inhibitor of angiogenesis, negatively regulated by oncogenes (e.g., c-Myc) and transcriptionally activated by the key tumor suppressor p53 (1). Yet in some cancers, such as colon adenocarcinomas, tumor progression accompanied by the loss of p53 does not result in TSP-1 down-regulation (2, 3). This paradox could involve promoter-independent mechanisms, for example microRNA-mediated regulation. In order to test this hypothesis, we conducted a global screen for microRNAs that regulate TSP-1 levels. Gain-of-function experiments and microarray data showed that in addition to the previously identified mir-17-92 cluster members (4, 5), several other miRs, including let-7, mir-199a-3p, mir-218 and miR-194, downregulate TSP-1. mir-194 was of particular interest, since its expression is known to be largely colon-specific (6), maintained by p53 and thus frequently reduced in advanced cancers (7). This reduced miR-194 expression could account for unexpectedly high TSP-1 levels in p53-null tumors. Indeed, mir-194 inhibition with antisense 2’-O-methyl ribonucleotides in p53-sufficient colon cancer cell lines strongly restored TSP-1 levels. Conversely, miR-194 mimics reduced TSP-1 expression at both RNA and protein levels. Furthermore, dual luciferase sensor assay demonstrated that this regulation was mediated by the single mir-194 site in the 3’UTR of the thbs1 gene. We then set up a retroviral transduction-based system to test the involvement of this miR-194 binding site in TSP-1 regulation by p53. Viruses expressing the thrombospondin-1 gene with 3’UTR in either wild type or miR-194-mutated configurations were introduced into HCT116-p53 sufficient cells (miR-194 levels are high) and their p53-null derivatives (miR-194 levels are low). In parental HCT116 cells, the mutation in the mir-194 site sharply increased TSP-1 levels, resulting in reduced angiogenesis in Matrigel plugs and tumor xenografts. However, the effects of this mutation disappeared in the absence of p53, resulting in lower TSP-1 expression levels. These results are consistent with the idea that miR-194 counterbalances transcriptional activation of TSP-1 by p53 and underscore the complex relationship between promoter-dependent and microRNA-mediated effects of p53. Grant Support: R01 [CA122334][1] (ATT) and T32 [CA009140][2] (PS) 1. K. M. Dameron, O. V. Volpert, M. A. Tainsky, N. Bouck, Science 265, 1582 (1994). 2. S. Kaiser et al., Genome Biol 8, R131 (2007). 3. D. A. Notterman, U. Alon, A. J. Sierk, A. J. Levine, Cancer Res 61, 3124 (2001). 4. M. Dews et al., Nat Genet 38, 1060 (2006). 5. M. Dews et al., Cancer Res 70, 8233 (2010). 6. K. Hino et al., RNA. 14, 1433 (2008). 7. C. J. Braun et al., Cancer Res 68, 10094 (2008). 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 3275. doi:10.1158/1538-7445.AM2011-3275 [1]: /lookup/external-ref?link_type=GEN&access_num=CA122334&atom=%2Fcanres%2F71%2F8_Supplement%2F3275.atom [2]: /lookup/external-ref?link_type=GEN&access_num=CA009140&atom=%2Fcanres%2F71%2F8_Supplement%2F3275.atom