Microrna-221,-222 And-181 Regulates Tamoxifen Resistance In Breast Cancer By Downregulating Timp3 And Facilitating Growth Factor Signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL We have shown earlier that microRNA (miR)-221 and -222 are upregulated in tamoxifen (TAM) resistant breast cancer cell line MCF-7 (OHTR), and in Her2 positive primary human breast tumors compared to the Her2 negative tumors1. In addition, expression of miR-181b was 3.5-fold higher in OHTR cells compared to the MCF-7 cells and endocrine resistant primary tumors. To determine the underlying mechanism of miR-mediated regulation of TAM resistance, we focused on identification of target genes regulated by these miRs. Bioinformatics search revealed presence of seed sequences for all three miRs on 3’-UTR of TIMP3, a tissue metalloprotease inhibitor. Luciferase assay confirmed TIMP3 to be a direct target of these miRs and was found to be regulated at both RNA and protein levels. In situ hybridization and immunohistochemical analysis demonstrated reciprocal relationship between TIMP3 and miR 221/222 expression in primary human tumors, where invasive ductal carcinomas expressed high level of the miRs and normal or non-invasive cancers expressed TIMP3, but not the miRs. Cell proliferation and wound healing assay demonstrated increased sensitization of the TAM resistant cells to the drug upon ectopic expression of TIMP3. In addition, mouse mammary tumors induced with TIMP3 overexpressing cells were found to be more sensitive to TAM compared to the tumors initiated with control cells. Conversely, knocking down TIMP3 by siRNA reduced sensitivity to TAM, both in cell cultures and in mouse mammary tumors. As TIMP3 is an inhibitor of matrix metalloproteases involved in positive regulation of growth factor signaling, we speculated that reduction in TIMP3 due to high miR-221/222/181 expression could facilitate growth factor signaling in OHTR cells. Indeed, EGF-induced MAPK phosphorylation was significantly higher in OHTR cells compared to MCF-7 cells and miR-221/222 overexpressing MCF-7 cells mimicked this phenomenon. On the contrary, phosphMAPK level was reduced when TIMP3 was overexpressed in OHTR cells and increased upon knocking down of TIMP3 in MCF-7 cells. Similar differences in MAPK phosphorylation was also observed in cells treated with low level estrogen or TAM, agonists for TAM resistant cells. Based on this data we conclude that overexpression of miR-221/222 and -181b facilitates growth factor signaling in TAM resistant cells by downregulating TIMP3. 1. Miller et. al. J. Biol. Chem 2008; 283 (44): 29897 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 1154. doi:10.1158/1538-7445.AM2011-1154