Tgf Beta Regulates Mir-182 Control Of Brca1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Transforming growth factor beta (TGFβ) is a pluripotent cytokine whose activity is significantly increased in epithelial cancers and induced by radiation and chemotherapy. Downregulation of BRCA1 or alterations in BRCA1 related pathways may contribute to response to therapy. However, the mechanism(s) underlying BRCA1 downregulation is not well understood. Our prior work indicated that inhibition of TGFβ canonical signaling reduces BRCA1 mRNA levels (Maxwell et al., Cancer Res. 68(20):8304-11, 2008). Here, we used the non-malignant human epithelial MCF10A cell line and murine Tgfb1 heterozygote (HT) and wildtype fibroblast cell lines to investigate TGFβ regulation of BRCA1. Pharmaceutical inhibition of TGFβ type I receptor kinase or incubation with pan-isoform neutralizing antibody, 1D11, resulted in significant down regulation of both BRCA1 mRNA and protein levels in MCF10A. Likewise, Brca1 protein and mRNA was decreased by >50% in cell cycle synchronized Tgfb1 HT fibroblasts compared to wildtype fibroblasts. Consistent with decreased Brca1, Tgfb1 HT fibroblasts exhibited defective DNA damage response as measured by 53BP1 foci following UV radiation treatment. We next investigated mechanisms regulating protein levels in MCF10A cells, which together indicated that TGFβ signaling regulates BRCA1 protein stability, prevents its degradation, and increases BRCA1 promoter activity. Surprisingly, TGFβ treatment rapidly (<30 minutes) increased BRCA1 protein and mRNA, which led us to also consider control at the level of mRNA stability since BRCA1 is known to be controlled by miR-182 (Moskwa et al., Mol Cell. 41(2):210-20, 2011). TGFβ treatment downregulates miR-182, conversely, miR-182 increases when TGFβ is inhibited by either small molecule inhibition of the type I receptor kinase or by ligand capture with neutralizing antibody in human cells. miR-182 increased 18-fold in Tgfb1 HT murine fibroblasts. Notably, MCF10A do not show changes in other known miRNAs that target BRCA1 for degradation, miRNA 146a and 146b (Garcia et al., EMBO Mol Med. 3(5):279-90, 2011), are not affected by TGFβ treatment or inhibition. Transfection of Tgfb1 HT fibroblasts with a miR-182 antagomir restored BRCA1 mRNA, protein and functional deficits. Further studies show that addition of TGFβ for short periods of time affects miRNA processing by decreasing pre-miR182 up to 50%, while TGFβ inhibition affects miR-182 translation. These data indicate that TGFβ exerts control over BRCA1 at several levels: protein stability, transcriptional control of the promoter, and translational control via miR-182. As many cancer cells maintain the ability to respond to TGFβ and TGFβ inhibition is clinically viable, we propose that TGFβ inhibition provides a therapeutic opportunity by impeding BRCA1 dependent DNA damage responses. Note: This abstract was not presented at the meeting. Citation Format: Haydeliz Martinez-Ruiz, Sangeetha Vijayakumar, Mary H. Barcellos-Hoff. TGFβ regulates miR-182 control of BRCA1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5218. doi:10.1158/1538-7445.AM2014-5218