The Fanconi anemia (FA)-BRCA pathway is a DNA damage response/repair pathway critical for genomic stability, tumor suppression and cellular resistance to interstrand DNA crosslinking agents. At the subcellular level, the FA-BRCA pathway is required for the localization of DNA response/repair proteins (e.g. FANCD2, RAD51) to sites of DNA damage where they form nuclear foci. While several mechanisms of pathway regulation have been identified (e.g. post-translational modifications), regulation of the FA-BRCA pathway by microRNAs is currently unknown. MicroRNAs are short non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. MicroRNAs have been implicated in the regulation of several biological processes and are often deregulated in diseases such as cancer. We hypothesize that the FA-BRCA pathway is regulated by microRNAs, based on our finding that several microRNAs inhibit ionizing radiation (IR)-induced nuclear foci formation of FANCD2 and/or RAD51. We developed a cell-based screen to identify microRNAs that negatively regulate the FA-BRCA pathway. Using IR-induced FANCD2/RAD51 nuclear foci formation as a readout, we screened the human miRIDIAN microRNA Mimics library (Dharmacon), which contains over 400 human microRNA mimics. HeLa cells were transfected with microRNA mimics, irradiated and immunostained for FANCD2/RAD51 foci formation. MicroRNA mimics that decreased IR-induced FANCD2/RAD51 foci formation were scored as positives. Through the initial screen, we identified 45 microRNAs that inhibited IR-induced FANCD2 and/or RAD51 foci formation in HeLa cells. Seven of these microRNAs were found to inhibit foci formation without significantly altering cell cycle progression. We focused on two of these microRNAs, miR-103 and miR-107, because they are overexpressed in a subset of human cancers. We found that miR-103 and miR-107 inhibited IR-induced RAD51 foci formation, reduced the efficiency of homologous recombination repair, and sensitized tumor cell lines to cisplatin. Furthermore, miR-103 and miR-107 downregulated protein levels of RAD51 itself and those of RAD51D, a RAD51 paralog required for RAD51 foci formation, in several cell lines. Both RAD51 and RAD51D contain putative miR-103 and miR-107 binding sites in the 3’-UTRs of their respective transcripts and may therefore be direct targets of these microRNAs. In conclusion, our study identified 7 microRNAs that inhibited IR-induced FANCD2 and/or RAD51 foci formation without altering cell cycle progression. We further show that two of these microRNAs, miR-103 and miR-107, regulate homologous recombination repair and chemosensitivity to interstrand cross-linking agents. Furthermore, miR-103 and miR-107 were found to regulate RAD51 and RAD51D expression. These findings are consistent with the hypothesis that microRNAs regulate the FA-BRCA pathway and promote chemosensitivity and genomic instability in cancer cells.

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 1951.