Abstract 1111: Reprogramming the breast cancer microenvironment using microRNAs that target DNA repair

Abstract

Preclinical and clinical studies have revealed that tumor endothelium is abnormal, resistant to genotoxic stress, and as such, functions as a key determinant of therapeutic responses to radiation and chemotherapy. While it is well established that radiation and chemotherapy cause DNA damage in tumor vasculature, the molecular mechanisms leading to subsequent cell cycle arrest, apoptosis or senescence in vascular cells are poorly understood. Therefore, identifying and understanding factor(s) that mediate DNA damage responses in tumor endothelial cells will provide potential targets for sensitizing tumor vasculature to radiation and other DNA damaging agents and improve their therapeutic efficacy in cancer. Recent data indicates that microRNAs (miRs) are potent regulators of DNA damage responses in the tumor microenvironment. miRs are short 20-22 nucleotide (nt) RNA molecules that regulate gene expression by binding to partially complementary sites in target mRNAs. Since miRs mediate several physiological processes in endothelial cells, we hypothesized that miRs regulate endothelial (EC) DNA damage responses. We used an expression screen to identify miRs induced by radiation, cisplatin or hydrogen peroxide in human ECs and identified seven specific miRs unique to intrinsic EC apoptosis pathways regulated by genotoxic stress. In vitro gain-of-function assays show that three of them, miR-21, miR-99b and miR-494 lead to endothelial senescence by impairing telomerase function and inhibit sprouting angiogenesis in vitro, in a 3D assay. Strikingly, we observed that these three miRs each target every member of the MRN (Mre11a-Rad50 and NBS1) complex, a critical part of the cellular DNA repair machinery. MRN complex plays a vital role in DNA ds break repair, replication, and telomere maintenance. Pulldown of a mutant RNA Induced Silencing Complex (RISC) from cells transfected the miR mimics enriched for the MRN mRNAs suggesting direct miRNA-MRN complex mRNA binding. Consistent with these results, knockdown of the MRN complex recapitulated the effects of the miRs, reproducing the senescence phenotype, angiogenesis inhibition and also impaired telomerase activity. Since MRE-11a is upregulated in human breast cancer patients, we asked if there was any differential expression of miR-494 in either the tumor ECs or tumor cells. Interestingly, ISH of a breast cancer tissue array revealed a significant reduction in tumor miR-494 levels compared with the adjacent normal tissue. Furthermore, ectopic expression of miR-494 diminished breast cancer cell proliferation in 2D and 3D. Our observations indicate that miR-494 behaves as a tumor suppressor microRNA by targeting the MRN complex, inducing senescence, cell cycle arrest and decreases angiogenesis. Therefore, we propose that restoration of these miRs targeting the MRN complex in breast cancer is likely to synergize with DNA damaging agents and decrease tumor burden. Citation Format: Cristina Espinosa-Diez, RaeAnna Wilson, Nathan Kanner, Rebecca Ruhl, Christina M. Hipfinger, Sudarshan Anand. Reprogramming the breast cancer microenvironment using microRNAs that target DNA repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1111.