MicroRNA-199a-3p exerts tumor suppressive functions by downregulating BRCA1 in triple negative breast cancer

Abstract

Breast cancer is the most common cancer and leading cause of cancer deaths among women globally. Triple-negative breast cancer (TNBC) accounts for 10-20% of all breast cancers and is characterized by a more aggressive phenotype and poor clinical outcomes. No targeted therapy is available to date for TNBC patients, whilst surgery and systemic treatment of chemotherapy remain the recommended clinical options, however, incomplete pathological response, drug resistance and disease recurrence are common. TNBC onsets have been correlated with the loss-of-function of breast cancer susceptibility gene BRCA1, which is a tumor suppressor gene essential for maintenance of genomic stability, cell cycle checkpoint arrests and homologous recombination (HR) DNA repair. On the other hand, germline BRCA1 mutation and HR defects were linked with better responses to platinum-based chemotherapy and poly-ADP ribose polymerase (PARP) inhibitors. Increasing evidence have suggested the roles of microRNAs (miRNAs) in mediating cancer progression and therapeutic sensitivity. In previous studies, miR-199a-3p expression was shown to differentiate between TNBC and non-TNBC patients. Also, it was in silico predicted as a BRCA1-targeting miRNA, which led to the speculation of its role in TNBC by downregulating BRCA1. In this study, an inverse correlation between miR-199a-3p and BRCA1 expressions was observed in TNBC tumor tissues. Prediction algorithms and dual-luciferase reporter assays demonstrated that BRCA1 is a target of miR-199a-3p, which suppressed the luciferase activity mediated by BRCA1 3’-untranslated region with miR-199a-3p seed binding sequence. Ectopic expression of miR-199a-3p and BRCA1 silencing in TNBC cell lines (MDA-MB-231, cisplatin-resistant MDA-MB-231/cisR and MDA-MB-468) resulted in downregulated BRCA1 protein levels, and inhibited cell proliferation, migration and clonogenic abilities. Moreover, miR-199a-3p overexpression was showed to suppress in vivo tumor growth in NOD-SCID mouse xenograft model. Next, the effects of miR-199a-3p-mediated BRCA1 dysfunction on drug sensitivity were investigated. DNA-damaging platinum agent, namely cisplatin, induced BRCA1 expression and suppressed miR-199a-3p in TNBC cells. Restoration of miR-199a-3p level and BRCA1 silencing reversed the acquired cisplatin resistance in MDA-MB-231/cisR cells, and also enhanced drug sensitivities of TNBC cells to carboplatin and PARP inhibitors. Moreover, miR-199a-3p sensitized the xenograft tumors to cisplatin treatment in vivo. To study the underlying mechanism of miR-199a-3p induction on drug sensitivity, the effects of miR-199a-3p on DNA damage repair, cell apoptosis and cancer stem cells were assessed. Using host cell reactivation assay, miR-199a-3p was demonstrated to suppress the DNA repair capability in TNBC cells. Ectopic miR-199a-3p expression combined with cisplatin treatment showed an inductive effect on cell cycle arrest and apoptosis. Lastly, miR-199a-3p suppressed the stem cell-like population in cisplatin-resistant cells, as indicated by the reduction of aldehyde dehydrogenase (ALDH)-positive cell population in ALDEFLUOR assay, and inhibited tumor initiating capability in mice xenografts. Taken together, these results suggested that miR-199a-3p exerted a tumor suppressive role in TNBC, and correlated with BRCA1 dysfunction to improve therapeutic efficacy against platinum-based agents and PARP inhibitors. MiR-199a-3p might hence show potential prognostic and therapeutic values in the treatment of TNBC.