miR-199a-3p targets stemness-related and mitogenic signaling pathways to suppress the expansion and tumorigenic capabilities of prostate cancer stem cells

Human cancers exhibit significant cellular heterogeneity featuring tumorigenic cancer stem cells (CSCs) in addition to more differentiated progeny with limited tumorinitiating capabilities. Recent studies suggest that microRNAs (miRNAs) regulate CSCs and tumor development. A previous library screening for differential miRNA expression in CD44⁺ (and other) prostate CSC vs. non-CSC populations identified miR- 199a-3p to be among the most highly under-expressed miRNAs in CSCs. In this study, we characterized the biological functions of miR-199a-3p in CD44⁺ prostate cancer (PCa) cells and in tumor regeneration. Overexpression of miR-199a-3p in purified CD44⁺ or bulk PCa cells, including primary PCa, inhibited proliferation and clonal expansion without inducing apoptosis. miR-199a-3p overexpression also diminished tumor-initiating capacities of CD44⁺ PCa cells as well as tumor regeneration from bulk PCa cells. Importantly, inducible miR-199a-3p expression in pre-established prostate tumors in NOD/SCID mice inhibited tumor growth. Using target prediction program and luciferase assays, we show mechanistically that CD44 is a direct functional target of miR-199a-3p in PCa cells. Moreover, miR-199a-3p also directly or indirectly targeted several additional mitogenic molecules, including c-MYC, cyclin D1 (CCND1) and EGFR. Taken together, our results demonstrate how the aberrant loss of a miRNAmediated mechanism can lead to the expansion and tumorigenic activity of prostate CSCs, further supporting the development and implementation of miRNA mimics for cancer treatment.