Abstract 276: Mir-139 regulates autophagy in prostate cancer cells through Beclin-1 and mTOR signaling proteins

Abstract

Abstract Background: Prostate cancer represents the second leading cause of cancer death in men and develops as a result of the accumulation of genetic and epigenetic alterations. We previously identified a panel of five miRNAs associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using NGS-based whole miRNome sequencing and qPCR-based validation analysis. We have shown that expression of miR-139, one of the downregulated miRNAs from this panel, was associated with improved prognosis in patients with localized prostate cancer. Furthermore, miR-139 overexpression inhibited prostate cancer cell growth through signaling pathways mediated in part through downregulation of AXL and IGF1R. Here, we further investigated the molecular mechanisms by which miR-139 could inhibit prostate cancer cell growth. Methods: We examined miR-139 transfected PC3, DU145 and LNCaP cells by morphology as well as by cell-based assays, confocal microscopy and immunoblotting. Results and Discussion: We found that treatment of prostate cancer cells with miR-139 resulted in phenotypic changes characteristic of autophagic cells. Conversion of the microtubule-associated protein light chain 3 (LC3-I to LC3-II) was used to monitor autophagy. We observed that the treatment of PC3 and LNCaP cells with miR-139 increased the conversion of LC3-I to LC3-II protein that was specifically inhibited by addition of miR-139 antagomir. The upregulation of LC3 II was further confirmed by confocal microscopy of PC3 cells treated with miR-139. Mammalian target of rapamycin (mTOR) and Beclin1 are two important autophagy-related molecules playing significant roles in different stages. mTOR negatively regulates autophagy machinery. Treatment of cells with miR-139 inhibited both activation and expression of mTOR. We also observed increased levels of phospho-Beclin 1 in PC3 cells treated with mir-139. Beclin 1 is a scaffold protein that assembles components for promoting or inhibiting autophagy and its phosphorylation controls autophagy. The cargo adaptor protein, p62/SQSTM1, interacts with autophagic substrates, delivering them to the autophagosome for degradation, and is degraded during autophagy. We found that during early treatment of cells with miR-139, p62 expression is inhibited, while at later stages, p62 expression accumulates, suggesting that autophagic flux may be blocked. These results suggest that miR-139 is regulating autophagy in prostate cancer cells at least in part through the mTOR and Beclin-1 proteins. Citation Format: Tania C. Benatar, Robert Nam, Christopher Sherman, Yutaka Amemiya, Arun Seth. Mir-139 regulates autophagy in prostate cancer cells through Beclin-1 and mTOR signaling proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 276.