Tumor suppressive miR-99b-5p as an epigenomic regulator mediating mTOR/AR/SMARCD1 signaling axis in aggressive prostate cancer

Abstract

IntroductionAfrican American (AA) men exhibited 2.3-fold higher PCa incidence and 1.7-fold higher PCa mortality rates when compared to the European American (EA) men. Besides the socioeconomic factors, emerging evidence has highlighted that biological risk factors may play critical roles in the AA PCa disparities. Previously, we have shown that downregulated miR-99b-5p and upregulated mTOR cooperatively promotes the AA PCa aggressiveness and drug resistance. MethodsIn this study, we aimed to explore the miR-99b-5p/mTOR/AR/SMARCD1 signaling axis in AA PCa aggressiveness. The analyses used in the study included immunofluorescence, western blot, in-vitro functional assays (TUNEL, colony forming, and MTT), and chromatin immunoprecipitation (ChIP)-qPCR assays in 2D and/or 3D culture model of EA PCa and AA PCa cell lines.ResultsSpecifically, the immunofluorescence staining, and western blot analysis has revealed that nuclear mTOR, AR, and SMARCD1 were highly expressed in AA PCa (MDA PCa 2b) compared to EA PCa (LNCaP) cell line. Western blot analysis further revealed that miR-99b-5p inhibited protein levels of mTOR, AR/AR-V7 and SMARCD1 in cytoplasm and nuclei of EA and AA PCa. The in-vitro functional (MTT, TUNEL, and clonogenic) assays have demonstrated that miR-99b-5p effectively inhibited cell proliferation/survival and induced cell apoptosis in EA and AA PCa cells. Moreover, combination of miR-99b-5p and enzalutamide (Enz) synergistically enhances the cytotoxicity against aggressive AA PCa and castration-resistant prostate cancer (CRPC). mTOR ChIP-qPCR assays further demonstrated that miR-99b-5p or miR-99b-5p/Enz significantly reduces the recruitment of mTOR to the genes involved in the metabolic reprogramming in CRPC.DiscussionTaken together, miR-99b-5p may function as an epigenomic driver to modulate the mTOR/AR/SMARCD1 signaling axis in AA PCa and resistant CRPC.

Organoids: An Emerging Precision Medicine Model for Prostate Cancer Research

“…Additionally, we established PCa organoids from LNCaP, C4-2B, 22Rv1 and MDA PCa 2b cells for exploring the mTOR/AR signaling in the in African American (AA) PCa disparities and CRPC progression. The results suggested that downregulation of tumor suppressive miR-99b-5p mediates the upregulation of the mTOR/AR/SMARCD1 signaling axis, consequently promoting tumor aggressiveness and metabolic reprogramming in AA PCa and CRPC [143]. These two studies highlighted the importance of PCa spheroids and organoids as efficient 3D models for evaluating drug effects and molecular mechanisms underlying PCa.…”

Combination of miR-99b-5p and Enzalutamide or Abiraterone Synergizes the Suppression of EMT-Mediated Metastasis in Prostate Cancer

“…The transfected PCa cells were then grown overnight and treated with vehicle, Enz, or Abi for an additional 48 h. The treatment groups were as follows: NC (transfection of nonsense control, and vehicle treatment), miR-99b-5p (transfection of miR-99b-5p mimic, and vehicle), Enz (transfection of nonsense RNA, and 20 µM of Enz), Abi (transfection of nonsense RNA and 10 µM Abi), miR-99b-5p/Enz (combination of miR-99b-5p mimic and 20 µM of Enz), and miR-99b-5p/Abi (combination of miR-99b-5p mimic and 10 µM of Abi). All the cells were grown in 5% CO 2 incubator at 37 • C. A total of 20 µM of Enz and 10 µM of Abi were the concentrations used in the experiments, according to previous studies [34,48].…”

“…Western blot assays were performed using the protocol as previously described [33,34,49]. The PCa cells were collected, and total proteins were extracted using M-PER with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…Previously, we identified dozens of reciprocal miRNAs/mRNA pairings and associated miRNA-mRNA regulatory networks in African American (AA) PCa disparities [31,32]. Among these miRNA-mRNA pairings, the downregulation of miR-99b-5p and upregulation of MTOR has been implicated as a critical epigenetic event that contributes to tumor aggressiveness in AA PCa and the progression of CRPC [33,34]. Furthermore, our studies have shown that miR-99b-5p simultaneously targets and inhibits the expression of MTOR, AR, and SMARCD1 (encoding SMARCD1, an AR coregulator), consequently blocking the translocation of the mTOR/AR/SMARCD1 complex from the cytoplasm to the nucleus, inhibiting the expression of mTOR/AR target genes, reversing metabolic rewiring, and sensitizing AA PCa and CRPC to Enz [34].…”

“…Additionally, AR signaling has also been implicated in the activation of EMT in PCa [42,43]. Due to the central/unique role of the reciprocal miR-99b-5p/MTOR pairing (downregulated/upregulated in AA PCa) in coordinating PI3K/AKT/mTOR, HIF1α, and AR signaling [31,33,34], we hypothesized that the miR-99b-5p/mTOR/AR/SMARCD1 signaling axis may play a critical functional role in regulating EMT-mediated metastasis in AA PCa and CRPC. To test this hypothesis, we performed a series of functional and biochemical experiments to examine whether transfection/overexpression of miR-99b-5p mimic could inhibit the expression of EMT-TF (Snail) and mesenchymal markers (N-cadherin and Vimentin) and restore the expression of epithelial marker (E-cadherin), ultimately inhibiting the metastatic capacities of AA PCa and CRPC cells.…”