Abstract B008: PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer

Introduction and Objectives: The dysfunctions of androgen receptor (AR) and TGF-β signaling contribute to prostate tumorigenesis and cancer progression. Our laboratory has identified PMEPA1 as an androgen responsive gene with prostate abundance, as well as PMEPA1/NEDD4 and AR negative feedback loop in the regulation of AR levels in prostate cancer cells. In addition, it has also been established by other groups that PMEPA1 inhibits TGF-β signaling via a similar negative feedback loop as a TGF-β responsive gene. Five isoforms are transcribed from distinct promoters within the PMEPA1 locus. PMEPA1 isoforms were shown to have variations at the N-terminus of the protein. This study focuses on understanding of the expression and biologic functions of PMEPA1 isoforms in CaP. Methods: The PMEPA1 isoforms were evaluated for expressions in multiple CaP cell lines, trensfectant derivatives, and prostate tumors. LNCaP cells were treated with R1881 (0, 0.1, 1.0 nM) and DU-145 and PC-3 cells were treated with TGF-β (0, 5 and 25 ng/ml) for 24 hours. PMEPA1 isoform specific plasmids and siRNAs were transfected into LNCaP, DU-145, and PC-3 cells individually. Cell proliferation was analyzed by cell counting, cell plating efficiency assay, and soft agar assay. The protein levels of PMEPA1 isoforms, AR, PSA, TGF-β receptor I, Smad2 were detected by immunoblotting, and the transcript levels of KLK3 (PSA), TGF-β responsive genes including THBS1, NEDD9 and COL1A1 were evaluated by QRT-PCR. The TGF-β signaling activity was measured by SMAD reported dual-luciferase assay. The transcript levels of PMEPA1-252, PMEPA3-287-STAG1, AR, PSA in 120 matched human benign and malignant frozen tissue were evaluated with Q-PCR. Results: The expressions of PMEPA1-252 and PMEPA2-344 isoforms were restricted to androgen-responsive prostate cancer cells in comparison to broader expression pattern of other isoforms (PMEPA3-287/STAG1, PMEPA4-259 and PMEPA5-237). The expression of PMEPA1-2 was androgen regulated, whereas expression of PMEPA 3-5 was regulated by TGF-β. Only PMEPA1-252 inhibited cell growth of LNCaP, DU-145 and PC-3 cells. In contrast, PMEPA2-4 promoted cell growth of DU-145 and PC-3 cells. Only PMEPA1-252 mediated AR protein degradation and inhibited AR signaling. In contrast, PMEPA3-287-STAG1 and PMEPA4-259 inhibited TGF-β signaling luciferase activity and transcript levels of THBS1, NEDD9 and COL1A1. All PMEPA1 isoforms were found to have no effects on protein level of TGF-β receptor I. Moreover, the deletion mutants of N-terminus and transmembrane domains interrupted the isoform specific inhibitory effects on AR/TGF-β signaling. The transcript level of PMEPA1-252 was higher than PMEPA3-287-STAG1 in benign prostate tissue. Different from PMEPA1-252, the expression of PMEPA3-287-STAG1 was not found to decrease in prostate tumor tissue. There was no significant correlation between the expression of PMEPA1-252 and PMEPA3-287-STAG1 in prostate tissue. The transcript of PMEPA1-252 was associated with PSA, but the expression of PMEPA3-287-STAG1 was found not to be associated with PSA or AR in CaP cells. Conclusions: The PMEPA isoforms appear to underscore distinct biologic functions in the context of androgen and TGF-β signaling. Intensively studied PMEPA1-252 was specific for AR degradation in prostate cancer cells and was consistent with previous observations of association of AR upregulation with loss of PMEPA1 in prostate cancer. The roles of PMEPA1 isoforms need to be better defined in prostate cancer and other cancers. Funding: This study was supported by CPDR, USUHS, HU0001-10-2-0002 to I.L.R. Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Gyorgy Petrovics, Yongmei Chen, Alagarsamy Srinivasan, Inger Rosner, Albert Dobi, Shiv Srivastava. PMEPA1 isoform specific regulation of androgen and TGF beta signaling in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B008.