Background: Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. Methods: Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. Results: Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. Conclusion: Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.
Bibliographical noteFunding Information:
Science Foundation of China (Grant No. 81502213, to D. Li), KU Leuven grant GOA/15/017 (to FC), and the National Cancer Institute (CA092236, to D. Liao). This work was also supported by the Focused Research and Development Program of Shandong Province (grant No: 2016GSF201171, to D. Li). Dawei Li and Jia Wang were supported by a scholarship from China Scholarship Council (CSC).
Florida Breast Cancer Foundation; Bankhead-Coley Cancer Research Program, Grant numbers: 09BB-11, 09BW-05, 4BF02, 6BC03; KU Leuven, Grant number: GOA/15/ 017; The National Natural Science Foundation of China, Grant number: 81502213; James and Esther King Biomedical Research Program, Grant number: 6JK03; National Cancer Institute, Grant number: CA092236; Focused Research and Development Program, Grant number: 2016GSF201171
We thank Arnold Berk, Hancheng Guan, Jianrong Lu, and David Ornelles for providing reagents. The work was supported by grants from Bankhead-Coley Cancer Research Program, and James and Esther King Biomedical Research Program, Florida Department of Health (09BB-11, 09BW-05, 4BF02, 6BC03, and 6JK03 to D. Liao), the Florida Breast Cancer Foundation (to D. Liao), the National Natural
- AR splice variants (AR-Vs)
- PI3K-kinase (PI3K)-Akt-mTOR signaling
- androgen receptor
- prostate cancer gene therapy
- transcriptional regulation