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Setiawan, Josie

University of British Columbia

Background: Advanced prostate cancer continues to pose a significant health burden due to therapeutic resistance. Although androgen deprivation and antiandrogen therapies directed at the androgen receptor (AR) C-terminal ligand-binding domain (LBD) initially benefit patients, they eventually develop resistance and progress to lethal metastatic castration-resistant prostate cancer. AR N-terminal domain (NTD) inhibitors were developed to overcome resistance mechanisms related to the AR-LBD, such as mutations and splice variants lacking the LBD (AR-V7). While LBD mutations have been well-characterized, the impact of NTD mutations remains unclear. Tryptophan residues W435 and W397 are essential in the binding site for the AR-NTD inhibitor EPI-7386, currently in clinical trials. We hypothesize that tryptophan mutations within the EPI-7386 binding site will alter AR transcriptional activity and reduce the potency of AR-targeting therapeutics. Methods: Site-directed mutagenesis was used to create full-length AR (FL-AR) and AR-V7 expression vectors with point mutations at W435 and W397. FL-AR plasmids were co-transfected into CV-1 or PC-3 cells with an AR-driven luciferase reporter. The mammalian two-hybrid system assay was used to assess the interaction between AR’s NTD and LBD (N/C interaction). AR-V7 expression plasmids were co-transfected into LNCaP cells with an AR-V7 specific reporter. IC50s for a panel of AR inhibitors were generated to assess the impact of tryptophan mutations in FL-AR and AR-V7. Results: Tryptophan mutations in FL-AR led to reporter-specific and cell-specific increases in transcriptional activity in response to androgen, notably W397G, W435L and W435C. In particular, W435L enhanced AR N/C interactions. These mutations reduced the potency of EPI-7386 to inhibit ARR3tk- and PSA-luciferase activities, but not other EPI analogs. In AR-V7, the W397G and W435G mutations impacted the potencies of EPI-7386 and another AR-NTD inhibitor, EPI-7170. Conclusions: Tryptophan residues in the AR-NTD play an important role in FL-AR and AR-V7 transcriptional activities. Key distinctions were revealed between AR inhibitors that suggest distinct mechanisms of drug action. Future work will investigate the recruitment of coregulatory proteins to the NTD region in which W397 and W435 reside. Ultimately, this work provides valuable insight into the development of novel treatment options for the terminal stage of prostate cancer where existing therapies fail.

Text

2024-03-28

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87649

Pathology and Laboratory Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/