MOLECULAR CANCER THERAPEUTICS | CANCER BIOLOGYAND TRANSLATIONAL STUDIES V-ATPase Inhibition Decreases Mutant Androgen Receptor Activity in Castrate-resistant Prostate Cancer

◥ Prostate cancer is critically dependent on androgen receptor (AR) signaling. Despite initial responsiveness to androgen deprivation, most patients with advanced prostate cancer subsequently progress to a clinically aggressive castrate-resistant prostate cancer (CRPC) phenotype, typically associated with expression of splicevariant or mutant AR forms. Although current evidence suggests that the vacuolar-ATPase (V-ATPase), a multiprotein complex that catalyzes proton transport across intracellular and plasma membranes, influences wild-type AR function, the effect of V-ATPase inhibition on variant AR function is unknown. Inhibition of V-ATPase reduced AR function in wild-type and mutant AR luciferase reporter models. In hormone-sensitive prostate cancer cell lines (LNCaP, DuCaP) and mutant AR CRPC cell lines (22Rv1, LNCaP-F877L/T878A), V-ATPase inhibition using bafilomycin-A1 and concanamycin-A reduced AR expression, and expression of AR target genes, at mRNA and protein levels. Furthermore, combining chemical V-ATPase inhibition with the AR antagonist enzalutamide resulted in a greater reduction in AR downstream target expression than enzalutamide alone in LNCaP cells. To investigate the role of individual subunit isoforms, siRNA and CRISPR-Cas9 were used to target the V1C1 subunit in 22Rv1 cells. Whereas transfection with ATP6V1C1-targeted siRNA significantly reduced AR protein levels and function, CRISPR-Cas9– mediated V1C1 knockout showed no substantial change in AR expression, but a compensatory increase in protein levels of the alternate V1C2 isoform. Overall, these results indicate that V-ATPase dysregulation is directly linked to both hormone-responsive prostate cancer and CRPC via impact on AR function. In particular, V-ATPase inhibition can reduce AR signaling regardless of mutant AR expression. Introduction Prostate cancer was diagnosed in 1.28 million men and caused 359,000 deaths in 2018 (1). Initially, prostate cancer cell growth is dependent on androgen receptor (AR) activation. Advanced disease is therefore treated with permanent androgen deprivation therapy (ADT), to limit cancer cell growth (2). However, subsequent progression from a hormone-sensitive prostate cancer (HSPC) to a more aggressive castrate-resistantprostate cancer (CRPC)phenotype is almost inevitable. MetastaticCRPC treatment options include hormonal therapy (abiraterone, enzalutamide), chemotherapy, and radium-223. However, CRPC outcomes remain poor with a median survival of around 2 years (2). CRPC prognosis is partly attributed to persistent AR signaling through adaptive alterations to the AR itself. AR alterations are relatively rare at prostate cancer diagnosis but become more prevalent through ADT/hormonal therapy exposure and represent a key mechanism for transition to CRPC (3). Gain-of-function point mutations, particularly in the ligand-binding domain (LBD), may provide growth advantage despite a castrate state. For example, the F877LARmutation converts some second-generation antiandrogens (e.g., enzalutamide, apalutamide), to agonist function (4). The F877L mutation has been observed in enzalutamide-resistant prostate cancer xenograft models (5) and detected in plasma DNA of patients with CRPC previously treatedwith apalutamide (4). T878A is a further well-characterized AR point mutation (6) that converts AR antagonists (e.g., flutamide) to agonists (7), and broadens AR ligand binding specificity, for example to progesterone (8). This mutation was found in abiraterone-treated patients with CRPC producing a progesterone-activated abirateroneresistant state (9). AR splice variants (AR-Vs) are truncated AR forms, resulting in ligand-independent constitutive activation, through loss of an LBD portion and represent a further CRPC transitionmechanism (10). The most common, AR-V7, is associated with enzalutamide and abiraterone resistance (11), and reduced survival (12). AR-V7 expression is heterogeneous, and may heterodimerise with full-length AR, or form homodimers, to bind androgen response elements (AREs) to facilitate a protumorigenic transcriptome (13). AR-V7 mRNA expression has been found 20 times higher in patientswithCRPC than in patientswith HSPC (14), and protein levels of AR-V7 have been shown to increase on development of CRPC (15). Vacuolar-ATPase (V-ATPase) is a multiprotein complex that catalyzes ATP-dependent proton transport across intracellular and plasma membranes. The resulting acidification of organelle lumens or extracellular space influences a diverse range of cellular processes, many of which are dysregulated in cancers (16). V-ATPase is composed of two domains, the cytosolic V1 domain and the integral membrane complex, VO. V1, which is responsible for ATP hydrolysis, is composed of eight subunits (A–H), whereas VO, responsible for Cancer Sciences Unit, Southampton General Hospital, Southampton, United Kingdom. Cancer Research UK Centre, University of Southampton, Southampton General Hospital, Southampton, United Kingdom. School of Biological Sciences, University of Southampton, Southampton, United Kingdom. School of Life Sciences, University of Sussex, Falmer, Brighton, United Kingdom. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Simon J. Crabb, Cancer Sciences Unit, University of Southampton, Southampton General Hospital, Southampton, Hampshire SO16 6YD, United Kingdom. Phone: 44 238 120 5170; Fax: 44 844 774 0621; E-mail: s.j.crabb@southampton.ac. uk Mol Cancer Ther 2021;20:739–48 doi: 10.1158/1535-7163.MCT-20-0662 2021 American Association for Cancer Research. AACRJournals.org | 739 on April 18, 2021. © 2021 American Association for Cancer Research. mct.aacrjournals. org Downloaded from Published OnlineFirst February 9, 2021; DOI: 10.1158/1535-7163.MCT-20-0662