Molecular analysis of androgen receptor splice variant AR-V3 reveals eminent ambiguity regarding activity and clinical utility

Background: Androgen Receptor (AR) splice variants (AR-Vs) have emerged as potential resistance mechanisms to AR-targeted agents (ARTAs) in prostate Cancer (PC), particularly in the context of castration-resistant disease. Among them, AR-V3 has been frequently detected, yet its biological function remains unclear due to conflicting results from initial studies. This study aimed to comprehensively investigate the molecular structure, activity, and clinical relevance of AR-V3.

Methods: We constructed plasmids encoding two AR-V3 isoforms-one newly identified isoform matching the human reference genome (AR-V3^ref) and the Other based on the 22Rv1 cell line sequence (AR-V3^22Rv1)-and transfected them into AR-negative PC-3 cells alone or co-expressed with AR full-length (AR-FL). Localization, transcriptional activity (via luciferase assays), and RNA Sequencing were performed. Protein structure modeling was conducted using AlphaFold2. Nonsense-mediated decay (NMD) was assessed through pharmacological inhibition. Clinically, AR-V3 expression in circulating tumor cells (CTCs) from 65 patients starting ARTA treatment was analyzed in relation to progression-free survival (PFS) and overall survival (OS).

Results: RNA-seq of AR-FL + AR-V3 vs. AR-FL alone showed no AR-V3-specific gene expression. Structure modeling revealed poor overall prediction confidence, particularly in the N-terminal domain, with no consistent structural features differentiating AR-V3^ref and AR-V3^22Rv1. Both isoforms localized mainly to the cytoplasm, regardless of hormonal stimulation or AR-FL co-expression. Neither isoform showed Androgen Receptor element (ARE) binding activity unless co-expressed with AR-FL. NMD analysis indicated neither isoform was degraded by this pathway. Clinically, AR-V3 + patients had significantly shorter OS (median 13 vs. 23 months; p = 0.02) among CTC + patients but showed no difference in PSA response or PFS under ARTA treatment.

Conclusions: Our data demonstrate that both AR-V3 isoforms are functionally inactive, lacking autonomous nuclear translocation or transcriptional activity. AR-V3 is not a substrate for NMD, and its protein structure remains poorly defined. While associated with worse overall survival, AR-V3 lacks predictive value for ARTA response, underscoring its limited utility as a biomarker. These findings emphasize the need for functional validation before integrating putative biomarkers into clinical practice.

Androgen receptor; Androgen receptor splice variant 3; Androgen receptor targeted agents; Biomarker; Liquid biopsy; Metastatic castration resistant prostate cancer; Predictive; Prognostic.