Abstract 2294: Examining the effect of second-generation antiandrogens in androgen receptor-overexpressing prostate cancer cells

Abstract Antiandrogens inhibit androgen receptor (AR) signaling, a critical driver of prostate cancer (PCa) growth and survival. While these agents are initially effective, prolonged therapy frequently results in resistance, often associated with AR overexpression or the emergence of constitutively active splice variants. To better understand how AR overexpression influences antiandrogen efficacy, we examined cell proliferation and AR target gene expression under second-generation antiandrogen treatment in AR-high PCa cells. In this study, we used darolutamide, enzalutamide, and apalutamide and systematically compared their effects on cell proliferation and AR-mediated gene expression using identical experimental setups. LNCaP-ARhi cells, expressing 4-6-fold higher AR than their parental cell line LNCaP-pcDNA3.1, were hormone-deprived and treated with increasing concentrations (0.001 nM-10 nM) of the synthetic androgen, R1881. Cell proliferation and morphology were monitored with Incucyte phase-contrast imaging for 7 days. Direct AR target gene expression (KLK3 and FKBP5) was quantified by qPCR. For co-treatment, we used two R1881 concentrations (0.01 nM and 0.1 nM): the lower promoted proliferation, the higher suppressed it, and both significantly increased KLK3 expression in AR-overexpressing cells. The cells were hormone-deprived, then treated with DMSO, 0.01 nM or 0.1 nM R1881, and antiandrogens (1 μM, 10 μM, and 25 μM). Cell proliferation and morphology were monitored for 5 days, and AR target gene expression was measured as previously described. Our results support previous findings that AR overexpression enhances androgen responsiveness and sustains cell growth even under low androgen concentrations (0.01 nM R1881). We also found that LNCaP-ARhi cells shifted from elongated, spindle-shaped cells to a flattened, polygonal morphology at R1881 0.03 nM-10 nM. Co-treatment with R1881 and antiandrogens restored the original morphology. All tested second-generation antiandrogens reduced R1881-stimulated growth and KLK3/FKBP5 gene expression in both cell lines, suggesting effective AR signaling inhibition in a dose-dependent manner. However, in LNCaP-ARhi cells, KLK3 expression remained elevated compared to DMSO control even at high antiandrogen concentrations. Overall, second-generation antiandrogens suppress AR signaling and growth in PCa cells, but AR overexpression may limit inhibitory efficacy, highlighting the need for strategies targeting AR-high conditions. Citation Format: Kia Vaalavirta, Hanna Rauhala, Tapio Visakorpi, . Examining the effect of second-generation antiandrogens in androgen receptor-overexpressing prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2294.