Tolerance of SOX2 and BRN2 in Prostate Cancer Cell Lines: Effects on Cell Cycle and Lineage Transformation

Prostate cancer (PCa) is the most prevalent malignancy in American men excluding skin cancer. In metastatic disease, resistance to androgen pathway inhibitors (APIs) is a major obstacle to curative treatment. One such resistance mechanism is the histological transformation from adenocarcinoma to a neuroendocrine (NE) phenotype, driven by transcription factors like SOX2 and BRN2. Of note, SOX2 is typically overexpressed in PCa lacking functional Rb and p53 and can regulate cell cycle proteins including Cyclin D1. These findings warrant an investigation into a potential connection between SOX2, the cell cycle, and tumor suppressors to further establish molecular mechanisms of neuroendocrine differentiation (NED). We also plan to investigate the potential role of BRN2 in this process. Lentiviral Tet-On® systems were cloned and transduced into the PCa cell lines LNCaP (WT RB1, WT TP53) and DU145 (RB1 -/-, TP53 mutant) for the inducible expression of SOX2 (iSOX2) and BRN2 (iBRN2). In vitro growth assays were performed by plating 5, 000 cells/well in a 96 well plate and adding doxycycline the following day (DOX). Western blots were performed to determine protein expression. Cell cycle analysis was performed by PI staining and flow cytometry. Inducible models showed robust induction of SOX2 or BRN2 after 24h induction. At 4d, iBRN2 cell lines demonstrated NE marker upregulation, specifically synaptophysin. SOX2, but not BRN2, induction decreased Cyclin D1 expression irrespective of Rb and p53 status. Intriguingly, LNCaP iSOX2 cells were less viable following induction and had distinct morphological changes including the loss of their stellate shape. LNCaP iBRN2 cells had less profound, but apparent, changes in morphology. No significant morphological changes were identified in DU145. Cell cycle analysis found a greater percentage of LNCaP iSOX2 cells in the sub-G1 state and a lesser percentage in the G2/M phase versus the empty vector. These results suggest that SOX2 overexpression (OE) may only be tolerated in an Rb and/or p53 deficient setting. Further research will seek to determine if this phenomenon is due to SOX2-induced repression of Cyclin D1. Subsequent experiments will use RB1 and/orTP53 KO cell lines to generate new SOX2 and BRN2 inducible systems. Novel experiments will seek to determine if RB1 and/or TP53 KO increases tolerance to SOX2 OE. Connecting this knowledge of the SOX2 protein to the cell cycle and hormonal signaling in PCa may offer insight into the interplay between emerging cell cycle-targeting therapeutics and the NED process. Connor Purcell, Praveen R. Srinivasan, Shengliang Zhang, Maximilian Pinho-Schwermann, William J. MacDonald, Lanlan Zhou, Andrea Schmidt, Claire Lin, Xiaobing Tian, Tyler J. Roady, Vida Tajiknia, Viva Voong, Wafik El-Deiry. Tolerance of SOX2 and BRN2 in prostate cancer cell lines: effects on cell cycle and lineage transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1458.