Abstract 6798: Targeting SMAD4<i>L535fs</i> in LNCaP prostate cancer cells: Implications for therapy resistance and sensitivity

Abstract Introduction: Prostate cancer is the most common form of cancer and a leading cause of cancer-related death in men worldwide. This disease progresses from an androgen-dependent, non-metastatic state to a highly aggressive, metastatic, androgen-independent phenotype through various intrinsic genetic alterations. One crucial genetic alteration involves SMAD4, a tumor suppressor in the TGF-β signaling pathway, which plays a significant role in promoting aggressive tumor progression. Recent studies have reported that the knockdown of SMAD4 in prostate cancer cells, such as LNCaP, promotes resistance to therapies, including androgen deprivation therapy (ADT) and chemotherapy. The SMAD4L535fs mutation has been implicated in altering cellular proliferation dynamics in LNCaP prostate cancer cells. This study investigates the impact of the SMAD4L535fs mutation on LNCaP prostate cancer cell proliferation and response to chemotherapeutic agents. Methods: LNCaP prostate cancer cells expressing SMAD4WT and mutant SMAD4L535fs were used to evaluate cell proliferation and viability. Cells were cultured under androgen deprivation conditions and treated with enzalutamide, gemcitabine, or ferroptosis inducers (erastin, ferrostatin-1). Cell viability and spheroid proliferation were assessed using CCK-8 and CellTiter-Glo assays over 72 hours. Apoptosis induction by enzalutamide was evaluated using Annexin V/PI staining. To assess the effects of DNA damage, LNCaP cells were treated with gemcitabine (10 μM), a DNA-damaging agent. For ferroptosis assessment, LNCaP cells were treated with ferroptosis inducers, including erastin (10 μM) and ferrostatin-1 (10 μM), for 72 hours. Results: SMAD4L535fs significantly enhanced the proliferation of LNCaP spheroids compared to the wild type under androgen deprivation and enzalutamide treatment.Viability assays demonstrated increased resistance to gemcitabine in SMAD4L535fs cells, suggesting a role for SMAD4L535fs in mediating chemoresistance.LNCaP cells treated with ferroptosis inducers showed differential responses, indicating that SMAD4L535fs may influence susceptibility to ferroptosis. Conclusion: SMAD4L535fs enhances the proliferation and chemoresistance of LNCaP prostate cancer cells, providing insight into potential therapeutic targets for overcoming drug resistance. Further studies are required to elucidate the molecular mechanisms underlying SMAD4L535fs-mediated ferroptosis resistance. Acknowledgment: This project is supported by the Research Impact Fund (Ref. No.: R4015-19) from the Research Grants Council in Hong Kong Citation Format: Chizhe Yi, Cindy Sin Ting Chow, Stephen Kwok Wing Tsui. Targeting SMAD4 L535fs in LNCaP prostate cancer cells: Implications for therapy resistance and sensitivity [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 6798.