Abstract Background: Prostate cancer (PCa) remains a significant health burden in the US, making it the second most frequently diagnosed malignancy in men. While the 5-year survival rate for localized tumors is 98%, metastatic prostate cancer presents a marked difference, with a 5-year survival rate of only 32%. The disparity in treatment effectiveness is largely due to low response rates and rapid resistance to docetaxel. Increasing evidence suggests that epigenetic mechanisms, particularly DNMT1-mediated hypermethylation, play a critical role in developing and maintaining this resistance by suppressing tumor-suppressor gene expression, leading to increased invasion, migration, and chemoresistance.Previous studies have shown that 5-azacytidine (5-AZA) can decrease epithelial-mesenchymal transition (EMT)-related markers and aggressive phenotypes in prostate cancer models. However, its effects in 3D spheroid systems, which better mimic the in vivo tumor microenvironment, remain unclear. Methods: To evaluate the impact of 5-AZA on epigenetic regulation, EMT markers, and aggressive phenotypes in 3D spheroids generated from PC3 and PC3-DR cells, spheroids were formed in ultra-low attachment plates and treated with 1-2 µM of 5-AZA. Protein expression of DNMT1 and resistance related markers was evaluated through Western blot. Results: PC3-DR spheroids in Matrigel showed strong invasion at 24 hours with outward cellular extensions and interaction with the matrix. Treatment with 5-AZA reduced this invasion in a dose-dependent manner. Protein analysis revealed significant suppression of DNMT1 in nuclear extracts, confirming effective targeting of epigenetic factors, whereas MDR1 remained abundant in cytosolic fractions, consistent with its role in chemoresistance. 5-AZA induced the re-expression of GSTP1 and shifted EMT programs toward an epithelial phenotype, corroborating previously reported findings in 2D models. Notably, PC3-DR spheroids demonstrated a stronger response to treatment than the parental PC3 cells, suggesting a greater reliance on methylation-driven pathways. Conclusions: Overall, 5-AZA appears to reverse aggressive phenotypes in 3D spheroid models of docetaxel-resistant prostate cancer by reducing DNMT1 activity, restoring epithelial identity, and suppressing invasion. These findings support the use of epigenetic modulation as a potential therapeutic strategy to overcome chemoresistance and emphasize the importance of 3D systems for modeling treatment-resistant diseases. Future studies integrating genome-wide methylation profiling and in vivo validation are needed to establish 5-AZA as a viable strategy to counteract docetaxel resistance in metastatic prostate cancer. Sponsored by U54 PHSU-MCC Grants: U54CA163071 NIH-NIMHD Grant MD007579, and NIH-NIGMS Grant U54GM133807. Citation Format: Alanis Paola Torres Rosado, Gabriela Castro Morales, Lenin J. Godoy, Jong Y. Park, Gilberto Ruiz Deya, Carmen M. Ortiz-Sanchez. Epigenetic modulation with 5-Azacytidine reduces aggressive phenotypes in 3D spheroid models of docetaxel-resistant prostate cancer 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 1970.
Rosado et al. (Fri,) studied this question.