Epigenetic reprogramming via DNMT1 inhibition reduces invasion and migration in docetaxel-resistant prostate cancer

Background: Metastatic castration-resistant prostate cancer (mCRPC) is associated with poor prognosis and high mortality, largely due to the emergence of androgen and chemotherapy resistance. Resistance to docetaxel occurs in nearly 50% of patients and remains a major clinical challenge. DNA methylation is known to contribute to therapy resistance through gene silencing and reprogramming of cellular pathways. This study investigated whether global DNA hypomethylation achieved through DNMT1 inhibition could reduce cell invasion and motility in docetaxel-resistant (DR) prostate cancer (PCa) cells. Methods: Parental and docetaxel-resistant cells (22Rv1, PC3, and DU145) were treated with 5-AZA (0.5-16 µM, 72 h). Viability was measured by MTT. Protein markers (AR, ARv7, MDR1, GSTP1, DNMT1) were assessed by Western blot; invasion and migration by Matrigel and wound-healing assays; and EMT markers ( CDH1 , SNAI2 , VIM ) by RT-PCR. Results: Treatment with 5-AZA reduced cell viability in a dose-dependent manner in all cell models except DU-145DR cells. DNMT1 inhibition was confirmed in all cell lines, while re-expression of GSTP1, indicating successful DNA hypomethylation, was observed in DU145-DR and PC3/PC3-DR cells. No changes were observed in AR, AR-V7, or MDR1 protein expression. Invasion capacity was significantly reduced in DU145-DR (1 µM: p<0.05; 2 µM: p<0.0001) and PC3-DR (1 µM: p<0.0001; 2 µM: p<0.01) cells. Migration capacity was also reduced in DU145-DR and PC3-DR cells following 5-AZA treatment. 5-AZA induced upregulation of CDH1 (DU145-DR: p<0.05; PC3-DR: p<0.01) and downregulation of TJP1 (DU145-DR: p<0.01; PC3-DR: p<0.01) compared to parental cells. Additionally, SNAI2 (p<0.05) and VIM (p<0.01) were significantly downregulated only in DU145-DR cells, suggesting a cell line-specific modulation of epithelial-mesenchymal transition (EMT) markers. Conclusions: These findings demonstrate that DNMT1 inhibition modulates EMT-related pathways, leading to reduced invasion and migrations in docetaxel-resistant PCa cells. The effect of 5-AZA appears to be mediated by upregulation of CDH1 and suppression of SNAI2 and VIM , highlighting the role of DNA methylation as a regulator of metastatic potential. This study provides mechanistic insight into the epigenetic regulation of therapy resistance and supports the potential of DNMT1 inhibition as a strategy to mitigate aggressive behavior in docetaxel-resistant mCRPC. Funding: Sponsored by U54 PHSU-MCC Grants: U54CA163071 & U54CA163068; NIH-NIMHD Grant MD007579, and NIH-NIGMS Grant U54GM133807.