PO.MCB06.02 · 分子与细胞生物学

通过抑制DNMT1进行表观遗传重编程可降低多西他赛耐药前列腺癌的侵袭和迁移

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

编号 1968 展板 20 时间 4/20 09:00–12:00 区域 Section 22 主讲 Carmen Ortiz-Sanchez, PhD

分会场 DNA Methylation

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作者与单位 Authors & Affiliations

Carmen M. Ortiz-Sanchez¹, Gabriela Castro², Shakira Abad², Solimar Esteves¹, Valerie Rodriguez¹, Stephanie Montalvo³, Rodniel Aviles¹, Ralphdy Vergne¹, Gustavo Alayón¹, Caleb Santiago¹, Lenin Godoy¹, Kosj Yamoah⁴, Gilberto Ruiz-Deya¹, Jong Y. Park⁴

¹Ponce Health Sciences University, Ponce, Puerto Rico,²Pontificia Universidad Católica de Puerto Rico, Ponce, Puerto Rico,³University of Puerto Rico at Mayaguez, Mayaguez, Puerto Rico,⁴H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL

摘要 Abstract

中文摘要

背景：转移性去势抵抗性前列腺癌（mCRPC）预后差、死亡率高，主要归因于雄激素和化疗耐药的出现。近50%的患者会发生多西他赛耐药，这仍是一项重大的临床挑战。已知DNA甲基化通过基因沉默和细胞通路重编程促进治疗耐药。本研究探讨了通过抑制DNMT1实现的DNA整体低甲基化能否降低多西他赛耐药（DR）前列腺癌（PCa）细胞的侵袭和运动能力。方法：对亲代细胞和多西他赛耐药细胞（22Rv1、PC3和DU145）使用5-AZA（0.5–16 µM，72 h）处理。采用MTT法测定活力。通过Western blot评估蛋白标志物（AR、ARv7、MDR1、GSTP1、DNMT1）；通过Matrigel和划痕愈合实验评估侵袭和迁移；通过RT-PCR评估EMT标志物（CDH1、SNAI2、VIM）。结果：除DU-145DR细胞外，5-AZA处理在所有细胞模型中均以剂量依赖方式降低细胞活力。在所有细胞系中均确认了DNMT1的抑制，而在DU145-DR和PC3/PC3-DR细胞中观察到GSTP1的重新表达，表明DNA低甲基化成功实现。AR、AR-V7或MDR1蛋白表达未见变化。DU145-DR（1 µM：p<0.05；2 µM：p<0.0001）和PC3-DR（1 µM：p<0.0001；2 µM：p<0.01）细胞的侵袭能力显著降低。5-AZA处理后，DU145-DR和PC3-DR细胞的迁移能力也降低。与亲代细胞相比，5-AZA诱导CDH1上调（DU145-DR：p<0.05；PC3-DR：p<0.01）和TJP1下调（DU145-DR：p<0.01；PC3-DR：p<0.01）。此外，仅在DU145-DR细胞中SNAI2（p<0.05）和VIM（p<0.01）显著下调，提示上皮-间质转化（EMT）标志物存在细胞系特异性的调节。结论：这些发现表明，DNMT1抑制可调节EMT相关通路，从而降低多西他赛耐药PCa细胞的侵袭和迁移。5-AZA的作用似乎是通过上调CDH1以及抑制SNAI2和VIM介导的，凸显了DNA甲基化作为转移潜能调控因子的作用。本研究为治疗耐药的表观遗传调控提供了机制层面的见解，并支持将DNMT1抑制作为减轻多西他赛耐药mCRPC侵袭性行为的策略的潜力。资助：由U54 PHSU-MCC资助：U54CA163071 与 U54CA163068；NIH-NIMHD资助 MD007579，以及NIH-NIGMS资助 U54GM133807。

查看英文原文 English abstract

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.

利益披露 Disclosure

C. M. Ortiz-Sanchez, None.. G. Castro, None.. S. Abad, None.. S. Esteves, None.. V. Rodriguez, None.. S. Montalvo, None.. R. Aviles, None.. R. Vergne, None.. G. Alayón, None.. C. Santiago, None.. L. Godoy, None.. K. Yamoah, None.. G. Ruiz-Deya, None.

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