DNA-PKc inhibition overcomes taxane resistance by promoting taxane-induced DNA damage in prostate cancer cells
DNA-PKc inhibition overcomes taxane resistance by promoting taxane-induced DNA damage in prostate cancer cells
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DOI：
10.1002/pros.24200
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发表时间：
2021-07-23
期刊：
影响因子：
2.8
通讯作者：
Goodman, Oscar B., Jr.
中科院分区：
文献类型：
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作者：
Chao, Olivia S.;Goodman, Oscar B., Jr.
背景 克服紫杉烷耐药性仍然是转移性去势抵抗性前列腺癌（mCRPC）的主要临床挑战。 DNA 修复蛋白的缺失与抗微管药物的耐药性相关。我们提出 DNA 损伤反应 (DDR) 途径的改变有助于紫杉烷耐药，并且这些改变的鉴定可能提供潜在的治疗靶点，使多西紫杉醇难治性 mCRPC 对基于紫杉烷的治疗重新敏感。方法通过 DDR 途径特异性聚合酶链式反应阵列和免疫印迹测定源自 DU-145 细胞的多西他赛耐药前列腺癌细胞系模型 (DU145-DxR) 中 DDR 基因表达的变化。编码 DNA-PKc（DNA 依赖性蛋白激酶催化单元）的 PRKDC 基因被注意到过度表达，并评估了其在多西他赛耐药性中的作用。用三种不同的抑制剂（NU7441、LTURM34 和 M3814）对 DNA-PKc 进行药理学抑制后，评估多西紫杉醇处理的 DU145-DxR 细胞的细胞活力和克隆存活率。还测试了 DNA-PKc 抑制对二线细胞毒性药物、卡巴他赛和依托泊苷的反应。通过彗星试验和双链断裂标记、γ H2AX 和 Rad51 分析评估 DNA-PKc 上调对 DNA 损伤修复的影响。最后，通过罗丹明 123 外排测定评估 DNA-PKc 抑制剂对 MDR1 活性的影响。结果 DDR 通路特异性基因分析显示 DU145-DxR 细胞中 PRKDC 和 CDK7 显着上调，MSH3 下调。与亲代 DU145 相比，当暴露于依托泊苷和多西紫杉醇时，DU145-DxR 细胞遭受的 DNA 损伤显着减少。使用所有三种抑制剂对 DNA-PKc（NHEJ 修复机制的一个组成部分）进行药理学抑制，可显着使 DU145-DxR 细胞对多西紫杉醇重新敏感。此外，DNA-PKc 抑制还使 DU145-DxR 对卡巴他赛和依托泊苷重新敏感，这表现出交叉耐药性。 DNA-PKc 的抑制导致依托泊苷和多西紫杉醇处理的 DU145-DxR 细胞中 DNA 损伤增加。最后，DNA-PKc 抑制不会影响 MDR1 活性，表明 DNA-PKc 抑制剂通过独立于 MDR1 的机制使紫杉烷耐药细胞重新敏感。结论 本研究支持 DDR 基因，特别是 DNA-PKc 在促进 mCRPC 中紫杉烷类耐药性中的作用。靶向前列腺 DNA-PKc 可能提供一种新策略来恢复紫杉烷难治性 mCRPC 中的紫杉烷敏感性。
Background Overcoming taxane resistance remains a major clinical challenge in metastatic castrate-resistant prostate cancer (mCRPC). Loss of DNA repair proteins is associated with resistance to anti-microtubule agents. We propose that alterations in DNA damage response (DDR) pathway contribute to taxane resistance, and identification of these alterations may provide a potential therapeutic target to resensitize docetaxel-refractory mCRPC to taxane-based therapy. Methods Alterations in DDR gene expression in our prostate cancer cell line model of docetaxel-resistance (DU145-DxR) derived from DU-145 cells were determined by DDR pathway-specific polymerase chain reaction array and immunoblotting. The PRKDC gene encoding DNA-PKc (DNA-dependent protein kinase catalytic unit), was noted to be overexpressed and evaluated for its role in docetaxel resistance. Cell viability and clonogenic survival of docetaxel-treated DU145-DxR cells were assessed after pharmacologic inhibition of DNA-PKc with three different inhibitors-NU7441, LTURM34, and M3814. Response to second-line cytotoxic agents, cabazitaxel and etoposide upon DNA-PKc inhibition was also tested. The impact of DNA-PKc upregulation on DNA damage repair was evaluated by comet assay and analysis of double-strand breaks marker, gamma H2AX and Rad51. Lastly, DNA-PKc inhibitor's effect on MDR1 activity was assessed by rhodamine 123 efflux assay. Results DDR pathway-specific gene profiling revealed significant upregulation of PRKDC and CDK7, and downregulation of MSH3 in DU145-DxR cells. Compared to parental DU145, DU145-DxR cells sustained significantly less DNA damage when exposed to etoposide and docetaxel. Pharmacologic inhibition of DNA-PKc, a component of NHEJ repair machinery, with all three inhibitors, significantly resensitized DU145-DxR cells to docetaxel. Furthermore, DNA-PKc inhibition also resensitized DU145-DxR to cabazitaxel and etoposide, which demonstrated cross-resistance. Inhibition of DNA-PKc led to increased DNA damage in etoposide- and docetaxel-treated DU145-DxR cells. Finally, DNA-PKc inhibition did not affect MDR1 activity, indicating that DNA-PKc inhibitors resensitized taxane-resistant cells via an MDR1-independent mechanism. Conclusion This study supports a role of DDR genes, particularly, DNA-PKc in promoting resistance to taxanes in mCRPC. Targeting prostatic DNA-PKc may provide a novel strategy to restore taxane sensitivity in taxane-refractory mCRPC.