The homologous recombination (HR) pathway is involved in DNA damage response (DDR), which is crucial to cancer cell survival after treatment with DNA damage agents. O6-methylguanine DNA methyltransferase (MGMT) is associated with cisplatin (CDDP) resistance in cancer cells; however, the underlying mechanisms remain unclear. Here, we explored the interactions between MGMT and the HR pathway in CDDP-activated DDR and their clinical implications in nasopharyngeal carcinoma (NPC). Human NPC cells were assessed using loss-of-function approaches in vitro. The expression correlations between MGMT and major proteins of the HR pathway were analyzed through Western blotting, quantitative real-time PCR, and bioinformatic analysis by using a public database. The physical interactions between MGMT and HR proteins were studied using co-immunoprecipitation and immunofluorescence analyses. Cell comet tails and γ-H2AX expression levels were examined to evaluate double-strand break (DSB) formation. Established immunofluorescence and reporter analyses were conducted to measure HR activity. Xenograft and cell viability studies were used to assess the therapeutic potential of MGMT inhibition in combination with CDDP and poly(ADP-ribose) polymerase (PARP) inhibitor, respectively. Among major proteins of the HR pathway, MGMT suppression inhibited CDDP-induced RAD51 expression. Bioinformatic analyses showed a positive correlation between MGMT and RAD51 expression in patients with NPC. Moreover, MGMT physically interacted with BRCA1 and regulated CDDP-induced BRCA1 phosphorylation (ser 988). In functional assays, MGMT inhibition increased CDDP-induced DSB formation through attenuation of HR activity. NPC xenograft studies demonstrated that MGMT inhibition combined with CDDP treatment reduced tumor size and downregulated RAD51 expression and BRCA1 phosphorylation. Furthermore, MGMT suppression increased PARP inhibitor–induced cell death and DSB formation in NPC cells. MGMT is crucial in the activation of the HR pathway and regulates DDR in NPC cells treated with CDDP and PARP inhibitor. Thus, MGMT is a promising therapeutic target for cancer treatments involving HR-associated DDR.

中文翻译：

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O6-甲基鸟嘌呤-DNA甲基转移酶通过靶向鼻咽癌的同源重组途径调节顺铂诱导的DNA双链断裂

同源重组 (HR) 途径参与 DNA 损伤反应 (DDR)，这对用 DNA 损伤剂治疗后的癌细胞存活至关重要。O6-甲基鸟嘌呤 DNA 甲基转移酶 (MGMT) 与癌细胞中的顺铂 (CDDP) 抗性有关；然而，潜在的机制仍不清楚。在这里，我们探讨了 MGMT 与 CDDP 激活的 DDR 中 HR 途径之间的相互作用及其在鼻咽癌 (NPC) 中的临床意义。在体外使用功能丧失方法评估人类 NPC 细胞。MGMT 与 HR 通路主要蛋白之间的表达相关性通过蛋白质印迹、实时定量 PCR 和生物信息学分析使用公共数据库进行分析。使用免疫共沉淀和免疫荧光分析研究了 MGMT 和 HR 蛋白之间的物理相互作用。检查细胞彗尾和 γ-H2AX 表达水平以评估双链断裂 (DSB) 的形成。进行已建立的免疫荧光和报告基因分析以测量 HR 活性。异种移植和细胞活力研究分别用于评估 MGMT 抑制与 CDDP 和聚（ADP-核糖）聚合酶 (PARP) 抑制剂组合的治疗潜力。在 HR 通路的主要蛋白质中，MGMT 抑制抑制了 CDDP 诱导的 RAD51 表达。生物信息学分析显示 NPC 患者中 MGMT 和 RAD51 表达之间呈正相关。此外，MGMT 与 BRCA1 物理相互作用并调节 CDDP 诱导的 BRCA1 磷酸化（ser 988）。在功能分析中，MGMT 抑制通过降低 HR 活性增加了 CDDP 诱导的 DSB 形成。NPC 异种移植研究表明，MGMT 抑制结合 CDDP 治疗可减小肿瘤大小并下调 RAD51 表达和 BRCA1 磷酸化。此外，MGMT 抑制增加了 NPC 细胞中 PARP 抑制剂诱导的细胞死亡和 DSB 形成。MGMT 在 HR 通路的激活中至关重要，并在用 CDDP 和 PARP 抑制剂处理的 NPC 细胞中调节 DDR。因此，MGMT 是涉及 HR 相关 DDR 的癌症治疗的有希望的治疗靶点。MGMT 抑制增加了 NPC 细胞中 PARP 抑制剂诱导的细胞死亡和 DSB 形成。MGMT 在 HR 通路的激活中至关重要，并在用 CDDP 和 PARP 抑制剂处理的 NPC 细胞中调节 DDR。因此，MGMT 是涉及 HR 相关 DDR 的癌症治疗的有希望的治疗靶点。MGMT 抑制增加了 NPC 细胞中 PARP 抑制剂诱导的细胞死亡和 DSB 形成。MGMT 在 HR 通路的激活中至关重要，并在用 CDDP 和 PARP 抑制剂处理的 NPC 细胞中调节 DDR。因此，MGMT 是涉及 HR 相关 DDR 的癌症治疗的有希望的治疗靶点。