National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
Department of Oncology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
J Biomed Sci. 2021 Jan 4;28(1):2. doi: 10.1186/s12929-020-00699-y.
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.
同源重组（HR）途径参与 DNA 损伤反应（DDR），这对于 DNA 损伤剂治疗后癌细胞的存活至关重要。O6-甲基鸟嘌呤 DNA 甲基转移酶（MGMT）与癌细胞中顺铂（CDDP）耐药有关；然而，其潜在机制尚不清楚。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 NPC 细胞。通过 Western blot、定量实时 PCR 和公共数据库的生物信息学分析分析 MGMT 与 HR 途径主要蛋白之间的表达相关性。使用免疫共沉淀和免疫荧光分析研究 MGMT 与 HR 蛋白之间的物理相互作用。通过细胞彗星尾巴和γ-H2AX 表达水平评估双链断裂（DSB）的形成。进行了已建立的免疫荧光和报告分析，以测量 HR 活性。分别使用异种移植和细胞活力研究来评估 MGMT 抑制与 CDDP 和聚（ADP-核糖）聚合酶（PARP）抑制剂联合治疗的治疗潜力。
在 HR 途径的主要蛋白中，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 表达呈正相关。此外，MGMT 与 BRCA1 发生物理相互作用，并调节 CDDP 诱导的 BRCA1 磷酸化（ser988）。在功能测定中，MGMT 抑制通过减弱 HR 活性增加了 CDDP 诱导的 DSB 形成。NPC 异种移植研究表明，MGMT 抑制联合 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 对于 CDDP 和 PARP 抑制剂处理的 NPC 细胞中 HR 途径的激活和 DDR 调节至关重要。因此，MGMT 是涉及 HR 相关 DDR 的癌症治疗的有前途的治疗靶标。
