Shang-Hung Chen1,2, Wen-Tsung Huang3, Wan-Chen Kao3, Sheng-Yen Hsiao3,4, Hsin-Yi Pan1, Chin-Wen Fang1, Yow-Ling Shiue5, Chia-Lin Chou6,7, Chien-Feng Li8,9,10. 1. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan. 2. Department of Oncology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan. 3. Division of Hematology-Oncology, Department of Internal Medicine, Chi Mei Medical Center, Liouying, Tainan, Taiwan. 4. Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan. 5. Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan. 6. Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan. clchou3@gmail.com. 7. Division of Colon and Rectal Surgery, Department of Surgery, Chi Mei Medical Center, No. 901, Zhonghua Rd., Yongkang Dist., Tainan, 71004, Taiwan. clchou3@gmail.com. 8. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan. angelo.p@yahoo.com.tw. 9. Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan. angelo.p@yahoo.com.tw. 10. Institute of Precision Medicine, National Sun Yat-sen University, No.70, Lien-hai Rd., Kaohsiung, 80424, Taiwan. angelo.p@yahoo.com.tw.
Abstract
BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: 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). METHODS:HumanNPC 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. RESULTS: 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. CONCLUSION: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.
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