Hormones and Cancer Division, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, Australia.
Oncogene. 2014 Jan 2;33(1):85-96. doi: 10.1038/onc.2012.538. Epub 2012 Nov 26.
Following exposure to radiation and chemotherapeutic agents, the epidermal growth factor receptor (EGFR) can modulate the repair of DNA double-strand breaks (DSB) by forming protein complexes that include the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). This is one of the key mechanism by which tumors become resistant to DNA-damaging therapies. Our previous studies have shown that insulin-like growth factor binding protein-3 (IGFBP-3) is a substrate for DNA-PKcs, and can transactivate EGFR. We therefore questioned whether IGFBP-3 might interact with the EGFR-DNA-PK complex that regulates the DNA damage response. The aim of this study was to delineate the role of IGFBP-3 in the response of breast cancer cells to DSB-inducing chemotherapeutic agents. In the estrogen receptor-negative breast cancer cell lines MDA-MB-468 and Hs578T, which express IGFBP-3 highly, nuclear localization of EGFR and IGFBP-3 was enhanced by treatment with cytotoxic drugs etoposide or doxorubicin and reduced by the EGFR kinase inhibitor gefitinib. Enhanced association among IGFBP-3, EGFR and DNA-PKcs, following the exposure to DNA-damaging drugs was supported by both co-immunoprecipitation analysis and direct visualization by proximity ligation assay. The activation of DNA-PKcs at Ser2056, DNA repair as measured by a nonhomologous end-joining assay, and the increase in EGFR and DNA-PKcs interaction induced by DNA-damaging agents, were all decreased by IGFBP-3 silencing, suggesting that IGFBP-3 has an obligatory role in the DNA repair response to DNA-damaging therapy. In conclusion, IGFBP-3 co-translocation to the nucleus of breast cancer cells and its formation of a complex with DNA-PKcs and EGFR in response to DNA damage shows its potential involvement in the regulation of DNA repair. This suggests the possibility of a therapeutic approach for sensitizing breast cancer to chemo- or radiotherapy by targeting the DNA repair function of IGFBP-3.
在暴露于辐射和化疗药物后，表皮生长因子受体 (EGFR) 可以通过形成包括 DNA 依赖性蛋白激酶 (DNA-PK) 的催化亚基的蛋白复合物来调节 DNA 双链断裂 (DSB) 的修复。这是肿瘤对 DNA 损伤治疗产生耐药性的关键机制之一。我们之前的研究表明，胰岛素样生长因子结合蛋白-3 (IGFBP-3) 是 DNA-PKcs 的底物，并且可以转激活 EGFR。因此，我们质疑 IGFBP-3 是否可以与调节 DNA 损伤反应的 EGFR-DNA-PK 复合物相互作用。本研究旨在阐明 IGFBP-3 在乳腺癌细胞对诱导 DSB 的化疗药物的反应中的作用。在雌激素受体阴性的乳腺癌细胞系 MDA-MB-468 和 Hs578T 中，IGFBP-3 表达水平较高，细胞毒药物依托泊苷或阿霉素处理后 EGFR 和 IGFBP-3 的核定位增强，而 EGFR 激酶抑制剂吉非替尼处理后则减弱。共免疫沉淀分析和邻近连接分析直接可视化均支持 DNA 损伤药物暴露后 IGFBP-3、EGFR 和 DNA-PKcs 之间的增强关联。DNA-PKcs 在 Ser2056 的激活、非同源末端连接测定法测量的 DNA 修复以及 DNA 损伤诱导的 EGFR 和 DNA-PKcs 相互作用的增加，均因 IGFBP-3 沉默而降低，表明 IGFBP-3 在 DNA 损伤修复反应中具有必需的作用。总之，IGFBP-3 向乳腺癌细胞核的共易位及其在 DNA 损伤后与 DNA-PKcs 和 EGFR 形成复合物，表明其可能参与 DNA 修复的调节。这表明通过靶向 IGFBP-3 的 DNA 修复功能，有可能为乳腺癌对化疗或放疗的敏感性提供一种治疗方法。
