EGFR/ERCC1 相互作用在辐射诱导的 DNA 损伤后的重要性。

Importance of EGFR/ERCC1 interaction following radiation-induced DNA damage.

机构信息

Authors' Affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, United Kingdom.

Authors' Affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, United Kingdom

出版信息

Clin Cancer Res. 2014 Jul 1;20(13):3496-506. doi: 10.1158/1078-0432.CCR-13-2695. Epub 2014 Apr 29.

DOI:10.1158/1078-0432.CCR-13-2695

PMID:24780295

Abstract

PURPOSE

The epidermal growth factor receptor (EGFR) plays an important role in cellular response to chemotherapy and radiotherapy through modulation of DNA repair. EGFR activates DNA-dependent protein kinase (DNA-PK) stimulating repair of DNA strand breaks (SB) and interstrand crosslinks (ICL). We investigated the role of EGFR in repair of ionizing radiation (IR)-induced SB independently of DNA-PK.

EXPERIMENTAL DESIGN

The EGFR interactome was investigated via mass spectrometry. IR-induced EGFR-ERCC1 binding was validated biochemically and via proximity ligation assay in different cell lines including the M059K and M059J glioma cell lines, proficient and deficient for the expression of DNAPKcs, respectively. EGFR-ERCC1 functional significance following IR-induced SB was investigated in knockdown experiments with the Comet and γH2AX foci assays. The effect of this interaction was tested with EGFR-ERCC1 knockdown in combination with gefitinib and NU7026 using the MTT and apoptosis assays.

RESULTS

This study demonstrates that EGFR inhibition further impairs IR-induced DNA repair in cells lacking expression of DNAPKcs or in combination with the DNAPK inhibitor NU7026. Our data suggest a role for EGFR in DNA repair independent of DNAPKcs but dependent on ERCC1. Alkaline comet and γH2AX foci assays in cells depleted of EGFR, ERCC1, or EGFR-ERCC1 expression demonstrated involvement of this interaction in DNA repair. Cellular survival and apoptosis data correlate with levels of residual DNA damage underlying the importance of this complex following SB.

CONCLUSION

These data emphasize the importance of understanding the various mechanisms by which EGFR modulates DNA repair to optimize targeted therapy for patients with cancer.

摘要

目的

表皮生长因子受体（EGFR）通过调节 DNA 修复，在细胞对化疗和放疗的反应中发挥重要作用。EGFR 激活 DNA 依赖性蛋白激酶（DNA-PK），刺激 DNA 链断裂（SB）和链间交联（ICL）的修复。我们研究了 EGFR 在独立于 DNA-PK 的情况下修复电离辐射（IR）诱导的 SB 的作用。

实验设计

通过质谱法研究 EGFR 相互作用组。通过生化和邻近连接分析在不同细胞系中验证了 EGFR-ERCC1 结合，包括 M059K 和 M059J 神经胶质瘤细胞系，分别对 DNA-PKcs 的表达具有功能和缺乏功能。在使用 Comet 和 γH2AX 焦点分析的敲低实验中，研究了 IR 诱导的 SB 后 EGFR-ERCC1 的功能意义。用 EGFR-ERCC1 敲低与 gefitinib 和 NU7026 联合作用，用 MTT 和凋亡分析检测这种相互作用的效果。

结果

本研究表明，在缺乏 DNA-PKcs 表达的细胞中，或与 DNA-PK 抑制剂 NU7026 联合使用时，EGFR 抑制进一步损害了 IR 诱导的 DNA 修复。我们的数据表明，EGFR 在 DNA 修复中发挥作用，独立于 DNA-PKcs，但依赖于 ERCC1。在 EGFR、ERCC1 或 EGFR-ERCC1 表达被耗尽的细胞中的碱性彗星和 γH2AX 焦点分析表明，这种相互作用参与了 DNA 修复。细胞存活和凋亡数据与潜在 DNA 损伤水平相关，这凸显了该复合物在 SB 后对 DNA 修复的重要性。