Brd4A调节DNA损伤应答影响食管癌放疗敏感性的机制研究

Emerging evidence shows DNA damage response (DDR) is the main reason of tumor radiosensitivity. In our previous studies, we found that IGFBP-3/EGFR pathway plays an important role in the regulation of esophageal squamous cell carcinoma (ESCC). Bromodomain 4 (Brd4), which encodes two common isoforms A and C in ESCC, can modulate the signaling response to DNA damage response, but the mechanism is not well understood. Further preliminary experiments showed that Brd4A high expression in ESCC tissues can lead to a poor radiosensitivity. Furthermore, Brd4A is able to modulate DNA-PKcs expression, interact with IGFBP-3 and DNA-PKcs in ESCC cells, and also bind to chromatin in mitotic cells. As mentioned above, we put forward the scientific hypothesis that Brd4A promotes IGFBP-3/EGFR/DNA-PKcs complex formation by upregulating and collecting DNA-PKcs and participates irradiation-induced DNA damage repair in mitotic ESCC cells. To gain the proof of Brd4A modulating DDR in ESCC cells, we will perform GST pull-down, co-immunoprecipitation, FICCI system and construct Brd4 knockout ESCC cells by CRISPR/Cas9. We aim to provide new viewpoints for improvement of therapeutic approaches by targeting Brd4A to improve ESCC radiosensitivity. Meanwhile, we will lay the foundation for the subsequent development of specific drugs targeting on both IGFBP-3/EGFR pathway and Brd4A.

DNA损伤应答（DDR）与肿瘤放疗敏感性相关。我们前期发现IGFBP-3/EGFR是调节食管癌放疗敏感性的重要通路。文献报道溴结构域蛋白Brd4影响放疗诱导的DDR，但Brd4A和Brd4C亚型调节DDR的差异及机制不明。预实验证明：①食管鳞癌组织Brd4A高表达导致放疗敏感性下降；②内源性Brd4A影响DNA-PKcs表达，并与IGFBP-3和DNA-PKcs形成复合物，富集于有丝分裂期细胞染色质中。由此我们提出“Brd4A上调并募集DNA-PKcs，促进IGFBP-3/EGFR/DNA-PKcs复合物形成，参与有丝分裂期食管癌放疗诱导的DDR进程”的假说。我们进一步采用免疫共沉淀、FUCCI系统、CRISPR/Cas9基因编辑等技术，获得Brd4A调控食管癌放疗诱导DDR的证据，为靶向Brd4A的食管癌放疗增敏及后续 “IGFBP-3/EGFR通路和Brd4A”双靶点药物治疗奠定基础。

DNA损伤应答与肿瘤放疗敏感性相关。文献报道溴结构域蛋白Brd4影响放疗诱导的DNA损伤应答，但具体机制不明。我们对食管癌组织标本及细胞株进行Western blot、免疫共沉淀、双荧光素酶等实验，结果表明：①Brd4在食管癌细胞中高表达促进放疗后DNA损伤的修复，减少细胞凋亡；②Brd4作用于ATF3基因启动子，促进ATF3在食管癌细胞中的表达；③ATF3调节丝氨酸和核苷酸代谢通路中关键合成酶（PHGDH、PSAT1、PSPH、SHMT1、SHMT2、MTHFD1、MTHFD2、MTHFD11）的表达，促进食管癌细胞放疗后DNA损伤的修复。由此我们得出“Brd4调节ATF3表达促进食管癌放疗后的DNA损伤应答，进而影响食管癌放疗敏感性”的结论。靶向Brd4分子为增加食管癌放疗敏感性，提高放疗疗效，改善患者生存提供了新的思路和重要理论基础。