双靶点抑制Chk1和Aurora-A协同放射增敏三阴性乳腺癌的机制研究

Triple negative breast cancers (TNBC), which are defined by lack of expression of estrogen,progesterone,or HER2 receptors, represent approximately 15% of all breast cancers. Patients with these tumors have high risk of local recurrence and poor prognosis. In addition to aggressive biological characteristics, poor outcomes in TNBC relate to a distinct lack of therapeutic options. There is a critical need to assess molecularly targeted therapeutics as radiosensitizers in this disease. Chk1 and Aurora-A are commonly overexpressed in TNBC tumors, their expressions are correlated with advanced tumor stage, poor prognosis and with tumor radioresistance. It has been shown shown that MK8776, a small molecule Chk1 kinase inhibitor, as well as MLN8237, a small molecule Aurora-A kinase inhibitor, can abrogate the G2/M checkpoint in cancer cells, sensitizing tumor cells to DNA-damaging agents. Some studys reported that the simultuneous Chk1 and Aurora-A inhibition in combination with chemotherapy resulted in a synergistic cytotoxic effect on colon cancer cells. However, the radiosensitizing effect by dual inhibition with MK8776 and MLN8237 has not been investigated. In our previous study, we discovered that the dual inhibition of the Chk1 and Aurora-A with MK8776 and MLN8237 enhances radiosensitivity synergistically in p53-mutant TNBC cells. Thus, the purpose of the present study was to comfirm the synergistic ability of MK8776 and MLN8237 to radiosensitize TNBC cells in vitro and in vivo, and to explore their molecular mechanisms. Our results would add new data to the application of irradiation in combination with MK8776 and MLN8237 as a new therapeutic strategy to improve local control in the treatment of TNBC.

三阴性乳腺癌（TNBC）缺乏已知的治疗靶点、预后差，是乳腺癌治疗的难点，其放疗效应需要进一步的提高。近期研究显示双靶点抑制细胞周期G2期检测点Chk1蛋白和M期纺锤体检测点Aurora-A蛋白对肿瘤化疗有协同增敏效应，但迄今尚无联合抑制二者对TNBC放射敏感性影响的研究报道。我们预实验研究发现：联合应用Chk1抑制剂MK8776和Aurora-A抑制剂MLN8237对p53突变型TNBC细胞有协同放射增敏作用，而对p53野生型TNBC细胞和正常乳腺细胞无放射增敏效应。本项目将在预实验基础上，通过分子、细胞和动物水平的相关研究，围绕不同p53状态的细胞周期变化及损伤修复展开相关分子机制的探索，以阐明双靶点抑制Chk1和Aurora-A协同放射增敏TNBC细胞的机理。预期结果有助于深入理解将细胞周期检测点作为新兴放射增敏靶点的分子机制，为TNBC新型放射增敏剂的研发提供新的思路。

三阴性乳腺癌（TNBC）缺乏已知治疗靶点、局部复发率高，是乳腺癌治疗的难点。放射治疗是乳腺癌重要的局部治疗手段之一，其在TNBC中的效应需要进一步的提高。近期研究显示靶向抑制细胞周期G2/M期检测点Chk1蛋白和M期纺锤体检测点Aurora-A蛋白对多种肿瘤具有显著抗肿瘤效应，联合抑制Chk1和Aurora-A蛋白对DNA损伤化疗药物具有协同效应，但迄今尚无联合抑制二者对TNBC放射敏感性影响的研究报道。我们研究结果发现：Chk1抑制剂MK8776和Aurora-A抑制剂MLN8237分别对TNBC细胞有放射增敏作用，联合应用两药具有协同放射增敏效应。两药联合引起TNBC细胞G2/M 周期阻滞障碍，同时显著增加射线对TNBC细胞的DNA损伤作用，相应的γH2AX含量及数量、细胞微核显著增加。当我们用TNBC细胞接种裸鼠成瘤细胞接种裸鼠成瘤，在体内观察药物的增敏作用也可得到相似的结果：射线照前MK-8776和/或MLN8237处理裸鼠，可观察到两药药物联合射线照组移植瘤体积显著小于其它处理组。为了探讨药物放射增敏的可能机制，我们检测了三株细胞系不同干预处理组凋亡水平，实验结果表明射线可以引起TNBC细胞凋亡水平显著升高。综上，双靶点抑制CHK1和Aurora-A可通过调控细胞周期增加射线导致的DNA损伤，增加细胞凋亡，进而增加TNBC细胞的放射敏感性。本项目所得结果有助于深入理解将细胞周期检测点作为新兴放射增敏靶点的分子机制，为TNBC中新型放射增敏剂的研发提供了新的方向和依据。