小分子化合物A33诱导磷酸酶SHP2降解的分子机制及其在结肠癌治疗中的意义

Our previous research has proved that the small molecule A33 can directly bind to phosphatase SHP2 to promote its degradation, and has a good inhibitory effect on colon cancer. This project intends to investigate the new regulatory mechanism for SHP2 degradation, including : explore the structural basis for degradation of SHP2 by A33; identify the E3 ubiquitin ligase which mediate the SHP2 degradation under the action of A33 and its possible ubiquitination sites, further to examine the interaction process of A33-SHP2-E3 ubiquitination ligase ; explore the efficacy of A33 in the treatment of colon cancer and verify the related mechanisms in vitro and vivo by using RNA- seq and other techniques. On this basis, comparing expression of SHP2 and E3 ubiquitin ligase in the colorectal cancer clinical samples and tumor adjacent tissues, and analyzing their correlation with tumor stage and PFS, to confirm whether SHP2 is highly expressed in colon cancer and its relationship with the degradation mechanism, and further confirm the meanings of targeted degradation of SHP2 in the treatment of colon cancer. This project is expected to clarify the new regulatory mechanism of SHP2 degradation and provide new strategies and candidate compounds for the treatment of colon cancer.

课题组前期研究证实小分子A33可直接结合磷酸酶SHP2促进其降解，对结肠癌有很好的抑制作用。本项目拟针对A33诱导SHP2降解这一新作用方式展开研究，采用计算机模拟、表面等离子共振、肽段截短及点突变、圆二色谱等探讨A33结合并诱导SHP2蛋白降解的结构基础；采用免疫共沉淀及质谱鉴定A33作用下介导SHP2降解的E3泛素化连接酶及其可能的泛素化位点，并考察A33-SHP2-E3泛素化连接酶的相互作用过程；结合RNA测序等手段从细胞和动物水平探讨A33治疗结肠癌的药效并验证机制。在此基础上，比较结肠癌与癌旁组织中SHP2及介导其降解的E3泛素化连接酶的表达水平，进行二者与肿瘤患者的分期及PFS的相关性分析，确证结肠癌中SHP2高表达与其降解机制缺失的关系，进一步确证靶向降解SHP2在结肠癌治疗中的意义。本项目的实施预期阐明SHP2降解的新调控机制，为结肠癌等肿瘤的治疗提供新的策略和候选化合物。

蛋白酪氨酸磷酸酶SHP2抑制剂是肿瘤药物治疗的重要靶点。本研究发现小分子A33——阿帕替尼通过与SHP2直接结合，诱导SHP2蛋白降解。免疫共沉淀实验验证了阿帕替尼诱导SHP2的泛素化水平增加，蛋白质谱结果表明与SHP2结合的E3泛素化链接酶可能是TRIM21/TRIM25。同时我们也通过CCK8、克隆形成、细胞周期、细胞划痕以及Trans-well细胞侵袭实验，明确阿帕替尼可以抑制结肠癌细胞的增殖并且促进其凋亡，诱导G0/G1细胞周期阻滞，阻碍结肠癌细胞的迁移和侵袭。CT26小鼠移植瘤模型也说明阿帕替尼可以抑制结肠癌生长。其具体机制为：阿帕替尼通过显著抑制了GSK3β（ser9）的磷酸化水平，从而导致β-catenin的泛素化降解增加，入核减少，从而抑制结肠癌的生长。但过表达SHP2后阿帕替尼对结肠癌细胞抑制作用下降，说明阿帕替尼对于结肠癌的抑制作用可能与诱导SHP2降解密切相关。本研究充分探究了阿帕替尼对结肠癌的抑制作用及其机制，为临床上结肠癌的治疗提供了新策略；同时提出了一种SHP2降解新的调控机制，为靶向SHP2的药物开发提供了新的思路。