雷公藤红素逆转EGFR靶向治疗耐药在转移性结直肠癌中的分子机制

Primary resistance and acquired resistance to the EGFR (epidermal growth factor receptor)-targeted therapy in metastatic colorectal cancer (mCRC) are key questions await investigations. Genetic mutation of KRAS is the most common underlying mechanism for the resistance to The resistance to EGFR block in metastasis colorectal cancer (mCRC) is mainly caused by RAS/RAF mutation, and the effective measurement is to find the synthetic genes which can kille the CRC cells with activated KRAS mutation but not the CRC cells with wild type KRAS. We have found that Cetuximab, a EGFR blocker, can effectively kill the CRC with activated KRAS mutation when combined with celastrol, even a small dosage use, which named synthetic lethality. However, the mechanism and the targets interacted with celastrol are all unclear. Recently, we have taken SHOC2 and SH2B1 as the candidates to celastrol by the combined use of chemical probes and protein arrays. Interestingly, the previous evidence from the reported papers also supported that the molecules may active the signaling pathway of RAS and PI3K. Therefore, we proposed that SHOC2 and SH2B1 have the main contribution to the synthetic lethality of CLS and CTX. In this project, we do the work as followed: 1) to validate the physical interactions between CLS and the genes and to evaluate the similarity at the biological function and the response gene signature; 2) to elucidate the biological function of the genes with in vivo mice model and corresponding molecular mechanisms; 3) to evaluate the predictive values of the genes and key molecules among their downstream signaling pathway by using the multiple methods including tissue microarrays, immunohistochemistry and data mining. Our project will discover the physical targets to celastrol and the mechanism of the synthetic lethality above, and provide the potential markers for the prediction of the resistance to EGFR block and the prognosis in colorectal cancer.

RAS/RAF突变是EGFR单抗治疗转移性结直肠癌(mCRC)耐药的主要原因，寻找RAS突变协同致死基因并针对性配伍用药是逆转其耐药性的重要策略。申请人发现：低剂量雷公藤红素(CLS)可显著增强西妥昔单抗(CTX)对KRAS突变CRC的致死效应，然介导该效应的关键靶点不明；利用化学探针-蛋白芯片技术，发现SH2B1、SHOC2等蛋白与CLS高效结合；以往文献表明二者参与RAS和PI3K信号,故推测它们介导CLS和CTX协同致死作用。围绕该假说，本研究拟：1)利用免疫共沉淀、体外功能评价及表达谱相似性等技术验证CLS与SHOC2、SH2B1之间是否存在相互作用；2)阐明SHOC2、SH2B1单独或联合抑制对KRAS突变CRC细胞体内成瘤及转移的作用，揭示其分子机制；3)围绕SHOC2、SH2B1及其下游信号关键承接点,利用组织芯片和信息挖掘技术评价其在CRC预后和CTX耐药预测方面的价值。

RAS/RAF突变是EGFR单抗治疗转移性结直肠癌(mCRC)原发耐药的主要原因，寻找与RAS突变协同致死的基因并针对性配伍用药是逆转其耐药性的重要策略。我们发现低剂量雷公藤红素可显著增EGFR抑制剂对KRAS突变CRC的致死效应，然而介导该效应的关键靶点及其分子机制不清楚。在本项目中，我们发现：（1）EGFR信号阻断与雷公藤红素联用对CRC细胞的抑制作用显著强于任一单药的抑制效果；（2）雷公藤红素与EGFR等RTKs下游分子（如EPHA2, FAK, SHP2）的抑制剂联合对CRC细胞具有显著的协同抑制效果；（3）CRC对SHP2抑制剂的耐药作用可能与AKT信号的反馈性激活有关、进一步发现SHP2抑制剂SHP099与AKT、FAK联用对CRC具有较强的协同致死效应；（4）相对与癌旁组织，SH2B1在CRC中表达升高，且其高表达指示CRC患者不良预后，SH2B1是一个潜在的CRC预后预测标志；（5）发现雷公藤红素通过抑制Shoc2的功能来影响ERK信号的活性，Shoc2基因的下调导致细胞增殖和移动性的显著降低。总之，本课题围绕雷公藤红素+EGFR抑制剂对KRAS 突变 CRC 的协同致死效应和分子机制，为克服 CRC的EGFR靶向耐药的治疗提供新的潜在新靶点，证明SH2B1、SHOC2、SHP2是治疗CRC的关键分子。这些结果为今后深入研究雷公藤红素在抗CRC中的作用提供了重要依据，为两药联用方案治疗CRC积累了更多基础工作。