RAS模拟小分子Rigosertib联合西妥昔单抗治疗RAS突变型结直肠癌的作用及机制研究

Cetuximab which targeted the epidermal growth factor receptor (EGFR) had proven to be efficacious in RAS wild-type colorectal cancers, but its effect was limited in RAS-mutant colorectal cancer. Combined treatment of Cetuximab and RAS down-stream signaling inhibitors was supposed to be a promising therapeutic method. Rigosertib (RGS) which was reported to be RAS-mimetic small molecule could bind to the RAS-binding domains (RBDs) of RAS effectors, and then blocked the RAS-signaling. In our previous work, RGS showed anti-proliferative and pro-apoptotic effects in RAS-mutant colorectal cancer cells; RGS also inhibited growth of Patient-derived Tumor Xenograft (PDX) in nude mice. We found that RGS could block the RAS down-stream signaling, however, its inhibition of RAS signaling induced feedback activation of other EGFR down-stream signaling. We supposed that the combined treatment of Rigosertib and Cetuximab could block the EGFR and RAS signaling pathway, which might be a therapeutic strategy in RAS-mutant colorectal cancer. In our research, we will verify its curative effects in colorectal cancer subcutaneous tumor xenograft model, hemi-splenic liver metastasis model, and patient-derived tumor xenograft model; then, we will analyze its effect in EGFR down-stream signaling pathway by phosphorylated protein mass spectrometry, western blot and immunohistochemistry.

抗EGFR的西妥昔单抗是治疗RAS野生型结直肠癌的有效药物，但对RAS突变型结直肠癌无效。西妥昔单抗联合RAS信号通路抑制剂的治疗方案有望解决此问题。Rigosertib（RGS）能模拟RAS与其下游效应蛋白结合，从而阻断RAS信号通路。我们前期研究发现RGS通过抑制增殖和促进凋亡显著杀伤RAS突变型结直肠癌细胞，小鼠移植瘤（PDX）模型实验也证实RGS对RAS突变型结肠癌有抗癌能力。我们注意到RGS虽能有效抑制RAS信号通路，但反馈激活EGFR下游其他信号通路。据此推测联用西妥昔单抗和RGS抑制EGFR和RAS信号通路，将是治疗RAS突变型结直肠癌的有效方案。本研究将利用皮下成瘤模型、半脾肝转移动物模型和PDX模型验证二者联合干预价值，并通过磷酸化蛋白质谱、免疫印迹、免疫组化等手段分析二者联合对EGFR下游各信号通路的抑制情况。本研究有望为提高RAS突变型结直肠癌的疗效提供新的解决方案。

RAS突变型结直肠癌患者预后较差，且缺乏针对性的治疗手段。小分子药物Rigosertib（RGS）是一种RAS模拟物，可阻断RAS下游通路的异常活化。本课题组在前期研究中发现，RGS 在RAS突变型病人来源肿瘤组织的裸鼠移植瘤（Patient-derived tumor xenograft，PDX）模型中具有显著抗肿瘤作用，提示RGS在RAS突变型结直肠癌中的治疗前景。本研究旨在探索RGS在RAS突变型结直肠癌中抗癌效应与机制。首先，通过检测经RGS处理后结直肠癌细胞增殖活性、克隆形成能力、细胞凋亡比例以及细胞周期分布等变化，我们发现：相比于RAS野生型结直肠癌，RGS能更有效杀伤RAS突变型结直肠癌，并诱导其细胞凋亡并阻滞其细胞周期于G2/M期。通过PDX模型以及裸鼠皮下成瘤模型，进一步证实RGS针对RAS突变型结直肠癌的特异性杀伤效应；并且，在一例原发性化疗耐药患者的PDX模型中，RGS的抗肿瘤效应强于目前临床常用的化疗方案。分析其机制，我们证实RGS可显著抑制RAS突变型结直肠癌中RAS下游信号通路的异常活化，且该阻断效应不依赖于RGS诱导的氧化应激。其次，我们还探究了RGS联合西妥昔单抗杀伤RAS突变型结直肠癌的可能性，并发现：在体外实验中，RGS联合西妥昔单抗确可更有效阻断RAS下游信号通路并杀伤结直肠癌细胞；但在动物实验中，却未能证实该联合抗肿瘤效应存在。最后，基于RAS突变型结直肠癌细胞系的裸鼠皮下成瘤模型，我们证实：尽管伴随骨髓抑制等毒副作用，联合应用RGS和奥沙利铂对RAS突变型结直肠癌具有更强的抗肿瘤效应。本研究完善了针对RGS的抗肿瘤效应及其与靶向药物与化疗药物联合应用的系统评价，有望为提高RAS突变型结直肠癌的疗效提供新的解决方案。