Skp2泛素化RIPK1参与结直肠癌细胞5-Fu耐药的分子机制

Colorectal cancer is the fifth leading cause of cancers in mortality in China, and chemotherapy resistance of 5-Fu is one of the major reasons which caused colorectal cancer clinic treatment failure. Our previous studies revealed that 5-Fu induced necroptosis was significantly disparate between sensitive and resistant colorectal cancer cells. In this study, we will illustrate the molecular mechanism of E3 ligase skp2 mediated 5-Fu chemotherapy resistance, and explore the underlying mechanism which involved in Skp2 regulated RIPK1 expression and activation via ubiquitination pathways. More importantly, we conducted xenograft model to evaluate the in vivo effect of Skp2-RIPK1 mediated necroptosis inhibition in colorectal cancer 5-Fu chemotherapy resistance. Our study will provide new ideas for the chemotherapy of colorectal cancer. Besides, it will have an important clinical significance and application prospects for targeted therapy and sensitizing chemotherapy.

结直肠癌是我国第五大致死性恶性肿瘤，5-Fu化疗耐药是导致结直肠癌治疗失败的重要原因。本项目前期研究发现在5-Fu敏感和耐药结直肠癌细胞内，5-Fu诱导的程序性坏死发生了明显改变。本项目将以泛素E3酶Skp2调控RIPK1的泛素化为切入点，从RIPK1的蛋白表达及功能活化两个方面研究Skp2对结直肠癌细胞5-Fu耐药的调控。确定Skp2通过影响RIPK1的生物学功能，从而抑制程序性坏死，导致结肠癌细胞5-Fu耐药的分子机制。并利用裸鼠移植瘤模型，在体内证实Skp2-RIPK1介导的程序性坏死抑制对结直肠癌5-Fu耐药的重要性。本研究将从程序性坏死及5-Fu化疗抵抗的角度阐明Skp2的生物学功能，为克服结直肠癌5-Fu耐药的临床治疗提供理论基础和实验证据。

Skp2 (S-phase kinase-associated protein 2) 属于F-box蛋白家族，是E3连接酶SCF Skp2复合物的重要组成成分，具有识别底物并诱导其泛素化的生物学功能 。文献报道Skp2在乳腺癌，前列腺癌，肝癌和结直肠癌等多种肿瘤中高表达，并与病人预后不良相关。Skp2高表达促进肿瘤细胞的增殖，凋亡抑制及侵袭转移等。Skp2参与结直肠癌的化疗耐药的分子机制还未见报道。本项目研究发现，Skp2在多株5-Fu耐受的结直肠癌细胞内过表达。Skp2与RIPK1在结直肠癌耐药细胞系的表达负相关，程序性坏死抑制剂可以部分缓解5-Fu诱导的细胞死亡，提示5-Fu可以激活程序性坏死促进肿瘤细胞死亡。本项目通过细胞增殖实验，软琼脂克隆形成实验，免疫共沉淀，免疫印迹，体内泛素化实验系统探讨了过表达Skp2与结直肠癌5Fu耐药的分子机制。利用裸鼠移植瘤实验，进一步证实了过表达Skp2对结直肠癌细胞体内化疗耐受的重要性。本项目取得的创新性发现为结直肠癌的临床化疗增敏提供了新的思路。