SRPK1诱导结肠癌奥沙利铂耐药的机制研究

Oxaliplatin (L-OHP) resistance is the main cause of treatment failure in the middle and late stage colorectal cancer patients. Identifying key proteins that participate in the chemotherapy resistance and elucidating the mechanism would be of great value for late stage colorectal cancer. Our previously study in 347 paraffin-embedded colorectal cancer tissue section by IHC showed that over expression of SRPK1 was associated with L-OHP resistance significantly. The up-regulation of SRPK1 could promote aberrant alternative splicing of cancer stem-cell like marker CD44v3 and expression level of anti-apoptosis protein Bcl-xL and Mcl-1L, enhance the CSCs-like phenotype and EMT, and activate Wnt and NF-κB signaling pathway. Furthermore, SRPK1 might interact with GSK3β directly and increased the phosphorylation of Ser9 of GSK3β, which in turn promoted translocation of β-catenin to the nucleus. The results above demonstrated that SRPK1-GSK3β signal pathway related to L-OHP resistance closely. In this project we aim to explore the effect of SRPK1 on aberrant alternative splicing of drug-resistant genes and activation of NF-κB/Wnt signal pathway and reveal the underlying mechanism of L-OHP resistance as well by using colorectal cell lines with L-OHP resistance trait and PDX (patient-derived xenograft model) mice model. The implementation of this project may provide a theoretical basis for the diagnosis and treatment of chemotherapeutic drug resistance in colorectal cancer and new targets for drug development.

奥沙利铂耐药是中晚期结肠癌治疗失败的主要原因，寻找化疗耐药通路的关键蛋白并阐明其功能及分子机制，对晚期结肠癌的诊治具有重要意义。我们前期工作发现 347例结肠癌切片中丝精氨酸蛋白激酶SRPK1的高表达与奥沙利铂的耐药显著相关，上调SRPK1的表达可增加干性标志物CD44v3和抗凋亡蛋白Bcl-xL、Mcl-1L的异常可变剪接，增强结肠癌细胞的干细胞表型和EMT，激活Wnt和NF-κB信号通路。同时发现SRPK1与GSK3β的直接互作可以增强GSK3βS9的磷酸化，促进β-catenin入核。提示我们SRPK1-GSK3β通路与结肠癌细胞奥沙利铂的耐药密切相关。本项目将以结肠癌细胞耐药株和鼠肠原位PDX模型，深入探讨SRPK1对耐药基因的异常可变剪接及SRPK1对NF-κB和Wnt通路的激活，揭示奥沙利铂耐药的分子机制。本课题的实施可为结肠癌化疗耐药的诊治提供理论基础和新的药物治疗靶点。

奥沙利铂（Oxaliplatin，L-OHP）是II/III期结直肠癌的一线治疗药物，奥沙利铂联合氟尿嘧啶/亚叶酸钙（5-FU/LV，FOLFOX）的治疗方案可使III期结肠癌复发危险减少24%，II期结肠癌复发危险减少25%，治疗晚期结直肠癌有效率32%~58%，但仍有近40% 患者因出现奥沙利铂耐药导致治疗失败。对奥沙利铂耐药机制的研究已成为临床上亟待解决的问题。寻找化疗耐药通路的关键蛋白并阐明其功能及分子机制，对结肠癌的临床诊治具有重要意义。.课题按照国自然申请书的“年度研究计划”顺利展开。课题收集临床结肠癌样品148例，分析SRPK1与样品临床特征、奥沙利铂耐药和患者预后间的相关性，并明确高表达SRPK1可以促进非耐药细胞向耐药细胞转变以及抗凋亡的生物学功能；课题进一步探讨了SRPK1 通过p65 及GSK3β的核累积对NF-κB 转录活性调控的分子机制，揭示了SRPK1 激活NF-κB 信号通路促进化疗耐药的分子机制。课题继续研究了SRPK1 与GSK3β 的互作，明确了SRPK1在体外和体内通过直接相互作用对GSK3β 进行磷酸化修饰，验证了SRPK1促进GSK3β Ser9自磷酸化增强。课题进一步通过截断型分析，首次提出，SRPK1的非激酶活性导致GSK3β Ser9位点的磷酸化增强，明确了SRPK1的Spacer domain 在耐药信号传递中的关键作用。综上，SRPK1 通过Akt和 GSK3β及对抗凋亡相关基因执行异常可变剪接促进结肠癌细胞奥沙利铂耐药，SRPK1的非激酶区域扮演重要的信号传递作用，揭示了SRPK1/Akt/GSK3β 轴调控奥沙利铂耐药的分子机制。.基于已有的SRPK1的功能和机制研究基础，我们已经申请相关专利，并委托南京金斯瑞有限公司靶向SRPK1的Spacer domain设计并生产3-5种针对该对接槽的小分子短肽。后续将进行下一步的转化研究工作。.课题按预期在SCI期刊 Cell Death&disease、Cell Death Discovery、Journal of Translational Medicine 发表研究论文4篇。指导硕士研究生11名，博士研究生1名，毕业硕士研究生3名。指导青年教师3名（副研究员2名王志峰、宋军伟；研究助理1名黄景强），并于2020年和2021年分别获得国家自然科学青年基金项目。