肿瘤微环境调控结直肠癌细胞重新获得干细胞特性新分子ARRDC4作用机制研究

Colorectal cancer stem cells (CCSCs) contribute greatly to the invasion, metastasis and recurrence in colorectal cancer (CRC).Tumor microenvironment has been implicated in promoting CRC cells to dedifferentiate and regain stemness characteristic to reprogram into CCSCs. Thus, it becomes a premise for target-eradicating CCSCs to clarify how tumor microenvironment promotes CRC reprogramming. However, the molecular mechanisms remain poorly understood. We previously identified a new molecule named ARRDC4 using shRNA retrovirus libraries. Transcription of ARRDC4 was significantly suppressed by glucose deprivation or adding reactive oxygen species or recombinant CXC chemokines. Knockdown of ARRDC4 resulted in obvious increase in spheres forming ability of CRC cells and proportion of CCSCs in vitro, indicating that ARRDC4 is an important negative-regulatory molecule in tumor microenvironment promoting CRC reprogramming. But the molecular mechanisms of ARRDC4 remain unclear. Based on previous results, our current study is aimed to screen and identify the critical candidate molecules of ARRDC4 by affinity chromatography tandem massspectrometry/yeast two-hybrid systems, clarify the molecular mechanisms of ARRDC4 regulating CRC reprogramming, and investigate the interaction between tumor microenvironment and transcription of ARRDC4. This study will lay a solid foundation for the development of new molecular targeted drugs to eradicate cancer stem cells in the future.

肿瘤干细胞是结直肠癌浸润转移和复发的主要原因之一。肿瘤微环境可促进普通肿瘤细胞去分化和重新获得干细胞特性(重编程)而成为肿瘤干细胞。阐明肿瘤微环境促进结直肠癌细胞重编程机制是进而靶向清除结直肠癌干细胞的基础，然目前对结直肠癌细胞重编程分子机制尚需明了。我们先前用shRNA逆转录病毒文库鉴定了一个新分子ARRDC4，发现剥夺葡萄糖、添加氧自由基和趋化因子等可明显抑制ARRDC4的转录；沉默ARRDC4显著增加结直肠癌细胞的体外克隆成球能力和肿瘤干细胞比例，提示ARRDC4是肿瘤微环境调控结直肠癌细胞重新获得干细胞特性的重要负性分子，然目前对ARRDC4分子作用机制尚不明了。本研究拟在先前基础上通过亲和串联质谱/酵母双杂交筛选和鉴定ARRDC4作用蛋白并阐明其参与结直肠癌细胞重编程分子机制；探讨微环境与ARRDC4转录的相互作用。本研究为今后研制清除肿瘤干细胞新的分子靶向药物打下扎实的基础。

为发现肿瘤微环境促进结直肠癌细胞重新获得干细胞特性过程中新的代谢重编程分子及其作用机制，本课题在前期工作基础上通过构建人结直肠癌移植瘤PDX小鼠模型、类器官培养(Organoid)和cDNA病毒文库的筛检方法，发现了一个新的代谢重编程相关分子—ARRDC4。乳酸、葡萄糖剥夺、缺氧、ROS、细胞因子(TGF-b和IFN-g)和化疗药物诱导性衰老等刺激可明显诱导ARRDC4的表达，并且发现结直肠癌干细胞也高表达ARRDC4；ARRDC4在普通结直肠癌细胞中仅少量定位于线粒体，而在结直肠癌干细胞中多数ARRDC4定位于线粒体；Halo-tag免疫共沉淀结合LC-MS/MS氨基酸测序示ARRDC4能被ANKRD22共沉淀，提示ARRDC4可能通过ANKRD22参与代谢重编程。我们发现表达ARRDC4增强了ANKRD22与有氧糖酵解关键酶PDK/PDH的结合，促进了结直肠癌细胞的有氧糖酵解；并且在结直肠癌干细胞中ARRDC4还协同ANKRD22和E-Syt1将脂质转运到线粒体和增强脂肪酸的b-氧化。进一步还发现WWP1是ARRDC4作用的另一个关键靶蛋白，WWP1与ARRDC4的相互作用不仅控制了结直肠癌(干)细胞外泌体的分泌水平，还改变了外泌体中miRNA的表达谱。提示ARRDC4/WWP1介导的通路可能在结直肠癌细胞外泌体影响肿瘤细胞转移能力过程中起重要作用，ANKRD22和WWP1可能是结直肠癌转移干预的潜在作用靶点。