TGFβ-AMPKα-SOX2轴在胃癌循环肿瘤干细胞转移中的机制研究

Some studies have suggested targeting circulating cancer stem cells (CCSCs) is the best effetive tumor therapeutic strategy for tumor metastasis. But we must first isolate and culture CCSCs, and get to know the key moleculars that regulate characteristics of CCSCs. In previous work, we firstly isolated and cultured circulating gastric cancer stem cells (CGCSCs), and we found knock-down of TGFβRⅡby RNA interference caused the metastatic capacity of CGCSCs was sharply attenuated. Results from proteomic profiling showed the phosphorylated level of TAK1、AMPKα and YAP was signficantly increased, but SOX2 was down regulated. These proteins are corelated to metastasis in CGCSCs? And relationship exists among them? Which the role of SOX2 in CGCSCs? All above questions are unclear. Our work will investigate the relationship among TAK1、AMPKα、YAP and SOX2 by using RNA interference, overexpression and co-IP; and confirm YAP regulates SOX2 transcriptional expression by using EMSA、Chip and luciferase assay; and research the metastatic role of these genes by using single cell sphere formation and tail intravenous injection; finally IHC was performed in gatric cancer tissues to examine their role in metastasis and analyze the prognostic factors with gastric cancer. Our work will highlight the mechanism of CCSCs metastasis.

研究提示靶向循环肿瘤干细胞（CCSCs）的治疗是最有效的肿瘤转移治疗方式，前提是必须先分离培养出CCSCs，并找到调控其生物学特性的关键分子。前期工作中，我们率先分离培养出了胃癌循环肿瘤干细胞（CGCSCs），并发现TGFβRⅡ干扰后，CGCSCs的转移能力急剧下降，蛋白质谱结果显示，TAK1、AMPKα和YAP等磷酸化水平显著升高，SOX2的表达明显下降，它们是否与CGCSCs的转移有关？它们之间的关系又是什么？SOX2在CGCSCs中究竟起什么作用，这些问题都有待回答。本课题拟通过RNA干扰、过表达，免疫共沉淀等技术阐明它们之间的上下游关系；利用EMSA、Chip和荧光素酶报告基因分析证实YAP对SOX2的转录调控作用；采用单细胞成球培养和尾静脉注射等研究它们在转移中的作用，并在胃癌患者的肿瘤组织中进行验证，分析它们与预后的关系。本研究结果将对揭示循环肿瘤干细胞转移的机制具有重要意义。

肿瘤的治疗到底是肿瘤转移的治疗，因此，深入了解肿瘤转移的特性具有重大的临床治疗意义。对TGFβRⅡ进行干扰后，我们发现胃癌循环肿瘤干细胞（circulating cancer stem cells，CCSCs）的体外侵袭能力和在裸鼠肺部的转移能力急剧下降，说明TGFβRⅡ对CGCSCs的转移能力起着重要的作用。进一步研究发现TGFβRⅡ通路的激活可以升高Smad2/3和YAP等磷酸化的水平，降低SOX2的表达。通过TGFβRⅡ激活剂、抑制剂和shRAN干扰等进一步明确了TGFβRⅡ通路对Smad2/3、YAP和SOX2的作用。上述结果使我们推测：TGFβRⅡ通路的活化降低SOX2的表达是通过升高p-YAP水平实现的？那么YAP又是怎么调控SOX2的表达的？利用EMSA、过表达和shRNA干扰等实验技术我们发现YAP可以特异结合SOX2转录表达调控区域，从而影响SOX2的表达，SOX2是YAP的下游信号分子。单细胞成球实验证明YAP不影响CGCSCs的干性；Transwell实验和动物实验证明YAP影响了CGCSCs的迁移和转移，而这种效应可以被低表达的SOX2所抵消，提示SOX2是最终影响CGCSCs转移的因素。通过SOX2-shRNA和过表达等体内外实验再次证实了SOX2对胃癌循环肿瘤干细胞的转移具有重要作用。最后，我们在胃癌患者的肿瘤组织中检测了p-AMPK，p-Smad4，p-Tgf  receptor 2和SOX2等的表达，并分析了它们与预后的关系，而统计分析结果未能证明它们与患者预后有明显的差异。