Sirt4 在肝癌起始细胞中的分子机制与功能

The existence of cancer stem cells is one of the basic reasons of the tumor recurrence and chemotherapy resistance in HCC. Appearing abnormal glucose metabolism of cancer stem cells on the growth and spread of the tumor and chemotherapy tolerance are closely linked. In recent years, research on tumor stem cells and glucose metabolism is rare. We according to reveal the intensive property between the characteristics of the tumor stem cells and glycolysis metabolism, in order to find the highway of interference measures, that can inhibit tumor spread and recurrence, and improve the sensibility of chemotherapy, that for the purpose of a strategy for the treatment against HCC. Previously, the expression of Sirt4 high expression of acetylate family members was found in liver cancer stem cells using a novelty monoclonal antibody 1B50-1 which was defined the specific identification for HCC stem cells. We have adopted a series of cellular and molecular biology experiments and animal models to reveal that Sirt4 can not only maintain the tumor cell stem cell properties, such as self-renewal ability, strong aggressive, tumorigenicity and drug-resistance, etc. Moreover, Sirt4 has a new regulatory effect on acetylation of key enzyme ENO1 in glycolysis, which leads to abnormal glucose metabolism and is not conducive to the implementation of chemotherapy. Therefore, we contacted the tumor stem cells and the relationship between glucose metabolism to find the problem of liver cancer chemotherapy tolerance from a new hypothesis, that puts forward the Sirt4 is a key functional molecules glucose metabolism of liver cancer stem cells and contributes to reverse the malignant biological behavior of HCC as well.

肝癌肿瘤干细胞的存在是肿瘤复发和化疗耐受的原因之一，而肿瘤干细胞的异常糖代谢对肿瘤侵袭和生长播散和化疗耐受有着紧密联系。近年来，对肿瘤干细胞和糖代谢的研究并不多见。我们针对肿瘤细胞干性维持特点与糖代谢展开深入研究，以期发现两者联系，如果采用干扰措施，就能抑制肿瘤的播撒和复发，提高化疗的敏感性，为肝癌治疗提供一种策略。先前在肝癌肿瘤干细胞中发现去乙酰化酶家族成员Sirt4高表达的表象。采用了一系列的细胞分子生物学实验和动物模型，发现Sirt4不但可维持肿瘤细胞干细胞特征，如自我更新能力，强侵袭性，致瘤性和耐药性等，而且对糖酵解中关键酶ENO1存在去乙酰化的负调控作用，导致糖代谢异常而不利于化疗实施。因此，我们联系肿瘤干细胞和糖代谢的关系，从新角度发现解决肝癌复发和化疗耐受的问题，提出Sirt4是肝癌干细胞糖代谢的关键功能分子而维持肿瘤细胞干性的假说。若采取联合干预措施可逆转其恶性生物学行为。

肿瘤起始细胞出现的异常糖代谢是研究肿瘤起始细胞新的热点。近年来，对肿瘤起始细胞和糖代谢的研究并不多见。本项目从糖酵解关键分子的蛋白质修饰角度，揭示了肿瘤起始细胞的存在是肝癌发生、发展及治疗措施的关键事件。我们采用了一系列的细胞、分子生物学实验和动物模型，诸如软琼脂集落形成实验、耐药检测MTT 法、Boyden Chamber 实验、质谱分析，流式细胞学分析分选，自我更新能力的微球形成实验，NOD/SCID 小鼠有限稀释和连续传代实验，发现SIRT4不但能够维持肿瘤细胞干性特征，如自我更新能力，侵袭性，致瘤性和耐药性等，而且SIRT4对糖酵解中关键酶ENO1和PKM2，进行了多重位点蛋白修饰，包括乙酰化和磷酸化的调控。项目计划按期完成，并在已经研究的基础上拓展了肿瘤的癌种研究。我们设计并生产了这两个分子的乙酰化位点修饰抗体（ENO1-K358）和磷酸化位点修饰抗体（PKM2-T454）。在动物模型中得到很好验证。同时，阐明了肝癌索拉非尼的耐药机制是通过肿瘤干细胞中CBX4/YAP1信号通路的激活而产生的。进一步，阐述了以α2δ1为标志物的肝癌肿瘤起始细胞一系列的调控途径，包括外基质交联-整合素-FAK-ERK通路；Hippo通路；miRNA机制等，在这些途径中，SIRT4发挥了关键的作用。以上的科学发现成为课题结题的重要数据。我们在国际期刊上发表了6篇相关文章，获批了一项国家发明专利，培养多名研究生。既是对本项目顺利结题提供了依据，也为下一步的课题研究提供了方向。