代谢应激下AMPK磷酸化修饰PROX1在肝癌发展中的作用及机制研究

A large number of studies have shown that aberrant expression of PROX1 plays an important role in tumor development. We have previously found that PROX1 promote the hepatocellular carcinoma(HCC) growth and metastasis, and confirmed that HCC with high expression of PROX1 has the characteristics of poor prognosis, high recurrence rate and insensitive to targeted drug. Therefore, PROX1 has been a valid target for the HCC treatment. The regulation mechanism of the abnormal expression of PROX1 is almost unknown. We have recently found that PROX1 is a key molecule regulated under glucose deprivation by using stable isotope labeling (SILAC). Functional studies have found that PROX1 protein levels are significantly downregulated under glucose starvation and its negatively regulated dependent on the AMP activated protein kinase (AMPK). Depth studies have found that AMPK directly interacts with PROX1 and phosphorylates PROX1, suggesting that PROX1 may be a novel substrate of AMPK. This study intends to investigate the molecular mechanisms of AMPK phosphorylation modification PROX1 promoting its protein degradation, In vitro, in vivo assays and clinical specimens clarify that the function of PROX1 phosphorylation modification in proliferation, invasion and metastasis of HCC cells, To of providing the theoretical basis and intervention targets for developing more effective means of treatment of HCC.

大量研究表明PROX1的异常表达在肿瘤的发生发展中起重要作用。申请人前期发现PROX1促进肝癌生长与转移，并证实高表达PROX1的肝癌具有预后差、复发率高和靶向药物不敏感的特征。因此，PROX1已成为肝癌治疗的有效靶点。目前PROX1异常表达的调控机制几乎不清楚。近期我们通过稳定同位素标记技术(SILAC)，发现PROX1是感应葡萄糖浓度变化的关键分子。功能研究发现葡萄糖饥饿条件下PROX1蛋白水平显著下调，并受能量代谢总开关-腺苷酸活化蛋白激酶(AMPK)的负向调控。深入研究发现AMPK与PROX1直接相互作用，并且磷酸化修饰PROX1，提示PROX1可能是AMPK的新型底物分子。本课题拟研究AMPK磷酸化修饰PROX1引发其蛋白质降解的分子机制，并通过体外、体内实验及临床标本阐明PROX1磷酸化修饰在肝癌细胞增殖、侵袭和转移中的功能，为研发更有效的肝癌治疗手段提供理论基础和干预靶点。

前期研究表明PROX1的高表达与肝癌患者预后差、复发率高和靶向药物治疗不敏感密切相关，但PROX1异常表达的调控机制几乎不清楚。本项目我们系统研究代谢应激下AMPK磷酸化修饰PROX1在肝癌发展中的作用及分子机制，研究首次发现了PROX1是一个感应葡萄糖浓度变化的关键分子，发现AMPK可以促进PROX1蛋白的下调，证实PROX1作为一种反馈机制，在营养缺乏时驱动肝癌细胞的代谢可塑性。机制上研究发现AMPK可以直接磷酸化修饰PROX1第79位的丝氨酸从而介导PROX1蛋白被CUL4-DDB1蛋白酶体降解，代谢应激所介导的PROX1蛋白下调可以启动肝癌细胞中BCAA代谢重编程，研究还证实PROX1主要通过维持肿瘤细胞内的BCAA含量从而激活mTOR通路最后驱动肝癌恶性转化过程。更有意思的是我们发现二甲双胍可以显著抑制高表达PROX1肝癌的疾病进展过程。该研究为以肿瘤代谢重编程为突破口探寻更有效的肝癌治疗方法提供依据，为临床肝癌治疗提供有效的药物作用靶点和理论基础，最后为肝癌的个体化精准防治提供精准分型方案。