细胞ATP生成异常- - Warburg效应的机理研究

It has been well established that metabolic reprogramming coincides with every stage of cancer development ranging from initiation,through promotion, all the way to metastatic progression. Among all of the metabolically adaptive events sequentially emerged during cancer development, the enhancement of glycolysis even in the presence of ample oxygen, which is also defined as Warburg effect, is the longest standing enigma and most controversial issue in the history of cancer biology. Even today, the detail mechanism of Warburg effect still remains largely elusive. In our previous published paper (Fang M.,et al.,Cell, 143:711), ENTPD5, an endoplasmic reticulum UDP hydrolase has been demonstrated to be involved in the continuous upregulation of glycolysis, which is further recapitulated by the much higher expression level of ENTPD5 in human prostate cancer cell line LNCaP，PTEN knock-out MEF cells as well as tumour samples from human prostate cancer patients. Further more, the decrease of cell proliferating rate and the level of multiple cell growth factor receptors in LNCaP cells as well as the shrinkage of LNCaP xenografts in nude mice were achieved by specifically knocking-down of endogenous ENTPD5. The distinctive role of ENTPD5 in cancer development and Warburg effect underlines the fact that glycolytic regulators other than enzymes themselves are able to serve as a new therapeutic target. To unravel the major puzzles of Warburg effect, based on our previous research and unique techniques, current application will devote efforts to establish an in vitro functional assay which faithfully mimics the glycolysis pathway in vivo; purify any components which are responsible for dysregulated ATP generation in cancer cells; and further validate the functional role of purified components during cancer initiation, invasion as well as metastasis. By utilizing our established recombinant system, we eventually seek to identify the specific therapeutic target in terms of dysregulated ATP metabolism in cancer cells; therefore provide alternative approaches for further improving the efficacy of current chemotherapy.

细胞在癌变过程中发生了系统性的代谢重构（Metabolic reprogramming）,尤其表现在糖酵解途径的特异性强化（即Warburg效应），其机制远未阐明。申请人在前期工作中发现内质网UDP水解酶ENTPD5参与细胞糖酵解途径的调控，ENTPD5水平在前列腺癌细胞系（LNCaP）和前列腺肿瘤病例中显著升高。下调ENTPD5表达水平后，LNCaP细胞增殖能力降低、细胞膜表面多种生长因子受体减少、裸鼠移植瘤变小。这说明以ENTPD5为代表的细胞糖酵解途径的调节蛋白，可能作为治疗癌症的新靶点。基于申请人前期的工作基础，为解决有关Warburg效应的关键科学问题，本立项拟首先在体外重建糖酵解途径的无细胞体系，然后纯化和分离癌细胞中能够导致Warburg效应的关键调节因子，再通过裸鼠移植瘤模型验证这些因子在肿瘤发生中的作用，为进一步寻找新的化疗靶点和药物筛选提供理论支持。

作为多类代谢反应的辅酶，胞内NAD、NADH水平及其比例的调控与细胞代谢异常密切相关，但是其调控机制以及与癌细胞Warburg效应的关系还很不清楚。利用糖酵解的无细胞体系，发现糖酵解速率高的细胞中缺失下调胞内NAD水平的NAD水解酶。生化分析表明，该NAD水解由串联的两步酶反应组成：首先NAD水解成游离ADPR和Nicotinamide；第二步ADPR水解为AMP和磷酸核糖，因而不同于已往报道的NAD消耗活性。在多种不同类别的癌细胞中，该NAD水解活性与糖酵解活性成负相关，受到生长因子负调控，可被细胞接触抑制活化，提示该活性可能参与Warburg效应的调控。. 除NAD的水解外，我们还研究了胞内NADH水平及NAD/NADH比例的调控。与正常肺上皮细胞相比，多种肺癌细胞系中存在依赖胞内还原剂GSH和硫锌酸的NAD还原酶。利用经典生化的方法纯化了这个NAD还原酶活性，鉴定为醛脱氢酶1A1和3A1(ALDH1A1/3A1)。ALDH1A1/3A1的还原剂依赖的NAD还原活性不被醛脱氢酶抑制剂抑制，也不受醛脱氢酶突变的影响，因而是不同于醛脱氢酶的新活性。功能研究表明，ALDH1A1/3A1的还原剂依赖的NAD还原活性上调胞内NADH水平，降低胞内NAD/NADH比例，可引起一系列代谢变化，从而促进肺癌移植瘤的生长。. PTEN-PI3K-AKT信号转导通路的异常活化可导致癌细胞的代谢重编程，因而PTEN-PI3K-AKT信号通路的调控是非常重要的问题。在这条信号通路的主要组成者中，PTEN、AKT受到泛素化调控，催化其泛素化的E3 连接酶已经被报道，但是PI3K是否也受到类似的调控还很不清楚。本研究中我们鉴定了两个PI3K催化亚基多聚泛素化的E3连接酶，其中NEDD4L催化的多聚泛素化与PI3K蛋白的稳定性和动力学有关；而TRAF6催化的多聚泛素化与PTEN-PI3K-AKT信号通路的信号转导有关。抑制NEDD4L导致PI3K催化亚基蛋白水平增高，而抑制TRAF6对其蛋白水平没有影响，但都会导致AKT的活化降低，Warburg效应减弱，肿瘤细胞生长抑制。. 另外，我们还证明去乙酰酶抑制剂丁酸钠通过增强抑癌基因P53 120位点乙酰化提高Apaf-1基因转录，因而增强细胞对线粒体凋亡通路的敏感性。