ALDH6A1缺损重塑糖脂代谢促进肝细胞癌发生的机制研究

Metabolic rewiring of glucose and fat is one of hallmarks of hepatocellular carcinoma (HCC). However, the regulatory mechanism collaboratively promoting HCC remains to be further addressed. Mitochondria Aldehyde dehydrogenase family 6 member A1 (ALDH6A1) catalyzes dehydrogenation of methylmalonate semialdehyde, which is an intermediate product of pyrimidine and valine catabolism. Analysis of public database, tissue chip and freshly collected HCC patient samples showed that ALDH6A1 was downregulated in HCC. ALDH6A1 expression level was negatively correlated to pathological stage of HCC and positively correlated to prognosis of HCC patients. ALDH6A1 functioned to suppresse HCC proliferation, migration and the ability to form subcutaneous tumor in nude mice. ALDH6A1 also affected mitochondria function evidenced by increase of NADH and ROS production but reduction of oxygen consumption rate and mitochondria membrane potential. ALDH6A1 also decreased glucose 6-phosphate production as well as fat accumulation. It also interacted with NDUFS7 and MDH2, components of mitochondria complex I and tricarboxylic acid (TCA) cycle. These observations suggest that ALDH6A1may alter mitochondria function to regulate metabolic reprogramming of glucose and fat and promote HCC occurrence. We propose to dissect the effect of ALDH6A1 on glycolysis, TCA cycle and oxidative phosphorylation by using Seahorse and metabolomic mass spectrometry techniques; verify the in vivo function of ALDH6A1 in suppressing HCC by using HCC mouse models related to metabolism; unveil the regulatory network by integral analysis of RNA-Seq and targeted metabolome data. This study will further unveil the hardcore mechanism of the disturbed metabolic homeostasis and metabolic reprogramming of glucose and fat in HCC pathogenesis. It also provides new insights and evidence for the development of novel strategy for HCC therapy.

糖脂代谢重塑是肝细胞癌HCC的基本特征，其协同调控HCC发生的核心机制尚待进一步阐明。线粒体醛脱氢酶ALDH6A1催化嘧啶和缬氨酸代谢中间产物甲基丙二酸半醛脱氢生成乙酰辅酶A、丙酰辅酶A与NADH，进入三羧酸循环和氧化磷酸化。前期公共数据库、组织芯片和新收肝癌样本的研究分析显示ALDH6A1表达在肝癌中显著下调；其表达水平与肝癌病理分级负相关，与病人预后正相关。ALDH6A1抑制肝癌细胞增殖、迁移和裸鼠体内皮下成瘤的能力。ALDH6A1显著影响线粒体功能，导致NADH和ROS增加，但耗氧率和线粒体膜势能降低。ALDH6A1降低肝癌细胞的乳酸水平，减少了脂质堆积。ALDH6A1还能与线粒体复合物I的组分NDUFS7以及三羧酸循环中的MDH2相互作用。这些前期研究提示ALDH6A1可能通过影响线粒体功能，从而调控糖脂代谢重塑、抑制HCC发生。我们计划采用Seahorse与代谢组质谱等技术系统分析ALDH6A1对糖酵解、TCA循环和氧化磷酸化的影响；利用代谢相关小鼠HCC模型，体内验证ALDH6A1在糖脂代谢重塑和HCC发生中的功能；RNA-Seq和代谢组整合解析ALDH6A1作用的机制和网络。本研究将进一步揭示代谢稳态失衡、糖脂代谢重塑在HCC发生发展中的核心作用，为研发HCC治疗新策略提供科学依据。

多种代谢物的改变体现肝细胞癌（HCC）代谢重编程的不同特征。然而，包括丙酰辅酶A（Pro-CoA）在内的大多数代谢物在代谢重编程和肝癌发生中的功能都是未知的。本研究旨在剖析Pro-CoA代谢如何影响在肝癌发生中的这些过程。来自于TCGA和新收肝癌样本的数据用于分析ALDH6A1介导的Pro-CoA代谢及其与HCC的相关性。靶向质谱法测定HCC病人血清和肝癌组织中的多种代谢物。通过HCC细胞增殖、迁移、裸鼠异种移植模型和小鼠原发性肝癌模型评估ALDH6A1生成的Pro-CoA的功能。非靶向代谢组学和靶向能量代谢组学分析结合后续多种生化检测剖析潜在机制。我们发现，由于 ALDH6A1 下调导致的Pro-CoA及其衍生物丙酰-L-肉碱 （PLC） 的减少与 HCC 密切相关。在功能上，ALDH6A1介导的Pro-CoA代谢体外抑制了小鼠的HCC细胞增殖并在体内损害肝癌发生。醛脱氢酶活性对于 ALDH6A1 功能是不可或缺的，而Pro-CoA羧化酶通过消除Pro-CoA来拮抗 ALDH6A1 功能。从机制上讲，ALDH6A1 导致癌症中枢碳代谢的特征富集和能量代谢受损：ALDH6A1 产生的Pro-CoA抑制了柠檬酸合酶 （CS） 活性，随后降低了 TCA 循环通量，损害了线粒体呼吸和膜电位，并减少了 ATP 的产生。此外，Pro-CoA代谢产生2-甲基柠檬酸（MCA），模拟Pro-CoA对CS的抑制作用，抑制线粒体呼吸和HCC细胞增殖。我们的研究揭示了 ALDH6A1 介导的Pro-CoA代谢下降有助于代谢重塑和促进肝癌发生的新特征。Pro-CoA、PLC和MCA可作为诊断和治疗肝细胞癌的新型代谢生物标志物。Pro-CoA代谢可能为开发针对HCC的新策略提供潜在的靶点。

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