BCAT1调节支链氨基酸代谢在胃癌中的作用及机制研究

Reprogrammed cellular metabolism is a common characteristic observed in various cancers. The abnormal branched-chain amino acids (BCAAs) metabolism and high levels of BCAA transaminase 1(BCAT1) have recently been associated with aggressiveness in several cancer entities. We have reported the metabolic profiling of tissue samples from lymph node metastasis (LNM)-positive gastric cancer (GC) patients (n=40), LNM-negative GC patients (n=40) and normal controls (n=40) based on untargeted 1H nuclear magnetic resonance (NMR) metabolomics approach. Surprisingly, we found the levels of BCAAs (leucine, isoleucine and valine) not only were increased in GC tissue specimens, but also were up-regulated during the progression of GC. Moreover, we also showed that BCAT1 was aberrantly activated in GC tissue samples. Knockdown of the BCAT1 expression in GC cells led to sharp decrease of cell proliferation, migration and invasion, induced cell apoptosis and inhibited cell cycle progression in vitro. However, whether BCAAs metabolism and BCAT1 directly regulate gastric cancer (GC) occurrence and development remains poorly understood. In this study, we will continue to verify the biological effect of BCAA metabolism and BCAT1 in vitro and vivo experiments. Moreover, the stable-isotope tracer (13C-labelled BCAAs and 15N-labelled BCAAs) experiments combined with targeted GC/MS metabolites analysis are used to demonstrate the intracellular production of BCAAs metabolism by BCAT1. Furthermore, to identify the molecular mechanism of BCAT1, we will analyze the histone methylation enzymes co-expression with BCAT1 using the data of GC methylation array from the TCGA database combined with experimental verification. This study not only get a major breakthrough in the underlying mechanism study of GC metabolic regulation, but also hit a novel strategy and target towards to GC therapy in the future.

细胞代谢重编程是多种癌症的共同特征，支链氨基酸（BCAAs）代谢异常、支链氨基酸氨基转移酶1（BCAT1）的高表达和多种癌症恶变有关。前期研究利用非靶向代谢组学方法，检测了40例正常对照、40例淋巴结转移阴性和40例淋巴结转移阳性的胃癌组织代谢谱，发现BCAAs含量随着胃癌恶性进程明显上调，且BCAT1在胃癌组织中异常高表达，沉默BCAT1显著抑制胃癌细胞的增殖、迁移和侵袭，并诱导细胞凋亡和周期阻滞。然而，BCAAs代谢和BCAT1是如何调节胃癌发生发展还所知甚少。本项目拟：①通过体内外实验检测BCAT1在胃癌中的生物学效应；②通过稳定同位素示踪结合GC/MS的靶向代谢物检测确定BCAAs在胃癌中的主要代谢流向；③通过TCGA数据库分析并结合实验验证BCAT1的调控机制。本研究不仅将阐明BCAAs在胃癌中的代谢变化及调控机制，而且为个体化医疗及小分子抑制剂的开发提供新的靶点及理论支持。

胃癌是影响我国居民健康的常见恶性肿瘤之一，给家庭、社会和国家造成了沉重的经济负担和损失，已成为一个亟待解决的公共卫生问题。本课题组前期利用非靶向代谢组学方法，检测了40例正常对照、40例淋巴结转移阴性和40例淋巴结转移阳性的胃癌组织代谢谱，发现支链氨基酸（BCAAs）含量随着胃癌恶性进程明显上调，支链氨基酸氨基转移酶1（BCAT1）在胃癌组织中异常高表达且与患者的生存期显著相关。本项目以BCAT1为切入点，研究BCAAs代谢在胃癌发生发展中的作用及其机制。首先采用siRNA抑制BCAT1的表达，明显降低了胃癌细胞的体外增殖、迁移和侵袭能力，随后利用慢病毒筛选了稳定敲低和过表达BCAT1的单克隆细胞株，体外实验和上述结果保持一致，尤其是胃癌细胞的转移能力变化显著。我们随之检测了和细胞转移密切相关的EMT化标志物，发现抑制BCAT1的表达，细胞间质标志物N-cadherin、β-catenin、Vimentin表达水平较对照组明显降低，上皮标志物E-cadherin表达升高，而过表达BCAT1后，上述标志物的表达正好相反。最后利用稳定敲低BCAT1的单克隆细胞株进行蛋白质组和代谢组学联合分析，并加以实验验证，发现敲低BCAT1后，BCAAs（Leu/Ile，Val）的含量明显降低，说明胃癌中BCAAs的代谢流向应是从BCKAs（支链氨基酮酸）转化成BCAAs，而BCAAs的减少抑制了mTOR通路，进而影响了和迁移、侵袭相关的一些蛋白质的合成，最终抑制胃癌细胞的转移。此外，BCKAs的堆积使得BCKDH酶活性增强，Acetyl CoA合成增多，但大量的Acetyl CoA作为底物进入了脂质代谢，导致TCA降低，能量合成减少，也抑制了胃癌细胞的发生发展。总之，本研究明确了BCAAs在胃癌中的代谢变化及调控机制，为胃癌的个体化医疗及小分子抑制剂的开发提供理论支持。