Molecular Basis of Cancer Cell Metabolic Reprogramming

 描述(申请人提供)：核受体是肝脏疾病的重要调节因子。癌症中的代谢重排与表观遗传水平的变化密切相关，代谢和表观遗传学的相互作用构成了癌症生物学的一条新途径。暴露于高浓度无机砷会增加患肝癌的风险。然而，砷的毒性机制还不是很清楚。我们过去的研究已经建立了核受体小异二聚体伙伴(SHP)对DNA甲基转移酶(DNMT)表达的反馈调节。利用无偏见的基因组筛选方法和SHP-/-小鼠，我的实验室最近发现了线粒体蛋白丙酮酸脱氢酶激酶4(PDK4)作为一种新的假定的肿瘤抑制因子，它介导了对肝癌代谢重新编程的表观遗传控制。总体目标是阐明PDK4和砷在肝癌细胞代谢和生长中的一种新的串扰。中心假说是，PDK4是哺乳动物雷帕霉素靶(MTOR)途径下游的关键营养感受器，通过抑制谷氨酰胺分解中的谷氨酸脱氢酶(GDH)和TCA循环中的丙酮酸脱氢酶(PDH)而发挥双刃剑的作用，这两条途径是促进癌症代谢的两条中心途径。此外，我们假设砷通过DNMT促进PDK4的表观遗传沉默，从而促进肝癌的生长。具体目标：1)确定营养素介导的PDK4在调节线粒体谷氨酰胺分解中的表达；2)确定PDK4引起的代谢重编程和线粒体功能的改变；以及3)确定砷对PDK4的表观遗传调控和肝癌发生的影响。拟议的实验建立在我们长期研究核受体对肝细胞癌发展的调控和适当使用基因敲除小鼠模型方面的经验。这项研究所需的关键试剂和方法正在进行中，包括稳定的PDK4过表达和击倒肝癌细胞，原位移植肝癌模型，以及PDK4-/-小鼠。高通量转录组学(RNA-seq)、代谢组学(GC/MS)、基因组学(MeDIP)和蛋白质组学(LC-MS/MS)将被用于拟议的研究，这使得该提案全面而可行。因此，这一应用在概念上和在实验方法的实施方面都是创新的。这项研究将营养物质介导的癌细胞代谢与表观遗传调控这一新兴的、竞争非常激烈的领域联系在一起，因此有望极大地促进我们对肝癌的基本理解。此外，PI还组建了一支具有适当专业知识的优秀研究团队，包括蛋白质组学(Schwartz，LAM)、癌细胞代谢(Ayer)、代谢组学(COX)和环境毒物(Delker)等领域。PI和她的合作者的专业知识结合在一起，进一步确保了这个项目的成功。

 DESCRIPTION (provided by applicant): Nuclear receptors are critical regulators of liver diseases. Metabolic rewiring in cancer is tightly connected to changes at the epigenetic level, and the interaction of metabolism and epigenetics constitutes a new avenue of cancer biology. Exposure to high concentrations of inorganic arsenic increases the risk of liver cancer. However, the mechanisms of arsenic toxicity remain less well understood. Our past studies have established a feedback regulation of DNA methyltransferase (Dnmt) expression by nuclear receptor small heterodimer partner (SHP). Using unbiased genomic screening approaches and SHP-/- mice, my laboratory recently uncovered the mitochondrial protein pyruvate dehydrogenase kinase 4 (PDK4) as a new putative tumor suppressor that mediates epigenetic control of HCC metabolic reprogramming. The overall objective is to elucidate a novel crosstalk between PDK4 and arsenic in HCC cell metabolism and growth. The central hypothesis is that PDK4 serves as a key nutrient sensor downstream of the mammalian target of rapamycin (mTOR) pathway, acting as a double- edged sword by inhibiting glutamate dehydrogenase (GDH) in glutaminolysis and pyruvate dehygrogenase (PDH) in TCA cycle, two central pathways that fuel cancer metabolism. Furthermore, we postulate that arsenic promotes HCC growth by enhancing epigenetic silencing of PDK4 through Dnmts. Specific Aims: 1) Determine nutrient-mediated PDK4 expression in modulation of mitochondrial glutaminolysis; 2) Determine metabolic reprogramming and altered mitochondrial function caused by PDK4; and 3) Determine the effect of arsenic on epigenetic regulation of PDK4 and hepatocarcinogenesis. The proposed experiments build on our long-standing experience in studying nuclear receptor regulation of HCC development and the appropriate use of knockout mouse models. Key reagents and methodologies required for the study are in hand, including stable PDK4 overexpression and knockdown HCC cells, an orthotopic transplanted liver cancer model, and PDK4-/- mice. High throughout transcriptomics (RNA-seq), metabolomics (GC/MS), genomics (MeDIP), and proteomics (LC-MS/MS) analyses will be employed for the proposed study, which makes the proposal comprehensive, yet feasible. Thus this application is innovative both conceptually and in the implementation of experimental approaches. This study links the emerging, very competitive field of nutrient mediated cancer cell metabolism with epigenetic regulation, and as such is expected to substantially advance our basic understanding of HCC. In addition, the PI has assembled an outstanding research team with appropriate expertise, including the areas of proteomics (Schwartz, Lam), cancer cell metabolism (Ayer), metabolomics (Cox), and environmental toxicants (Delker). The combined expertise of the PI and her collaborators further ensures the success of this project.