S-Adenosylmethionine in Protein Posttranslational Modifications and HCC Treatment

 描述（由申请人提供）：S-腺苷甲硫氨酸（SAMe）是主要的生物甲基供体，是多胺和GSH的前体。SAMe在所有哺乳动物细胞中通过甲硫氨酸腺苷转移酶（MAT）产生。在肝脏中，SAMe生物合成通过MAT 1/III同工酶（由MAT 1A编码的α 1的四聚体和二聚体）进行。慢性肝病患者的肝脏MAT活性和SAMe水平降低。我们发现，缺乏MAT 1A的小鼠表现出增加的氧化应激和自发发展肝细胞癌（HCC）。SAMe在美国广泛用作健康补充剂，在欧洲和亚洲用于治疗。尽管其用途广泛，但其作用的分子机制在很大程度上尚不清楚。我们已经报道，SAMe的许多行动是模仿其代谢产物甲硫腺苷（MTA）。特别是，两者都是选择性地促肝癌和结肠癌细胞凋亡，并抑制促炎细胞因子的表达。SAMe是甲基供体，但MTA抑制甲基化。我们积累的结果显示，三种关键的翻译后蛋白质修饰，甲基化、类小泛素化和磷酸化，在慢性肝SAMe耗竭的情况下失调，可以通过SAMe校正。这些途径之间存在有趣的串扰和相互作用，这些途径在HCC中经常失调，并且通过SAMe和MTA的药理学给药靶向。该应用程序旨在研究肝脏SAMe缺乏如何影响这些蛋白质翻译后修饰（PTM）以及药理学SAMe/MTA如何作用于这些途径。提出了四个具体目标：1。确定SAMe耗竭对类小泛素化机制和蛋白质PTM的影响。我们将阐明肝脏SAMe缺乏对SUMO化机制和SUMO-，Ser/Thr-磷酸化和甲基化蛋白质组变化的影响。2.检查SAMe消耗如何影响MAPK、受体酪氨酸激酶（RTK）和Tyr-磷酸蛋白质组的活性。我们的初步数据表明几种MAPK和RTK的活性受SAMe水平的影响。我们将阐明所涉及的机制以及SAMe水平如何影响Tyr-磷酸化蛋白质组。3.检查SAMe和MTA对蛋白质PTM的药理作用。药理学SAMe和MTA减少泛素缀合酶9（Ubc 9），其是用于sumoylation的唯一E2酶，并抑制ERK和Akt信号传导。我们将确定负责的分子，涉及的机制，并检查它们对肝癌细胞系和正常人肠上皮细胞中蛋白质PTM的影响。4.检查SAMe/MTA或索拉非尼单独或联合治疗HCC的有效性。我们将检查口服SAMe或MTA ±索拉非尼与索拉非尼单独治疗HCC生长和复发的疗效。还将在肿瘤组织中检查这些治疗对蛋白质PTM的影响。这些目标的成功完成应该增强我们对SAMe调控的蛋白质PTM之间复杂相互作用的了解，并评估SAMe/MTA在HCC治疗中的疗效，这些主题与公共卫生高度相关。

 DESCRIPTION (provided by applicant): S-adenosylmethionine (SAMe) is the principal biological methyl donor, precursor of polyamines and GSH. SAMe is made in all mammalian cells via the enzyme methionine adenosyltransferase (MAT). In liver, SAMe biosynthesis is carried out by MATI/III isoenzymes (tetramer and dimer of a1 encoded by MAT1A). Patients with chronic liver disease have reduced hepatic MAT activity and SAMe levels. We showed that mice lacking MAT1A exhibit increased oxidative stress and develop hepatocellular carcinoma (HCC) spontaneously. SAMe is widely available in the United States as a health supplement and is used therapeutically in Europe and Asia. Despite its wide usage, the molecular mechanisms of its actions are largely unclear. We have reported that many of SAMe's actions are mimicked by its metabolite methylthioadenosine (MTA). In particular, both are selectively pro-apoptotic in liver and colon cancer cells and inhibit the expression of pro-inflammatory cytokines. SAMe is a methyl donor but MTA inhibits methylation. Our accumulated results show three key post- translational protein modifications, methylation, sumoylation and phosphorylation, are dysregulated in the setting of chronic hepatic SAMe depletion that can be corrected by SAMe. There is interesting crosstalk and interplay between these pathways, which are frequently dysregulated in HCC, and are targeted by pharmacologic administration of SAMe and MTA. This application is designed to examine how hepatic SAMe deficiency impacts on these protein posttranslational modifications (PTMs) and how pharmacologic SAMe/MTA work on these pathways. Four specific aims are proposed: 1. Define the effect of SAMe depletion on sumoylation machinery and protein PTMs. We will elucidate the effect of hepatic SAMe deficiency on sumoylation machinery and changes in the SUMO-, Ser/Thr-phospho- and methyl-proteomes. 2. Examine how SAMe depletion affects activity of MAPKs, receptor tyrosine kinases (RTKs) and Tyr-phospho- proteome. Our preliminary data indicate the activity of several MAPKs and RTKs are affected by SAMe level. We will elucidate the mechanisms involved and how SAMe level impacts on the Tyr-phosphoproteome. 3. Examine pharmacologic SAMe and MTA actions on protein PTMs. Pharmacologic SAMe and MTA reduce ubiquitin-conjugating enzyme 9 (Ubc9), the sole E2 enzyme for sumoylation, and inhibit ERK and Akt signaling. We will determine the molecule responsible, mechanisms involved, and examine their effects on protein PTMs in liver cancer cell lines and normal human intestinal epithelial cells. 4. Examine effectiveness of SAMe/MTA or sorafenib alone or in combination in HCC treatment. We will examine the efficacy of oral SAMe or MTA ± sorafenib versus sorafenib alone in the treatment of HCC growth and recurrence. The effect of these treatments on protein PTMs will also be examined in tumor tissues. Successful completion of these aims should enhance our knowledge of the complex interplay between the protein PTMs that is regulated by SAMe and assess SAMe/MTA efficacy in HCC treatment, topics that are highly relevant to public health.