S-Adenosylmethionine in Protein Posttranslational Modifications and HCC Treatment

 说明(申请人提供)：S-腺苷蛋氨酸(同)是主要的生物甲基供体，多胺和谷胱甘肽的前体。在所有哺乳动物细胞中，蛋氨酸腺苷转移酶(MAT)也有同样的作用。在肝脏中，同样的生物合成是由MatI/III同工酶(由MAT1A编码的A1的四聚体和二聚体)进行的。慢性肝病患者肝组织MAT活性和水平均降低。我们发现，缺乏MAT1A的小鼠表现出氧化应激增加，并自发发展为肝细胞癌(HCC)。在美国，Same作为一种健康补充剂被广泛使用，在欧洲和亚洲也被用于治疗。尽管它被广泛使用，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的许多作用是被其代谢物甲硫腺苷(MTA)模仿的。特别是，两者都选择性地促进肝和结肠癌细胞的凋亡，并抑制促炎细胞因子的表达。甲基供体也是如此，但MTA抑制甲基化。我们的累积结果表明，翻译后三个关键的蛋白质修饰，即甲基化、总甲基化和磷酸化，在慢性肝功能衰竭的背景下是失调的，可以通过相同的方法纠正。这些通路之间存在着有趣的串扰和相互作用，这些通路在肝细胞癌中经常是失调的，并且是SAME和MTA的药物给药的靶点。这项应用旨在研究肝脏相同缺陷如何影响这些蛋白质翻译后修饰(PTM)，以及药理学上相同/MTA如何在这些途径上发挥作用。提出了四个具体的目标：1.定义相同的耗竭对总甲基化机制和蛋白质PTM的影响。我们将阐明肝脏同型缺乏症对SUMO机制的影响以及SUMO-、Ser/Thr-磷酸和甲基蛋白质组的变化。2.检测相同的缺失对MAPKs、受体酪氨酸激酶(RTKs)和酪氨酸磷酸化蛋白质组的活性的影响。我们的初步数据表明，几种MAPK和RTK的活性受到相同水平的影响。我们将阐明涉及的机制以及相同水平如何影响Tyr-磷酸蛋白质组。3.检测药理作用和MTA对蛋白质PTM的作用。药理相同和MTA减少泛素结合酶9(Ubc9)，并抑制ERK和Akt信号转导。Ubc9是唯一的用于苏莫化的E2酶。我们将确定负责的分子，涉及的机制，并检测它们对肝癌细胞系和正常人类肠道上皮细胞中蛋白质PTM的影响。4.观察SAME/MTA或索拉非尼单独或联合治疗肝癌的疗效。我们将比较口服SAME或MTA±索拉非尼与单用索拉非尼治疗肝癌生长和复发的疗效。这些治疗对蛋白质PTM的影响也将在肿瘤组织中进行检测。这些目标的成功实现将加强我们对受SAME调控的蛋白PTM之间复杂相互作用的了解，并评估SAME/MTA在肝癌治疗中的有效性，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.