Tumor suppressing pathways in renal cancer

肾癌的肿瘤抑制途径

• 批准号: 10252173
• 负责人: Maria F Czyzyk-Krzeska
• 金额: --
• 依托单位: CINCINNATI VA MEDICAL CENTER RESEARCH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10252173
• 关键词: ATP Synthesis Pathway; Affect; Age; Age-Years; Amino Acids; Anabolism; Aspartate; Autophagocytosis; Autophagosome; Bioenergetics; C-terminal; Cell Proliferation; Citric Acid Cycle; Clear cell renal cell carcinoma; Cleaved cell; Clinical; Complex; Coupled; Cytoplasm; Cytosol; Data; Databases; Digestion; Elements; Enzymes; Epidemiology; Felis catus; Gender; Genes; Genetic Transcription; Glucose; Glutamates; Glycolysis; Health; Human; Hydroxylation; Incidence; Investigational Therapies; Ions; Life; Light; Lipids; MAP1 Microtubule-Associated Protein; Maintenance; Malate-Aspartate Shuttle Pathway; Malates; Malignant Neoplasms; Mediating; Medical; Membrane; Metabolic; Metabolism; Military Personnel; Mitochondria; Modeling; Molecular; NADH; Natural regeneration; Oncogenic; Output; Oxidation-Reduction; Pathway interactions; Pentosephosphate Pathway; Pentosephosphates; Peptides; Persons; Population; Primates; Process; Procollagen-Proline Dioxygenase; Proline; Regulation; Renal Cell Carcinoma; Renal carcinoma; Risk; Risk Factors; Role; Structure; Testing; Tobacco smoking behavior; Tumor Suppressor Proteins; Veterans; Warburg Effect; Work; antiporter; cancer cell; caveolin 1; former smoker; gene repression; glucose metabolism; lysosomal proteins; male; military veteran; new therapeutic target; novel; oxidation; paralogous gene; prevent; service member; stemness; therapeutic target; tumor

Clear cell renal cell carcinoma (ccRCC) is a serious health concern for military personnel, particularly males beyond 40 years of age, including military veterans. According to The Defense Medical Epidemiology Database for 1995-2004 the incidence of RCC specifically for military members after the 4th decade of life is dramatically increased to 8.5 as compared to 1.5 cases per 100,000 person-years of the overall incidence. This proposal investigates direct mechanistic connection between selective autophagy and glucose metabolism in the context of renal cancer cells. Autophagy is a tightly regulated process of self-digestion. Formation of an autophagosome requires lipidation and insertion of microtubule associated protein 1 light chains A, B and C (MAP1LC3A, B, C, referred to as LC3A, LC3B, and LC3C) into the autophagosomal membrane. We established that LC3C autophagy is tumor suppressing and functions downstream form VHL, tumor suppressor lost in clear cell renal cell carcinoma. LC3C is an evolutionary late gene, present only in higher primates and humans, that contains a unique and conserved C-terminal 20 amino acid peptide that is cleaved during initiation of LC3C autophagy. The C-terminal peptide of LC3C has a proline hydroxylation motif similar to the canonical motifs in HIFαs, where prolines are hydroxylated by 2-oxyglutarate (2OG)-dependent EGLN proline hydroxylases. Our preliminary data indicate that P133 within the LC3C peptide undergoes hydroxylation by EGLN3 proline hydroxylase in an autophagy- dependent manner. Recently we discovered that LC3C autophagy requires glucose metabolic flux. In turn, loss of LC3C increases the steady-state levels of glycolytic and pentose phosphate metabolites, representing hallmarks of oncogenic form of metabolism (Warburg effect) particularly relevant in ccRCC. We determined that LC3C co-immunoprecipitates and targets for autolysosomal degradation malate/2-oxyglutarate(2OG) and aspartate /glutamate antiporters, SCL25A11 and SLC25A13, respectively that are part of the mitochondrial malate-aspartate shuttle (MAS). MAS transfers reducing equivalents between mitochondria and cytoplasm, yielding mitochondrial NADH for ATP synthesis while generating cytosolic NAD to sustain glycolysis. Additionally, the shuttle exchanges glutamate and aspartate that contributes to biosynthetic potential. We propose a novel, metabolism-coupled mechanism of tumor suppressing LC3C activity: LC3C autophagy targets MAS proteins for lysosomal degradation in the process of mitophagy. This acts as a checkpoint for glycolysis by regulating cytosolic NAD/NADH ratio, as well as for SLC25A13-mediated export of aspartate from mitochondria. That indicates that LC3C metabolically partners with transcriptional effects of VHL inhibiting glycolysis. Moreover, we hypothesize that selective activation of LC3C autophagy in the proximity to mitochondrial carriers is caused by 2OG derived from glucose through the TCA cycle and transported through the SLC25A11 which activates EGLN3, leading to P133 hydroxylation. Here we will mechanistically investigate metabolic inputs and functional output of LC3C autophagy in regard to MAS in the context of renal cancer. Aim 1 will determine role of LC3C structural elements in the autophagic degradation of SLC25A11/13 and in functional consequences for MAS activity. We hypothesize that LC3C C-terminal peptide and P133 are necessary and potentially sufficient for this activity. Aim 2 will identify metabolic effects of LC3C tumor suppressing activity. We hypothesize that LC3C will (i) inhibit NAD/NADH regeneration and glycolytic activity and (ii) suppress export of aspartate from mitochondria and its availability for biosynthetic pathways. Aim 3 will determine molecular mechanism by which glucose regulates LC3C activity. We propose that LC3C autophagy is activated by sensing mitochondrial 2OG levels through EGLN3-dependent hydroxylation of P133 in the C-terminal peptide. 2OG utilized by EGLN3-LC3C is derived from glucose and exits mitochondria through SLC25A11, indicating that glucose oxidation is critical for LC3C autophagic activity.

清除细胞肾细胞癌（CCRCC）是军事人员的严重健康问题，尤其是以外的男性 40岁，包括退伍军人。根据国防医学流行病学数据库1995- 2004年 在生命的第四个十年之后，专门针对军事成员的RCC事件急剧增加到8.5 相比之下，总体事件的每100,000人年为1.5例。该提案调查了直接 在肾癌的背景下，选择性自噬和葡萄糖代谢之间的机械联系 细胞。自噬是一个严格的自我消化过程。自噬体的形成需要 微管相关蛋白1光链A，B和C的脂质和插入（map1lc3a，b，c，引用 作为LC3A，LC3B和LC3C）进入自噬体膜。我们确定LC3C自噬是 抑制肿瘤和功能下游形成VHL，在透明细胞肾细胞癌中丧失的肿瘤抑制剂。 LC3C是一个进化的晚期基因，仅存在于高级灵长类动物和人类中，其中包含一个独特的和 在LC3C自噬开始期间裂解的保守C末端20氨基酸肽。 C末端 LC3C的肽的脯氨酸羟基化基序与HIFαs中的规范基序相似，其中脯氨酸为 由2-氧基戊二酸（2OG）依赖性EGLN脯氨酸羟基酶羟基羟基羟基化。我们的初步数据表明 LC3C剥离内的P133在自噬中通过EGLN3脯氨酸羟化酶经过羟基化 依赖方式。最近，我们发现LC3C自噬需要葡萄糖代谢通量。反过来，损失 LC3C的稳定水平增加了糖酵解和磷酸五磷酸磷酸盐代谢物，代表 代谢的致癌形式的标志（Warburg效应）在CCRCC中尤其重要。我们确定了这一点 LC3C自染色体降解苹果酸/2-氧气（2OG）和靶标的共释剂和靶标（2OG）和 天冬氨酸 /谷氨酸抗植物，SCL25A11和SLC25A13，分别是线粒体的一部分 苹果酸天冬氨酸班车（MAS）。 MAS转移减少线粒体和细胞质之间的当量， 产生线粒体NADH进行ATP合成，同时产生胞质NAD来维持糖酵解。此外， 班车交换有助于生物合成潜力的谷氨酸和天冬氨酸。我们提出了一本小说， 抑制LC3C活性的代谢耦合机制：LC3C自噬靶向MAS蛋白的MAS蛋白 线粒体过程中的溶酶体降解。通过调节，这是糖酵解的检查点 胞质NAD/NADH比以及SLC25A13介导的天冬氨酸的出口。那 表明LC3C代谢与VHL抑制糖酵解的转录作用的伴侣。而且，我们 假设在与线粒体载体附近的LC3C自噬的选择性激活是由 2og从葡萄糖通过TCA周期得出，并通过SLC25A11运输 EGLN3，导致P133羟基化。在这里，我们将机械研究代谢输入和功能 在肾癌的背景下，LC3C自噬的输出。 AIM 1将确定LC3C的作用 SLC25A11/13自噬降解的结构元素以及对MAS的功能后果 活动。我们假设LC3C C末端胡椒和P133是必要的，并且可能足以满足 活动。 AIM 2将确定LC3C抑制活性的代谢作用。我们假设LC3C将会 （i）抑制NAD/NADH再生和糖酵解活性，以及​​（ii）抑制从线粒体的天冬氨酸出口 及其对生物合成途径的可用性。 AIM 3将确定葡萄糖的分子机制 调节LC3C活性。我们建议通过传感线粒体2og水平激活LC3C自噬。 通过C末端肽中P133的EGLN3依赖性羟基化。 EGLN3-LC3C使用的2OG是 源自葡萄糖并通过SLC25A11退出线粒体，表明葡萄糖氧化对于 LC3C自噬活动。