Tumor suppressing pathways in renal cancer

肾癌的肿瘤抑制途径

• 批准号: 10426280
• 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/10426280
• 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; 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; 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.

透明细胞肾细胞癌 (ccRCC) 是军事人员的一个严重的健康问题，尤其是男性以外的男性 40岁以上，包括退伍军人。根据 1995-2004 年国防医学流行病学数据库 军事人员在 40 岁之后的 RCC 发病率急剧上升至 8.5 相比之下，总发病率为每 10 万人年 1.5 例。该提案直接调查 肾癌选择性自噬与葡萄糖代谢之间的机制联系 细胞。自噬是一个严格调控的自我消化过程。自噬体的形成需要 微管相关蛋白 1 轻链 A、B 和 C（MAP1LC3A、B、C，称为 LC3A、LC3B 和 LC3C）进入自噬体膜。我们确定 LC3C 自噬是 肿瘤抑制和VHL下游功能，肿瘤抑制因子在透明细胞肾细胞癌中丢失。 LC3C 是一种进化晚期基因，仅存在于高等灵长类动物和人类中，包含独特且 保守的 C 端 20 个氨基酸肽，在 LC3C 自噬启动过程中被切割。 C端 LC3C 的肽具有类似于 HIFα 中的典型基序的脯氨酸羟基化基序，其中脯氨酸是 被 2-羟基戊二酸 (2OG) 依赖性 EGLN 脯氨酸羟化酶羟基化。我们的初步数据表明 LC3C 肽中的 P133 在自噬过程中被 EGLN3 脯氨酸羟化酶羟化 依赖方式。最近我们发现LC3C自噬需要葡萄糖代谢流。反过来，损失 LC3C 增加了糖酵解和磷酸戊糖代谢物的稳态水平，代表 致癌代谢形式的标志（Warburg 效应）与 ccRCC 特别相关。我们确定 LC3C 免疫共沉淀和自溶酶体降解苹果酸/2-羟基戊二酸 (2OG) 和的靶标 天冬氨酸/谷氨酸逆向转运蛋白，分别是 SCL25A11 和 SLC25A13，它们是线粒体的一部分 苹果酸-天冬氨酸穿梭机（MAS）。 MAS 在线粒体和细胞质之间转移还原当量， 产生线粒体 NADH 用于 ATP 合成，同时产生胞质 NAD 以维持糖酵解。此外， 穿梭机交换谷氨酸和天冬氨酸，这有助于生物合成潜力。我们提议写一本小说， 肿瘤抑制 LC3C 活性的代谢耦合机制：LC3C 自噬靶向 MAS 蛋白 线粒体自噬过程中的溶酶体降解。这通过调节作为糖酵解的检查点 胞质 NAD/NADH 比率，以及 SLC25A13 介导的线粒体天冬氨酸输出。那 表明 LC3C 在代谢上与 VHL 抑制糖酵解的转录作用相配合。此外，我们 假设线粒体载体附近 LC3C 自噬的选择性激活是由 2OG 通过 TCA 循环从葡萄糖衍生而来，并通过激活 SLC25A11 进行运输 EGLN3，导致 P133 羟基化。在这里，我们将机械地研究代谢输入和功能 肾癌中与 MAS 相关的 LC3C 自噬输出。目标 1 将确定 LC3C 的作用 SLC25A11/13 自噬降解中的结构元件以及 MAS 的功能后果 活动。我们假设 LC3C C 端肽和 P133 是必要的，并且可能足以实现这一点 活动。目标 2 将确定 LC3C 肿瘤抑制活性的代谢效应。我们假设 LC3C 将 (i) 抑制 NAD/NADH 再生和糖酵解活性，以及​​ (ii) 抑制线粒体中天冬氨酸的输出 及其生物合成途径的可用性。目标 3 将确定葡萄糖的分子机制 调节 LC3C 活性。我们建议通过检测线粒体 2OG 水平来激活 LC3C 自噬 通过 C 端肽中 P133 的 EGLN3 依赖性羟基化。 EGLN3-LC3C 利用的 2OG 是 源自葡萄糖并通过 SLC25A11 离开线粒体，表明葡萄糖氧化对于 LC3C 自噬活性。