Tumor suppressing pathways in renal cancer

透明细胞肾细胞癌（ccRCC）是军事人员的严重健康问题，特别是男性， 40岁，包括退伍军人。根据1995-2004年国防医学流行病学数据库， 特别是在40岁以后的军事人员中，RCC的发病率急剧增加到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 肾癌中选择性自噬与糖代谢的机制联系 细胞自噬是一种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的脂化和插入（MAP 1 LC 3A、B、C，参见 称为LC 3A、LC 3B和LC 3C）进入自噬体膜。我们确定LC 3C自噬是一种 肿瘤抑制和VHL下游功能，透明细胞肾细胞癌中的肿瘤抑制因子丢失。 LC 3C是一种进化晚期基因，仅存在于高等灵长类动物和人类中， 在LC 3C自噬起始期间被切割的保守的C-末端20个氨基酸肽。c-末端 LC 3C的肽具有类似于HIFαs中的典型基序的脯氨酸羟基化基序，其中脯氨酸被 通过2-氧戊二酸（2 OG）依赖性EGLN脯氨酸羟化酶进行羟基化。我们的初步数据显示， LC 3C肽内的P133在自噬中通过EGLN 3脯氨酸羟化酶进行羟基化， 依赖的方式。最近我们发现LC 3C自噬需要葡萄糖代谢流。反过来，损失 LC 3C增加糖酵解和戊糖磷酸代谢物的稳态水平，代表 致癌代谢形式的标志（瓦尔堡效应）在ccRCC中尤其相关。我们确定 LC 3C共免疫沉淀物和自体溶酶体降解的靶点苹果酸/2-氧戊二酸（2 OG）和 天冬氨酸/谷氨酸反向转运蛋白，分别为SCL 25 A11和SLC 25 A13，它们是线粒体的一部分。 苹果酸-天冬氨酸穿梭（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，本发明还 穿梭体交换谷氨酸和天冬氨酸，这有助于生物合成潜力。我们要写一部小说， 肿瘤抑制LC 3C活性的代谢偶联机制：LC 3C自噬靶向MAS蛋白， 线粒体自噬过程中的溶酶体降解。这作为糖酵解的检查点，通过调节 细胞溶质NAD/NADH比率，以及SLC 25 A13介导的天冬氨酸从线粒体的输出。的 表明LC 3C在代谢上与VHL抑制糖酵解的转录作用相关。而且我们 假设LC 3C自噬在线粒体载体附近的选择性激活是由 2 OG通过TCA循环从葡萄糖衍生，并通过SLC 25 A11转运， EGLN 3，导致P133羟基化。在这里，我们将机械地研究代谢输入和功能 LC 3C自噬的输出与肾癌背景下的MAS有关。目标1将确定LC 3C的作用 SLC 25 A11/13自噬降解和MAS功能后果中的结构元件 活动我们假设LC 3C C-末端肽和P133是必要的，并且可能足以实现这一点。 活动目的2将鉴定LC 3C肿瘤抑制活性的代谢作用。我们假设LC 3C将 (i)抑制NAD/NADH再生和糖酵解活性和（ii）抑制天冬氨酸从线粒体输出 以及其在生物合成途径中的可用性。目的3将确定葡萄糖 调节LC 3C活性。我们认为LC 3C自噬是通过感知线粒体2 OG水平而激活的。 通过C末端肽中P133的EGLN 3依赖性羟基化。EGLN 3-LC 3C使用的2 OG是 来源于葡萄糖并通过SLC 25 A11离开线粒体，表明葡萄糖氧化对于 LC 3C自噬活性。

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.