Investigating the Role of Reduced Branched-Chain Amino Acid Catabolism in Clear Cell Renal Cell Carcinoma

项目总结 北美是世界上肾癌发病率最高的地区，其亚型也是最常见的 肾透明细胞癌(CcRCC)。令人震惊的是，慢性肾细胞癌的发病率在美国和 全球范围内。一旦发生转移，CcRCC的总体五年生存率只有10%-12%， 证明了新疗法的必要性。根据组织学和病理学研究，慢性肾细胞癌常见的是代谢功能障碍。 分子分析。治疗慢性肾细胞癌的新疗法可能针对其失调的代谢途径。这个 CcRCC的病因很复杂，但肥胖是一个主要的危险因素。高水平的支链氨基酸 (BCAA)存在于慢性肾细胞癌和肥胖症患者的血清中，这表明可能存在机制联系。 支链氨基酸(亮氨酸、异亮氨酸和缬氨酸)是必需氨基酸，其浓度受 饮食摄入和分解代谢。支链氨基酸代谢促进许多不同类型癌症的肿瘤生长，但 支链氨基酸在ccRCC中的作用尚不清楚。支链氨基酸的分解代谢主要通过前两种酶，支链氨基酸 转氨酶(BCAT)和支链酮酸脱氢酶(BCKDH)。BCKDH是一家 催化限速反应的酶复合体。支链氨基酸分解代谢的产物随后被氧化。 在线粒体内产生琥珀酰辅酶A和乙酰辅酶A，它们可以被TCA循环用于 失活和线粒体呼吸。 我的初步数据显示支链氨基酸分解酶BCAT2和BCKDH亚单位是 与正常邻近肾组织(NAT)相比，人ccRCC肿瘤中的表达经常减少。这一数字减少了 表达早在第一阶段就发生了，并与总存活率的降低有关。此外，BCAA和 其分解代谢产物在ccRCC中减少。这些结果表明支链氨基酸的分解代谢减少。 在ccRCC早期，并有助于ccRCC的攻击性。CcRCC可降低支链氨基酸代谢 线粒体呼吸和ROS，因为ccRCC由于大量的 细胞内的脂类可以发生过氧化。CcRCC通过减少线粒体呼吸和ROS 下调与脂肪酸氧化和氧化磷酸化有关的基因。我假设 支链氨基酸代谢降低促进ccRCC细胞生长和肿瘤发生，并通过 减少线粒体呼吸和ROS的产生。目标1将确定如何减少 支链氨基酸分解代谢促进肾细胞癌细胞体外生长。我将使用遗传学和药理学方法来 评估支链氨基酸代谢的增减对肾细胞癌和永生化肾上皮细胞增殖的影响 细胞系。目的2将确定减少支链氨基酸分解代谢如何在体内促进肾细胞癌的发生。这就做 利用新的小鼠遗传模型确定支链氨基酸分解代谢通量对肾上皮细胞功能的影响 和ccRCC肿瘤发生。总之，这些方法将确定BCAA通过哪些机制 分解代谢调节ccRCC肿瘤的发生，并确定新的治疗靶点来对抗这种疾病。

PROJECT SUMMARY North America has the highest incidence of renal cancer in the world with the most common subtype being clear cell renal cell carcinoma (ccRCC). Alarmingly, the incidence of ccRCC is on the rise in the U.S. and globally. The overall five-year survival rate for ccRCC is only 10-12% once it becomes metastatic, demonstrating the need for new therapies. Metabolic dysfunction is common in ccRCC based on histologic and molecular analysis. New therapies to treat ccRCC could target its dysregulated metabolic pathways. The etiology of ccRCC is complex but a major risk factor is obesity. High levels of branched-chain amino acids (BCAAs) are present in the serum of patients with ccRCC and obesity, suggesting a potential mechanistic link. BCAAs (leucine, isoleucine, and valine) are essential amino acids, whose concentrations are regulated by dietary intake and catabolism. BCAA metabolism promotes tumor growth in many different types of cancer, but the role of BCAAs in ccRCC is unknown. BCAA catabolism occurs primarily via the first two enzymes, BCAA transaminase (BCAT) and branched chain ketoacid dehydrogenase (BCKDH), respectively. BCKDH is an enzyme complex that catalyzes the rate-limiting reaction. The products of BCAA catabolism are then oxidized within the mitochondria to produce succinyl-CoA and acetyl-CoA, which can be used by the TCA cycle for anaplerosis and mitochondrial respiration. My preliminary data demonstrate that BCAA catabolic enzyme BCAT2 and BCKDH subunits are frequently reduced in human ccRCC tumors compared to normal adjacent kidney tissue (NAT). This decreased expression occurs as early as stage 1 and is associated with reduced overall survival. Additionally, BCAAs and their catabolic metabolites are decreased in ccRCC. These results suggest that BCAA catabolism is reduced early in ccRCC and contributes to ccRCC aggressiveness. ccRCC may reduce BCAA metabolism to decrease mitochondrial respiration and ROS because ccRCC is susceptible to ROS due to the large amount of intracellular lipids that can undergo peroxidation. ccRCC decreases mitochondrial respiration and ROS by downregulating genes involved in fatty acid oxidation and oxidative phosphorylation. I hypothesize that reduced BCAA metabolism promotes ccRCC cell growth and tumorigenesis, and does so by decreasing mitochondrial respiration and the production of ROS. Aim 1 will determine how reduced BCAA catabolism promotes ccRCC cell growth in vitro. I will use genetic and pharmacologic approaches to assess how gain or loss of BCAA metabolism affects proliferation of ccRCC and immortalized renal epithelial cell lines. Aim 2 will identify how reduced BCAA catabolism contributes to ccRCC tumorigenesis in vivo. I will use novel genetic mouse models to determine the role of BCAA catabolic flux on renal epithelial cell function and ccRCC tumorigenesis. Together, these approaches will identify the mechanisms by which BCAA catabolism regulates ccRCC tumorigenesis and identify novel therapeutic targets to combat this disease.