Tumor Suppressing Pathways in Kidney Cancer

 描述（由申请人提供）： 透明细胞肾细胞癌（ccRCC）是军事人员的严重健康问题，特别是40岁以上的男性，包括退伍军人。根据1995-2004年的国防医学流行病学数据库，与总发病率的每10万人-年1.5例相比，在生命的第4个十年之后，专门针对军事成员的RCC的发病率急剧增加到8.5例。我们的实验室研究了由von Hippel-Lindau（VHL）蛋白调节的分子途径，VHL蛋白是ccRCC中最常见的肿瘤抑制因子。在上一个资助期间，我们发表了关于自噬在ccRCC生长中作用的重要数据。这一竞争性更新是建立在这些实质性观察的基础上的。自噬是一种自我平衡的功能，通过这种功能，细胞处理自己的细胞器和蛋白质，以消除缺陷分子并回收营养物质。在肿瘤生长过程中，许多癌症依赖于自噬作为营养来源。此外，自噬被不同的癌症疗法激活，导致对治疗的抗性，并促进自噬抑制剂、氯喹衍生物在联合疗法中的使用。MAP 1 LC 3（LC 3）是形成自噬体所必需的蛋白质。在我们最近发表的工作和初步结果中，我们表明两个LC 3旁系同源物LC 3B和LC 3C对ccRCC的生长具有相反的影响。LC 3B依赖的自噬促进VHL缺失的RCC细胞形成的肿瘤的生长，而LC 3C依赖的自噬具有肿瘤抑制作用并且在VHL（-）细胞中不存在。该提案的重点是研究LC 3C依赖性自噬的活性。最重要的是，我们发现LC 3C，而不是LC 3B，自噬程序的氧化（限速酶，葡萄糖6-磷酸脱氢酶，G6 PD）和非氧化（转酮醇酶，TKT）戊糖磷酸途径（PPP）的降解酶的目标。LC 3C的作用对PPP具有特异性，不影响糖酵解途径。这一重要的癌症途径产生核糖-5-磷酸以产生核酸和NADPH以用于脂质合成和还原谷胱甘肽，所有这些都导致增强的细胞存活和生长。KIRC TCGA发表的无监督分析进一步支持了我们数据的重要性，该分析显示G6 PD，TKT水平与ccRCC的不良结局之间存在显著相关性。这一提议的主要假设是，通过HIF的诱导，VHL的损失减少了LC 3C，允许PPP的激活以支持肿瘤生长。在目标1中，我们将确定VHL诱导LC 3C基因表达的分子机制。我们的工作假设是HIF通过诱导抑制转录因子的表达来抑制LC 3C。在目标2中，我们将使用遗传和代谢组学方法确定LC 3C在PPP的氧化和非氧化分支控制核酸合成和NADPH生成的活性中的作用。在目标3中，我们将确定PPP的氧化和非氧化分支在人肾ccRCC标本中原位异种移植肿瘤生长和PPP酶表达中的作用。在目标4中，我们将确定LC 3C而不是LC 3B特异性靶向TKT和G6 PD进行自噬降解的分子机制。初步数据支持我们的工作假设是，LC 3C效应的特异性是由LC 3C C-末端肽介导的。

 DESCRIPTION (provided by applicant): 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. Our laboratory studies molecular pathways regulated by the von Hippel-Lindau (VHL) protein, the most frequently lost tumor suppressor in ccRCC. During the previous funding period we have published important data regarding the role of autophagy in growth of ccRCC. This competitive renewal builds on these substantial observations. Autophagy is a homeostatic function by which cells process their own organelles and proteins in order to eliminate defective molecules and to recycle nutrients. During tumor growth many cancers become addicted to autophagy as a source of nutrients. Moreover, autophagy is activated by different cancer therapies, causing resistance to treatments, and promoting use of autophagic inhibitors, chloroquine derivatives in combination therapies. MAP1LC3s (LC3) are essential proteins for the formation of an autophagosome. In our recently published work and preliminary results, we show that two LC3 paralogs, LC3B and LC3C, have opposite effects on growth of ccRCC. LC3B-dependent autophagy promoted growth of tumors formed by RCC cells with lost VHL, while LC3C-dependent autophagy was tumor suppressive and absent in VHL(-) cells. This proposal focuses on investigations to understand the activity of LC3C-dependent autophagy. Most importantly, we found that LC3C, but not LC3B, autophagic program targets of degradation enzymes of oxidative (rate limiting enzyme, glucose 6-phosphate dehydrogenase, G6PD) and non-oxidative (transketolase, TKT) pentose phosphate pathway (PPP). The effect of LC3C is specific for PPP and does not affect the glycolytic pathway. This important cancer pathway produces ribose-5-phophate for the production of nucleic acids and NADPH for the lipid synthesis and reduction of glutathione, all resulting in enhanced cell survival and growth. The importance of our data is further supported by unsupervised analysis published by KIRC TCGA, which showed significant correlation between levels of G6PD, TKT and poor outcome in ccRCC. The leading hypothesis for this proposal is that loss of VHL through indction of HIF reduces LC3C, permitting activation of PPP to support tumor growth. In Aim 1 we will determine the molecular mechanism by which VHL induces LC3C gene expression. Our working hypothesis is that HIF suppresses LC3C by inducing expression of repressing transcription factors. In Aim 2 we will determine the role of LC3C in the activities of the oxidative and non-oxidative branches of PPP in controlling nucleic acid synthesis and generation of NADPH using genetic and metabolomics approaches. In Aim 3 we will determine the role of the oxidative and non-oxidative branches of PPP in orthotopic xenograft tumor growth and expression of PPP enzymes in human kidney-ccRCC specimens. In Aim 4 we will determine the molecular mechanism by which LC3C, but not LC3B, specifically targets TKT and G6PD for autophagic degradation. Our working hypothesis, supported by preliminary data is that the specificity of LC3C effects is mediated by the LC3C C-terminal peptide.