Molecular Therapeutics of Kidney Cancer: VHL Gene and Fumarate Hydratase Gene

肾癌的分子治疗- vhl基因和富马酸水合酶基因了解导致肾癌的基因为开发这种疾病的分子治疗方法提供了机会。我们已经确定了3种导致肾癌的基因：VHL基因（透明细胞肾细胞癌）；c-Met基因（乳头状1型肾癌）；以及BHD基因（嫌色性肾癌）。泌尿肿瘤科的科学家们正在深入研究VHL基因的损伤（突变）如何导致VHL和散发性肾癌患者的表现。最近，研究表明VHL蛋白与其他蛋白形成复合物，包括长链蛋白C和B以及CUL-2蛋白，该复合物以缺氧诱导因子（hif1 α和hif2 α）的α亚基为靶点，进行泛素介导的降解。这通常是一个缺氧介导的过程，即在缺氧条件下，HIF不会被VHL复合物降解。HIF是一种转录因子，可调节许多下游癌症重要基因的转录，如VEGF、Glut 1、TGFalpha和PDGF。当VHL基因发生突变时，在VHL患者的种系或透明细胞肾癌患者的肿瘤组织中，hif不能被降解，其结果是VEGF、Glut1、TGFalpha和PDGF的过转录。评估靶向VHL通路的药物在VHL和透明细胞肾癌中的作用的一种方法是测定体外和体内阻断VEGF和TGFalpha/EGFr通路的药物的活性。透明细胞RCC的另一种分子治疗方法是使用格尔达霉素类似物，它可以破坏HIF与HSP-90的结合。体外研究表明，即使在VHL -/-细胞系中，17AAG格尔达霉素类似物也能降解HIF。我们从人体材料中开发的肾癌模型正在进行体外和体内研究，以评估阻断这种癌症基因通路的药物作为治疗透明细胞肾癌的潜在方法的作用。评估格尔达霉素类似物以及靶向VEGF/EGFr受体和VHL通路其他部分的药物作用的临床试验目前正在进行中。克雷布斯循环酶，富马酸水合酶（FH），是遗传性平滑肌瘤型肾细胞癌（HLRCC）的基因。HLRCC患者有发展为皮肤和子宫平滑肌瘤以及一种非常侵袭性的2型乳头状肾癌的风险。我们在95%的HLRCC家族的种系中发现了FH基因突变，在HLRCC相关的肾癌中发现了FH基因的杂合性缺失。为了了解克雷布斯循环酶的突变如何导致肾癌，我们对hlrcc相关的肾肿瘤进行了缺氧诱导因子1 α (hif1 α)和缺氧诱导因子2 α (hif2 α)的染色。我们发现HIF1alpha和HIF2alpha在HLRCC肾肿瘤中均升高。我们正在开发新的人类肿瘤体外模型，并评估体外和体内系统的生长情况。在体外模型中，我们发现当富马酸水化酶（用SiRNA）失活时，富马酸增加，富马酸的增加抑制脯氨酸羟化酶。脯氨酸羟化酶的抑制阻止了正常的vhl介导的HIF降解，为HLRCC肾癌中HIF降解失调提供了一种不依赖vhl的机制。这些研究为开发靶向治疗方法治疗hlrc相关肾癌提供了理论依据。体外和体内研究正在进行中，以评估阻断这种癌症基因通路的药物作为治疗hlrc相关和散发性2型乳头状肾癌的潜在方法的作用。

Molecular Therapeutics of Kidney Cancer-VHL Gene and Fumarate Hydratase Gene Understanding the genes that cause kidney cancer provides the opportunity to develop approaches for molecular therapeutics for this disease. We have identified 3 genes that cause cancer of the kidney: the VHL gene (clear cell renal cell carcinoma); the c-Met gene (papillary type 1 renal carcinoma); and the BHD gene (chromophobe renal carcinoma). Targeting the VHL Clear Cell Kidney Cancer Gene Pathway Urologic Oncology Branch scientists are studying intensively how damage (mutation) to the VHL gene leads to the manifestations in VHL and sporadic renal carcinoma patients. Recently, it is has been shown that the VHL protein forms a complex with other proteins, including elongin C and B and the CUL-2 protein, and this complex targets the alpha subunit of hypoxia inducible factors (HIF1alpha and HIF2alpha) for ubiquitin-mediated degradation. This is a hypoxia-mediated process normally, i.e., under hypoxic conditions HIF is not degraded by the VHL complex. HIF is a transcription factor that regulates the transcription of a number of downstream genes important for cancer, such as VEGF, Glut 1, TGFalpha and PDGF. When the VHL gene is mutated, in the germline of VHL patients or in tumor tissue from patients with clear cell renal carcinoma, the HIFs cannot be degraded and the result is the over-transcription of VEGF, Glut1, TGFalpha and PDGF. One approach to evaluating the role of agents targeting the VHL pathway in VHL and clear cell renal carcinoma is to determine the activity of agents which block the VEGF and TGFalpha/EGFr pathways in-vitro and in-vivo. Another approach for molecular therapeutics of clear cell RCC is by use of agents such as geldanamycin analogues, which disrupt the binding of HIF to HSP-90. In-vitro studies have shown that the 17AAG geldanamycin analogues can degrade HIF even in VHL -/- cell lines. In-vitro and in-vivo studies are underway in kidney cancer models that we have developed from human material to evaluate the role of agents which block this cancer gene pathway as a potential approach for the treatment of clear cell kidney cancer. Clinical trials evaluating the role of geldanamycin analogues as well as agents which target the VEGF/EGFr receptors and other parts of the VHL pathway are currently in progress. Targeting the Fumarate Hydratase Gene: Type 2 Papillary Kidney Cancer The Krebs cycle enzyme, fumarate hydratase (FH), is the gene for Hereditary Leiomyomatosis Renal Cell Carcinoma (HLRCC). HLRCC patients are at risk for the development of cutaneous and uterine leiomyomas as well as a very aggressive form of type 2 papillary kidney cancer. We have found mutations of the FH gene in the germline of 95% of our HLRCC families and loss of heterozygosity of the FH gene in HLRCC-associated kidney cancer. In order to understand how mutation of a Krebs cycle enzyme could cause kidney cancer we stained HLRCC-associated kidney tumors for the presence of hypoxia induced factor 1alpha (HIF1alpha) and hypoxia induced factor 2alpha (HIF2alpha). We found both HIF1alpha and HIF2alpha to be elevated in the HLRCC kidney tumors. We are developing novel in-vitro models from human tumors and evaluating growth in in-vitro and in-vivo systems. In in-vitro models we found that when fumarate hydratase was inactivated (with SiRNA), fumarate increased and the increase in fumarate inhibited prolyl hydroxylase. The inhibition of prolyl hydroxylase prevented normoxic VHL-mediated HIF degradation, providing a VHL-independent mechanism for dysregulation of HIF degradation in HLRCC kidney cancer. These studies provided the rationale for the development of a targeted therapeutic approach for the treatment of HLRCC-associated kidney cancer. In-vitro and in-vivo studies are underway to evaluate the role of agents which block this cancer gene pathway as a potential approach for the treatment of HLRCC-associated as well as sporadic type 2 papillary kidney cancer.