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个导致肾癌的基因：VHL基因(肾透明细胞癌)、c-Met基因(乳头状1型肾癌)和BHD基因(嫌色肾癌)。针对VHL透明细胞肾癌基因通路，泌尿外科肿瘤科科学家正在深入研究VHL基因的损伤(突变)如何导致VHL和散发性肾癌患者的临床表现。最近，研究表明，VHL蛋白与其他蛋白形成复合体，包括细长蛋白C和B以及CuL-2蛋白，该复合体针对缺氧诱导因子的α亚基(HIF1和HIF2)，以实现泛素介导的降解。这通常是一个低氧介导的过程，即在低氧条件下，HIF不被VHL复合体降解。HIF是一种转录因子，它调节一些对癌症重要的下游基因的转录，如血管内皮生长因子、GLUT 1、转化生长因子和血小板衍生生长因子。当VHL基因发生突变时，无论是在VHL患者的胚系中，还是在透明细胞肾癌患者的肿瘤组织中，HIFs都不能被降解，结果是血管内皮生长因子、谷氨酸、转化生长因子和血小板衍生生长因子的过度转录。评估靶向VHL途径的药物在VHL和透明细胞肾癌中的作用的一种方法是确定在体外和体内阻断血管内皮生长因子和转化生长因子/EGFR途径的药物的活性。透明细胞肾癌分子治疗的另一种方法是使用格尔达霉素类似物，破坏HIF与HSP-90的结合。体外研究表明，17AAG格尔达霉素类似物即使在VHL-/-细胞系中也能降解HIF。我们从人类材料开发的肾癌模型正在进行体外和体内研究，以评估阻断这一癌症基因途径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血管内皮生长因子/EGFR受体和VHL途径其他部分的药物的临床试验目前正在进行中。靶向富马酸水合酶基因：2型乳头状肾癌Krebs循环酶富马酸水合酶(FH)是遗传性平滑肌瘤病肾细胞癌(HLRCC)的基因。HLRCC患者有发生皮肤和子宫肌瘤的风险，以及一种侵袭性很强的2型乳头状肾癌。我们在95%的HLRCC家系中发现了FH基因突变，并在HLRCC相关肾癌中发现了FH基因杂合性缺失。为了了解Krebs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低氧诱导因子1和低氧诱导因子2的检测。我们发现HLRCC肾肿瘤中HIF1和HIF2均升高。我们正在开发新的人体肿瘤体外模型，并在体外和体内系统中评估生长情况。在体外模型中，我们发现当富马酸水合酶失活(使用siRNA)时，富马酸增加，富马酸增加抑制Pro羟基酶。抑制Pro羟基酶可阻止常氧VHL介导的HIF降解，为HLRCC肾癌中HIF降解的失调提供了VHL非依赖的机制。这些研究为开发治疗HLRCC相关肾癌的靶向治疗方法提供了理论基础。体外和体内研究正在评估阻断这一癌症基因途径的药物作为治疗HLRCC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 (HIF1α and HIF2α) 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, TGFα 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, TGFα 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 TGFα/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 1α (HIF1α) and hypoxia induced factor 2α (HIF2α). We found both HIF1α and HIF2α 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.