HIF-2α release and HIF-interactions in tubular cells and clear cell renal cell carcinoma

肾透明细胞癌(CcRCC)是最常见的肾癌亚型，占肾肿瘤的70%以上。肾小管上皮细胞中的von Hippel-Lindau(VHL)基因突变被认为是肾癌进化的主干事件，最早发生在出生后的第二个十年。VHL综合征患者携带一个VHL等位基因的遗传性突变，并在一生中获得第二个等位基因的额外突变。根据潜在的第一个突变，这可能会极大地增加发生ccRCC和其他肿瘤的风险，这些肿瘤是这种综合征的特征，包括嗜铬细胞瘤、血管母细胞瘤和视网膜血管瘤。VHL的缺失导致低氧诱导转录因子(HIF)的稳定，并触发其转录活性。缺氧诱导因子-2α亚型在肾小管缺陷肾小管上皮细胞中从头表达，并在肾细胞癌中具有致癌作用。全基因组关联研究已经发现了HIF-2α基因座非蛋白编码区的多态。这些基因的多态改变了患肾癌的风险。这表明转录失调在很大程度上促进了HIF-2α表达的释放和肿瘤的形成。我们的初步观察表明，在未转化的肾小管上皮细胞中，低氧诱导因子可通过直接的α相互作用调节低氧诱导因子-2α的表达。然而，调控元件和转录因子在这个基因组座位上促进这种表达并调节表观遗传格局的谱系尚不清楚。该区域与肾细胞癌相关的SNPs可能与低氧诱导因子信号相互作用，从而促进低氧诱导因子-2α的表达。我们将利用高端测序和细胞培养技术，准确地确定肾小管细胞、早期癌病变和进展期肿瘤中HIF-2α调控的表观遗传动力学，从而确定对致癌HIF-2α表达的新机制。我们将求助于大量的组织标本以及原代肾小管和癌细胞的生物库来执行我们的分析。此外，我们计划修改从肾癌综合征患者尿液中分离的肾小管细胞中的低氧诱导因子信号，以模拟肾癌进展的早期步骤。鉴于目前对缺氧诱导因子-2α抑制剂治疗肾癌的临床评估，有必要对表观遗传学变化进行全面的分子分析。这项拟议的工作将仔细定义在早期肾癌发展过程中操纵HIF-2α表达的机制。它将提供一个范例，通过整合遗传和阶段特定的遗传和表观遗传事件，对肿瘤启动子的调控进行详细的机制分析。

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer, accounting for more than 70% of kidney tumors. Mutation of the von Hippel-Lindau (VHL) gene in renal tubular cells has been identified as the truncal event in renal cancer evolution with first hits often occurring as early as in the second decade of life. Patients with VHL syndrome carry an inherited mutation in one VHL allele and acquire additional mutations in the second allele during life. Depending on the underlying first mutation this can dramatically increase the risk of developing ccRCC and additional tumors which characterize this syndrome including pheochromocytomas, hemangioblastomas, and retinal angiomas. Loss of VHL leads to stabilization of hypoxia inducible transcription factors (HIF) and triggers their transcriptional activity. The HIF-2α isoform is expressed de novo in VHL-defective tubular cells and appears to have an oncogenic role in the context of ccRCC. Genome-wide association studies have identified polymorphisms in non-protein coding regions in the HIF-2α gene locus. These polymorphisms modify the risk of developing renal cancer. This suggests that transcriptional dysregulation contributes substantially to the release of HIF-2α expression and tumor formation. Our preliminary observations indicate that HIF can regulate the mRNA expression of HIF-2α via direct DNA interactions at the HIF-2α locus in non-transformed renal tubular cells. However, the repertoire of regulatory elements and transcription factors contributing to this expression and modulating the epigenetic landscape at this genomic locus is unclear. It seems also plausible that RCC-associated SNPs in this region may interact with HIF-signalling to promote HIF-2α expression. We will employ high-end sequencing and cell culture technology to exactly define epigenetic dynamics of HIF-2α regulation in renal tubular cells, early cancerous lesions and progressed tumors, thereby identifying novel mechanistic insights into oncogenic HIF-2α expression. We will resort to a large biobank of tissue specimens as well as primary renal tubular and cancer cells to perform our analyses. Furthermore, we plan to modify HIF-signalling in tubular cells isolated from the urine of patients with VHL-syndrome to model early steps of renal cancer evolution.Given the current clinical evaluation of HIF-2α inhibitors for the treatment of VHL-associated renal cancer, a comprehensive molecular analysis of epigenetic changes, is needed. The proposed work will carefully define mechanisms that manipulate expression of HIF-2α during early renal cancer development. It will provide a paradigm for a detailed mechanistic analysis of the regulation of a tumor promoter by integrating inherited and stage specific genetic and epigenetic events.