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

透明细胞肾细胞癌（ccRCC）是肾癌最常见的亚型，占肾脏肿瘤的70%以上。肾小管细胞中的von Hippel-Lindau（VHL）基因突变已被确定为肾癌演变中的干细胞事件，首次发生通常早在生命的第二个十年。患有VHL综合征的患者在一个VHL等位基因中携带遗传突变，并且在生活中在第二个等位基因中获得额外的突变。取决于潜在的第一突变，这可以显著增加发展ccRCC和表征该综合征的其他肿瘤的风险，包括嗜铬细胞瘤、血管母细胞瘤和视网膜血管瘤。VHL的缺失导致缺氧诱导转录因子（HIF）的稳定并触发其转录活性。HIF-2α亚型在VHL缺陷型肾小管细胞中重新表达，似乎在ccRCC中具有致癌作用。全基因组关联研究已经确定了HIF-2α基因位点非蛋白质编码区的多态性。这些多态性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表达的释放和肿瘤形成。我们的初步观察表明，HIF可以通过直接DNA相互作用在HIF-2α基因座的非转化肾小管细胞中调节HIF-2α mRNA的表达。然而，调控元件和转录因子的剧目，有助于这种表达和调节表观遗传景观在这个基因组位点是不清楚的。该区域的RCC相关SNPs可能与HIF信号相互作用促进HIF-2α表达，这似乎也是合理的。我们将采用高端测序和细胞培养技术来准确定义肾小管细胞、早期癌性病变和进展性肿瘤中HIF-2α调控的表观遗传动力学，从而确定致癌HIF-2α表达的新机制。我们将求助于一个大型生物样本库的组织标本以及原代肾小管和癌细胞来进行我们的分析。此外，我们计划修改从VHL综合征患者尿液中分离的肾小管细胞中的HIF信号传导，以模拟肾癌演变的早期步骤。鉴于目前HIF-2α抑制剂治疗VHL相关肾癌的临床评价，需要对表观遗传变化进行全面的分子分析。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。它将提供一个详细的机制分析的肿瘤启动子的调节整合遗传和阶段特异性遗传和表观遗传事件的范例。

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.