Exploration of ZHX2 as a novel substrate of pVHL and an oncogenic driver of renal cancer

项目摘要 透明细胞肾细胞癌（ccRCC）约占所有肾癌的85%，具有耐药性。 对多种癌症治疗都有很大的作用，而且是高度致命的ccRCC的一个标志是von Hippel基因失活， Lindau（VHL）肿瘤抑制基因，其在高达90%的肿瘤中通过突变或超甲基化而失活。 关于ccRCC虽然pVHL作为E3泛素连接酶衔接蛋白发挥作用，促进E3泛素的降解，但pVHL的功能与E3泛素连接酶衔接蛋白不同。 低氧诱导（HIFα）转录因子，其他关键的pVHL底物只是现在才出现，它们的 在肾癌中的功能作用仍然不明确。在这里，我们确定了一个锌指/同源结构域蛋白ZHX 2 作为潜在的新型pVHL底物，通过使用新开发的全基因组体外表达策略 再加上GST结合筛选。我们的初步数据表明，pVHL与 ZHX 2以羟化酶活性依赖的方式，类似于HIFα蛋白的调节。在功能上， ZHX 2的敲低阻断pVHL-缺陷的细胞的细胞增殖、软琼脂生长和异种移植肿瘤生长 肾癌细胞重要的是，与对照组相比，在ccRCC肿瘤中ZHX 2表达水平强烈升高。 正常的病人。ZHX 2与NF-κ B的RelA/p65亚基相互作用， pVHL缺失诱导RelA/p65核定位。ChIP-Seq和微阵列的综合分析也揭示了 ZHX 2在ccRCC中调节NF-κ B依赖性和非依赖性途径。因此，我们假设 ZHX 2通过NF-κ B依赖和非依赖途径促进肾肿瘤发生 VHL失活后。这是第一项针对ZHX 2的促癌功能的研究， 关注其在肾癌中pVHL损失下游的关键作用。在具体目标1中，我们将研究 ZHX 2以羟基化依赖性方式被pVHL调节的机制。在具体目标2中， 将确定pVHL缺陷型肾癌中ZHX 2失调的功能意义。具体目标 3、我们将确定ZHX 2调节NF-κ B依赖性和非依赖性信号传导的机制， 以及pVHL丧失后的肾肿瘤发生。成功完成这一提案将提供重要的新 对与大多数ccRCC相关的致癌机制的分子见解以及 针对这种典型致命疾病的新疗法的潜力。

Project Summary Clear cell renal cell carcinoma (ccRCC), which accounts for approximately 85% of all renal cancers, is resistant to a variety of cancer therapies and is highly lethal. A hallmark of ccRCC is the inactivation of the von Hippel Lindau (VHL) tumor suppressor gene, which is inactivated either by mutation or hypermethylation in up to 90% of ccRCC. While pVHL functions as an E3 ubiquitin ligase adaptor protein that promotes the degradation of hypoxia inducible (HIFα) transcription factors, other key pVHL substrates are only now emerging and their functional roles in renal cancer remains undefined. Here we identify a zinc-finger/homeodomain protein ZHX2 as a potential novel pVHL substrate by using a newly developed genome-wide in vitro expression strategy coupled with GST-binding screening. Our preliminary data suggests that pVHL interacts with and degrades ZHX2 in a hydroxylase activity-dependent manner, similar to regulation of the HIFα proteins. Functionally, knockdown of ZHX2 blocks cell proliferation, soft agar growth and xenograft tumor growth of pVHL-deficient renal cancer cells. Importantly, ZHX2 expression levels are strongly elevated in ccRCC tumors compared to normal patients. Mechanistically, ZHX2 interacts with the RelA/p65 subunit of NF-kB and positively regulates pVHL-loss induced RelA/p65 nuclear localization. Integrated analyses of ChIP-Seq and microarray also reveal that ZHX2 regulates both NF-kB-dependent and independent pathways in ccRCC. Therefore, we hypothesize that ZHX2 promotes renal oncogenesis through both NF-kB dependent and independent pathways following VHL inactivation. This is the first study directed at a pro-oncogenic function for ZHX2, with the focus on its key role downstream of the loss of pVHL in renal cancer. In Specific Aim 1, we will study the mechanism by which ZHX2 is regulated by pVHL in a hydroxylation-dependent manner. In Specific Aim 2, we will determine the functional significance of deregulated ZHX2 in pVHL-deficient renal cancer. In Specific Aim 3, we will determine the mechanisms by which ZHX2 regulates NF-kB-dependent and -independent signaling and renal tumorigenesis upon pVHL loss. Successful completion of this proposal would provide significant new molecular insight into oncogenic mechanisms associated with the great majority of ccRCC as well as the potential for new therapies for this typically lethal disease.