HIF-2alpha mediated mitophagy and lipogenesis in clear cell renal cell carcinoma

描述(申请人提供)：肾透明细胞癌(CcRCC)是世界上最常见的肾脏恶性肿瘤。虽然转移性肾细胞癌对传统的细胞毒药物和放射治疗无效，但通过了解这种疾病的分子发病机制，已经出现了延长生命的靶向治疗方法。特别是，超过80%的肾细胞癌病例表现出依赖于缺氧诱导因子(HIF)的转录反应的结构性激活，这种激活是由HIF-β亚基(HIF-1或HIF-22β)和HIF-1β的异二聚体介导的。在ccRCC的情况下，很明显，疾病进展需要HIF-2，而不是HIF-1。虽然在氧气、血清和营养充足的条件下，HIF-2不是体外细胞增殖所必需的，但异种移植小鼠模型显示，HIF-2对体内肿瘤的生长是必不可少的，表明HIF-2在体内肿瘤微环境中介导了重要的细胞应激反应。然而，HIF-2 S促肿瘤活性的下游效应机制仍不完全清楚。利用基因表达谱，我通过siRNA强烈抑制HIF-2的活性，确定了CcRCC中HIF-2调控的候选途径。这些结果表明，HIF-2调控介导有丝分裂(NIX)和从头脂肪生成的必需基因(SREBF1、ACLY、ACACB、FASN、SCD1)。NIX依赖的有丝分裂吞噬作用通过消除受损的线粒体在预防氧化应激中发挥着至关重要的作用，线粒体是细胞内活性氧物种(ROS)的主要内源性来源。虽然HIF-2已被证明能增强对电离辐射诱导的氧化应激和细胞凋亡的抵抗力，但其确切机制尚不清楚。因此，我的第一个假设(AIM1)是，依赖于HIF-2的有丝分裂是维持氧化还原稳态和支持肿瘤细胞代谢所必需的。此外，包括乳腺癌和前列腺癌在内的多种恶性肿瘤的生存和生长都需要结构性新生脂肪生成。尽管观察到ccRCC是一种载脂的表型，但在异种移植瘤模型中，HIF-2抑制后失去了这种表型，但从头开始脂肪生成在ccRCC中的作用尚不清楚。值得注意的是，依赖HIF-2上调转化生长因子-β的表达是血清非依赖性体外增殖所必需的--这种情况可能需要合成新的脂肪酸。因此，我的第二个假设(AIM2)是，HIF-2β介导的转化生长因子-β的表达激活了SREBP1依赖的从头脂肪生成，以支持肿瘤细胞的存活和增殖。为了验证这些假设，我将利用细胞培养、鼠原位肾肿瘤模型和原始患者样本来研究HIF-2对每个过程的调节，评估抑制这些途径(从遗传学和药理学上)的抗肿瘤潜力，并评估这些过程在人类肿瘤中的激活。这些发现将为研究肾细胞癌的分子发病机制、低氧诱导因子生物学以及潜在的抗肿瘤治疗提供新的见解。

DESCRIPTION (provided by applicant): Clear cell renal cell carcinoma (ccRCC) is the most common renal malignancy worldwide. While metastatic ccRCC is refractory to conventional cytotoxic drugs and radiation, life-extending targeted therapies have emerged from an understanding of the molecular pathogenesis of this disease. In particular, over 80% of ccRCC cases exhibit constitutive activation of Hypoxia Inducible Factor (HIF)-dependent transcriptional responses, mediated by heterodimers of HIF-¿ subunits (HIF-1¿ or HIF-22¿) and HIF-1¿. In the case of ccRCC, it is clear that HIF-2¿, but not HIF-1¿, is required for disease progression. While HIF-2¿ is not required for in vitro cell proliferation under oxygen, serum, and nutrient-replete conditions, xenograft mouse models have revealed that HIF-2¿ is essential for in vivo tumor growth, suggesting that HIF-2¿ mediates vital cellular stress responses within the in vivo tumor microenvironment. However, the downstream effectors of HIF-2¿'s tumor-promoting activity remain incompletely understood. Using gene expression profiling, I have identified candidate HIF-2¿ regulated pathways in ccRCC by acutely suppressing HIF-2¿ activity by siRNA. These results indicate that HIF-2¿ regulates essential genes mediating mitophagy (NIX) and de novo lipogenesis (SREBF1, ACLY, ACACB, FASN, SCD1). NIX-dependent mitophagy plays a vital role in preventing oxidative stress by eliminating damaged mitochondria, the primary endogenous source of cellular reactive oxygen species (ROS). While HIF-2¿ has been demonstrated to promote resistance to ionizing radiation-induced oxidative stress and apoptosis, the precise mechanism is not clear. Thus, my first hypothesis (AIM1) is that HIF-2¿-dependent mitophagy is required to maintain redox homeostasis and support tumor cell metabolism. Additionally, constitutive de novo lipogenesis is needed for viability and growth in multiple malignancies, including breast and prostate carcinoma. Despite the observation that ccRCC are lipid laden, a phenotype lost upon HIF-2¿ suppression in xenograft tumor models, the role of de novo lipogenesis in ccRCC is unknown. Notably, HIF-2¿-dependent upregulation of TGF-¿ expression is required for serum-independent in vitro proliferation-a condition that likely requires the synthesis of new fatty acids. Thus, my second hypothesis (AIM2) is that HIF-2¿-mediated TGF-¿ expression activates SREBP1-dependent de novo lipogenesis to support tumor cell viability and proliferation. To test these hypotheses, I will utilize cell culture, a muine orthotopic renal tumor model, and primary patient samples to study the regulation of each process by HIF-2¿, evaluate the anti-tumor potential of suppressing these pathways (genetically and pharmacologically), and assess for activation of these processes in human tumors. These findings will provide novel insights into molecular pathogenesis of ccRCC, HIF biology, and potential anti-tumor therapies against ccRCC.