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

描述（申请人提供）：透明细胞肾细胞癌（ccRCC）是世界范围内最常见的肾脏恶性肿瘤。虽然转移性ccRCC对传统的细胞毒性药物和放射治疗是难治的，但由于对这种疾病的分子发病机制的了解，延长生命的靶向治疗已经出现。特别是，超过80%的ccRCC病例表现出缺氧诱导因子（HIF）依赖性转录反应的组成性激活，由HIF-亚基（HIF-1¿或HIF-22¿）和HIF-1¿的异源二聚体介导。在ccRCC的情况下，很明显HIF-2¿是疾病进展所必需的，而不是HIF-1¿。虽然HIF-2¿在氧气、血清和营养充足的条件下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¿对体内肿瘤生长至关重要，这表明HIF-2¿在体内肿瘤微环境中介导重要的细胞应激反应。然而，HIF-2促进肿瘤活性的下游效应物仍不完全清楚。利用基因表达谱，我通过siRNA急性抑制HIF-2活性，确定了ccRCC中HIF-2调控的候选途径。这些结果表明HIF-2¿调节介导线粒体自噬（NIX）和新生脂肪生成的必需基因（SREBF1， ACLY， ACACB， FASN, SCD1）。nix依赖性线粒体自噬通过消除受损线粒体（细胞活性氧（ROS）的主要内源性来源）在预防氧化应激中起着至关重要的作用。虽然HIF-2¿已被证明可以促进对电离辐射诱导的氧化应激和细胞凋亡的抵抗，但其确切机制尚不清楚。因此，我的第一个假设（AIM1）是HIF-2依赖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需要组成性新生脂肪生成。尽管观察到ccRCC是脂质负载的，在异种移植肿瘤模型中HIF-2抑制导致表型丢失，但新生脂肪生成在ccRCC中的作用尚不清楚。值得注意的是，HIF-2依赖性的TGF-表达上调是血清不依赖的体外增殖所必需的——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-2介导的TGF-表达激活srebp1依赖性的新生脂肪生成，以支持肿瘤细胞的活力和增殖。为了验证这些假设，我将利用细胞培养、小鼠原位肾肿瘤模型和原发性患者样本来研究HIF-2¿对每个过程的调节，评估抑制这些途径（遗传和药理学）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ccRCC的分子发病机制、HIF生物学以及针对ccRCC的潜在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.