Regulation of the Hypoxia Inducible Factor (HIF)

描述（由申请人提供）：哺乳动物细胞采用缺氧反应途径来感知和响应氧气可用性的变化。这一途径既有助于机体对缺氧胁迫的生理适应，也有助于许多氧气供应受损的疾病状态的进展，包括缺血和癌症。缺氧诱导转录因子（Hypoxia Inducible transcription Factor， HIF）是这一途径的核心，它介导了与肿瘤发生相关的几个靶标的表达，包括糖酵解、细胞存活/增殖、血管生成和侵袭/转移相关的基因。最近，我们的实验室和其他人已经表明，HIF的稳定性和辅激活剂的募集分别受到氧依赖性脯氨酸和天冬酰胺羟化酶的强烈调节。虽然HIF可以作为肿瘤内缺氧的一种功能而被诱导，但在癌症中，HIF活性经常通过多种氧无关机制被上调，这些机制由激活癌基因或灭活肿瘤抑制基因的遗传改变引起。这项建议的目标是双重的。(1)我们将研究HIF活性与候选肿瘤抑制基因之间的关系，生长家族成员4抑制剂（ING4），据报道在胶质母细胞瘤中抑制血管生成和肿瘤生长。本研究提出了一种组合方法来研究(a) ING4介导的HIF抑制(b) ING4通过HIF脯氨酰羟化酶被募集到HIF的机制(c)确定被募集到HIF的ING4相关因子和其他转录因子，以及(d)人类肿瘤中ING4表达、HIF关联和HIF活性之间的关系。(2) HIF由两个亚基组成，每个亚基都包含两个PAS结构域，这两个结构域在HIF功能中的作用尚未完全阐明。以最近确定的HIF PAS- b结构域的溶液结构为出发点，我们将(a)通过体外实验和细胞培养模型研究HIF PAS结构域的结构/功能关系；(b)通过改变细胞培养和小鼠肿瘤模型中的PAS结构域来证明破坏HIF功能的潜在治疗效用；(c)追求直接结合PAS的小分子HIF拮抗剂的开发域。总之，这些研究将增加我们对HIF调控的理解，并可能为治疗干预提供新的途径。

DESCRIPTION (provided by applicant): Mammalian cells employ a hypoxic response pathway to sense and respond to changes in oxygen availability. This pathway contributes to both the physiological adaptation to hypoxic stress as well as the progression of a number of disease states in which oxygen supply is compromised, including ischemia and cancer. The Hypoxia Inducible transcription Factor (HIF) lies at the heart of this pathway and mediates expression of several targets relevant to tumorigenesis including genes involved in glycolysis, cell survival/proliferation, angiogenesis, and invasion/metastasis. Recently, our laboratory and others have shown that both HIF stability and coactivator recruitment are acutely regulated by oxygen-dependent prolyl and asparaginyl hydroxylases, respectively. While HIF can be so induced as a function of intratumoral hypoxia, HIF activity is frequently upregulated in cancers by a variety of oxygen-independent mechanisms resulting from genetic alterations that activate oncogenes or inactivate tumor suppressors. The objectives of this proposal are twofold. (1) We will investigate a relationship we've identified between HIF activity and a candidate tumor suppressor gene, Inhibitor of Growth family member 4 (ING4), reported to repress angiogenesis and tumor growth in glioblastomas. Proposed in this Aim is a combination approaches for the study of (a) ING4-mediated repression of HIF (b) the mechanism by which ING4 is recruited to HIF via the HIF prolyl hydroxylase (c) the identification of ING4-associated factors recruited to HIF and perhaps other transcription factors and (d) the relationship between ING4 expression, HIF association, and HIF activity in human tumors. (2) HIF is composed of two subunits, each of which contains two PAS domains whose roles in HIF function have not yet been fully elucidated. Using insights derived from the recently determined solution structures of the HIF PAS-B domains as a starting point we will (a) investigate the structure/function relationships of the HIF PAS domains via in vitro assays and cell culture models (b) demonstrate the potential therapeutic utility of disrupting HIF function through alterations to the PAS domains both in cell culture and in mouse tumor models and (c) pursue the development of small molecule HIF antagonists that directly bind the PAS domains. Together, these studies will increase our understanding of HIF regulation and may provide new avenues for therapeutic intervention.