Regulation of Hypoxia-inducible Factor 1 by Cyclin Dependent Kinases

描述（由申请人提供）：缺氧诱导因子1（HIF-1）转录因子是细胞对缺氧反应的关键介质。什么？HIF-1亚基在氧气存在下迅速降解，但在缺氧条件下稳定，并激活促进血管生成、存活、侵袭和葡萄糖代谢的基因的转录。HIF-1可通过基因改变激活，从而增强HIF-1？的转录或抑制其降解。例如，von Hippel-Lindau（VHL）病使患者易患侵袭性肾系统肿瘤，其特征在于HIF-1？的稳定。HIF-1的组成性表达？导致这些肿瘤的发展，这些肿瘤具有高度的血管生成性并且对治疗有抵抗力。因此，HIF-1抑制代表了治疗缺氧和VHL缺陷型肿瘤患者的有希望的策略。而HIF-1？已经被很好的研究，缺氧诱导因子-1的非氧依赖性调节？还没有完全被理解。我们有证据表明HIF-1的稳定性？由细胞周期蛋白依赖性激酶（CDK）调节，特别是CDK 1和CDK 4。推动这项研究的中心假设是，HIF-1？表达受CDK调节，鉴定这种调节的分子机制将产生抑制肿瘤存活、血管生成和与HIF-1活化相关的治疗抗性的新策略。我们将通过以下目标来解决这个假设：1）确定CDKs调节HIF-1的机制？表情2]检测CDK抑制对HIF-1？表达肿瘤，并鉴定与批准的抗癌疗法的协同组合。这一建议依赖于三个主要的方法：1]监测表达水平和活性的HIF- 1在响应CDK抑制，2]使用质谱法来确定新的翻译后修饰，改变HIF-1？稳定性和 3]在HIF-1中使用CDK抑制剂？与标准疗法组合表达肿瘤。初步数据表明，抑制CDK激酶活性显着降低HIF-1的稳定性？在蛋白质水平上。因此，我们计划进行质谱和生化分析，以确定新的HIF-1的翻译后修饰？并确定CDKs调节HIF-1a表达的机制。接下来，我们将研究CDK抑制作为一种新的治疗方法，以靶向组成型表达HIF-1的肾肿瘤的疗效？由于VHL的丢失。首先，将使用体外测定来评估 CDK抑制剂的疗效，并确定与标准抗癌疗法的有前途的组合。然后，在体内肿瘤模型将用于确定是否CDK介导的抑制HIF-1？抑制肿瘤生长和血管生成，并使HIF-1？促使肿瘤在标准疗法下经历细胞死亡。最终，该提案的目标是促进CDK抑制剂的临床翻译，用于具有组成性活性IF-1的肿瘤，同时还产生有关HIF-1？稳定性由CDK调节。

DESCRIPTION (provided by applicant): The hypoxia-inducible factor 1 (HIF-1) transcription factor is a critical mediator of the cellular response to oxygen deprivation. The ? subunits of HIF-1 are rapidly degraded in the presence of oxygen, but are stabilized under hypoxic conditions and activate the transcription of genes that promote angiogenesis, survival, invasion, and glucose metabolism. HIF-1 can be activated by genetic alterations that enhance the transcription or inhibit the degradation of HIF-1?. For example, von Hippel-Lindau (VHL) disease predisposes sufferers to aggressive tumors of the renal system, which are characterized by the stabilization of HIF-1?. The constitutive expression of HIF-1? drives the development of these tumors, which are highly angiogenic and resistant to therapy. Thus, HIF-1 inhibition represents a promising strategy for the treatment of patients with hypoxic and VHL-deficient tumors. While the oxygen-dependent regulation of HIF-1? has been well studied, the oxygen- independent regulation of HIF-1? is not completely understood. We have evidence that the stability of HIF-1? is regulated by cyclin dependent kinases (CDKs), specifically CDK1 and CDK4. The central hypothesis driving this research is that HIF-1? expression is regulated by CDKs, and identification of the molecular mechanisms underlying this regulation will produce novel strategies to inhibit tumor survival, angiogenesis, and therapeutic resistance associated with HIF-1 activation. We will address this hypothesis via the following aims: 1] Identify the mechanism by which CDKs regulate HIF-1? expression. 2] Test the anti-tumor efficacy of CDK inhibition on HIF-1? expressing tumors, and identify synergistic combinations with approved anticancer therapies. This proposal relies on three major approaches: 1] monitoring the expression levels and activity of HIF- 1 in response to CDK inhibition, 2] the use of mass spectrometry to identify of novel post-translational modifications that alter HIF-1? stability, and 3] utilizing CDK inhibitors in HIF-1? expressing tumors in combination with standard therapies. Preliminary data indicate that inhibition of CDK kinase activity significantly decreases the stability of HIF-1? at the protein level. Therefore, we plan to perform mass spectrometry and biochemical analyses to identify novel post-translational modifications on HIF-1? and determine the mechanism through which CDKs regulate HIF-1a expression. Next, we will investigate the efficacy of CDK inhibition as a novel therapeutic approach to target renal tumors that constitutively express HIF-1? due to the loss of VHL. First, in vitro assays will be used to assess the efficacy of CDK inhibitors and identify promising combinations with standard anticancer therapies. Then, in vivo tumor models will serve to determine if CDK-mediated repression of HIF-1? inhibits tumor growth and angiogenesis, and sensitizes HIF-1?-driven tumors to undergo cell death in response to standard therapies. Ultimately, the goal of this proposal is to facilitate the clinical translation of CDK inhibitors for use in tumors with constitutively active IF-1 while also yielding biological information about how HIF-1? stability is regulated by CDKs.