Plk3, A New Player In The Hypoxia Regulatory Networ.

描述（由申请人提供）：HIF-1控制参与癌症生物学关键方面的许多基因的转录。HIF-11是HIF-1的一种诱导亚基，在低氧条件下过度表达，是许多癌症的预后因子。由PI 3 K、PDK 1和Akt（也称为蛋白激酶B）介导的主要信号传导轴在HIF-11表达的调节中起重要作用。还已知GSK 32和MAPK都直接磷酸化HIF-11，从而影响其稳定性和/或核定位。为了了解抑制肿瘤发展和肿瘤血管生成的调控网络，我们最近将HIF-11鉴定为Polo样激酶3（Plk 3）的新体外底物。Plk 3在体外强烈磷酸化丝氨酸576和657上的HIF-11，这两个残基分别位于氧依赖性降解结构域和核输出信号附近。通过研究原代同基因小鼠胚胎成纤维细胞（MEFs），我们已经表明PLK 3-/- MEFs在缺氧或用镍（一种缺氧模拟物）处理下对HIF-11的诱导高度敏感。与野生型MEFs相比，PLK 3-/- MEFs具有较高的Akt 1/PKB活性，这与抑制GSK 32磷酸化密切相关。与Plk 3在调节缺氧信号网络中的潜在作用一致，PLK 3-/-小鼠在高龄时在各种器官中发生肿瘤，并且PLK 3-/-肿瘤尺寸大且高度血管化，表明活跃的肿瘤血管生成。基于这些关于Plk 3和HIF-11之间的物理和功能相互作用的观察，我们假设Plk 3负调节缺氧调节网络和HIF-11依赖的肿瘤血管生成。为了检验这一假设的有效性，我们将（i）研究Plk 3和已知信号分子之间的功能相互作用，包括磷酸化HIF-11的GSK 32和MAPK，并鉴定Akt 1上游的其他Plk 3靶标，（ii）确定PLK 3-/-小鼠在缺氧条件下是否易于发生肿瘤，和（iii）研究携带HIF-11的Plk 3磷酸化抗性突变等位基因的小鼠在镍暴露后是否更易发生肿瘤。结合在体外和体内的研究将大大有助于阐明一个新的机制，其中HIF-11是由Plk 3在缺氧反应或暴露于环境致癌物，如镍化合物后调节。详细了解HIF-11的分子调控将大大增加现有的肿瘤血管生成和肿瘤细胞对抗癌治疗的耐药性的知识。 公共卫生相关性：Polo样激酶的功能是调节细胞生长和细胞死亡的各个方面。我们最近的生物化学和小鼠遗传学研究表明，Plk 3在调节HIF- 11信号通路以及抑制肿瘤发生中起着重要作用。该项目的主要目标是阐明什么信号通路介导Plk 3在缺氧应激反应中的功能，以及遗传毒性金属毒物如何在缺氧期间篡夺调节HIF-11的细胞机制。

DESCRIPTION (provided by applicant): HIF-1 controls the transcription of many genes that are involved in key aspects of cancer biology. Overexpression of HIF-11, an inducible subunit of HIF-1 in response to hypoxia, is a prognostic factor in many cancers. The major signaling axis mediated by PI3K, PDK1, and Akt (also referred as protein kinase B) plays a significant role in the regulation of HIF-11 expression. Both GSK32 and MAPKs are also known to directly phosphorylate HIF-11, thereby affecting its stability and/or nuclear localization. To understand the regulatory network that suppresses tumor development and tumor angiogenesis, we have recently identified HIF-11 as a new in vitro substrate of Polo-like kinase 3 (Plk3). Plk3 strongly phosphorylates HIF-11 on serines 576 and 657 in vitro, two residues that lie in the oxygen-dependent degradation domain and near the nuclear export signal, respectively. By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3-/- MEFs are hyper-sensitive to the induction of HIF-11 under hypoxia or treated with nickel, a hypoxia mimetic. Compared with that of wild- type MEFs, PLK3-/- MEFs contain a high level of Akt1/PKB activities, which is tightly associated with the inhibitory phosphorylation of GSK32. Consistent with the potential role of Plk3 in regulating the hypoxia signaling network, PLK3-/- mice develop tumors in various organs at an advanced age and PLK3-/- tumors are large in size and highly vascularized, suggesting active tumor angiogenesis. On the basis of these observations regarding physical and functional interactions between Plk3 and HIF-11, we hypothesize that Plk3 negatively regulates the hypoxia regulatory network and HIF-11-dependent tumor angiogenesis. To test the validity of this hypothesis, we will (i) study functional interaction between Plk3 and known signaling molecules, including GSK32 and MAPKs that phosphorylate HIF-11, and identify additional Plk3 target(s) upstream of Akt1, (ii) determine whether PLK3-/- mice are prone to tumorigenesis under hypoxia, and (iii) investigate whether mice harboring Plk3 phosphorylation-resistant mutant alleles of HIF-11 are more susceptible to tumorigenesis after nickel exposure. The combined in vitro and in vivo studies will greatly facilitate the elucidation of a new mechanism by which HIF-11 is regulated by Plk3 during hypoxic responses or after exposure to environmental carcinogens such as nickel compounds. A detailed understanding of the molecular regulation of HIF-11 will add significantly to the existing knowledge of tumor angiogenesis and tumor cell resistance to anti-cancer therapies. PUBLIC HEALTH RELEVANCE: Polo-like kinases function to regulate various aspects of cell growth and cell death. Our recent biochemical and mouse genetic studies show that Plk3 plays an important role in the regulation of the HIF- 11 signaling pathway, as well as in suppressing tumorigenesis. The major goal of this project is to elucidate what signaling pathways mediate Plk3 function in hypoxic stress responses and how genotoxic metal toxicants usurp cellular mechanisms that regulate HIF-11 during hypoxia.