Role of elF2a phosphorylation and ER stress in hypoxia tolerance and tumor growth

描述（由申请人提供）：缺氧是实体瘤微环境的一个特征良好的组成部分，它会促进对放疗和化疗的抵抗，并与较差的总体预后相关。肿瘤细胞在低氧环境中通过HIF依赖性和HIF非依赖性两种机制来适应和克服低氧应激。通过在先前资助的申请的特定目标下进行的实验和本提案的初步结果，我们已经表明，缺氧/缺氧快速激活由内质网（ER）激酶PERK激活和翻译起始因子eIF 2？磷酸化介导的翻译控制程序。该途径下调全球蛋白质合成，但同时上调选择的应激反应蛋白的表达。这些结果与来自其他组的结果一起确定了未折叠蛋白质反应，一种对ER应激的细胞适应机制，被缺氧激活，并且是缺氧耐受和肿瘤生长所必需的。在本申请中，我们建议扩展这些发现，以确定缺氧的UPR激活信号，确定PERK和eIF 2的下游效应子的作用？本发明的目的是通过研究UPR的磷酸化，表征由ER应激激活的促凋亡和抗凋亡途径，并测试缺氧肿瘤细胞对UPR激活的存活依赖性是否可以在治疗上被利用。在具体目标1中，我们将测试是否缺乏下游PERK效应物ATF 4表达抑制，以及ATF 4的过表达促进肿瘤生长，并且我们将鉴定和表征缺氧激活的ATF 4的转录靶点。在具体目标2下，我们将试图通过分析ER驻留折叠酶蛋白质二硫键异构酶和Erolp氧化酶上的-SH基团的状态来描述缺氧条件下PERK和UPR激活的机制。在特定目标3中，我们将研究ER靶向bcl-2和ER定位bax/巴克在UPR活性和缺陷细胞中通过缺氧/缺氧诱导ER依赖性凋亡中的作用。在具体目标4下，我们将测试对遗传毒性化疗剂具有抗性的缺氧肿瘤细胞是否对药理学ER应激源急性敏感，以及ER应激源与电离辐射或化疗剂的组合是否在动物肿瘤模型中导致更好的肿瘤生长抑制。实体瘤转移和对大多数已建立的化疗方案的耐药性是肿瘤发病率的关键因素，因此构成了重大的公共卫生问题。成功完成拟议的研究将揭示一种新的途径，有助于肿瘤发生和对治疗的抵抗，并可能提供新的靶点和方法来选择性地攻击这些耐药细胞，从而改善治疗结果。

DESCRIPTION (provided by applicant): Hypoxia is a well-characterized component of the solid tumor microenvironment that promotes resistance to radiation therapy and chemotherapy and is associated with a poorer overall prognosis. The hypoxic tumor cell elicits both HIF-dependent and HIF-independent mechanisms to adapt to and overcome the stress of low oxygen in tumors. Through experiments performed under the specific aims of the previously funded application and preliminary results in this proposal, we have shown that hypoxia/anoxia rapidly activates a translational control program mediated by activation of the endoplasmic reticulum (ER) kinase PERK and phosphorylation of the translation initiation factor elF2?. This pathway downregulates global protein synthesis but at the same time upregulates the expression of select stress-response proteins. These results together with those from other groups established that the Unfolded Protein Response, a cellular adaptation mechanism to ER stress, is activated by hypoxia and is required for hypoxia tolerance and tumor growth. In this application we propose to expand these findings to identify the signal for UPR activation by hypoxia, identify the role(s) of downstream effectors of PERK and elF2? phosphorylation, characterize pro- and anti- apoptotic pathways activated by ER stress and test whether the reliance of hypoxic tumor cells on UPR activation for survival can be therapeutically exploited. In Specific Aim 1 we will test whether lack of the downstream PERK effector ATF4 expression inhibits, and overexpression of ATF4 promotes, tumor growth and we will identify and characterize transcriptional targets of hypoxia-activated ATF4. Under Specific Aim 2 we will attempt to delineate the mechanism responsible for PERK and UPR activation under hypoxia by analyzing the status of-SH groups on the ER-resident folding enzymes Protein Disulfide Isomerase, and Erolp oxidase. In Specific Aim 3 we will investigate the role(s) of ER-targeted bcl-2 and ER-localized bax/bak in the induction of ER-dependent apoptosis by hypoxia/anoxia in UPR-proficient and -deficient cells. Under Specific Aim 4 we will test whether hypoxic tumor cells, which are resistant to genotoxic chemotherapeutic agents, are acutely sensitive to pharmacological ER stressors and whether the combination of ER stressors with ionizing radiation or chemotherapeutic agents results in better inhibition of tumor growth in animal tumor models. Solid tumor metastasis and resistance to most established chemotherapy regimens are key contributors to tumor morbidity and thus constitute a significant public health problem. Successful completion of the proposed studies will uncover a novel pathway that contributes to tumorigenesis and resistance to therapy and can potentially offer new targets and approaches to selectively attack these resistant cells and thus improve therapeutic outcome.