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

描述（由申请人提供）：缺氧是实体肿瘤微环境的一个很好的特征组成部分，它促进对放疗和化疗的抵抗，并与较差的总体预后相关。低氧肿瘤细胞诱导hif依赖性和hif非依赖性机制来适应和克服肿瘤中的低氧应激。通过在先前资助申请的特定目的下进行的实验和本提议的初步结果，我们已经表明缺氧/缺氧可以快速激活一个翻译控制程序，该程序是由内质网（ER）激酶PERK的激活和翻译起始因子elF2?的磷酸化介导的。该途径下调了整体蛋白合成，但同时上调了部分应激反应蛋白的表达。这些结果与其他研究小组的结果一起证实，未折叠蛋白反应是细胞对内质网应激的一种适应机制，在缺氧时被激活，并且是缺氧耐受性和肿瘤生长所必需的。在本应用中，我们建议扩展这些发现，以确定缺氧激活UPR的信号，确定PERK和elF2的下游效应物的作用。磷酸化，表征内质网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激活的依赖是否可以用于治疗。在Specific Aim 1中，我们将测试下游PERK效应物ATF4表达的缺乏是否抑制肿瘤生长，而ATF4的过表达是否促进肿瘤生长，我们将识别和表征缺氧激活ATF4的转录靶点。在Specific Aim 2中，我们将通过分析位于er的折叠酶蛋白二硫异构酶和Erolp氧化酶上sh基团的状态，试图描述缺氧条件下PERK和UPR激活的机制。在Specific Aim 3中，我们将研究er靶向的bcl-2和er定位的bax/bak在er精通和er缺乏细胞缺氧/缺氧诱导er依赖性凋亡中的作用。在Specific Aim 4下，我们将在动物肿瘤模型中测试耐基因毒性化疗药物的缺氧肿瘤细胞是否对内质网应激源急性敏感，以及内质网应激源与电离辐射或化疗药物联合使用是否能更好地抑制肿瘤生长。实体瘤转移和对大多数现有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。这些研究的成功完成将揭示一种有助于肿瘤发生和治疗耐药的新途径，并可能提供选择性攻击这些耐药细胞的新靶点和方法，从而改善治疗效果。

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.