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

描述(由申请人提供)：缺氧和营养缺乏条件是肿瘤微环境的动态特征，有助于癌症的进展和对治疗的抵抗。我们先前已经证明，低氧应激激活内质网(ER)激酶PERK，从而诱导翻译起始因子eIF2？在SER51上。后来，我们还证明了营养感应激酶GCN2类似地激活了实体瘤中eIF2的磷酸化，以响应氨基酸和葡萄糖的剥夺。EIF2的磷酸化？不仅减少了全球翻译等耗费能源的过程，还创造了一个环境，促进更有效地翻译应激反应基因，如ATF4，一种转录因子，上调参与适应内质网应激的基因。EIF2的磷酸化？而ATF4的上调代表了不同细胞应激激活的共同机制，因此被称为整合应激反应(ISR)。肿瘤细胞中ISR的破坏显著影响了它们在应激状态下的增殖和存活以及生长肿瘤的能力 在活体内。综上所述，我们的数据支持一种模型，在该模型中，转化细胞激活体内的ISR作为对氧气和营养剥夺压力的适应性反应，并且在几个步骤中破坏这一途径会损害应激下的细胞存活和肿瘤生长。 该建议的总体假设是，在肿瘤微环境应激条件下激活的ISR转导通路PERK和GCN2，激活了导致细胞存活和血管生成增加的通路，并有助于转移。为了验证这一假设，我们提出了以下三个具体目标：在目标1中，我们将确定细胞周期蛋白依赖的激酶抑制剂p21在介导ISR熟练和缺乏营养的细胞在低氧和营养剥夺反应中的细胞周期停滞和存活中的作用。在目标2中，我们将使用体外血管生成模型来研究GCN2和PERK在血管生成中的作用。我们还将使用抗体阵列和主动翻译的mRNAs的蔗糖沉淀分析来识别GCN2和PERK下游的血管生成介质。在目标3中，我们将使用转基因小鼠的纤维肉瘤模型，该模型将与GCN2+/+和GCN2-/-小鼠杂交。将在这些模型中研究血管生成和转移。这些目标的完成将确定ISR是否是肿瘤发生和转移的关键靶点，并确定这种活动的机制。PI的实验室和制药公司正在积极寻找PERK和GCN2的抑制剂。因此，这些数据可以促进先导化合物快速进入临床前动物试验阶段。 公共卫生相关性：肿瘤微环境在使肿瘤更具侵袭性和对治疗更具抵抗力方面发挥重要作用。这项提议延续了过去10年来所做的工作，以了解这些应激(如低氧和低糖)增加肿瘤生成新血管、生长和扩散到其他部位的能力的分子机制。成功完成这项提案中的目标可能会导致针对纤维肉瘤等侵袭性癌症的治疗干预的新目标。

DESCRIPTION (provided by applicant): Hypoxia and nutrient deprivation conditions are dynamic features of the tumor microenvironment that contribute to cancer progression and resistance to treatment. We have previously shown that hypoxic stress activates the endoplasmic reticulum (ER) kinase PERK thereby inducing phosphorylation of the translation initiation factor eIF2? on ser51. Later, we also demonstrated that the nutrient-sensing kinase GCN2, similarly activates eIF2 phosphorylation in solid tumors in response to both amino acid and glucose deprivation. Phosphorylation of eIF2? not only reduces energy expensive processes such as global translation, but also creates an environment that promotes the more efficient translation of stress-responsive genes, such as ATF4, a transcription factor that upregulates genes involved in adaptation to ER stress. The phosphorylation of eIF2? and the upregulation of ATF4 represent a common mechanism activated by different cellular stresses, thereby being termed the Integrated Stress Response (ISR). Disruption of the ISR in tumor cells dramatically affects their proliferation and survival under stress and their ability to grow tumors in vivo. Together, our data support a model in which transformed cells activate the ISR in vivo as an adaptive response to oxygen and nutrient deprivation stress and that disruption of this pathway at several steps compromises cellular survival under stress and tumor growth. The overall hypothesis of this proposal is that the ISR transducers PERK and GCN2 which are activated under conditions of tumor microenvironmental stress, activate pathways that lead to increased cell survival and angiogenesis and contribute to metastasis. To test this hypothesis, we propose the following three specific aims: In Aim 1, we will determine the role of the cyclin-dependent kinase inhibitor p21 in mediating cell-cycle arrest and survival in response to hypoxia and nutrient deprivation in ISR-proficient and deficient cells. In Aim 2, we will investigate the role of GCN2 and PERK in angiogenesis using in vitro angiogenesis models. We will also identify mediators of angiogenesis downstream of GCN2 and PERK using antibody arrays and sucrose sedimentation analysis of actively translated mRNAs. In Aim 3, we will use transgenic mouse models of fibrosarcoma which will be crossed to GCN2+/+ and GCN2-/- mice. Angiogenesis and metastasis will be investigated in these models. Completion of these aims will establish whether the ISR is a critical targets of tumorigenesis and metastasis and define the mechanism of such an activity. Inhibitors of PERK and GCN2 are being actively pursued by the PI's lab and by pharmaceutical companies. Therefore, such data could facilitate rapid movement of lead compounds into preclinical animal testing phase. PUBLIC HEALTH RELEVANCE: The tumor microenvironment plays important roles in making tumors more aggressive and more resistant to therapy. This proposal continues work performed over the last 10 years to understand the molecular mechanisms by which these stresses (such as low oxygen and low glucose) increase the ability of tumors to make new blood vessels, grow and spread to other sites. Successful completion of the aims in this proposal may lead to new targets for therapeutic intervention against aggressive cancers such as fibrosarcomas.