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eIF 2 α在ser 51上的磷酸化。后来，我们还证明了营养敏感激酶GCN 2，类似地激活eIF 2磷酸化在实体瘤中响应氨基酸和葡萄糖剥夺。eIF 2 α的磷酸化不仅减少了能量昂贵的过程，如整体翻译，而且还创造了一个环境，促进应激反应基因的更有效的翻译，如ATF 4，一种上调参与适应ER应激的基因的转录因子。eIF 2 α的磷酸化和ATF 4的上调代表了不同细胞应激激活的共同机制，因此被称为综合应激反应（ISR）。肿瘤细胞中ISR的破坏显著影响其在应激下的增殖和存活以及其生长肿瘤的能力 in vivo.总之，我们的数据支持一个模型，其中转化细胞在体内激活ISR作为对氧和营养剥夺应激的适应性反应，并且在几个步骤中破坏该途径会损害应激和肿瘤生长下的细胞存活。 该提议的总体假设是，在肿瘤微环境应激条件下被激活的ISR转换器PERK和GCN 2激活导致增加的细胞存活和血管生成并有助于转移的途径。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性激酶抑制剂p21在介导细胞周期阻滞和生存响应缺氧和营养剥夺ISR-熟练和缺陷细胞的作用。在目标2中，我们将使用体外血管生成模型研究GCN 2和PERK在血管生成中的作用。我们还将使用抗体阵列和蔗糖沉降分析活性翻译的mRNA来鉴定GCN 2和PERK下游的血管生成介质。在目标3中，我们将使用纤维肉瘤的转基因小鼠模型，其将与GCN 2 +/+和GCN 2-/-小鼠杂交。将在这些模型中研究血管生成和转移。这些目标的实现将确定ISR是否是肿瘤发生和转移的关键靶点，并确定这种活性的机制。PI实验室和制药公司正在积极研究PERK和GCN 2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。

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 eIF2α 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 eIF2α 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 eIF2α 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.