Pharmacological targeting of the Unfolded Protein Response as an antitumor strate

描述（由申请人提供）：低氧/缺氧是大多数实体肿瘤微环境的一个很好的特征组成部分。大量的实验和临床证据支持这一观点，即缺氧从根本上改变了肿瘤的生理机能，使其向更具侵略性的表型发展。未折叠蛋白反应（Unfolded Protein Response， UPR）是细胞在内质网（Endoplasmic Reticulum， ER）应激（包括肿瘤微环境中的生理应激）存在下促进细胞存活的应激适应途径。PERK/eIF2a/ATF4通路降低了蛋白质翻译的整体速率，从而减轻了缺氧下的代谢应激，同时诱导了重要伴侣蛋白和促生存基因的翻译上调。另一个UPR成分是激活内切酶/激酶IRE1及其直接效应物XBP1，其激活旨在增加内质网折叠能力的转录程序。最近来自两家co- pi实验室的体外和体内研究表明，与具有完整UPR的细胞生长的肿瘤相比，具有消融UPR反应的转化细胞在体外表现出对缺氧的耐受性降低，形成的肿瘤生长较慢，表明UPR激活有助于肿瘤发生。在初步研究中，我们开发了基于细胞和动物的UPR激活和小分子抑制实验。本提案的总体目标是验证UPR激活作为一个重要的抗肿瘤靶点，并使用新的体外和体内试验来确定这种反应的有效抑制剂作为新的化疗药物。在Aim 1中，我们将采用PERK活性的体外报告基因测定来鉴定PERK/eIF21/ATF4通路的特异性抑制剂，并测试这些抑制剂与IRE-1通路抑制剂（依雷他汀类药物）联合使用对常氧和缺氧条件下肿瘤细胞存活的影响。在Aim 2中，我们将研究PERK和IRE1通路抑制剂与蛋白酶体抑制剂硼替佐米在体外和体内杀死缺氧肿瘤细胞中的潜在协同作用。Aim 3下的研究将评估斑马鱼作为模型系统的使用，以分析PERK和IRE1途径抑制剂抑制异种移植人类肿瘤和抑制血管生成的能力，而不会引起显著的发育异常。最后，在Aim 4中，我们将在小鼠肿瘤模型中测试已鉴定化合物的功效和潜在毒性。我们期望这些努力将最终发展为特异性和强效的普遍定期审议抑制剂，这些抑制剂单独使用或与现有的抗肿瘤药物和模式相结合，将有效地减少临床前和临床恶性肿瘤的肿瘤负担。公共卫生相关性：实体瘤的一个特征是需要适应并最终克服肿瘤肿块生长过程中低氧、生长因子、葡萄糖和pH的应激环境。新血管生成支持肿瘤生长的需求现在已经得到了很好的确立，并且是几种有希望的抗肿瘤方式的基础。基于我们实验室和其他人发表的数据，我们提出未折叠蛋白反应在适应缺氧应激中也起着至关重要的作用，就像血管生成一样，代表了“肿瘤发展的包罗和稳定方面”，因此为治疗开发提供了独特的机会。本提案旨在确定靶向这种适应性反应关键成分的药物，这些药物有可能提供额外的和新颖的方法来靶向阻碍现有抗肿瘤治疗的压力，从而提高抗肿瘤治疗效果。

DESCRIPTION (provided by applicant): Hypoxia/anoxia is a well-characterized component of the microenvironment of most solid tumor. Considerable experimental and clinical evidence supports the notion that hypoxia fundamentally alters the physiology of the tumor towards a more aggressive phenotype. The Unfolded Protein Response (UPR) is a cellular stress adaptation pathway which promotes cell survival in the presence of Endoplasmic Reticulum (ER) stress, including physiological stress in the tumor microenvironment. The PERK/eIF2a/ATF4 pathway reduces the global rates of protein translation thereby alleviating metabolic stress under hypoxia while at the same time induces the translational upregulation of important chaperones and pro-survival genes. Another UPR component is the activation of the endonuclease/kinase IRE1 and its immediate effector XBP1, which activate a transcriptional program aimed at increasing the folding capacity of the ER. Recent in vitro and in vivo studies from the labs of the two co-PIs, have shown that transformed cells with ablated UPR responses exhibit reduced tolerance to hypoxia in vitro and form tumors that are slower growing compared to tumors grown from cells with an intact UPR, indicating that UPR activation contributes to tumorigenesis. In preliminary studies, we have developed cell-based and animal-based assays for UPR activation and its inhibition by small molecules. The overall goal of this proposal is to validate UPR activation as an important anti- tumor target and to use novel in vitro and in vivo assays to identify potent inhibitors of this response as novel chemotherapeutic agents. In Aim 1, we will employ in vitro reporter assays of PERK activity to identify specific inhibitors of the PERK/eIF21/ATF4 pathway and test the effect of combined administration of these inhibitors with inhibitors of the IRE-1 pathway (Irestatins) on tumor cell survival under normoxia and hypoxia. In Aim 2, we will investigate the potential synergy between inhibitors of the PERK and IRE1 pathways with the proteasome inhibitor Bortezomib in killing hypoxic tumor cells in vitro and in vivo. Studies under Aim 3, will evaluate the use of Zebrafish as a model system to analyze the ability of inhibitors of the PERK and IRE1 pathways to inhibit xenotransplanted human tumors and to inhibit angiogenesis without causing significant developmental abnormalities. Finally, in Aim 4 we will test the efficacy and potential toxicity of identified compounds in mouse tumor models. We expect that these efforts will culminate in the development of specific and potent inhibitors of the UPR which alone, or in combination with existing antitumor agents and modalities will be effective in reducing tumor burden in preclinical and clinical malignancies. PUBLIC HEALTH RELEVANCE: A hallmark of solid tumors is the requirement to adapt to, and eventually overcome the stressful environment of low oxygen, growth factors, glucose and pH in the growing tumor mass. The requirement for neoangiogenesis to support tumor growth is now well established and is the basis for several promising anti-tumor modalities. Based on published data from our labs and others, we propose that the Unfolded Protein Response also plays a crucial role in adaptation to hypoxic stress, and like angiogenesis, represents an "encompassing and stable aspect of tumor development" and thus provides a unique opportunity for therapeutic exploitation. This proposal aims to identify agents that target key components of this adaptive response has the potential to offer additional and novel approaches to target the very stresses that hinder existing anti-tumor treatments and thereby improve antitumor treatment efficacy.