Pharmacologic /Genetic Inhibition of XBP1 as Hypoxia Targeted Therapeutic Strateg

XBP1 的药理学/基因抑制作为缺氧靶向治疗策略

基本信息

批准号：
7558955
负责人：
ALBERT KOONG
金额：
$ 25.77万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7558955
关键词：
Antineoplastic Agents Binding Proteins Biochemical Biological Assay Boxing Cell Line Cells Dimerization Dominant-Negative Mutation Endoplasmic Reticulum Gene Targeting Genetic Growth Hypoxia Image Integral Membrane Protein Lethal Dose 50 Link Luciferases Malignant neoplasm of pancreas Mediating Methods Modeling Monitor Northern Blotting Nude Mice Phosphotransferases Proteins RNA Splicing Reporter Reverse Transcriptase Polymerase Chain Reaction Role Signal Pathway Specificity Stress Tetracycline Tetracyclines Therapeutic Transfection Tumor Cell Invasion base chemotherapy endonuclease gemcitabine high throughput screening in vivo inhibitor/antagonist matrigel neoplastic cell pancreatic neoplasm research study response small hairpin RNA small molecule libraries subcutaneous therapeutic target transcription factor tumor tumor growth

项目摘要

Hypoxic and endoplasmic reticulum (ER) stress are two essential components of the tumor microenvironment linking the cellular response to these factors with tumor growth. We have previously shown that hypoxia activates the unfolded protein response (DPR), a signaling pathway that is triggered in response to ER stress. X-box binding protein (XBP-1) is an important regulator of the transcriptional branch of this response and is spliced into its active for by IRE1, an ER transmembrane protein. Using XBP-1 deficient cells, we demonstrated that XBP-1 mediates survival under hypoxia and is critical for tumor growth. Given its role in regulating survival under hypoxia and its requirement for tumor growth, we hypothesize that targeting XBP-1 will be an effective therapeutic strategy. However, there are few examples of anti-cancer drugs that can effectively inhibit transcription factor activation. Therefore, our strategy for inhibiting XBP-1 is to block the activity of IRE1, an ER transmembrane protein responsible for splicing XBP-1 into its active form. IRE1 is activated by dimerization and autophosphorylation through its kinase domain. The endonuclease activity of IRE1 depends upon having an intact kinase domain, and to date, XBP-1 is the only described substrate for the endonuclease function of IRE1. In this proposal, we will investigate the effects of inhibiting XBP-1 using a pharmacologic and genetic strategy. We have identified a group of XBP-1 inhibitors (termed irestatins) in a high throughput screen of a 66,000 compound small molecule library. We have also generated several cell lines in which we inhibit XBP- 1 using dominant negative inhibitors of IRE and XBP-1 as well as a tetracycline regulated XBP-1 shRNA expressing cell line. We will monitor in vivo XBP-1 splicing activity within tumors using bioluminescent imaging of a luciferase reporter regulated by XBP-1 splicing. We will examine the effects of XBP-1 inhibition by multiple methods in a using a subcutaneous and orthotopic model of pancreatic cancer. We will use biochemical cell based assays to define the mechanism of action of the irestatins, investigate the consequences of XBP-1 inhibition on pancreatic tumor growth, and combine XBP-1 inhibition with other promising therapies for pancreatic cancer.

缺氧和内质网（ER）应激是肿瘤的两个重要组成部分将细胞对这些因素的反应与肿瘤生长联系起来的微环境。我们之前有过研究表明，缺氧会激活未折叠蛋白反应（DPR），这是一种在对 ER 应激的反应。 X-box结合蛋白（XBP-1）是转录分支的重要调节因子 IRE1（一种内质网跨膜蛋白）将其剪接成其活性。使用XBP-1 通过对缺陷细胞的研究，我们证明了 XBP-1 介导缺氧条件下的存活，并且对于肿瘤生长至关重要。鉴于其在调节缺氧条件下生存的作用及其对肿瘤生长的要求，我们假设以XBP-1为靶点将是一种有效的治疗策略。但抗癌的例子却很少能有效抑制转录因子激活的药物。因此，我们抑制XBP-1的策略是阻断 IRE1 的活性，IRE1 是一种内质网跨膜蛋白，负责将 XBP-1 剪接成其活性形式。 IRE1 通过其激酶结构域通过二聚化和自磷酸化来激活。这 IRE1 的核酸内切酶活性取决于是否具有完整的激酶结构域，迄今为止，XBP-1 是唯一描述了 IRE1 核酸内切酶功能的底物。在本提案中，我们将研究使用药理学和遗传学抑制 XBP-1 的效果战略。我们在高通量筛选中鉴定出了一组 XBP-1 抑制剂（称为 irestatins） 66,000个化合物小分子库。我们还产生了几种细胞系，其中我们抑制 XBP- 1 使用 IRE 和 XBP-1 的显性失活抑制剂以及四环素调节的 XBP-1 shRNA 表达细胞系。我们将使用生物发光监测肿瘤内 XBP-1 体内剪接活性由 XBP-1 剪接调节的荧光素酶报告基因的成像。我们将检查 XBP-1 抑制的效果通过多种方法使用胰腺癌的皮下和原位模型。我们将使用基于生化细胞的测定法来定义易司他汀的作用机制，研究 XBP-1 抑制对胰腺肿瘤生长的影响，并将 XBP-1 抑制与其他抑制相结合胰腺癌的有希望的治疗方法。