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

• 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
• 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

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.

低氧和内质网（ER）应激是肿瘤的两个必不可少的成分 将细胞反应与这些因素与肿瘤生长联系起来的微环境。我们以前有 表明缺氧激活展开的蛋白质反应（DPR），这是一种触发的信号传导途径 对ER应力的反应。 X-box结合蛋白（XBP-1）是转录分支的重要调节剂 在这种响应中，并将其插入其活性，以by IRE1，一种ER跨膜蛋白。使用XBP-1 缺乏细胞，我们证明XBP-1在缺氧下介导生存率，对于肿瘤生长至关重要。 鉴于其在调节缺氧下的生存及其对肿瘤生长的需求中的作用，我们假设 靶向XBP-1将是一种有效的治疗策略。但是，很少有反癌的例子 可以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 为了阻止IRE1的活性，ER跨膜蛋白负责将XBP-1插入其活性 形式。 IRE1通过其激酶结构域通过二聚化和自磷酸化激活。这 IRE1的核酸内切酶活性取决于具有完整的激酶结构域，迄今为止，XBP-1是唯一的 描述了IRE1的核酸内切酶函数的底物。 在此提案中，我们将研究使用药理和遗传学抑制XBP-1的影响 战略。我们已经确定了一组XBP-1抑制剂（称为irestatins） 66,000个复合小分子库。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的显性负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的体内XBP-1剪接活性 由XBP-1剪接调节的荧光素酶报告器的成像。我们将检查XBP-1抑制作用 通过使用胰腺癌的皮下和原位模型的多种方法。 我们将使用基于生化细胞的测定法来定义iRestatins的作用机理，研究 XBP-1抑制胰腺肿瘤生长的后果，并将XBP-1抑制与其他 胰腺癌的有前途的疗法。