Pharmacologic /Genetic Inhibition of XBP1 as Hypoxia Targeted Therapeutic Strateg

低氧和内质网应激是肿瘤的两个基本组成部分 将细胞对这些因子的反应与肿瘤生长联系起来的微环境。我们之前已经 研究表明，低氧激活未折叠蛋白反应(DPR)，这是一种在 对内质网压力的反应。X-box结合蛋白(XBP-1)是转录分支的重要调节因子 并被内质网跨膜蛋白IRE1拼接到其活性形式中。使用XBP-1 我们证明了XBP-1在低氧条件下介导存活，并且对肿瘤生长至关重要。 考虑到它在低氧条件下调节生存的作用以及它对肿瘤生长的要求，我们假设 靶向XBP-1将是一种有效的治疗策略。然而，抗癌的例子很少。 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 为了阻断IRE1的活性，IRE1是一种ER跨膜蛋白，负责将XBP-1剪接到其活性中 形式。IRE1通过其激活域被二聚化和自磷酸化激活。这个 IRE1的核酸内切酶活性依赖于具有完整的激酶结构域，到目前为止，XBP-1是唯一的 描述了IRE1的内切酶功能的底物。 在这项提案中，我们将用药理学和遗传学方法研究抑制XBP-1的效果。 策略。我们已经在高通量筛选中确定了一组XBP-1抑制剂(称为irestatins)。 6.6万个化合物小分子文库。我们还产生了几个细胞系，在其中我们抑制了XBP- 使用IRE和XBP-1的显性负性抑制剂以及四环素调控的XBP-1 shRNA 表达细胞系。我们将使用生物发光技术在体内监测肿瘤内XBP-1的剪接活性 XBP-1剪接调控的荧光素酶报告的成像。我们将研究XBP-1的抑制作用 采用多种方法建立皮下原位胰腺癌模型。 我们将使用基于生化细胞的分析来定义irestatins的作用机制，研究 抑制XBP-1对胰腺肿瘤生长的影响及与其他药物联合应用 治疗胰腺癌的有希望的疗法。

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.