Pharmacologic /Genetic Inhibition of XBP1 as Hypoxia Targeted Therapeutic Strateg

低分化和内质网（ER）应激是肿瘤的两个重要组成部分 微环境将细胞对这些因子的反应与肿瘤生长联系起来。我们先前已经 显示缺氧激活未折叠蛋白反应（DPR），这是一种信号通路， 对ER应激的反应。X-box结合蛋白（XBP-1）是转录分支的重要调控因子 并通过一种ER跨膜蛋白IRE 1剪接成其活性形式。使用XBP-1 缺陷的细胞，我们证明了XBP-1介导缺氧下的生存，是肿瘤生长的关键。 考虑到它在调节缺氧条件下的存活中的作用及其对肿瘤生长的要求，我们假设， 靶向XBP-1将是有效的治疗策略。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 阻断IRE 1的活性，IRE 1是一种负责将XBP-1剪接成其活性的ER跨膜蛋白， form. IRE 1通过其激酶结构域的二聚化和自磷酸化被激活。的 IRE 1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IRE 1内切核酸酶功能的底物。 在这个建议中，我们将研究抑制XBP-1的作用，使用药理学和遗传学方法， 战略我们已经在一个高通量筛选中鉴定了一组XBP-1抑制剂（称为伊瑞他汀）， 66，000个化合物小分子库。我们还产生了几种抑制XBP的细胞系- 1使用IRE和XBP-1的显性负性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技术监测肿瘤内XBP-1的体内剪接活性， XBP-1剪接调控的荧光素酶报告基因的成像。我们将研究XBP-1抑制剂的作用， 通过多种方法在皮下和原位胰腺癌模型中进行。 我们将使用基于生化细胞的测定来确定伊瑞他汀的作用机制，研究伊瑞他汀的生物学活性。 XBP-1抑制对胰腺肿瘤生长的影响，以及将联合收割机XBP-1抑制与其他抑制组合， 胰腺癌的治疗方法

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.