Hypoxic and endoplasmic reticulum (ER) stress are two essential components of the tumormicroenvironment linking the cellular response to these factors with tumor growth. We have previouslyshown that hypoxia activates the unfolded protein response (DPR), a signaling pathway that is triggered inresponse to ER stress. X-box binding protein (XBP-1) is an important regulator of the transcriptional branchof this response and is spliced into its active for by IRE1, an ER transmembrane protein. Using XBP-1deficient 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 thattargeting XBP-1 will be an effective therapeutic strategy. However, there are few examples of anti-cancerdrugs that can effectively inhibit transcription factor activation. Therefore, our strategy for inhibiting XBP-1 isto block the activity of IRE1, an ER transmembrane protein responsible for splicing XBP-1 into its activeform. IRE1 is activated by dimerization and autophosphorylation through its kinase domain. Theendonuclease activity of IRE1 depends upon having an intact kinase domain, and to date, XBP-1 is the onlydescribed substrate for the endonuclease function of IRE1.In this proposal, we will investigate the effects of inhibiting XBP-1 using a pharmacologic and geneticstrategy. We have identified a group of XBP-1 inhibitors (termed irestatins) in a high throughput screen of a66,000 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 shRNAexpressing cell line. We will monitor in vivo XBP-1 splicing activity within tumors using bioluminescentimaging of a luciferase reporter regulated by XBP-1 splicing. We will examine the effects of XBP-1 inhibitionby 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 theconsequences of XBP-1 inhibition on pancreatic tumor growth, and combine XBP-1 inhibition with otherpromising therapies for pancreatic cancer.
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