Abstract
Abstract The Integrated Stress Response (ISR) is a program elicited by mammalian cells to attenuate global translation in response to a variety of stressors. The GCN2 arm of the ISR is activated in response to amino acid deprivation, resulting in the upregulation of the transcription factor ATF4. ATF4 modulates the expression of genes involved in amino acid transport and synthesis. We have previously demonstrated that the expression of both GCN2 and ATF4 is increased in tumor cells compared to normal tissue, and the ablation of ATF4 or GCN2 significantly decreases tumor growth in mice. WE are interested in the contribution of the GCN2-ATF4 pathway in cell migration and autophagy, two processes that contribute to tumor growth and metastasis. The impact of ATF4 expression on the migration of human fibrosarcoma cells (HT1080) was assessed using scratch assays and transwell migration analysis, and invasion was analyzed using Matrigel-coated inserts. Preliminary results indicate that decreased expression of ATF4 or GCN2 is associated with diminished migration and invasion in HT1080 cells. We can exploit this finding therapeutically through the pharmacological inhibition of ATF4 To this end, we have employed a small molecule screen to identify inhibitors of ATF4 via a reporter construct with the 5’-UTR of ATF4 fused to the luciferase gene in HT1080 cells. When ATF4 reporter cells were treated with Thapsigargin, we observed more than a four-fold induction of luciferase over control cells in a PERK-dependent manner. Using a chemical library of over 160,000 diverse small molecules and the NIH Molecular Libraries Small Molecule Repository we performed a primary screen on the HT1080 ATF4-luciferase cells. We have identified 4 small molecules based on the inhibition of ATF4 induction, and low µM or high nM IC50 values. The compounds are currently undergoing validation in secondary screens for the specific inhibition of ATF4 and not CMV-luciferase or XBP-1-luciferase reporters. We plan to identify structural families and perform structure-activity relationship (SAR) analysis to identify related compounds. Validated compounds are currently being analyzed for the ability to reduce tolerance to amino acid deprivation through cell proliferation, cell survival, and in vitro and in vivo tumorigenicity assays. Interestingly, a compound which appears to activate ER stress was identified from the screen. The effects of this drug on autophagy and cell migration are currently being assessed. Our findings represent a novel translational strategy by which the ISR is exploited to sensitize tumors to microenvironmental stressors, such as amino acid deprivation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2072. doi:10.1158/1538-7445.AM2011-2072
Published Version
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