EFFECTS OF TNFAIP8 KNOCKDOWN ON EGFR AND IGF-1R SIGNALING AND CYTOTOXICITIES OF TARGETED DRUGS IN NON-SMALL CELL LUNG CANCER CELLS
Day, Timothy Francis
Lung cancer is the most commonly diagnosed cancer and leading cause of cancer-related deaths worldwide. Non-small cell lung carcinoma (NSCLC) accounts for 85% of lung cancer cases. Molecular therapies targeting epidermal growth factor receptor (EGFR) have been developed for NSCLC. However, resistance of NSCLC to various therapies is a major clinical challenge. Previous studies from our laboratory and others have shown that tumor necrosis factor-alpha-inducible protein 8 (TNFAIP8), the first discovered member of a highly conserved TNFAIP8 family, is an oncogenic and metastatic molecule. TNFAIP8 (T8) expression is induced by NF-kB and is high in many cancers including NSCLC. This thesis project investigates the effects of T8 shRNA (shT8) on EGFR signaling in NSCLC cells. Since acquired resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs) has been associated with activation of insulin-like growth factor-1 receptor (IGF-1R), the effects of shT8 on IGF-1R signaling were also studied. The shRNA silencing of T8 resulted in inhibition of growth factor (EGF, IGF-1, FGF-1, VEGF)-stimulated cell migration in A549 lung cancer cells. T8 knockdown cells showed increased expression of sorting nexin 1 (SNX1), a regulator of EGFR expression through endosomal trafficking, and siRNA silencing of SNX1 partially restored EGFR expression in shT8 cells. Decreased levels of EGF-inducible pEGFR and pERK were observed in shT8 cells as compared to control cells. T8 knockdown cells also showed increased expression of IGF-1 binding protein 3 (IGFBP3), a negative regulator of IGF-1R signaling. Consistently, IGF-1-inducible expression of pIGF-1R and pAKT was decreased, and siRNA silencing of IGFBP3 resulted in increased pIGF-1R and pAKT levels in shT8 cells. In preliminary studies, cytotoxic effects of EGFR and IGF-1R TKIs, gefitinib and AG-1024, were enhanced in shT8 cells. Aberrant regulations of EGFR and IGF-1R signaling are hallmarks of several other tumors including breast, melanoma and pancreatic cancers. Present studies suggest that TNFAIP8 plays an important role in EGFR and IGF-1R signaling in lung cancer cells, and offer a rationale for development of T8 knockdown models of a wide range of TKI-resistant tumor cells. Ultimately, these efforts may lead to a new strategy for preventing or delaying resistance to EGFR and IGF-1R-targeted therapeutics.
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