Analysis of TWEAK Receptor (Fn14) Expression and Function in Non-Small Cell Lung Cancer Cells

Abstract

The cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a TNF superfamily member that is expressed by multiple cell types and is involved in many functions including proliferation, migration, survival, differentiation, de-differentiation, or cell death. It is the only ligand for the TNF receptor (TNFR) superfamily member Fibroblast Growth Factor-Inducible 14 (Fn14). The TWEAK:Fn14 signaling axis mediates multiple cellular processes including inflammation, angiogenesis, cell growth and death, and progenitor differentiation to aid in wound repair. Fn14 is overexpressed in over a dozen solid tumor types and constitutive signaling of the receptor is thought to be involved in tumor growth and metastasis. We previously showed that Fn14 levels are elevated in non-small cell lung cancer (NSCLC) tumors and NSCLC cell lines expressing constitutively activated Epidermal Growth Factor Receptor (EGFR) mutants. Also, we found that treatment of EGFR-mutant cells with erlotinib, (an EGFR tyrosine kinase inhibitor that is FDA-approved for use in the treatment of advanced NSCLC) decreases Fn14 levels and that Fn14 levels regulate NSCLC cell migration in vitro. In the present study, we extended these findings by showing that Fn14 levels also regulate NSCLC cell invasion. We also provide evidence that EGFR-mutant NSCLC cells that express high levels of Fn14 exhibit constitutive activation of the cytoplasmic tyrosine kinase Src. We found that inhibition of Src activity in NSCLC cells by dasatinib decreases Fn14 gene expression at both the mRNA and protein levels. Src depletion in NSCLC cells by siRNA also downregulates Fn14 protein expression. Finally, we show that Fn14 expression is significantly higher in an NIH 3T3 cell line engineered to express the constitutively active v-Src oncoprotein in comparison to parental NIH 3T3 cells, and that the NIH 3T3/v-Src cells require Fn14 expression for full invasive capacity. Taken together, these data demonstrate a functional role for Fn14 in NSCLC cell invasion and identify the Src tyrosine kinase as a new regulator of Fn14 gene expression.