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

      Cheng, Emily; Winkles, Jeffrey Allan (2014)
      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.
    • Regulation of Class III Beta-Tubulin by Src-Mediated Tyrosine Phosphorylation

      Alfano, Alan; Qiu, Yun (2015)
      Prostate cancer (PCa) is the second most lethal cancer in men, accounting for an estimated 30,000 deaths in 2013. For diagnoses of advanced disease, the standard treatment is androgen deprivation therapy (ADT). Despite initial success of ADT, many PCa patients relapse into an incurable androgen-insensitive disease termed Castration Resistant Prostate Cancer (CRPC). Taxane compounds (such as docetaxel [DTX]) + Steroid (such as Prednisone) is a first-line treatment, but DTX resistance is common in patients with advanced CRPC. Tubulins are an integral part of the cytoskeleton, and play a pivotal role in cell signaling, migration, and division. They exist in vivo either as soluble monomers or as α-/β-tubulin heterodimers. These dimers are either recycled into monomers, or polymerized into microtubules (MTs). Microtubules serve a range of functions including structural support of the cell body, scaffolding for signaling molecules, and mitotic spindle support. β-tubulin is also the molecular target for taxane compounds. High expression levels of Class III β-tubulin (TUBB3, a primarily neural isoform of β-tubulin) correlate with taxane resistance and poor prognosis in several human cancers, including ovarian cancer (serous adenocarcinoma), breast cancer, non-small-cell lung cancer (NSCLC), and prostate cancer (PCa). It is known that c-Src (a known proto-oncogene) phosphorylates β-tubulins during both hematopoietic and neural differentiation. This has also been observed in leukemia cells. Despite recent advances, the relationship between Src-mediated tyrosine phosphorylation and microtubule modulation is still poorly understood, especially in the context of advanced solid malignancies. Herein we show that activated Src-kinase is able to phosphorylate TUBB3 at tyrosine 340 (Y340), and that tyrosine phosphorylation of βIII-tubulin at Y340 by Src-family kinases is critical in stabilization of TUBB3, and also plays a role in regulation of mitotic spindles. Given the clinical utility of TUBB3 as a biomarker of poor prognosis, characterizing cancer-specific post-translational modification (PTM) of TUBB3 in aggressive cancer cell types could lead to more specified biomarkers of patient outcome or therapeutic response. Additionally, a deeper understanding of the complex interplay between the Src family of kinases (SFKs) and microtubules could enable future researchers to design more specific and effective microtubule-targeted and/or SFK-targeted therapies.