• 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.
    • The Effects of Constitutive TWEAK/Fn14 Pathway Activation in Murine B16 Melanoma Cells In Vitro and In Vivo

      Armstrong, Cheryl Lynn; Winkles, Jeffrey Allan; 0000-0003-2484-2299 (2016)
      The cytokine tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a TNF superfamily member that binds exclusively to the cell surface receptor fibroblast growth factor-inducible 14 (Fn14). Fn14 also binds exclusively to TWEAK and mediates activation of multiple downstream signaling pathways that promote or inhibit cellular responses such as proliferation, migration, invasion, death and differentiation. Elevated TWEAK and Fn14 expression have been detected in multiple cancer tissues, and several groups have evaluated the role of TWEAK/Fn14 signaling in mouse models of tumor growth and metastasis. These previous studies suggest that TWEAK/Fn14 signaling promotes both tumorigenesis and metastasis; however, no precise mechanism for these effects has been elucidated. As TWEAK is known to stimulate a diverse range of cellular responses dependent upon cell type, we hypothesized that TWEAK/Fn14 signaling would promote tumor growth and metastasis via multiple mechanisms that would involve direct effects of TWEAK on cancer cells and host cells and indirect effects of TWEAK/Fn14 signaling in cancer cells on the tumor microenvironment. To test this hypothesis, we generated TWEAK-overexpressing B16 murine melanoma cell lines that are syngeneic with the immunocompetent C57BL/6 mouse. We evaluated the effects of TWEAK overexpression in B16 cells on cell growth, migration, invasion, survival, and microtentacle formation in vitro. We also evaluated the effect of TWEAK overexpression on subcutaneous tumor growth in the C57BL/6 mouse and lung colony formation in both immunocompetent C57BL/6 mice (wild-type, Fn14 knock-out, CCR2 knock-out) and immunodeficient athymic nude and NSG mice. We found that TWEAK overexpression in B16 cells inhibited cell proliferation and cell invasion in vitro, but had no effect on subcutaneous tumor growth or lung colony formation in the mouse. We further determined that TWEAK-regulated B16 cell invasion was mediated, at least in part, by the non-canonical NF-κB signaling pathway. As TWEAK activation of the non-canonical NF-κB signaling pathway has not previously been reported to negatively regulate cell invasion, we further evaluated the role of non-canonical NF-κB signaling in human DU145 prostate cancer cells, which have previously been reported to demonstrate a TWEAK-induced increase in invasion. We found that this same signaling pathway was also important for TWEAK-stimulated DU145 cell invasion. Therefore, even though TWEAK:Fn14 binding activates non-canonical NF-κB signaling in both B16 melanoma cells and DU145 prostate cancer cells, the same signaling pathway triggered different downstream outcomes; specifically, inhibition or stimulation of cell invasion, respectively. In contrast to the hypothesis of the study, TWEAK/Fn14 signaling in B16 melanoma cells did not stimulate pro-tumorigenic or pro-metastatic effects in vitro or in vivo. Conversely, TWEAK/Fn14 signaling inhibited B16 cell proliferation and invasion in vitro. Future work may be necessary to determine if these experimental results can be generalized to all melanoma cells.
    • The Fn14 Receptor is Up-regulated by HER2 Overexpression and Heregulin1-β1 (HRG) Signaling in Breast Cancer Cells and Contributes to HRG-MMP9-driven Cell Invasion.

      Asrani, Kaushal Vijay; Winkles, Jeffrey Allan (2012)
      Human epidermal growth factor receptor (HER)-2 overexpression occurs in ~25% of all breast cancers and is associated with increased metastatic potential and poor patient survival. Abnormal HER2 activation, either through HER2 overexpression or heregulin (HRG):HER3 binding, elicits the formation of potent HER2-HER3 heterodimers and drives breast cancer cell growth and metastasis. Fibroblast growth factor-inducible 14 (Fn14), a member of the TNF receptor superfamily, is overexpressed in human breast tumors, and high expression levels strongly correlate with both the invasive HER2+/ER- intrinsic subtype and indicators of poor prognosis. We report here that HER2 and Fn14 are also co-expressed in a transgenic mouse model of HER2-driven breast tumorigenesis. In consideration of these findings, we investigated whether HER2 activation in breast cancer cells could directly induce Fn14 gene expression. We found that transient or stable transfection of MCF7 cells with HER2 increased Fn14 protein levels and this effect was primarily due to an increase in Fn14 stability. Also, HRG1-beta1 treatment of MCF7 cells transiently induced Fn14 mRNA and protein expression. Both the HER2-and HRG1-beta1-induced increase in Fn14 expression in MCF7 cells as well as increased Fn14 expression in HER2 gene-amplified AU565 cells could be blocked by HER2 kinase inhibition with lapatinib or HER2/HER3 depletion using siRNA. We also report that Fn14-depleted, HER2-overexpressing MCF7 cells have reduced basal cell migration capacity and reduced HRG1-beta1-stimulated cell migration, invasion and matrix metalloproteinase (MMP)-9 expression. Furthermore, expression of Fn14 in Fn14-depleted HER2-overexpressing cells increased HRG1-beta1-stimulated invasion and MMP-9 expression. Preliminary results also suggest that Fn14 depletion in MCF7/HER2-18 and SKBR3 cells might be decreasing anchorage-dependent and -independent growth in vitro and tumor xenograft growth in vivo. Together, these results indicate that the Fn14 receptor may be an important downstream regulator of both HER2 overexpression- and HRG1-beta1-driven HER2/HER3 signaling in breast cancer cells.
    • Fn14-targeted, Tissue-penetrating Nanoparticles for Treatment of Primary and Metastatic Tumors

      Dancy, Jimena; Winkles, Jeffrey Allan; Kim, Anthony J.; Woodworth, Graeme; 0000-0002-7428-9682 (2018)
      Nanotherapeutics is a burgeoning field in cancer therapy that may address some of the issues associated with systemically administered chemotherapeutics, including a non-specific mechanism of action and a poor biodistribution profile, which results in dose-limiting toxicities. However, many of the nanoparticle (NP) formulations approved for clinical use in solid tumor therapy provide only modest improvements in patient survival. This is in part due to rapid clearance from the circulation, inability to efficiently target tumor cell drug uptake, and NP tumor penetration barriers, including a dense and complex extracellular matrix (ECM) and an elevated interstitial fluid pressure. These barriers hinder the penetration of drugs and NPs into and within tumors limiting therapeutic efficacy. Fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor receptor (TNFR) superfamily, is expressed at low levels in normal tissues but highly expressed in over 20 solid cancer types. Thus, Fn14 has the potential to be an ideal candidate for the development of targeted therapy and its down-regulation may contribute to positive disease outcomes. However, while targeting therapeutics to specific disease components may decrease some of the limitations mentioned, in order to fully capitalize on the potential benefits of targeting, low levels of non-specific adhesivity and off-target binding must be maintained in setting off an effective level of target-specific binding. My thesis project tests the hypothesis that biodegradable, polyethylene glycol (PEG)- and anti-Fn14 antibody-coated, drug-loaded NPs will exhibit high efficacy against Fn14-positive tumors due to their ability to penetrate tissue and effectively target Fn14-positive cancer cells. We tested this hypothesis in the following Specific Aims: 1) Determine the thresholds for NP size and PEG density for effective tumor penetration and examine the potential penetrative capacity of FDA-approved NPs used for breast cancer patients, 2) Determine if Fn14-targeted and/or non-targeted tissue-penetrating, paclitaxel (PTX)-loaded biodegradable NPs are more effective than the FDA-approved NP formulation Abraxane in reducing primary and metastatic breast cancer growth, and 3) Evaluate the potential use of our biodegradable NP formulation against glioblastoma (GBM), the most common and deadly form of adult brain cancer, by examining NP dispersion, cellular uptake, and tumor retention in a murine model of GBM. These findings will generate new knowledge related to the value of tumor-specific targeting for NP therapeutics.