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

Abstract

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.