The human Fn14 receptor gene is up-regulated in migrating glioma cells in vitro and overexpressed in advanced glial tumors

Abstract

Glioblastoma multiforme comprises the majority of human brain tumors. Patients with glioblastoma multiforme have poor survival rates, with an average life expectancy of <1 year. To assess possible mechanisms and to potentially target invasive glioma cells, we previously measured the gene expression profiles of glioma cells under migration-activated or passive states. One of the genes identified was Fn14, which encodes a cell surface receptor for the tumor necrosis factor superfamily member named TWEAK. In this study, we show that Fn14 gene expression is induced in migration-activated glioma cells in vitro and significantly increases according to tumor grade in vivo (P < 0.01), with highest levels in glioblastoma tissue specimens. The in situ expression pattern of Fn14 mRNA and protein was confined to primary glioma cells and the vascular endothelium, with no detection in adjacent normal brain. Conversely, TWEAK mRNA levels are low in glioblastoma samples relative to normal brain tissue. In addition, activation of the Fn14 receptor by addition of recombinant TWEAK resulted in increased glioma cell migration in vitro. These results suggest a positive role for TWEAK and Fn14 in glioma progression and indicate that Fn14 gene expression may serve as a marker for invasive glioma cells.

Figure 1.

Fn14 mRNA and protein expression in glioma cell lines cultured on tissue culture plastic or glioma-derived substrate. A: Northern blot analysis using RNA from each of the indicated cell lines cultured on tissue culture plastic (P) or glioma-derived ECM (M) was performed using the indicated cDNA probes. GAPDH was used as loading control and relative Fn14 mRNA expression was normalized to GAPDH and presented as fold induction over G112 cells cultured on tissue culture plastic. B: Immunoblot analysis of Fn14 expression in glioma cell lines. Total cell lysates were prepared from the indicated cell lines and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis using Fn14 polyclonal antiserum.

Figure 2.

Fn14 mRNA expression in various glial tissues. A: Quantitation of Fn14 mRNA expression using real-time RT-PCR. Total RNA was extracted from various glial tissues and analyzed by quantitative PCR for Fn14 mRNA and normalized to histone H3.3 expression. Fn14 expression for various glial tissues was then normalized to expression in normal brain. (NB, average Fn14 expression in three normal brain samples; PA, pilocytic astrocytoma; LGA, low-grade astrocytoma; OLIGO, oligodendroglioma; AA, anaplastic astrocytoma; GBM, glioblastoma multiforme). B: Assessment of Fn14 and histone H3.3 amplicon specificities after QRT-PCR. The PCR reaction was stopped at the exponential log phase and reaction products were separated on an agarose gel and detected by ethidium bromide staining.

Figure 3.

Immunohistochemical analysis of Fn14 expression in human glioblastoma tissue biopsies. Serial sections of paraffin-embedded glioma biopsies were used for peroxidase-based immunostaining for Fn14. Localization of Fn14 protein was detected in glioma cells (A) but not in normal adjacent brain (B). No immunoreactivity was detected when glioma biopsy tissue was stained with rabbit preimmune serum (C). Scale bar, 100 μm.

Figure 4.

TWEAK mRNA expression in glioma cell lines and glioma tissues. A: Northern blot analysis using RNA from each of the indicated cell lines cultured on tissue culture plastic (P) or glioma-derived ECM (M) was performed using the indicated cDNA probes. GAPDH was used as loading control and relative TWEAK mRNA expression was normalized to GAPDH and presented as fold induction over G112 cells cultured on tissue culture plastic. B: Total RNA was extracted from various glioma biopsy tissues and real-time quantitative RT-PCR was performed to analyze TWEAK mRNA expression. TWEAK mRNA expression was normalized to histone H3.3 mRNA expression, and the TWEAK expression in glioma tissues was further normalized to the average of TWEAK mRNA expression levels in three normal brain samples.

Figure 5.

TWEAK enhances glioma cell migration via Fn14 receptor activation. A: Glioma cells were plated onto 10-well glass slides precoated with laminin and then serum-deprived for 8 hours before addition of TWEAK. In some experiments, TWEAK was preincubated with Fn14-Fc decoy receptor or control mouse IgG before addition to cell monolayer. Cell migration was assessed throughout 48 hours. B: Glioma cells were treated with TWEAK and/or Fn14-Fc protein as described above and then the cells were washed with PBS and reincubated with TWEAK. Cell migration was assessed 24 hours later.

Figure 6.

Effect of TWEAK and Fn14-Fc on glioma cell viability. U118 and T98G cells were plated onto 10-well glass slides precoated with laminin and then serum-starved for 8 hours before addition of TWEAK and/or Fn14-Fc decoy receptor. In some experiments, cells were treated with TRAIL as a control for the cell viability assay. At the end of the migration intervals, the cells were washed three times with PBS and incubated with the Live/Dead solution. Live cells fluoresce green and dead cells fluoresce red. a and e, No treatment; b and f, TWEAK; c and g, TWEAK plus Fn14-Fc decoy; d and h, TRAIL.