Increased fibroblast growth factor-inducible 14 expression levels promote glioma cell invasion via Rac1 and nuclear factor-kappaB and correlate with poor patient outcome.

Abstract

Glial tumors progress to malignant grades by heightened proliferation and relentless dispersion throughout the central nervous system. Understanding genetic and biochemical processes that foster these behaviors is likely to reveal specific and effective targets for therapeutic intervention. Our current report shows that the fibroblast growth factor-inducible 14 (Fn14), a member of the tumor necrosis factor (TNF) receptor superfamily, is expressed at high levels in migrating glioma cells in vitro and invading glioma cells in vivo. Forced Fn14 overexpression stimulates glioma cell migration and invasion, and depletion of Rac1 by small interfering RNA inhibits this cellular response. Activation of Fn14 signaling by the ligand TNF-like weak inducer of apoptosis (TWEAK) stimulates migration and up-regulates expression of Fn14; this TWEAK effect requires Rac1 and nuclear factor-kappaB (NF-kappaB) activity. The Fn14 promoter region contains NF-kappaB binding sites, which mediate positive feedback causing sustained overexpression of Fn14 and enduring glioma cell invasion. Furthermore, Fn14 gene expression levels increase with glioma grade and inversely correlate with patient survival. These results show that the Fn14 cascade operates as a positive feedback mechanism for elevated and sustained Fn14 expression. Such a feedback loop argues for aggressive targeting of the Fn14 axis as a unique and specific driver of glioma malignant behavior.

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2006;66:9535-9542. Published online October 3, 2006. Cancer Res


Nhan L. Tran, Wendy S. McDonough, Benjamin A. Savitch, et al.

B and Correlate with Poor Patient Outcome
κ
Factor-
Levels Promote Glioma Cell Invasion via Rac1 and Nuclear
Increased Fibroblast Growth Factor-Inducible 14 Expression



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Increased Fibroblast Growth Factor-Inducible 14 Expression
Levels Promote Glioma Cell Invasion via Rac1 and Nuclear
Factor-KB and Correlate with Poor Patient Outcome
Nhan L. Tran,
Jeffrey A. Winkles,
Galen Hostetter,
Jennifer S. Michaelson,
Joseph C. Loftus,
1Wendy S. McDonough,
2Marc Symons,
1Dominique B. Hoelzinger,
4Linda C. Burkly,
5Luigi Mariani,
1Benjamin A. Savitch,
3Mitsutoshi Nakada,
1Jessica L. Rennert,
4Christopher A. Lipinski,
6and Michael E. Berens
1Shannon P. Fortin,
1Heather E. Cunliffe,
1
1
1
5
1
1Cancer and Cell Biology Division, Translational Genomics Research Institute, Phoenix, Arizona;
and the Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland;
Cell Biology, The Feinstein Institute for Medical Research at North Shore-Long Island Jewish Health System, Manhasset,
New York;
6University Hospital, Inselspital, Bern, Switzerland
2Departments of Surgery and Physiology
3Center for Oncology and
4Biogen Idec, Inc., Cambridge, Massachusetts;
5Mayo Clinic Scottsdale, Scottsdale, Arizona; and
Abstract
Glial tumors progress to malignant grades by heightened
proliferation and relentless dispersion throughout the central
nervous system. Understanding genetic and biochemical
processes that foster these behaviors is likely to reveal specific
and effective targets for therapeutic intervention. Our current
report shows that the fibroblast growth factor-inducible 14
(Fn14), a member of the tumor necrosis factor (TNF) receptor
superfamily, is expressed at high levels in migrating glioma
cells in vitro and invading glioma cells in vivo. Forced Fn14
overexpression stimulates glioma cell migration and invasion,
and depletion of Rac1 by small interfering RNA inhibits this
cellular response. Activation of Fn14 signaling by the ligand
TNF-like weak inducer of apoptosis (TWEAK) stimulates
migration and up-regulates expression of Fn14; this TWEAK
effect requires Rac1 and nuclear factor-KB (NF-KB) activity.
The Fn14 promoter region contains NF-KB binding sites,
which mediate positivefeedback causingsustainedoverexpres-
sion of Fn14 and enduring glioma cell invasion. Further-
more, Fn14 gene expression levels increase with glioma grade
and inversely correlate with patient survival. These results
show that the Fn14 cascade operates as a positive feedback
mechanism for elevated and sustained Fn14 expression. Such a
feedbacklooparguesforaggressivetargetingoftheFn14axisas
a unique and specific driver of glioma malignant behavior.
(Cancer Res 2006; 66(19): 9535-42)
Introduction
Survival of patients with glioblastoma multiforme (GBM) is
compromised by the proclivity of the tumor for local invasion into
the surrounding normal brain, causing most patients with GBM to
succumb to the disease in <1 year (1). An understanding of glioma
oncogenesis has steadily improved, but the molecular mechanisms
mediating glioma invasion are still nascent. Gene discovery strate-
gies have been used with the anticipation of identifying molecular
pathways that regulate glioma cell migration and invasion (2, 3).
Fibroblast growth factor inducible-14 (Fn14) is a type I
transmembrane protein of 102 amino acids in length after removal
of signal peptide, making it the smallest member of the tumor
necrosis factor (TNF) superfamily of receptors (4–6). The 29-
amino-acid cytoplasmic tail of the Fn14 receptor lacks a death
domain but contains a single TNF receptor-associated factor
(TRAF) binding site flanked by two conserved threonine residues
(6, 7). This TRAF site links Fn14 to the nuclear factor-nB (NF-nB)
and mitogen-activated protein kinase pathways to regulate
endothelial cell proliferation (8) and glioma cell survival (9). We
reported that Fn14 receptor is up-regulated in migrating glioma
cells (10) and that activation of the Fn14 receptor by addition of
TNF-like weak inducer of apoptosis (TWEAK), an exclusive ligand
for Fn14, enhances glioma cell survival (9). Additionally, Fn14
expression is very low in normal brain tissue but elevated in GBM
specimens (10). Furthermore, high expression of Fn14 has been
reported in other human cancers, including hepatocellular (5),
pancreatic (11), and breast carcinomas (12).
Dysregulation of cell migration contributes to malignant
processes, such as tumor invasion, metastasis, and angiogenesis
(13). Cell migration is dependent on continual reorganization of the
actin cytoskeleton (14). Members of the Rho family of small
GTPases, RhoA, Rac1, and Cdc42, are key mediators of such
reorganization and regulate cell migration (15, 16). RhoA mediates
the formation of stress fibers and focal adhesions (16), whereas
Rac1 stimulates actin assembly, resulting in the formation of
lamellipodia (16, 17).
Here, we describe a role for Rac1 in Fn14-mediated glioma
migration and invasion and show that TWEAK can induce Fn14
expression levels via the Rac1/IKKh/NF-nB pathway. This pathway
serves as a positive feedback loop that drives sustained Fn14
expression, thereby stimulating invasion. High expression of Fn14
mRNA correlates with increasing glial tumor grade and poor
clinical outcome. We show a critical role for Fn14 in the invasive
phenotype of glioma cells and suggest that Fn14 overexpression
imparts a poor prognosis to patients with GBM.
Materials and Methods
Cell culture conditions. Human astrocytoma cell lines T98G, U118, U87
(American Type Culture Collection, Manassas, VA), G112 (18), and SF767
Note: Supplementary data for this article are available at Cancer Research Online
(http://cancerres.aacrjournals.org/).
Requests for reprints: Michael E. Berens, Cancer and Cell Biology Division, The
Translational Genomics Research Institute, 445 North Fifth Street, Phoenix, AZ 85004.
Phone: 602-343-8760; Fax: 602-343-8844; E-mail: mberens@tgen.org.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-0418
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(19) were maintained in MEM supplemented with 10% heat-inactivated
fetal bovine serum (FBS; Hyclone Laboratories, Inc., Logan, UT). For
adenoviral infection, 1.5 ? 105cells were plated into a six-well plate and
cultured for 24 hours before infection. Before TWEAK addition, cells were
cultured in reduced serum (0.5% FBS) for 16 hours.
Preparation of recombinant adenoviruses and infection. The cDNA
encoding murine myc-tagged Fn14 wild-type protein (pSec/Tag2/Fn14wt;
ref. 7) was excised and subcloned into the adenoviral shuttle vector
pShuttle-CMV to prepare recombinant E1-deleted adenoviruses using the
Ad-Easy system as previously described (9). Adenoviruses expressing the
superrepressor InB mutant (S32A/S36A) and signal deficient myc-Fn14tCT
were previously described (9). Adenoviruses expressing Rac1N17 and
Rac1V12 were previously described (20). Cells were infected at matched
multiplicity of infections ranging from 5 to 20. Expression plasmids for
Cdc42N17, Rac1N17, and Rac1V12 were previously described (21, 22).
Antibodies, reagents, Western blot analysis, and immunofluores-
cence. Monoclonal antibodies to Rac and RhoA were purchased from BD
Transduction Labs (San Jose, CA). Monoclonal antibodies specific to
phospho-InBa (Ser32/36) and polyclonal antibodies to Cdc42, IKKh and
InBa, and phospho-IKKh were obtained from Cell Signaling (Beverly, MA).
A monoclonal antibody to a-tubulin was obtained from Upstate Biotech-
nology (Lake Placid, NY). Human recombinant TWEAK was purchased
from PeproTech (Rock Hill, NJ), and laminin from human placenta was
obtained from Sigma (St. Louis, MO). Anti-Fn14 polyclonal serum was
precviously described (4). Rhodamine-phallodin was obtained from
Invitrogen (Carlsbad, CA).
Immunoblotting and immunofluorescence microscopy experiments were
done as previously described (23). Immunofluorescent samples were
examined under LSM 5 Pascal Laser scanning confocal microscope (Zeiss,
Thornwood, NY) using the appropriate filters.
Immunoprecipitation and Rac and Rho activity assays. Cells were
infected with adenoviruses expressing control LacZ, myc-Fn14wt, or myc-
Fn14tCT, then cultured for an additional 24 hours. Immunoprecipitation
was carried out as previously described (23).
Activity assays for Rac1 and RhoA were done according to the protocol of
the manufacturer (Pierce, Rockford, IL). Where indicated, 2.5 Ag/mL soluble
Fn14-Fc decoy receptor or control mouse IgG (Sigma) were preincubated
with 100 ng/mL TWEAK at 37jC for 15 minutes before cell treatments as
described previously (10).
Radial cell migration assay. Cellular migration was quantified as
previously described (10).
Laser capture microdissection, RNA isolation, and quantitative
reverse transcription-PCR. Laser capture microdissection of tumor core
and invasive cells was done and total RNA was isolated as previously
described (3). PCR analysis of Fn14 and histone H3.3 mRNA levels was
conducted with LightCycler analysis software and quantified as previously
described (10).
Small-interfering RNA preparation and transfection. Small interfer-
ing RNA (siRNA) oligonucleotides specific for Rac1 and GL2 Luciferase
were previously described (24). siRNA sequences for Fn14 are as follows:
Fn14i-475 (bp 475-495; 5¶-CATCCATTCTAGAGCCAGTCT) and Fn14i-863
(bp 863-883; 5¶-TAGGAGGGCTGGCCCTAAGAT). Transient transfection of
siRNA was carried out as previously described (24). Fn14-directed siRNA
was transfected at 40 nmol/L, and no cell toxicity was observed. Maximum
inhibition of mRNA and protein levels was achieved 48 to 72 hours after
transfection.
Organotypic brain slice invasion assay. Preparation and culture of
adult rat brain slices were carried out as described previously (25). Glioma
cells (1 ? 105; T98G, U118, and U87) stably expressing green fluorescence
protein (GFP) were placed onto the putamen of the brain slice (0.5 AL
transfer volume). Glioma cell invasion into rat brain slices was quantified
using the LSM 5 confocal microscope and depth of invasion (z-axis stacks)
was calculated as previously described (25).
Biotinylated electrophoretic mobility shift assay. Glioma cells were
plated at a density of 3 ? 106in 100 mm2tissue culture dishes in normal
growth medium. After 12 hours, cells were cultured under reduced serum
(0.5% FBS) for an additional 16 hours before TWEAK (100 ng/mL) addition.
In certain experiments, TWEAK was preincubated with Fn14-Fc decoy
receptor (2.5 Ag/mL) or control mouse IgG. Isolation of cell nuclear protein
was carried out using the NE-PER kit (Pierce) according to the protocol
of the manufacturer. Protein-DNA complexes were detected using biotin
end-labeled double-stranded DNA 22-mer probes containing the NF-nB
binding sites within the Fn14 promoter (NF-nB-Fn14wt sense: 5¶-GAGATA-
AGGGAAATTCCTAGGC; antisense: 5¶-GCCTAGGAATTTCCCTTATCTC;
NF-nB-Fn14mt sense: 5¶-GAGATAAGCGAGAATCGTAGGC; antisense:
5¶-GCCTACGATTCTCGCTTATCTC). The binding reactions were done using
the LightShift kit (Pierce) according to the protocol of the manufacturer.
Where indicated, 200-fold molar excess of unlabeled NF-nB-Fn14wt
oligonucleotides or anti-p65 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA; 1 mg/mL) was included. The reaction products were resolved
by gel electrophoresis and detected by chemiluminescence according to the
protocol of the manufacturer (Pierce).
Microarray data, survival analysis, and Fn14 expression. Snap-frozen
nonneoplastic brain specimens from epileptogenic patients (n = 24) and
tumor (n = 160) specimens with clinical information were collected at
Hermelin Brain Tumor Center, Henry Ford Hospital (Detroit, MI). All
specimens were collected under an Institutional Review Board–approved
protocol and deidentified for patient confidentiality. Clinical information
was provided for all samples (29 astrocytomas, 82 glioblastomas, and 49
oligodendrogliomas).
Gene expression profiles of these brain specimens were captured using
Affymetrix U133 Plus 2 GeneChips according to the protocol of the
manufacturer (Affymetrix, Santa Clara, CA) at the Neuro-Oncology Branch
at the National Cancer Institute. Array data was processed according to
Affymetrix MAS5 (Microarray Suite 5) algorithm implemented in Affymetrix
GCOS (GeneChip Operating Software) and uploaded into GeneSpring 7.2 for
data management (Silicon Genetics, Redwood City, CA).
Expression values were filtered for highly variable (differentially
expressed) genes (coefficient of variation >30%) across samples producing
a list of 7,322 genes. Principal component (PC) analysis was done to
investigate the relationship between samples (i.e., to find clusters within the
data). Components are sorted from most to least amount of variation. Two
clusters were evident in a three-dimensional scatter plot of PC1, PC2, and
PC3. The three components accumulatively account for 46% of the variation
in the data set. Kaplan-Meier survival curves were developed for each
cluster. One cluster had a median survival time of 401 days and the other
cluster had a median survival time of 952 days. Box plots for Fn14
expression in each cluster derived from PC analysis were graphed.
Significance between the two populations was tested with a two-sample
t test assuming unequal variances.
Brain tumor tissue microarray and immunohistochemistry. A
glioma invasion TMA was assembled by Dr. D.H. Friedrich as previously
described (3). Immunohistochemical analysis for Fn14 was done using an
Fn14 monoclonal antibody, P4A8 (Biogen Idec, Inc., Cambridge, MA). P4A8
was generated in an Fn14 knockout mouse (26) immunized with Fn14-myc-
his recombinant protein. Anti-Fn14 monoclonal antibodies were screened
and selected for binding to Fn14-positive cell lines. Immunohistochemistry
was done as previously described using 2.5 Ag/mL of the P4A8 antibody
(10). A scoring system for chromophore was used to capture the outcome:
0, negative; 1, weak; 2, moderate; 3, strong staining.
Results
Fn14 expression is increased in migrating glioma cells
in vitro and invasive cells in vivo. We previously showed that
Fn14 expression increased when glioma cell lines were plated
on a migration-promoting substrate (10). Here, we examined if
the up-regulated expression of Fn14 arises coincident to the
migration behavior, irrespective of the specific substrate the cells
encounter. RNA was isolated from two subpopulations of cells
seeded on glioma-derived ECM: actively migrating cells at the
dispersing rim and migration-restricted cells atthe crowded cellular
core. Fn14 expression was assessed using quantitative reverse
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transcription-PCR (QRT-PCR). In three glioma cell lines, cells
migrating at the rim showed 4- to 19-fold induction compared with
core cells (Fig. 1A). RNA was also isolated from tumor core and
invasive rim glioma cells from human biopsies; QRT-PCR showed
4- to 6-fold induction of Fn14 in the GBM cells isolated at the rim
compared with matched core (Fig. 1B).
Forced Fn14 overexpression stimulates glioma cell migra-
tion and alters cellular morphology. Fn14 signaling can be
initiated by either TWEAK binding or forced Fn14 overexpression
(7). Our finding of elevated expression of Fn14 in migrating glioma
cells suggested that forced overexpression of Fn14 may stimulate
glioma cell migration. Glioma cell lines were infected with
adenoviruses expressing either a full-length Fn14 receptor (myc-
Fn14wt) or a signaling-deficient truncated Fn14 receptor (myc-
Fn14tCT; ref. 7). Overexpressed myc-Fn14wt in SF767 and T98G
cells caused a 2-fold induction of cell motility (Fig. 1C). Over-
expression of Fn14 in glioma cells also resulted in morphologic
changes, including neurite-like extensions, membrane ruffling, and
lamellipodia formation (Supplementary Data 1). Furthermore, dual
staining for myc-Fn14wt and F-actin shows extensive colocaliza-
tion near the cell perimeter and in the cellular extensions and
lamellipodia (Supplementary Data 1). Cells expressing the signal-
ing-deficient myc-Fn14tCT protein migrated poorly (Fig. 1C) and
showed no morphologic changes (data not shown), indicating that
Fn14 signaling is essential for Fn14-mediated motility.
Fn14 interacts with Rac1 and regulates Rac1 activity.
Because Rac1 is essential for lamellipodia formation in glioma
cells (15), our observation that Fn14 overexpression increased
lamellipodia suggests that Fn14 signaling involves Rac1. To test
this hypothesis, we examined whether TWEAK-mediated Fn14
signaling affects the activation of Rac1. T98G cells treated with
TWEAK showed high Rac1 activation compared with untreated
cells (Fig. 2A). Blocking TWEAK binding to Fn14 by an Fn14-Fc
decoy receptor prevented TWEAK-induced Rac1 activation. Over-
expression of myc-Fn14wt also induced Rac1 activation, whereas
no Rac1 activation was detected in cells expressing the signaling-
deficient receptor myc-Fn14tCT (Fig. 2A). The effect of Fn14 on
Rac1 is specific because Fn14 signaling did not affect Cdc42
activation (data not shown).
Rac1 coimmunoprecipitates with myc-Fn14wt, but not with
myc-Fn14tCT (Fig. 2B), indicating a physical interaction between
Fn14 and Rac1. This interaction is independent of Rac1 activation
because Fn14 interacts equally well with constitutively active and
dominant negative Rac1 (data not shown).
TWEAK treatment caused a decrease in RhoA activation
(Fig. 2C), and the Fn14-Fc decoy receptor antagonized TWEAK
Figure 2. Fn14 interacts with Rac1 and regulates Rac1 activity. T98G cells were
treated with either TWEAK alone, TWEAK preincubated with Fn14-Fc decoy
receptor, or TWEAK preincubated with mouse IgG. In certain experiments,
cells were infected with adenoviruses expressing either myc-Fn14wt or
myc-Fn14tCT. Cell extracts were assayed for active Rac1 using the PBD assay
(A) or active RhoA using the RBD assay (C). For coimmunoprecipitation,
T98G cells were infected with adenoviruses expressing control LacZ,
myc-Fn14wt, or myc-Fn14tCT and cultured for 24 hours. Fn14 bound proteins
were immunoprecipitated from total cellular lysates with a monoclonal
anti-myc antibody and immunoblotted for Rac1 (B) or RhoA (D).
Figure 1. Fn14 expression in glioma cell migration in vitro and invasion in vivo.
A, 24 hours after cell seeding (1 mm confluent disc) on 10-well glass slides
precoated with glioma-derived ECM, migrating cells at the rim (R) and migration-
restricted cells at the core (C) were isolated and total RNA was extracted.
Relative Fn14 mRNA expression was assessed by QRT-PCR, normalized to
histone H3.3, and presented as fold induction over SF767 core. B, QRT-PCR
analysis of Fn14 mRNA levels from laser capture microdissected glioma core
and rim cells from cryopreserved biopsy specimens. Levels of Fn14 mRNA were
normalized to histone H3.3 and expressed as proportions of the mRNA levels of
Fn14 in GBM12 core. C, migration of glioma cells infected with adenoviruses
expressing control LacZ, myc-Fn14wt, or myc-Fn14tCT over 48 hours. Columns,
mean of three independent experiments.
Fn14 Promotes Glioma Invasion
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suppression of RhoA activation. Overexpression of myc-Fn14wt
receptor also prevented RhoA activation, whereas signaling-
deficient myc-Fn14tCT did not change RhoA activity (Fig. 2C).
Immunoprecipitation of Fn14 failed to show a physical association
with RhoA (Fig. 2D). These results show that Fn14 interacts and
colocalizes with Rac1 and that the TWEAK-Fn14 signaling axis
controls Rac1 and RhoA GTPase activity in opposite directions.
Depletion of Rac1 expression by siRNA suppresses Fn14-
induced glioma cell migration and invasion. To determine the
role Rac1 plays in Fn14-induced glioma migration, we inhibited
Rac1 expression in two different glioma cell lines overexpressing
the myc-Fn14wt receptor. siRNA-mediated depletion of Rac1
expression in T98G and SF767 cells was f90% effective (Fig. 3A)
and caused a strong inhibition of Fn14-mediated cell migration
(Fig. 3B, lane d). This level of cell migration inhibition is similar to
the level caused by suppression of Fn14 signaling using the
Fn14tCT receptor (Fig. 3B, lane f).
The effect of either Fn14 or Rac1 knockdown on cell invasion
was examined using an ex vivo organotypic rat brain slice model
(24, 25). Invasion of GFP-expressing human glioma cells into the
rat brain slices over 48 hours was quantified by confocal
microscopy. Depletion of Fn14 (f80%; Fig. 3C) resulted in a 40%
to 50% decrease in cell invasion among the three glioma cell lines
examined (Fig. 3D; lanes d, h, and l) compared with LacZ controls
(lanes a, e, and i). Conversely, overexpression of myc-Fn14wt
receptor resulted in a 20% to 50% increase in cell invasion (Fig. 3D;
lanes b, f, and j); this invasion was suppressed by Rac1 depletion
(Fig. 3D; lanes c, g, and k). These data corroborate the cell
migration results and further support a role for Fn14 in glioma cell
invasion mediated through Rac1.
TWEAK regulation of Fn14 expression is dependent on the
Rac1/IKKB/NF-KB signaling pathway. The relationship between
Fn14 expression and cell motility raises questions about the
molecular and cellular upstream regulators of Fn14 transcription. It
was intriguing to contemplate a possible positive feedback loop
wherein activation of Fn14 signaling would lead to its own
overexpression; therefore, we investigated whether TWEAK could
induce Fn14 expression in glioma cells. Increased expression of
Fn14 mRNA was detected 2 hours post-TWEAK addition and
peaked between 4 and 8 hours (Fig. 4A). TWEAK induction of
elevated Fn14 protein (Fig. 4B) was detected with similar kinetics
to the changes in mRNA. Because Fn14 signals through Rac1 to
increase cell motility, we queried whether TWEAK regulation of
Fn14 expression was dependent upon Rac1. T98G cells were treated
with Rac1 siRNA, placed in reduced serum, and then TWEAK was
added. SiRNA-mediated depletion of Rac1 resulted in a strong
reduction of both Rac1 and Fn14 protein expression (Fig. 4C).
Depletion of Rac1 expression inhibited TWEAK-induced Fn14
expression (Fig. 4C), suggesting that Rac1 is essential for TWEAK
stimulation of Fn14 expression.
Activated Rac1 influences diverse signaling pathways, including
the activation of multiple intracellular signaling molecules, such as
Figure 3. Rac1 regulates Fn14-induced glioma migration in vitro and invasion ex vivo. A, Rac1 protein levels were determined in SF767 and T98G cells
transfected with siRNA targeting control luciferase (ctrl siRNA) or siRNA targeting Rac1 by Western blotting. a-Tubulin protein levels were also analyzed by Western
blotting to monitor gel loading. Representative of three independent experiments. B, glioma cells were transfected with siRNA targeting either luciferase or Rac1.
After 24 hours, cells were infected with adenoviruses expressing control LacZ, myc-Fn14tCT, or myc-Fn14wt and cultured for 16 hours. Cell migration was assessed
over 48 hours. Columns, average of three independent experiments. C, Fn14 protein levels were determined in T98G cells transfected with siRNA targeting control
luciferase or the indicated siRNAs targeting Fn14 by Western blotting. a-Tubulin protein levels were also analyzed by Western blotting to monitor gel loading.
Each panel is a representation of three independent experiments. D, glioma cells stably expressing GFP were transfected with siRNA directed against Rac1 for
24 hours and then infected with adenoviruses expressing myc-Fn14wt. In certain experiments, cells were either infected with adenoviruses expressing myc-Fn14wt or
transfected with siRNA directed against Fn14. Cells were implanted into the bilateral putamen on rat organotypic brain slices and observed at 48 hours. Depth
of invasion was then calculated from Z-axis images collected by confocal laser scanning microscopy. The mean value of the depth of invasion was obtained from
six independent experiments.
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the c-Jun NH2-terminal protein kinases (JNK; ref. 27), p38 kinases
(28), and the NF-nB transcription factor family (29). Inhibition
of JNK by SP-600125 or p38 by SB-239063 in TWEAK-treated
glioma cells did not inhibit TWEAK induction of Fn14 (data not
shown). However, inactivation of NF-nB in glioma cells by infection
with an adenovirus expressing the superrepressor InBa mutant
(InBaM) strongly diminished TWEAK-induced Fn14 mRNA and
protein expression (Fig. 4D and E). Similarly, infection of glioma
cells with the dominant-negative Rac1N17 adenovirus inhibited
TWEAK-induced Fn14 mRNA and protein expression (Fig. 4D
and E).
Because Rac1 has been shown to activate NF-nB via IKKh
phosphorylation (29), we examined the effect of Rac1 inhibition on
both IKKh and InBa phosphorylation following TWEAK stimula-
tion. T98G glioma cells treated with TWEAK showed a rapid
increase in phosphorylation of IKKh within 5 minutes, after which
phosphorylation levels diminished by 1 hour (Fig. 5A). Likewise,
InBa serine phosphorylation was detected within 5 minutes of
TWEAK treatment, but the phosphorylation level remained
constant for over 2 hours, whereas total InBa protein was
degraded (Fig. 5A). Inhibition of Rac1 function with Rac1N17
protein suppressed TWEAK-induced IKKh and InBa phosphoryla-
tion (Fig. 5B). Neutralizing TWEAK with the Fn14-Fc decoy
receptor also prevented TWEAK-induced IKKh and InBa phos-
phorylation (Fig. 5B). However, expression of dominant-negative
Cdc42N17 did not affect Fn14-induced IKKh phosphorylation (data
not shown).
The human Fn14 gene sequence upstream of exon 1 was
scanned using Ensemble7and Motif Library Search.8This region
contains a NF-nB consensus binding site at ?2167 to ?2176. We
examined NF-nB binding to this motif by analyzing nuclear
extracts from untreated or TWEAK-treated T98G cells. No
induction of NF-nB binding activity was observed in untreated
cells (Fig. 5C, lane a). Binding activity was robust 2 hours post-
TWEAK addition (lane b), whereas, no NF-nB DNA binding activity
was detected when TWEAK was first neutralized with the Fn14-Fc
decoy receptor. The specificity of this complex was determined by
competition assays using excess molar amounts of unbiotinylated
wild type (lane d) or normal amounts of biotinylated mutated
(lane e) NF-nB probe. In addition, an antibody to the p65 subunit of
NF-nB shifted the NF-nB-DNA binding complex to a more slowly
migrating form (a ‘‘supershift’’), confirming the presence of the p65
subunit of NF-nB in the binding complex (Fig. 5C, lane f). These
data indicate that TWEAK-induced Fn14 gene expression involves
promoter activation by NF-nB.
Fn14 mRNA expression levels correlate with brain tumor
grade and poor patient outcome. In an earlier report, we
evaluated Fn14 mRNA and protein expression levels in a limited
panel of normal brain and brain tumor specimens (10). Here, the
clinical relevance of Fn14 overexpression was investigated by
analysis of Fn14 mRNA expression in 24 nonneoplastic brain and
160 brain tumor specimens (provided by Dr. Howard Fine, Neuro-
Oncology Branch, National Cancer Institute, Bethesda, MD). In
normal brain specimens, Fn14 expression is relatively low, but is
significantly higher in GBM samples (n = 82) and seems to increase
according to tumor grade (Fig. 6A). Levels of TWEAK mRNA did
not vary across the clinical samples (data not shown). PC analysis
was used to investigate the relationship of Fn14 expression across
all tumor samples and patient outcome. Two separate clusters
emerged in a three-dimensional scatter plot of PC1, PC2, and PC3
(Supplementary Data 2A). To discern any clinical relevance of the
clusters, Kaplan-Meier survival curves were developed for the two
clusters. Median survival time of cluster 1 was 952 days (long term),
whereas cluster 2 had a median survival of 401 days (short term;
Supplementary Data 2B). Specifically, analysis of the Affymetrix
expression value for Fn14 in the GBM specimens for each cluster
showed that patients with GBM in the short-term survival cluster
had higher expression of Fn14 (11.6) than GBM patients in the
long-term survival cluster (3.6; P < 0.001; Fig. 6B). These data
suggests that high Fn14 expression levels correlate with poor
patient outcome.
Figure 4. TWEAK regulation of Fn14
expression is dependent on Rac1 and NF-nB
activity. T98G and U118 glioma cells were
treated with TWEAK for the indicated times
and total RNA and protein were isolated.
A, Fn14 and histone H3.3 mRNA levels were
analyzed by QRT-PCR. B, Fn14 and
a-tubulin proteins were analyzed by Western
blot. C, T98G cells were transfected with
siRNA targeting luciferase or siRNA
targeting Rac1 and cultured for 24 hours.
Cells were treated with or without TWEAK
and cultured for an additional 6 hours.
Cell lysates were collected and analyzed
using anti-Rac1, anti-Fn14, and anti-a-tubulin
antibodies. D and E, T98G cells were
infected with adenoviruses expressing
control LacZ, superrepressor InBaM,
dominant-negative Rac1N17, or
constitutively active Rac1V12. Six hours
after TWEAK addition, total RNA and protein
were isolated. Fn14 and histone H3.3 mRNA
levels were analyzed by QRT-PCR (D),
and Fn14 and a-tubulin protein levels were
analyzed by Western blotting (E). Columns,
mean of three independent experiments.
7http://www.ensembl.org.
8http://motif.genome.jp.
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Immunohistochemical validation of Fn14 protein expres-
sion using a brain tumor tissue microarray. We next examined
Fn14 protein expression in a series of brain tumor specimens
assembled on a brain tissue microarray containing 9 control cases
and 62 tumor cases. Examination of Fn14 levels in control non-
neoplastic brain specimens from epileptogenic patients (Fig. 6C,
a and d) showed 66.7% negative staining and 33.3% weak staining
(Supplementary Data 3), mainly found in normal vascular
endothelium and some reactive astrocytes. In contrast, the
majority of oligoastrocytomas showed negative to weak expression
and 18.2% showed moderate staining (Supplementary Data 3).
Similarly, in anaplastic astrocytoma, the majority of the cases
showed weak (66.7%) to moderate (33.3%) expression of Fn14. High
levels of Fn14 expression were observed in medulloblastoma cases,
with 57% moderate and 42% strong positive staining. Similarly, of
the 27 GBM cases, 1 tumor was negative (3.7%), 3 were weakly
positive (11.1%), 20 were moderately positive (74.1%), and 3 were
strongly positive (11.1%). Fn14 immunoreactivity was observed in
both glioblastoma tumor core (Fig. 6C, b and e; arrowhead) and
invading cells (Fig. 6C; e and f, arrow). Tumor vascular
endothelium (Fig. 6C, f, V) and normal endothelium (data not
shown) had moderate staining for Fn14.
Discussion
This study shows that Fn14 signaling promotes glioma cell
invasion and the small GTPase Rac1 is a key mediator of Fn14-
induced cell migration and invasion. We also found that TWEAK
binding to Fn14 stimulates transcription of Fn14 through a Rac1/
NF-nB–dependent mechanism. Indeed, NF-nB binds to the highly
conserved NF-nB consensus site upstream of the Fn14 transcrip-
tional start site. A model of Fn14-mediated self-regulation is shown
in Supplementary Data 4.
Elevated NF-nB activity is observed in various cancers including
GBM (30, 31). NF-nB activation may contribute to cellular resistance
to cytotoxic interventions and promote cell invasion by
up-regulating genes involved in cell survival (i.e., BCL-XL, A1), cell-
cell adhesion (i.e., intercellular adhesion molecule-1) and cell-
extracellular matrix (ECM) interactions (i.e., tenacin-C, laminin B2
chain; ref. 32). Our finding that NF-nB regulates Fn14 expression is
quite intriguing because we have also shown that Fn14 promotes
gliomacellsurvivalviatheNF-nBpathwaythatleadstoactivationof
BCL-XLand BCL-W (9). In glioma, genetic lesions that delete tumor
suppressor genes, such as phosphatase and tensin homologue
deleted on chromosome 10, oramplify oncogenes suchas epidermal
growth factor receptor, are likely to contribute to constitutive
activation ofNF-nB.Itseemstenablethatthetrigger forthispositive
promotion cycle begins with elevated NF-nB activity driven by these
well-described oncogenic changes that progressively lead to up-
regulated transcription of Fn14. Activation of Fn14 receptor, in turn,
signals to promote further activation of NF-nB and increased Fn14
levels,andconsequentlycellinvasionandrobustgliomacellsurvival.
In pilot studies, we note that inhibition of NF-nB activity by either
the superrepressor InBa mutant or the pharmacologic inhibitor
SN50suppressesFn14-inducedcellmigrationandinvasion(datanot
shown). We are currently probing the genetic signature of the Fn14-
NF-nBpathwayforpointsofvulnerabilityinitscontrolofgliomacell
invasion.
Rac1 is essential for various aspects of malignant transforma-
tion, including anchorage-independent growth, survival, invasion,
and metastasis (21, 33, 34). In glioma cells, depletion of Rac1
expression by siRNA oligonucleotides results in a decrease in cell
migration and invasion (33). Additionally, suppression of Rac1
activity via a dominant-negative form of Rac1 induces death in
glioma cell lines and primary GBM but not normal human adult
astrocytes (35). We report a critical role for Rac1 in Fn14-
stimulated migration and invasion of glioma cells. We show that
Fn14 activates Rac1 and inactivates RhoA. Rac1 can inhibit RhoA
activity (36), suggesting that the suppression of RhoA activity by
Fn14 signaling may be a consequence of Fn14-mediated activation
of Rac1. In glioma cells, Fn14 binds equally well to active and
inactive Rac1, which is in agreement with a previous study by
Figure 5. TWEAK stimulates IKKh and InBa
phosphorylation via Rac1, and NF-nB binds
to the Fn14 promoter. A, T98G cells were
treated with TWEAK for various time
periods and cell extracts were analyzed for
phospho-IKKh, total IKKh, phospho-InBa,
total InBa, and a-tubulin levels by Western
blotting. B, T98G cells were infected with
adenoviruses expressing Rac1N17 or
Rac1V12. Cells were then treated with
TWEAK, TWEAK preincubated with the
Fn14-Fc decoy receptor, or TWEAK
preincubated with mouse IgG. Cell lysates
were collected after 15-minute posttreatment
and analyzed for phospho-IKKh, total IKKh,
phospho-InBa, total InBa, and a-tubulin
levels by Western blotting. C, T98G cells
were treated with TWEAK, TWEAK
preincubated with the Fn14-Fc decoy
receptor, or TWEAK preincubated with
mouse IgG. Nuclear proteins were isolated 2
hoursposttreatmentandincubatedwithbiotin
end-labeled, wild-type Fn14 (Fn14wt)
oligonucleotides containing the NF-nB
consensusbinding region.Proteinswere also
incubated with biotin end-labeled Fn14
oligonucleotides containing mutated NF-nB
consensus binding region (Fn14mt). In
certain experiments, either 200-fold molar
excess of unlabeled NF-nB-Fn14wt
oligonucleotides or anti-p65 antibody was
incubated with the nuclear lysates from
TWEAK-treated cells.
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Tanabe et al. (37) using PC12 cells. Our immunoprecipitation
studies do not distinguish whether Rac1 associates with Fn14
directly or indirectly as a complex with other proteins. However,
the observation that Rac1 does not associate with the Fn14tCT
protein suggests either that Rac1 associates with Fn14 through
TRAF proteins or via signaling proteins that act downstream of
TRAF; the coimmunoprecipitation of Rac1 with Fn14 argues for the
former. A study by Min and Pober (38) showed that TRAF2
interacts with Rac1 to promote downstream JNK activation.
Additionally, yeast two-hybrid studies have shown that TRAF1,
TRAF2, TRAF3, and TRAF5 interact with Fn14 at the TRAF binding
region, which links the Fn14 receptor to transcription factors such
as NF-nB (7). Current studies are ongoing to explore the
mechanism of interaction between Fn14 and Rac1.
Although local dissemination of glioma cells is a principal
contributor to the poor clinical outcome for these patients, a
therapeutic approach targeting these invading glioma cells remains
elusive. The results of an extended investigation into the clinical
relevance of Fn14 point to Fn14 as an attractive candidate for such
targeted therapy. We report that the levels of expression of Fn14 in
glial tumors correlate with tumor grade; advanced GBM showed
the highest expression of Fn14 mRNA and protein. Examination of
Fn14 protein expression in situ portrays strong Fn14 staining in
cells at the site of active invasion compared with tumor core. This
notion of microregional changes in Fn14 expression driving
invasion is supported by the 4- to 6-fold increase in Fn14 mRNA
in invading cells collected by laser capture microdissection from
GBM specimens. This corroborates the in vivo immunohistochem-
istry observations. More compelling is the finding that Fn14
expression is an indicator of poor outcome, because GBM patients
in short-term survival expressed significantly increased Fn14 levels
compared with GBM patients with long-term survival (Fig. 6B).
Thus, the level of Fn14 expression may be a good predictor of
survival outcome, complimenting other prognostic indicators.
This study suggests that Fn14 may be a key driver of the
malignant invasive behavior for glial tumors. Fn14 activation
promotes dispersion of the tumor, whereas also engaging
biochemical mechanisms leading to enhanced cell survival. Our
discovery that Fn14 signaling includes the downstream mediators
Rac1 and NF-nB, whose activation by Fn14 subsequently triggers
transcription of Fn14, is among the first positive feedback signaling
events described for human malignancies, and offers a model for
Figure 6. Analysis of Fn14 expression in
various human glial tumors. A, expression
levels of Fn14 mRNA were mined in an
Affymetrix gene expression profile of 184
normal and brain tumor specimens. Box
plots for Fn14 expression for each tumor
type were graphed. Significance between
GBM and either nonneoplastic brain (NB),
oligoastrocytoma (OL), or anaplastic
astrocytoma (AA) was tested with a
two-sample t test assuming unequal
variances. B, expression values of Fn14
mRNA were analyzed in two clusters derived
from three-dimensional scatter plot of PC
analysis from the gene expression profiling
of normal and brain tumor specimens.
Kaplan-Meier survival curves were
developed for each cluster. Cluster one has
a survival mean of 401 days (short-term
survival) and cluster two has a survival mean
of 952 days (long-term survival). Box plots
for Fn14 expression in GBM specimens for
each cluster derived from PC analysis are
shown. Significance between the two
populations was tested with a two-sample
t test assuming unequal variances.
C, paraffin sections of invasive glioma tissue
microarray were immunostained with a
monoclonal antibody against Fn14. Top
(a, b, and c), ?10 magnification. Bar,
100 Am. Bottom (d, e, and f), ?20
magnification. Bar, 50 Am. a and d, control
tumor-free nonneoplastic brain. b and e,
a tissue punch from a GBM case containing
tumor core and invasive cells. Arrowheads in
e, Fn14 staining in the tumor core; arrow,
Fn14 staining in invading cells at the
adjacent tumor edge. c and f, tissue punches
obtained from GBM specimens at the far
invasive edge. Arrow in f, Fn14 staining in
invading glioma cells; V, Fn14 staining in
vessels.
Fn14 Promotes Glioma Invasion
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the biological basis of spontaneous malignant progression. We
argue that interference with Fn14 activation and its signaling are
rational therapeutic targets for glioma.
Acknowledgments
Received 2/1/2006; revised 6/9/2006; accepted 7/21/2006.
Grant support: NIH grants NS-42262 (M.E. Berens), HL-39727 (J.A. Winkles),
CA87567 (M. Symons), and CA103956 (J.C. Loftus); Ruth L. Kirschstein National
Research Service Award F32 CA112986-01 (N.L. Tran); and a Russell Becker-American
Brain Tumor Association grant (N.L. Tran).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Howard Fine for providing access to the gene expression profiles of
human nonneoplasmic and brain tumor specimens.
Cancer Research
Cancer Res 2006; 66: (19). October 1, 2006
9542
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