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The Journal of Neuroscience, April 1, 1998, 18(7):2370-2376
Expression of a Cleaved Brain-Specific Extracellular Matrix
Protein Mediates Glioma Cell Invasion In Vivo
Hong
Zhang,
Gail
Kelly,
Cynthia
Zerillo,
Diane M.
Jaworski, and
Susan
Hockfield
Section of Neurobiology, Yale University School of Medicine, New
Haven, Connecticut 06520-8001
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ABSTRACT |
Malignant gliomas (primary brain tumors) aggressively invade the
surrounding normal brain. This invasive ability is not demonstrated by
brain metastases of nonglial cancers. The brain-specific,
brain-enriched hyaluronan binding (BEHAB)/brevican gene, which encodes
an extracellular hyaluronan-binding protein, is consistently expressed
by human glioma and is not expressed by tumors of nonglial origin
(Jaworski et al., 1996 ). BEHAB/brevican can be cleaved into an
N-terminal fragment that contains a hyaluronan-binding domain (HABD)
and a C-terminal fragment (Yamada et al., 1995). Here, using antisera to peptides in the predicted N-terminal and C-terminal proteolytic fragments, we demonstrate that the BEHAB/brevican protein is cleaved in
invasive human and rodent gliomas. A role for this protein in glioma
cell invasion was tested by transfecting a noninvasive cell line with
the BEHAB/brevican gene. The noninvasive 9L glioma cell was transfected
with either full-length BEHAB/brevican or the HABD and tested for
invasion in in vitro and in vivo invasion assays. Although both constructs increased invasion in
vitro, only the HABD increased invasion by tumors growing
in vivo. Experimental intracranial tumors from
full-length transfectants showed no increase in invasion over control
tumors, whereas tumors from HABD transfectants showed a marked
potentiation of tumor invasion, producing new tumor foci at sites
distant from the main tumor mass. This work demonstrates a role for a
brain-specific extracellular matrix protein in glioma invasion, opening
new therapeutic avenues for a uniformly fatal disease.
Key words:
glioma; brain tumor; astrocytoma; tumorogenesis; motility; proteoglycan; BEHAB; brevican; invasion; extracellular
matrix
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INTRODUCTION |
Primary tumors of the CNS (gliomas)
are notoriously difficult to control, attributable in large measure to
their highly invasive behavior. The ability to invade into the
surrounding normal brain is essentially a property unique to primary
brain tumor cells; nonglial tumor cells that metastasize to brain, such
as those originating from primary tumors elsewhere (e.g., breast or
lung), grow as circumscribed masses with well-defined borders. The
composition of the extracellular matrix may be a critical factor in
determining the invasive potential of cancer cells, such that the
production of matrix elements by glioma cells might mediate their
invasion into normal tissue.
The composition of the extracellular matrix of the brain changes over
the course of development (Herndon and Lander, 1990; Hockfield, 1990;
Sheppard et al., 1991). Behaviors characteristic of cells in the
developing brain, such as cell proliferation and migration, neuronal
and glial process outgrowth, and the elaboration of the capillary
network, take place in a soluble matrix that is permissive for cell
movement. In contrast, there is little cell motility in the normal
mature brain. The matrix of the mature brain is relatively insoluble
compared with that of the immature brain and can stabilize mature
cell-cell relationships (Hockfield et al., 1990). A return to an
immature, more soluble matrix during tumor growth could facilitate
tumor cell motility and angiogenesis.
We recently cloned the gene for a brain-specific extracellular matrix
protein, BEHAB (brain-enriched hyaluronan binding) (Jaworski et al.,
1994), which was independently cloned in another laboratory and named
brevican (Yamada et al., 1994). BEHAB/brevican encodes a member of the
proteoglycan tandem-repeat family of proteins, and its predicted
product is a secreted protein with a hyaluronan-binding domain (HABD)
(Jaworski et al., 1994; Yamada et al., 1994). A GPI-anchored isoform of
this protein has also been reported (Seidenbecher et al., 1995). The
BEHAB/brevican gene is expressed at high levels during the period when
glial cells, which give rise to primary brain tumors, are first born
(Jaworski et al., 1995). BEHAB/brevican is also expressed in tumors
derived from glial cells (HABD) (Jaworski et al., 1996). In every one
of over 40 surgical samples of human glioma assayed to date, including
oligodendroglioma, all grades of astrocytoma, and gliosarcoma,
BEHAB/brevican mRNA is detected. By contrast, BEHAB/brevican is
undetectable in tumors that are not of glial origin, including CNS
lymphoma, meningioma, and carcinomas of the lung, colon, and breast, in
either primary locations or as metastases to the brain. Importantly,
BEHAB/brevican is expressed at very low levels in the normal adult
human brain and in brain samples from individuals with noncancer
neuropathologies (Jaworski et al., 1996). In rodent brain tumor models,
glioma cell lines that reproduce the invasive behavior characteristic
of human glioma express BEHAB/brevican mRNA, whereas glioma cell lines
that grow noninvasively do not express it (Jaworski et al., 1996). The
expression in invasive tumors, together with the predicted structure of
the protein, suggests that BEHAB/brevican plays a role in glioma cell invasion.
Here, BEHAB/brevican antibodies have been used to study the expression
of the protein in invasive human and rodent brain tumors. We show that
in these tumors, BEHAB/brevican is cleaved into an N-terminal fragment
containing the hyaluronan-binding domain (HABD) and a C-terminal
fragment. To test for a role for BEHAB/brevican in tumor invasion, we
transfected a noninvasive glioma cell line with either the full-length
BEHAB/brevican gene or with a construct encoding the N-terminal HABD.
Although both constructs increase invasion in vitro, in both
a Matrigel invasion assay and a motility assay, only the HABD fragment
increases the invasion of experimental tumors in vivo. These
results offer an explanation for the unusual invasive ability of brain
tumor cells and identify new therapeutic targets for a uniformly fatal
disease.
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MATERIALS AND METHODS |
Cell transfections. 9L gliosarcoma cells were
transfected either with a full-length cDNA encoding the secreted form
of rat BEHAB/brevican (generously provided by Dr. Yu Yamaguchi, Burnham Institute) (Yamada et al., 1995) or with a 1.1 kb cDNA (nucleotides 60-1172 of the full-length clone) encoding the HABD of BEHAB/brevican by either calcium phosphate coprecipitation or electroporation. The
cDNAs were cloned into the EcoRI site of the eukaryotic
expression vector pCDNA3 (Invitrogen, San Diego, CA). Transfectants
were selected in DMEM with 10% fetal bovine serum (FBS) and 1 mg/ml G418. After 10 to 14 d, G418-resistant colonies were isolated and
assayed for BEHAB/brevican or HABD expression by RNase protection and
Northern blot. Transfectants expressing an appropriately sized mRNA
that hybridized with a BEHAB/brevican-specific probe were subcloned and
used for further studies. As a control, 9L cells were transfected with
the pCDNA3 vector containing a cDNA insert encoding green fluorescent
protein (GFP) (generously provided by Dr. Thom Hughes, Yale
University). Stable transfectants were maintained in DMEM with 10% FBS
and 500 µg/ml G418. The rat CNS-1 glioma cell line was maintained in
RPMI with 10% FBS.
Western blot analysis. Five milliliters of OPTI-MEM (Life
Technologies) with 1% FBS were added to cultures when cells reached 80% confluence on 100 mm culture plates. After 48 hr, conditioned medium was collected, and cell debris was removed by centrifugation. For cell homogenates, cultures were rinsed in Dulbecco's PBS (DPBS; Life Technologies) with a cocktail of protease inhibitors (Boehringer Mannheim, Indianapolis, IN), and cells were collected by scraping. For
tumor samples, tissues were homogenized in DPBS with protease inhibitors. Samples were electrophoresed on either 8 or 10%
SDS-polyacrylamide gels, and proteins were then electrophoretically
transferred to nitrocellulose. Blots were incubated with specific
rabbit primary antisera (see below), followed by alkaline
phosphatase-conjugated goat anti-rabbit IgG secondary antibodies
(Promega, Madison, WI). Immunoreactive bands were visualized with nitro
blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma, St.
Louis, MO).
Matrigel invasion assay. To study the invasive ability of
tumor cells, we performed an in vitro Matrigel assay
(Mohanam et al., 1993). Briefly, 100 µl of a Matrigel solution
(Collaborative Research, Bedford, MA; 1 mg/ml in DMEM) was placed on a
Transwell insert (Costar, Cambridge, MA; 12 mm, 8 µm pore size) and
allowed to gel at 37°C for 30-40 min. Tumor cells were suspended in
medium (100 cells/µl of DMEM with 10% FBS), and 100 µl aliquots
were added to each Matrigel-coated Transwell insert. The lower chamber of the Transwell was filled with 500 µl of DMEM with 10% FBS to which either fibronectin (5 µg/ml) or hyaluronan (HA) (200 µg/ml) was added as a chemoattractant. Either 18 or 6 hr later, cultures were
fixed in acid alcohol and stained with Coomassie blue (0.1% in 50%
methanol with 7.5% acetic acid). Cells on the upper side of the insert
membrane were removed with a cotton swab, and the number of cells that
had migrated to the lower side of the membrane was counted. For each
membrane, eight random fields were selected, and the number of cells
was counted on an inverted microscope using a 20× objective lens.
Transwell motility assay. The assay was performed
essentially as described for the Matrigel assay (above), with the
exception that the Transwell insert was uncoated. Tumor cells (10,000 cells/well) were applied to the Transwell membrane, and the lower
chamber was filled with medium (DMEM with 10% FBS, supplemented with
HA at 200 µg/ml). Six hours later, cultures were processed and
analyzed as described above for the Matrigel assay.
Antibodies. Rabbit antisera to BEHAB/brevican were generated
to a peptide in the HABD (amino acids 253-279,
DLNGELFLGAPPGKLTWEEARDYCLER) or to a peptide in the C-terminal fragment
(amino acids 506-529, SPSPRPPRVHGPPAETLQPPREGS). Antisera were
affinity-purified, and specific immunoreactivity was confirmed by
blocking with specific peptides.
Intracranial grafts. Intracranial grafts were performed as
described previously (Jaworski et al., 1996). Briefly, cell suspensions were prepared in complete PBS (PBS supplemented with 1 µg/ml
MgCl2 and CaCl2 and 0.1% glucose) at 1-5 × 104 cells/µl. Using a Hamilton syringe, we
injected stereotaxically 3 µl of the cell suspension over a 4-5 min
period into the thalamus of a postnatal day 45 rat (Lewis for CNS-1
cells; Fischer 344 for 9L-transfected cell lines). Ten to fifteen days
after the injection, the rats were killed, and the brains were quickly
frozen on dry ice. Each brain was sectioned at 20 µm onto
gelatin-subbed slides, and the sections were stained with cresyl violet
to visualize tumor cells. Sections were also stained with an antibody
to nestin (monoclonal antibody Rat-401) (Hockfield and McKay, 1985)
that recognizes glioma cells. An identical distribution of tumor cells was seen in sections stained with either cresyl violet or Rat-401. Images of random sections through each tumor were captured on a
computer. Using the National Institutes of Health Image program, we
determined the border of the tumor with the underlying thalamus, and
the number of cell clusters at distances of 0.5-1 mm and over 1 mm
from the tumor border was counted in each section. The statistical analyses incorporated one random section from each of several independent tumors (n = 6 independent tumors for
9L-GFP; n = 12 for 9L-BEHAB/brevican; and
n = 14 for 9L-HABD).
Statistical analyses. All statistical analyses were
performed using the Student's t test; the level of
significance was set at p < 0.01.
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RESULTS |
To begin our studies to determine whether BEHAB/brevican might
play a role in tumor invasion, we assayed protein expression in rodent
and human brain tumors. Experimental brain tumors established from the
rat CNS-1 and 9L glioma cell lines, when implanted as intracranial
grafts in syngeneic hosts, show two different patterns of growth
(Jaworski et al., 1996). Intracranial grafts of the 9L gliosarcoma line
display properties characteristic of brain metastases of peripheral
tumors, in that they do not express BEHAB/brevican mRNA and they grow
as well-defined cell masses that do not invade the surrounding brain
tissue. In marked contrast, intracranial grafts of the CNS-1 glioma
cell line display properties characteristic of human glioma, in that
they express BEHAB/brevican mRNA and grow invasively. To study the
protein product of the BEHAB/brevican mRNA, we examined BEHAB/brevican
protein expression in experimental rodent brain tumors. Tumors
established from the 9L cell line do not express BEHAB/brevican
protein. In contrast, Western blots of brain tumors established from
CNS-1 cells show three major immunoreactive bands (Fig.
1A). The 140 kDa band
represents full-length BEHAB/brevican, and the 90 and 50 kDa bands
represent C- and N-terminal cleavage products, respectively, from a
predicted, conserved proteolytic site (Yamada et al., 1995). The 140 and 90 kDa forms correspond to the 140 and 80 kDa bands reported
previously (Seidenbecher et al., 1995; Yamada et al., 1995). Surgical
samples of human gliomas were also analyzed by Western blotting, and
these, like the invasive rodent tumors, showed both full-length and
cleaved protein products of the BEHAB/brevican gene (Fig.
1B) that were of slightly greater apparent molecular
mass.
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Figure 1.
Expression of BEHAB/brevican in rodent and human
brain tumors. A, Western blots of invasive rodent brain
tumors established from CNS-1 cells show full-length 140 kDa
BEHAB/brevican, as well as 90 and 50 kDa proteolytic fragments.
Full-length and cleaved 90 kDa bands are visualized with an antibody to
a peptide in the C-terminal portion of BEHAB/brevican; the 50 kDa band
is visualized with an antibody to a peptide in the N-terminal portion
of BEHAB/brevican (see Materials and Methods). B,
Western blots of surgical samples from neuropathologically diagnosed
glioblastoma multiforme show 150 kDa full-length BEHAB/brevican and a
97 kDa proteolytic fragment. These bands are immunoreactive with the
antibody to the C-terminal peptide; however, the antibody to the
N-terminal peptide does not recognize the human protein.
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To test whether BEHAB/brevican mediates invasion, we established
stable transfectants of the noninvasive 9L gliosarcoma cell line. One
set of transfectants, 9L-BEHAB/brevican, carried a construct encoding
full-length BEHAB/brevican (amino acids 1-883), and a second set,
9L-HABD, carried a construct encoding the N-terminal HABD (amino acids
1-371), which corresponds approximately to the 50 kDa cleavage
product. 9L cells transfected with the gene for the GFP served as a
control (9L-GFP). Expression of appropriate mRNA was demonstrated by
RNase protection and Northern blot analyses (data not shown). Western
blot analysis confirmed the expression of the predicted proteins (Fig.
2). 9L-GFP transfectants showed no
BEHAB/brevican immunoreactivity, whereas cell homogenates of 9L-BEHAB/brevican and 9L-HABD transfectants exhibited a 140 kDa- and a
50 kDa-immunoreactive species, respectively. Both 9L-BEHAB/brevican and
9L-HABD transfectants secreted the encoded proteins into the medium in
which they were maintained (Fig. 2, lanes 5, 6). The secreted proteins were more disperse and slightly larger in apparent molecular mass, most likely because of glycosylation. Immunoreactivity on Western blots of both 9L-BEHAB/brevican and 9L-HABD transfectants was blocked by specific, but not by irrelevant, peptides (data not
shown).
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Figure 2.
Transfected 9L cells express full-length
BEHAB/brevican or the HABD. Western blots of cell homogenates
(lanes 1-3) and conditioned media (lanes
4-6) are from transfected cells. 9L-GFP cell
homogenates (lane 1) and conditioned media (lane
4) show no immunoreactivity for BEHAB/brevican or its
cleavage products. 9L-BEHAB/brevican cell homogenates (lane
2) and conditioned media (lane 5) contain a 140 kDa-immunoreactive species. 9L-HABD cell homogenates (lane 3) and conditioned media (lane 6) contain
a 50 kDa species. The immunoreactive species in conditioned media from
both 9L-BEHAB/brevican and 9L-HABD transfectants are polydisperse
(lanes 5, 6), possibly reflecting
glycosylation.
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Transfected cells were first tested for invasive ability using the
Matrigel in vitro invasion assay (Albini et al., 1987; Merzak et al., 1994). Eighteen hours after plating onto Matrigel-coated Transwell inserts, the number of cells that had migrated through the
Matrigel to the bottom of the filter was counted (Fig.
3A). Two independent lines
from each construct were assayed. 9L-GFP transfectants showed little
migration under any condition tested. Both 9L-BEHAB/brevican and
9L-HABD transfectants showed a marked increase in invasive ability over
that seen with the control 9L-GFP cells. There was no statistically
significant difference between the invasive capacity of
9L-BEHAB/brevican and 9L-HABD transfectants. Invasion through Matrigel
was assayed using both fibronectin and hyaluronan as attractants in the
lower chamber of the Transwell apparatus, and there was no difference
in the response of either cell type to the different attractants. When
the Matrigel assay was performed using a 6 hr incubation period,
similar results were found; 9L-BEHAB/brevican and 9L-HABD cells showed
equivalent increases in invasiveness over 9L-GFP cells (data not
shown).
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Figure 3.
Full-length BEHAB/brevican and the HABD
increase invasion and motility of 9L cells in vitro.
A, Invasion in the Matrigel invasion assay is
potentiated by the expression of either full-length
BEHAB/brevican or the HABD. Two independent cell lines for
each of the constructs (B/b-1 and B/b-2
for 9L-BEHAB/brevican; HABD-1 and HABD-2
for 9L-HABD) were assayed for invasion in the Matrigel invasion assay (see Materials and Methods). Invasion is expressed relative to that
observed for the 9L-GFP transfectants. Both constructs markedly increased invasion. Invasion was equivalent when either fibronectin or
hyaluronan was the attractant in the lower chamber. B,
Motility in the absence of Matrigel is also increased by the expression of either full-length BEHAB/brevican or the HABD. As seen in the Matrigel assay, both constructs similarly increased motility in a
Matrigel-independent assay. The degree of invasion or motility seen for
all four transfected cell lines was statistically different from 9L-GFP
cells at the p < 0.01 level. There was not a
statistically significant difference between the behavior of
9LBEHAB/brevican versus 9L-HABD transfectants. Error bar indicates
SEM.
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To eliminate the possibility that the full-length BEHAB/brevican
was cleaved by proteases in the Matrigel, two experiments were
performed. First, heat-inactivated Matrigel (56°; 1 hr) was used for
the invasion assay, which gave identical results to those described
above. Second, another motility assay was performed that did not
require Matrigel. We performed a standard cell motility assay using a
Transwell apparatus (Koochekpour et al., 1995); cells were plated onto
the Transwell inserts without any Matrigel coating. Six hours later,
the number of cells that migrated to the lower side of the insert
membrane was counted. In the absence of Matrigel, both
9L-BEHAB/brevican and 9L-HABD cells showed a marked increase in
motility over 9L-GFP cells (Fig. 3B). As seen in the
Matrigel invasion assay, there was not a significant difference between
the motility of cells expressing either the full-length or the HABD
protein.
Matrigel provides a reproducible, uniform matrix; however, its
composition is quite different from that of the extracellular matrix of
brain. Matrigel and the extracellular matrix of most tissues contain
collagen, laminin, and fibronectin, constituents that are lacking or
present at extremely low abundance in the extracellular matrix of the
adult brain (Lander and Hockfield, 1998). Therefore, cell migration
through Matrigel may not accurately reflect the ability of cells to
invade within the matrix of the brain. To test the ability of
BEHAB/brevican to mediate invasion in situ, we used an
in vivo brain tumor invasion model. Transfected 9L cells
were injected into the diencephalon of adult rats. Ten to 15 d
after injection, the brains were sectioned to study tumor growth and
the movement of tumor cells away from the main tumor mass into the
underlying thalamus (Fig. 4). As in the
in vitro assay, two independent lines for each transfectant
were tested for invasion in vivo.
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Figure 4.
Intracranial tumors established
from 9L-HABD, but not from 9L-BEHAB/brevican, cells show increased
invasion into the surrounding brain. A,
A', Intracranial tumors (asterisks)
derived from 9L-GFP cells grow as compact cell masses, with little
infiltration into the surrounding brain. The border with the underlying
thalamus (A') is smooth, with very few clusters of cells
seen beyond the border between the tumor and the normal brain.
B, B', Intracranial tumors
(asterisks) derived from 9L-BEHAB/brevican cells showed identical behaviors to those of the control transfectants. These tumors
also grew as compact cell masses, with little infiltration of the
surrounding brain. Here again, very few cell clusters were observed in
the normal brain adjacent to the tumor (B').
C, C', Intracranial tumors
(asterisks) derived from 9L-HABD cells showed a marked
increase in invasive ability compared with the other two cell lines.
Although the main tumor mass was a compactgroup of cells, many cell
clusters were seen in the surrounding normal brain. The border
of the 9L-HABD tumors with the underlying thalamus (C')
was often interrupted by peninsulas of cells extending out from the
main tumor mass.
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Intracranial grafts of 9L-GFP transfectants (n = 6)
showed the same histological pattern reported previously for parental, untransfected 9L cells (Jaworski et al., 1996). That is, they grew as
highly compact cell masses, with relatively smooth borders (Fig.
4A,A'). Very few clusters of 9L-GFP
cells were found distal to the boundary between the tumor and the
underlying thalamus (Fig. 4A', Table
1). In all of the sections from 9L-GFP
tumors assayed, only two cell clusters were observed over 1 mm from the tumor border. The behavior of tumors resulting from
9LBEHAB/brevican transfectants (n = 12) was
indistinguishable from that of tumors resulting from 9L-GFP
transfectants. The 9L-BEHAB/brevican tumors also grew as compact cell
masses, with few cell clusters located outside of the main tumor mass
(Fig. 4B,B'). Of the few clusters of cells that were observed beyond the border of the tumor with the
underlying thalamus, almost all were located 0.5-1 mm from the tumor
border (Table 1). Western blots of 9L-BEHAB/brevican tumors detected
only the 140 kDa full-length protein (Fig.
5, lane 1). Therefore,
although the expression of full-length BEHAB/brevican can increase the
migration of 9L cells in an in vitro invasion assay, when
tested in vivo, expression of the full-length protein does
not confer on 9L cells the ability to migrate through normal adult
brain matrix.
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Figure 5.
Intracranial tumors established from
9L-BEHAB/brevican and 9L-HABD cells express full-length or HABD
proteins. Western blots of tumors produced after intracranial injection
of 9L-BEHAB/brevican (lane 1) or HABD (lane
2) transfectants are shown. In 9L-BEHAB/brevican tumors
(lane 1), immunoreactivity for BEHAB/brevican is at 140 kDa. No evidence of a cleavage product is seen. In 9L-HABD tumors (lane 2), only a 50 kDa product is observed.
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The lack of invasion by 9L-BEHAB/brevican transfectants in
vivo, along with the demonstration of proteolytic cleavage
products in human gliomas and invasive rat brain tumors, led us to test whether the HABD of BEHAB/brevican might mediate invasion in
vivo. The behavior of 9L-HABD transfectants (n = 14) was markedly different from that of 9L-GFP and
9L-BEHAB/brevican transfectants (Fig. 4C,C').
In 9L-HABD experimental tumors, the border between the tumor and the
underlying thalamus was interrupted by peninsulas of cells extending
from the main tumor mass (Fig. 4C'). In addition, there were
numerous cell clusters 0.5-1.0 mm from the tumor border, with many
cell clusters located over 1 mm from the main tumor mass (Table 1).
Western blots of 9L-HABD tumor samples showed the presence of the
50-55 kDa HABD product (Fig. 5, lane 2). Therefore, expression of the HABD of BEHAB/brevican can increase the ability of 9L
cells to migrate through the brain matrix in vivo.
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DISCUSSION |
We show here that, within the brain, invasive ability can be
conferred on a noninvasive glioma cell line by expression of an
N-terminal fragment of the BEHAB/brevican protein but not by expression
of the full-length protein. Because partial proteolytic cleavage of the
BEHAB/brevican protein occurs endogenously in both human and rodent
invasive brain tumors, these results provide an explanation for glioma
cell motility in the adult human brain. In addition, they suggest new
therapeutic possibilities for human brain tumor.
Many studies have suggested a central role for extracellular matrix
proteins in tumor growth and motility (Paulus et al., 1996; Haugland et
al., 1997; Merzak and Pilkington, 1997). HA and HA-binding proteins
have been implicated in tumor metastasis (Knudson and Knudson, 1993;
Entwistle et al., 1996). For example, the HA-binding protein RHAMM
increases the metastatic potential of fibroblast cell lines (Hall et
al., 1995). Our present results provide evidence that expression of the
extracellular, HA-binding, BEHAB/brevican protein by invasive rodent
and human brain tumors can increase the ability of glioma cells to
invade into the surrounding normal brain.
Although both 9L-BEHAB/brevican and 9L-HABD cells showed an equivalent
increase in invasive ability in the in vitro Matrigel invasion assay compared with control cells, only the 9L-HABD cells showed an increase in invasion when grown as intracranial grafts. Therefore, an increase in invasive ability observed in the Matrigel assay did not necessarily predict an increase in invasive ability in
the brain. One explanation for the invasive behavior of
9L-BEHAB/brevican cells in Matrigel, but not in the brain, is that
glioma invasion requires proteolysis of the BEHAB/brevican protein, and
the Matrigel might contain the necessary proteases. Our results
indicate that proteolysis is not likely to be responsible for the
migration of 9L-BEHAB/brevican cells through Matrigel. First, heat
inactivation of the Matrigel, which should inactivate protease
activity, did not alter the invasive ability of 9L-BEHAB/brevican
transfectants. Second, 9L-BEHAB/brevican cells also migrated in a
Matrigel-free motility assay. Therefore, it is unlikely that cleavage
of the protein explains the motility of the 9L-BEHAB/brevican
transfectants in the in vitro assays. A more likely
explanation is that, whereas the full-length BEHAB/brevican protein can
increase invasion and motility on extracellular matrix elements of
non-neural origin, it does not increase invasion or motility on the
extracellular matrix of the brain. This result suggests that caution be
exercised in the interpretation of results from in vitro
assays, because the behavior of cells in in vitro assays may
not accurately predict the behavior of cells in vivo. A
similar discrepancy has been reported for other tumor cell types, in
which increased invasiveness in a Matrigel assay was not paralleled by
increased invasiveness in vivo (Noel et al., 1991). Invasion
through normal brain tissue may be particularly difficult to model
in vitro because of the unusual and heterogeneous
composition of the extracellular matrix of the brain (Lander et al.,
1997).
In the normal adult human brain, the level of BEHAB/brevican expression
is extremely low. In the normal adult rat brain, however, BEHAB/brevican is expressed at easily detectable levels (Jaworski et
al., 1994; Yamada et al., 1994; Yamaguchi, 1996). It is important to
note that, although cells in the rat brain surrounding an invasive tumor express BEHAB/brevican, the level of expression by invasive tumors (such as those derived from either C6 or CNS-1 glioma cells) is
markedly higher than that in the surrounding normal brain (Jaworski et
al., 1996). Therefore, the level of cleaved BEHAB/brevican in the
surrounding brain may not be sufficient to permit invasion by the
9L-GFP or full-length 9L-BEHAB/brevican transfectants. Alternatively,
invasion may require expression of BEHAB/brevican by the tumor cells
themselves, perhaps involving an interaction of the cleavage product(s)
with the glioma cell surface (Yamada et al., 1997). Although expression
of an N-terminal HABD fragment of BEHAB/brevican clearly increases
the invasive ability of 9L cells, the pattern of invasion of 9L-HABD
transfectants does not fully mimic that of the highly invasive CNS-1
and C6 cell lines (Jaworski et al., 1996), suggesting that
additional molecules may act to facilitate glioma invasion further.
The BEHAB/brevican gene is expressed as two isoforms, one secreted and
one GPI-linked (Seidenbecher et al., 1995). In the normal rat brain
(Seidenbecher et al., 1995) and in human brain tumors (J. Gaw, V. Chang, and S. Hockfield, unpublished observations), the GPI-linked form
is expressed at very low levels compared with the secreted form. The
role, if any, of the GPI-linked form in tumor invasion is currently
under investigation. The most important modification of the
BEHAB/brevican protein product for cell invasion appears to be cleavage
into the N-terminal fragment containing an HABD and the C-terminal
fragment. A predicted site for proteolytic cleavage of
BEHAB/brevican has been mapped and is highly conserved with a
proteolytic site of another ECM protein, aggrecan (Yamada et al., 1995;
Yamaguchi, 1996). The observations that BEHAB/brevican is cleaved in
invasive human and rodent tumors and that full-length BEHAB/brevican
does not mediate invasion in vivo together indicate that
proteolytic cleavage is required for invasion. Moreover, cleavage of
the protein appears to require an association of the protease activity
with the cell that expresses the protein, because the protein produced
by 9L-full-length transfectants remains uncleaved (Fig. 5, lane
1), whereas the endogenous BEHAB/brevican expressed in the
surrounding brain is cleaved.
The results presented here are consistent with a two-component
mechanism for brain tumor cell invasion: first, an upregulation in the
expression of BEHAB/brevican by glioma cells, followed by the cleavage
of the protein into N-terminal HABD and C-terminal fragments. Earlier
work from our laboratory indicates that expression of BEHAB/brevican by
glioma cells is regulated by a brain-derived factor (Jaworski et al.,
1996). When maintained under standard cell culture conditions or when
grown as subcutaneous grafts outside of the brain, none of the 19 glioma cell lines we have tested expresses BEHAB/brevican. When glioma
cell lines are grown as intracranial grafts, however, cell lines that
grow with an invasive phenotype characteristic of human glioma are
induced to express BEHAB/brevican. Cell lines that grow as noninvasive
intracranial tumors do not express the gene. The identity of the
BEHAB/brevican-inducing factor has yet to be determined but appears to
be a soluble, brain-specific factor. In addition, the results presented
here provide evidence of a second required component, a protease that
cleaves BEHAB/brevican, the identity of which we are currently
pursuing. Candidate proteases include the matrix metalloproteinases
expressed in gliomas (Merzak and Pilkington, 1997), for which
BEHAB/brevican is a possible substrate. Regulated cleavage of
extracellular matrix proteins may be a general mechanism for the
control of cell motility; recent studies have demonstrated that
cleavage of laminin-5 is required for breast epithelial cell migration
(Giannelli et al., 1997) and that inhibition of matrix
metalloproteinase activity during embryogenesis blocks myoblast
migration into the tongue (Chin and Werb, 1997).
The aggressive invasion by malignant glioma into the surrounding normal
brain makes brain tumors highly refractory to regional therapies, such
as surgery or focal irradiation. BEHAB/brevican is expressed with
unprecedented specificity in glioma and presents a novel potential
target for the treatment of malignant primary brain tumor. Here, we
have shown that invasion by glioma cells into normal brain is
potentiated by a cleavage product of BEHAB/brevican. In addition to
targeting the BEHAB/brevican gene or its products, the present work
suggests another therapeutic target, the protease responsible for the
cleavage of BEHAB/brevican. BEHAB/brevican is brain-specific and is
expressed at almost undetectable levels in the normal adult human
brain; targeted disruption of either the expression or the proteolytic
cleavage of BEHAB/brevican would be predicted to have minimal
deleterious consequences on normal brain tissue. Functional inhibition
of BEHAB/brevican might slow the migration of tumor cells from the
original tumor site(s), thereby increasing the efficacy of regional
therapies.
| |
FOOTNOTES |
Received Nov. 10, 1997; revised Jan 13, 1998; accepted Jan. 15, 1998.
This work was supported by National Institutes of Health Grants EY06511
and NS35228 to S.H. We thank our colleagues for their generosity in
supplying reagents that were central to this work: Dr. William Hickey
for the CNS-1 cell line, Dr. Fred Gage for the 9L gliosarcoma cell
line, Dr. Thom Hughes for the GFP construct, and Dr. Yu Yamaguchi for
the full-length BEHAB/brevican construct. We also thank Drs. E. D. Gundelfinger and C. Seidenbecher for antisera to BEHAB/brevican, which
were used in preliminary experiments, and Drs. Sydney Gary and Cynthia
Lander for critically reviewing this manuscript. We also acknowledge
our ongoing collaboration in this work with Drs. Joseph Piepmeier,
Thomas Byrne, and Veronica Chang.
Correspondence should be addressed to Dr. Susan Hockfield, Section of
Neurobiology, Yale University, 333 Cedar Street, SHM C-405, New Haven,
CT 06510-8001.
Dr. Jaworski's present address: Department of Anatomy and
Neurobiology, University of Vermont School of Medicine, Burlington, VT
05405.
| |
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Top
Abstract
Introduction
Substance via MeSH
