From the Departments of Medicine, ¶ Surgery,
Pathology, ** Pharmacology and Cancer Biology, and
Radiation Oncology, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, November 11, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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We employed a genetically defined
human cancer model to investigate the contributions of two genes
up-regulated in several cancers to phenotypic changes associated with
late stages of tumorigenesis. Specifically, tumor cells expressing two
structurally unrelated bone-related genes, osteonectin and
osteoactivin, acquired a highly invasive phenotype when implanted
intracranially in immunocompromised mice. Mimicking a subset of
gliomas, tumor cells invaded brain along blood vessels and developed
altered vasculature at the brain-tumor interface, suggesting that
production of those two proteins by tumor cells may create a complex
relationship between invading tumor and vasculature co-opted during
tumor invasion. Interestingly, the same tumor cells formed massive
spontaneous metastases when implanted subcutaneously. This dramatic
alteration in tumor phenotype indicates that cellular microenvironment
plays an important role in defining the specific effects of those gene
products in tumor behavior. In vitro examination of tumor
cells expressing either osteonectin or osteoactivin revealed that there
was no impact on cellular growth or death but increased
invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic
inhibitors of MMP-2/9 and MMP-3 blocked the increased in
vitro invasion associated with osteoactivin expression, but only
MMP-3 inhibition altered the invasive in vitro
phenotype mediated by osteonectin. Results from this genetically
defined model system are supported by similar findings obtained from
several established tumor cell lines derived originally from human
patients. In sum, these results reveal that the expression of a single
bone-related gene can dramatically alter or modify tumor cell behavior
and may confer differential growth characteristics in different
microenvironments. Genetically defined human cancer models offer useful
tools in functional genomics to define the roles of specific
genes in late stages of carcinogenesis.
Gene expression analyses of human cancers have yielded tremendous
quantities of data. Unfortunately, the phenotypic consequences of many
changes in gene expression pattern between cancers and their
corresponding normal tissues are largely unclear. To address this
problem, we took a functional genomics approach by using a genetically
defined glioma model system to investigate genes involved
in the acquisition of malignant phenotype associated with late stages
of tumorigenesis. Particularly, we were interested in genes whose
expression is not associated with normal brain tissues or astrocyte
cultures but which are nevertheless overexpressed in gliomas. Among
those candidate genes, several bone-related genes were noticeably
overexpressed in a high proportion of gliomas. Of note, two
structurally unrelated genes, osteonectin and osteoactivin, have been
found previously to be overexpressed in several other types of cancers,
but their precise contribution to the development of specific cancer
phenotype has yet to be elucidated.
Osteonectin, also known as secreted protein, acidic and
rich in cysteine (SPARC) or BM-40, is a 43-kDa extracellular matrix protein. Osteonectin was originally discovered as one of the
most abundant non-collagenous components of bone, but it is also
expressed in a number of other cell types that are involved in active
remodeling of tissues (1). Thus, the primary physiological role of
osteonectin has been postulated to be an important modulator of
cell-extracellular matrix interactions during the processes of tissue
remodeling (2, 3). Osteonectin is also abnormally expressed in many cancers, including gliomas (4, 5), medulloblastomas (6), meningiomas
(7), and cancers of the gastrointestinal tract, breast, lung, kidney,
adrenal cortex, prostate, and bladder (3, 8-11). In gliomas,
osteonectin is expressed in all tumor grades, usually at the
tumor-brain margin and sites of neoangiogenesis (4), suggesting that
osteonectin expression may be involved in tumor cell invasion (14). In
contrast, increased osteonectin expression in other tumor types is
associated with a conversion to invasive and metastatic tumors and a
correlation in some instances with elevated expression of matrix
metalloproteinases (MMPs)1
that is linked to increased tumor malignancy (11-13). Importantly, reduced expression of osteonectin by an antisense approach was found to
correlate with a reduction in tumor formation by melanoma cells (15).
Thus, although the precise role of osteonectin in the pathological
process of carcinogenesis remains to be elucidated, osteonectin
overexpression is intimately correlated with the progression of
tumorigenesis of multiple types of human cancers.
Osteoactivin, also known as GPNMB or dendritic cell-associated, heparan
sulfate proteoglycan-integrin ligand (DC-HIL), is a type I
transmembrane glycoprotein that is localized to the cell surface and
lysosomal membranes (16), as well as in a secreted form (17). Highly
expressed in bone, the physiological function of osteoactivin is
postulated to be involved in the regulation of osteoblast maturation
(16). Interestingly, osteoactivin is found to be overexpressed in
melanomas (18), gliomas (19), and cancers of the breast, stomach, and
pancreas.2 Although the
precise role of osteoactivin in cancer development remains unknown, it
is tempting to suggest that the significant elevation of this
bone-related protein in gliomas has a role in brain tumor progression.
To determine the specific contributions of osteonectin and
osteoactivin expression to human cancer development, we employed a
genetically defined model system in which the genetic effects of
increased gene expression could be directly linked to phenotypic changes of tumorigenesis. In this system, transformed human astrocytes by the sequential introduction of the simian virus-40 large T antigen,
the human telomerase catalytic subunit (hTERT), and oncogenic Ha-Ras
display a phenotype mimicking that of low grade gliomas in the
formation of non-invasive tumor mass in immune-compromised animals
(20). The characteristics of this human tumor model system allowed us
to test specifically the phenotypic changes associated with tumor
progression derived from the expression of genes under investigation.
Consequently, we found that expression of osteonectin or osteoactivin
was associated with angiocentric intracranial invasion and increased
production of MMPs by the tumor cells. Furthermore, osteonectin and
osteoactivin expression caused the development of spontaneous
metastases systemically when the tumor cells were implanted
subcutaneously. Our results suggest that the expression of bone-related
genes in advanced human cancers may represent a novel mechanism by
which tumor cells acquire capabilities that are associated with the
phenotypic changes in late stages of tumorigenesis dependent on tumor
microenvironment. Moreover, the data strongly support the notion that
the genetically defined model of human cancers (20-23) offers a useful
tool for functional genomics in defining the pathological contributions of specific genes to late stages of tumorigenesis.
Generation of Cell Lines--
A genetically defined human
glioma cell line was generated as described previously (20). A 1.5-kb
cDNA fragment (a generous gift from Sandra Rempel, Henry Ford
Hospital) and a full-length cDNA fragment of GPNMB (a generous gift
from H. P. Bloemers, University of Nijmegen, The Netherlands) were
each cloned into a retroviral backbone with a bleo selection
marker. Cells underwent a positive selection with Zeocin (400 µg/ml, Invitrogen). U87MG, U251MG (American Type Culture Collection,
Manassas, VA), and D54MG (Duke University Medical Center) were infected
with a retrovirus expressing either a puromycin resistance gene or
osteonectin and puromycin resistance. Selection was undertaken with
puromycin (1 µg/ml). Early passage polyclonal cultures were used for
all experiments.
Western Analysis--
Cells were analyzed for the expression of
osteonectin, growth factor receptors, and MMPs by Western
analysis. A 10-cm plate was lysed, and 50 µg of total cellular
protein was used for each sample. Samples were subjected to SDS-PAGE
analysis and transferred to a PDVF membrane. The membrane was blocked
in Tris-buffered saline with 0.05% Tween 20 and 5% albumin. Primary
antibodies for anti-osteonectin (Hematologic Technologies, Essex
Junction, VT), anti-GPNMB (gift of Carol Wikstrand, Duke University),
anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-tubulin (Sigma), anti-MMP-9 (Calbiochem), anti-MMP-3
(Calbiochem), anti-MMP-2 (Calbiochem), anti-epidermal growth factor
receptor (EGFR) (gift of Carol Wikstrand, Duke University),
anti-platelet-derived growth factor receptor Growth Inhibition Assays--
Cells were plated into
12-well plates at a density of 2 × 104 cells per well
and labeled for the last 6 h with 4 µCi of
[3H]thymidine, fixed in 10% trichloroacetic acid, and
lysed in 0.2 N NaOH. [3H]thymidine incorporation into the
DNA was measured with a scintillation counter. Each measurement was
performed in triplicate.
Fluorescence-activated Cell Sorter Analysis--
Cells were
plated into 10-cm plates at a density of 5 × 105
cells per well, serum-starved overnight after attachment, and then fed
with media containing serum for 24 h. Cells were then trypsinized, fixed in 70% ethanol, washed once in phosphate buffered saline, and
resuspended in RNaseA (100 µg/ml) and propidium iodide (50 µg/ml).
Samples were analyzed on a FACScan (BD Biosciences) flow cytometer. Each experiment was performed in triplicate.
Soft Agar Assays--
For these assays, 35-mm plates were
prepared with a base layer of Dulbecco's minimal essential media with
10% calf serum (Invitrogen) and 0.6% bacto agar (BD Biosciences).
Cells were plated at a density of 5 × 104 cells per
plate in a mix of Dulbecco's minimal essential media with 10% calf
serum and 0.4% bacto agar. Plated cells were fed once a week with 0.5 ml of Dulbecco's minimal essential media plus 10% calf serum and
0.4% bacto agar. After 14 days, the plates were stained with 0.5 ml of
0.005% crystal violet. On each plate, colonies with more than 30 cells
were counted. Each measurement was performed in triplicate.
Tumor Formation Assays--
Intracranial tumor formation was
tested with SCID-beige mice injected with 1 × 106
glioma cells in 10 µl of Methocel (Dow Chemical Co., Midland, IL).
Mice were sacrificed when they developed any neurological abnormalities. Brains were serially sectioned and examined by histopathology. Immunohistochemistry was performed on all tumors with
hematoxylin and eosin, factor VIII, collagen IV, and Ki-67. Vascular
characteristics of tumors were compared through an examination of
vessel number and size in selected high power fields. Fields were
selected to represent maximal tumor diameters without significant areas
of necrosis. Internal organs were completely removed and evaluated
grossly for macroscopic metastases. Selected livers and lungs were
examined for microscopic metastases. SCID-beige mice (Taconic,
Germantown, NY) were subcutaneously injected in the flank with 10 × 106 glioma cells per mouse in 100 µl of Matrigel (BD
Biosciences) (20). Mice were regularly checked for tumor formation.
Tumor volume was calculated with the formula 0.5 × (length) × (width)2. Tumors were removed and examined by
immunohistopathology. Mice that were noted to have significant weight
loss were sacrificed regardless of primary tumor size. All internal
organs including the brain were removed at the time of sacrifice and
grossly examined for metastases. Selected lungs, livers, and brains
were sectioned and examined for microscopic metastases.
Matrigel Invasion Assays--
Kits were purchased from BD
Biosciences and used according to instructions. Briefly, 2.5 × 104 tumor cells were incubated in selected conditions
(serum-free media, 1% Me2SO control, 100 µM MMP-2 Inhibitor I (Calbiochem), 100 µM
MMP-2/9 Inhibitor I (Calbiochem), or 100 µM MMP-3
Inhibitor II (Calbiochem)) and then allowed to attach and invade. Cells were fixed 22 h after plating. The invasion was calculated as a
ratio of that shown by control (uncoated) inserts to that shown by
Matrigel-coated inserts. Experiments were performed in triplicate.
Statistical Analysis--
Wilcoxon rank sum test was used
in all analysis
Osteonectin and Osteoactivin Expression Induces Brain
Invasion Associated with Penetrating Vessels--
Differential
gene expression analyses of primary brain tumors and normal brain by
the SAGE technology have permitted the identification of numerous genes
that are increased in expression in malignant gliomas, including
osteonectin and osteoactivin (4, 19). To understand the role of these
two genes in the pathogenesis of gliomas, we determined the effects of
overexpressing osteonectin or osteoactivin by using a genetically
defined human glioma cell line (hereafter referred to as the THR glioma
line) developed in our laboratory (20). When implanted intracranially
in immune-compromised mice, THR cells display a non-invasive phenotype.
This phenotype is in contrast to that of gliomas in patients that are
universally invasive, suggesting that additional genetic alterations
beyond those already present in this cell line are required to develop an invasive phenotype in vivo. Thus, the THR cells represent
an ideal model system in which to study the contributions of specific genetic changes to late stages of glioma development. Through the use
of a retroviral system with an independent selectable marker, we
generated polyclonal THR glioma cells that ectopically expressed either
osteonectin (Fig. 1A) or
osteoactivin (Fig. 1B). Early passage cells were used in all
experiments to minimize genetic changes associated with simian virus 40 (SV40) T antigen expression.
To determine the potential impact of osteonectin or osteoactivin
expression on tumor development, we implanted these engineered THR cell
lines intracranially in suspensions. In this nature environment, the
genetically defined glioma cells formed large extra-axial masses with
histologic features consistent with a malignant neural tumor including
pseudopallisading necrosis (Fig. 1C), regardless of whether
the tumors expressed the vector, osteonectin, or osteoactivin. Tumors
derived from vector control cells were located predominantly within the
subarachnoid space, and rare foci superficially invaded the
Virchow-Robin spaces but extended no deeper than the molecular layer of
the superficial cerebral cortex. The Virchow-Robin spaces and
vasculature remained delicate without expansion by tumor, increased
numbers of vessels, or hypertrophy of the endothelium (not shown).
However, in 23 of 33 mice implanted with the osteonectin-expressing
glioma cells, tumors were located within the subarachnoid space and
displayed a striking phenotype of invasion (Fig. 1D), as
well as expansion of the perivascular Virchow-Robin spaces by masses of
tumor cells that extended into the deep layers of the cerebral cortex
(Fig. 1E) similar to previous reports of glioma invasion in
mouse models (24). In sharp contrast to the tumors derived from vector
control glioma cells, blood vessels associated with the tumors were
increased in size and number (Fig. 1F). These features
readily contrasted with adjacent brain tissues not invaded by the
tumor. Neoplastic cells in rare nests and single cells were seen
adjacent to the Virchow-Robin spaces in the parenchyma, a finding that
may represent either true invasion of brain parenchyma or formation of
an angiocentric perivascular zone outside the limits of the pia-glial
membrane. The prominent tumor associated vasculature was readily
detected by immunohistochemical staining of the factor VIII antigen,
which showed enhanced proliferation and endothelial hypertrophy of the
osteonectin-expressing glioma tumors in comparison with the normal
vasculature seen in adjacent brain, as well as the tumors derived from
the vector control cells.
Osteoactivin-expressing cells formed intracranial tumors in a fashion
similar to those expressing osteonectin but with a lower rate of brain
invasion (Fig. 1, D and G) and fewer associated vascular alterations (Fig. 1H). Taken together, our data
suggest that the expression of osteonectin and osteoactivin induce
glioma invasion in the context of brain environment associated with
penetrating blood vessels.
Osteonectin and Osteoactivin Expression Induces an Invasive
Phenotype in Vitro--
Having documented the dramatic effects of
osteonectin and osteoactivin expression on tumor invasion in xenograph
experiments, we next investigated the mechanism by which these genes
exert tumorigenic effects. Although in vitro systems are
incomplete models of tumor invasion and metastasis as they lack normal
stromal interactions, they allow selected analysis of potential changes at the cellular level, including cell proliferation, apoptosis, and
invasion. Previously, it was reported that osteonectin inhibits cell
proliferation (25) and induces apoptosis in some ovarian carcinoma cell
lines (26). However, we find that expression of osteonectin or
osteoactivin in our THR glioma cells did not cause any significant
changes in cell proliferation (Fig.
2A), apoptosis (Fig.
2A), or DNA synthesis (Fig. 2B). Disruption of osteonectin expression has been linked to loss of IGF-1R expression (27), but we found no differences in the expression levels of IGF-1R,
PDGFR- Osteonectin and Osteoactivin Expression Is Associated with
Increased MMP Expression--
To further explore the mechanisms that
mediate the invasive effects of osteonectin and osteoactivin, we
examined the expression profiles of proteins intimately associated with
tumor invasion, specifically the MMPs. As shown in Fig. 3C,
we found that osteonectin and osteoactivin expression significantly
increased the production of MMP-9 and MMP-3 by the tumor cells with
minimal changes in MMP-2 expression. To test whether this up-regulation
of MMPs is directly linked to the invasive behavior of tumors
expressing osteonectin and osteoactivin, we examined the effect of
specific MMP inhibitors in the Matrigel invasion assay. Treatment of
cells expressing osteonectin with an MMP-2/9 inhibitor had no effect (Fig. 3D), suggesting that the increase in MMP-9 expression
was not a major contributing factor to the changes in invasive behavior by the osteonectin-expressing tumor cells. However, MMP-3 inhibitor treatment substantially reduced invasion through Matrigel by the same
tumor cells (Fig. 3D), suggesting an important role for
MMP-3 in mediating the in vitro invasive behavior of these
cells. Consistent with this observation, treatment of parental THR
cells with exogenous osteonectin induced the production of MMP-3 within
4 h (Fig. 3E), whereas the expression of MMP-2 and
MMP-9 did not change (data not shown). Similarly, invasion of Matrigel
by THR cells expressing osteoactivin was also most sensitive to a
blockage of MMP-3 activity, although this activity was partially
inhibited by inhibitors specific for MMP-2 and MMP-2/9 (Fig.
3B). Taken together, the data suggest that osteonectin and
osteoactivin may promote tumor invasion, at least in part, by
increasing the production of specific MMPs.
Cells Expressing Osteonectin and Osteoactivin Display a Spontaneous
Metastatic Phenotype--
Since osteonectin and osteoactivin are
expressed in other types of advanced cancers, we investigated the
potential effects of these two genes on tumor progression in a
different growth microenvironment. To do this, osteonectin-expressing
and osteoactivin-expressing THR glioma cells were subcutaneously
injected into the flanks of SCID-beige mice. The primary subcutaneous
tumors derived from the glioma cells expressing osteonectin have an
identical latency period (~29 days) to a vector control cell line but
grow to a larger volume (Fig.
4A). Tumors from the
osteoactivin-expressing cell line display a longer latency (~39 days)
but subsequently grow to, on average, a larger size than vector
controls (Fig. 4A). Tumors expressing osteonectin and
osteoactivin did not exhibit a difference in angiogenic features from
the control cells (data not shown). Strikingly, both types of tumor
cells developed massive spontaneous intrathoracic and/or
intraperitoneal metastases (Fig. 4, B and C).
Metastases were found in 50% (16 of 32) of mice injected with
osteonectin-expressing glioma cells and 14% (3 of 15) of osteoactivin-expressing glioma cells (Fig. 4D). In contrast,
the THR vector control cell line exhibited a single small, discrete metastasis in only 1 of 15 mice, and no metastases developed in 25 mice
implanted with the THR parental cell line (not shown). Interestingly,
the development of metastases was independent of primary tumor size.
Three of 16 mice that developed metastases from osteonectin-expressing
cells had no grossly evident subcutaneous tumors at the initial site of
injection at the time of euthanasia. Therefore, the data suggest that
the development of a metastatic phenotype from osteonectin-expressing
glioma cells is associated with an early stage of tumor development
inside the animals.
Upon close pathological examination, we found that tumors derived from
osteonectin-expressing glioma cells display a phenotype of solid and
circumscribed neoplasms capable of invading the pancreas, peripancreatic soft tissues, subcutaneous tissues, and liver (Fig. 4, E and F). Examination of hematoxylin- and
eosin-stained sections of tumors derived from osteonectin-expressing
cells revealed a malignant phenotype of poorly differentiated neoplasm.
Within the tumor mass, there were numerous areas of atypical mitoses and necrosis pseudopalisaded by neoplastic cells. Taken together, histological analyses of tumors derived from osteonectin-expressing cells revealed typical pathological features that are consistent with
metastatic disease. Interestingly, we found that the metastasized tumors did not possess the features of prominently increased
vasculature and hypertrophic endothelium that are often associated with
the invasive brain tumors. Consistent with this observation,
lymphatic/vascular invasion and perineural extension were not detected
in the metastatic sites.
To test whether the effects of osteonectin described above were cell
line-specific, we investigated the behavior of tumor development by the
human glioma cell lines D54MG, U87MG, and U251MG engineered to express
higher levels of osteonectin (Fig. 4G). Importantly, we
found that ectopic expression of osteonectin by these glioma cell lines
also induced spontaneous metastases when they were injected
subcutaneously in the flanks of SCID-beige mice (Fig. 4D).
These results suggest that osteonectin expression can induce a
non-metastatic cancer cell line to adopt a metastatic phenotype with
the alteration in the expression of only a single gene.
Technological improvements now permit comprehensive analysis
of gene expression patterns in cancer specimens. The resulting data
have created a wealth of information to be mined, but utilization of
the data remains difficult due to a lack of functional information for
specific genes. Here we demonstrate the use of a genetically defined
human cancer model system to investigate the function of specific genes
differentially expressed in human cancers. Previously developed model
systems of cancer have included cell lines created from human
tumors and murine models. Although each model system has been of great
benefit to our understanding of the contributions that specific genetic
alterations play in the development of specific phenotypes of cancer,
each system has significant drawbacks. Human cell lines are generally
derived from advanced cancers that have widespread genetic changes and
genomic instability as well as changes from long passages in cell
culture. Murine models permit genetic control but are labor-intensive
and suffer from potential species-specific differences in the process
of transformation and gene function (28). Thus, a genetically defined
human cancer model system offers a useful tool to determine the roles
of specific genes in carcinogenesis, particularly the late stages of
this multistep process.
Using this system, we investigated genes that normally appear to be
highly expressed in bone without presence in brain tissues but have
significantly increased expression in human gliomas. Although the
expression of both osteonectin and osteoactivin has been linked to
tumor progression in previous studies, their precise contributions to
late stages of tumorigenesis remain unclear. Through an ectopic
expression strategy in this model system, we revealed that osteonectin
expression induced a highly malignant phenotype with significantly
increased brain invasion associated with vascular proliferation and
spontaneous systemic metastasis, whereas osteoactivin expression
resulted in a more modest increase in malignant phenotype with less
frequent metastases and lower degrees of vascular change. Although the
highly metastatic characteristics displayed by the tumor cells in our
study do not fully replicate the precise behavior of gliomas in humans,
the data strongly support the notion that expression of osteonectin and
osteoactivin could significantly alter or modify tumor behavior.
Considering the fact that these genes are also overexpressed in other
types of human cancers that do display a metastatic phenotype, this
finding is clearly relevant and significant to our understanding of the mechanism underlying metastasis associated with those types of human cancers.
Furthermore, our results indicate that the cellular microenvironment
potently modulates the phenotypic behavior of cancer cells that express
those two genes since glioma cells expressing osteonectin, and to a
lesser extent, osteoactivin, formed large spontaneous metastases with
subcutaneous implantation but remained localized to the brain with
intracranial implantation. The vascular and extracellular environment
in the brain is radically different from that of other organs, with the
presence of the blood-brain barrier, absence of lymphatics, and
brain-specific extracellular matrix and cell-cell interactions
associated with neuronal and glial migration during development. These
differences may partially account for the differential behavior of
cells expressing osteonectin implanted in the brain versus other parts
of the body. This finding strongly suggests that contributions to the
development of specific tumorigenic phenotypes by the expression of
specific genes are dependent on the specific characteristics of the
microenvironment associated with tumor progression, adding another
layer of complexity to the molecular mechanisms underlying late stages
of tumorigenesis.
Another potentially important finding is that osteonectin and
osteoactivin appear to promote a specific invasive phenotype intracranially involving tumor cell invasion along pre-existent blood
vessels in the Virchow-Robin spaces and development of altered vasculature at the brain-tumor interface. This phenotype mimics the
behavior of a subset of human gliomas and many invasive
medulloblastomas. Osteonectin has been reported to have an effect on
angiogenesis through regulation of VEGF activity (29). In our studies,
neither osteonectin nor osteoactivin induced significant changes in the vasculature as measured by vessel number or diameter within the primary
tumors, whether formed subcutaneously or intracranially. In contrast,
intracranial tumor cells expressing osteonectin and osteoactivin grow
along penetrating blood vessels, and the blood vessels at the invasive
front were markedly abnormal with vessel hypertrophy and hyperplasia.
These results suggest that the production of osteonectin and
osteoactivin by glioma cells may create a complex relationship between
invading tumor and normal vasculature that may be co-opted during tumor
invasion, consequently allowing expansion of the tumor mass without the
induction of angiogenesis at a significant level.
The malignant phenotypes mediated by osteonectin and osteoactivin
likely involve multiple mechanisms at the molecular level, with the
induction of MMP expression as an important component. The rapid
increase in MMP-3 protein levels in response to osteonectin treatment
by the THR tumor cells strongly suggests that osteonectin may regulate
the production of this specific MMP through a more direct mechanism.
Taken together, our results validate the use of a genetically defined
human cancer model system in investigating the contributions of
specific genes to late stages of tumorigenesis. This in vivo system permits the application of a functional genomics approach in
defining the specific activities of genes that have been identified to
be abnormally expressed in human cancers, particularly those associated
with tumor invasion, metastasis, and angiogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(PDGFR
) (Santa Cruz
Biotechnology), anti-PDGFR
(Santa Cruz Biotechnology), or
anti-insulin-like growth factor-1 receptor (IGF-1R) (Calbiochem)
antibodies were used. Secondary antibodies were either goat anti-rabbit
(Bio-Rad) or sheep anti-rabbit antibodies (Amersham Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Osteonectin and osteoactivin
expression increases brain invasion. As shown in A, a
genetically defined human glioma cell line was engineered to
overexpress osteonectin. Western analysis of conditioned media (30 µl
of media/sample) revealed that cells appropriately expressed and
secreted osteonectin. Par, parental; Vec, vector
control; ON, osteonectin. As shown in B, a
genetically defined human glioma cell line was engineered to
overexpress osteoactivin. Cellular lysates were resolved by SDS-PAGE
and analyzed for osteoactivin expression by Western analysis. As shown
in C, genetically defined glioma cells were injected
intracranially in immunocompromised mice. Histology of the tumors
expressing osteonectin reveals necrosis (*) with pseudopallisading
tumor cells consistent with a grade IV glioma. As shown in
D, the rate of brain invasion of vector control tumor cells
(VEC), osteonectin-expressing cells (ON), and
osteoactivin-expressing cells (OA) was measured on serial
sections. The total number of brains examined per point (N)
is indicated. Invasion was graded as no invasion (None),
limited invasion into the molecular layer (Min), or
widespread and/or deep brain penetration (Sig). *,
p = 0.011; **, p = 0.15. As shown in
E-H, in general, tumors grew largely separately, but those
tumors expressing osteonectin developed brain invasion (indicated by
arrows in E, hematoxylin and eosin) following
penetrating blood vessels (indicated by arrows in
F, factor VIII staining). Osteoactivin expression was
associated with significant tumor invasion into normal brain (indicated
by arrows in G, hematoxylin and eosin) but with
fewer significant vascular changes than osteonectin (indicated by
arrows in H, factor VIII staining).
or -
, or EGFR between control and osteonectin-expressing cells (Fig. 2C). Osteoactivin expression was associated with
an increase in EGFR expression but a decrease in PDGFR-
expression and no change in PDGFR-
or IGF-1R expression (Fig. 2C).
In addition, the ectopic expression of osteonectin or osteoactivin in
human astrocytes expressing SV40 T antigen and human telomerase
catalytic subunit failed to provide a mitogenic stimulus to transform
these cells in the absence of oncogenic Ras as measured by
soft agar colony formation assays (Fig. 2D). These results
suggest that osteonectin and osteoactivin expression is mainly
associated with the acquired abilities by the tumor cells in invasion
rather than tumor initiation. To test this hypothesis, we examined the
impact of osteonectin and osteoactivin expression on tumor cell
invasion in vitro. As shown in Fig.
3, A and B,
osteonectin or osteoactivin expression significantly increased cell
invasion in all cell lines tested as measured by the Matrigel invasion
assay. Constitutive expression of osteonectin or osteoactivin appeared
to increase the ability of tumor cells to degrade components of the
extracellular matrix and increased motility, a critical aspect of
invasive cancers.
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Fig. 2.
Phenotype associated with expression with
osteonectin and osteoactivin. Genetically defined glioma cells
were engineered to express vector control (VEC), osteonectin
(ON), or osteoactivin (OA). As shown in
A, cell cycle analysis by flow cytometry revealed that the
expression of osteonectin and osteoactivin was associated with minimal
changes in the cell cycle fractions. As shown in B, DNA
synthesis measured by thymidine incorporation revealed minimal impact
by osteonectin or osteoactivin expression. As shown in C,
equal amounts of protein from genetically defined human glioma cells
expressing osteonectin or osteoactivin were subjected to Western
analysis. Osteonectin expression was not associated with changes
relative to vector control, whereas osteoactivin expression was
associated with a moderate increase in EGFR expression and decrease of
PDGFR- expression. As shown in D, transformation of human
astrocytes expressing SV40 T antigen and the human telomerase catalytic
subunit with vector control, osteonectin, osteoactivin, or oncogenic
Ha-ras (RAS) was tested in a soft agar colony formation
assay.
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Fig. 3.
Osteonectin and osteoactivin expression is
associated with increased invasion and expression of MMP-9 and
MMP-3. As shown in A, Matrigel invasion assays were
performed with the genetically defined THR glioma cell line, D54MG,
U87MG, and U251MG, each engineered to express osteonectin
(ON). The expression of osteonectin was clearly linked to
increased invasion relative to vector control (VEC). *,
p = 0.01. As shown in B, Matrigel analysis
of glioma cells expressing osteoactivin with specific MMP-2, MMP-2/9,
and MMP-3 inhibitors ablated the increase in invasion associated with
osteoactivin expression (OA) relative to vector
controls. CON, control. *, p = 0.01 relative to vector control; **, p = 0.01 relative to
untreated osteoactivin-expressing tumor. As shown in C,
Western analysis of a genetically defined glioma cell line with vector
control (V), osteonectin, or osteoactivin expression
revealed increased expression of gelatinase B (MMP-9) and stromelysin-1
(MMP-3) but minimal change of gelatinase A (MMP-2). As shown in
D, Matrigel analysis of glioma cells expressing either
vector control or osteonectin showed no change in invasion with an
MMP-2/9 inhibitor, but an MMP-3 inhibitor ablated the increase in
invasion associated with osteonectin expression (*, p = 0.01 relative to untreated). As shown in E, parental
genetically defined THR glioma cells were treated with 50 µg/ml
purified human osteonectin (Hematologic Technologies, Essex, VT).
Conditioned media were collected simultaneously with either no
osteonectin or after treatment with osteonectin for specific times. The
media (50 µl) was resolved by SDS-PAGE and immunoblotted for
stromelysin-1 (MMP-3).
View larger version (87K):
[in a new window]
Fig. 4.
Osteonectin and osteoactivin expression
induces spontaneous metastases. As shown in A, a
genetically defined human glioma cell line was engineered to
overexpress a vector control (VEC), osteonectin
(ON), or osteoactivin (OA). 107 cells
were implanted subcutaneously into the right flank of SCID-beige mice.
Tumor volumes were measured twice a week and calculated by a
formula of 0.5(width)2(length). Tumor volumes were
plotted from mice that were not sacrificed due to development of
metastases as the tumors of these mice were frequently small at the
time of euthanasia. As shown in B and C, a
genetically defined glioma cell line engineered to ectopically express
osteonectin (B) or osteoactivin (C) developed
large spontaneous metastases (indicated by white arrows) in
the thorax and peritoneum when implanted subcutaneously in the flanks
of immunocompromised mice. As shown in D, osteonectin and
osteoactivin expression increased the rate of spontaneous metastases of
human glioma cell lines. The total number of mice examined per point is
indicated. *, p < 0.0001; **, p = 0.2;
***, p = 0.15; ****, p = 0.058. As
shown in E and F, pathology was consistent with a
malignant neural tumor (E, with normal pancreas) and with a
high Ki-67 index (F). The percentage of Ki-67-positive cells
was not significantly different between the vector control and
osteonectin- or osteoactivin-expressing tumors (data not shown). As
shown in G, expression of osteonectin was assayed by Western
analysis of cellular lysates of human glioma cell lines engineered to
overexpress osteonectin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank G. J. Riggins, A. Hjelmeland, J. Herndon, and R. McLendon for helpful discussions, S. Rempel for osteonectin cDNA, C. Wikstrand for GPNMB antibody, and S. Keir, Y. Yu, and R. Nelson for technical assistance. J. Parsons provided editorial support.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants K08 NS02055 (to J. N. R.), R01 CA83770 (to X.-F. W.), R01 CA94184 and CA82481 (to C. M. C.), and NS20023 (to D. D. B.); a grant from the Pediatric Brain Tumor Foundation (to J. N. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed: Duke University Medical Center, Box 2900, Durham, NC 27710. Tel.: 919-681-1693; Fax: 919-684-6514; E-mail: rich0001@mc.duke.edu and wang0011{at}mc.duke.edu.
§§ A Leukemia and Lymphoma Society Scholar.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M211498200
2 NCBI SAGE Genie web site.
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; IGF-1R, insulin-like growth factor-1 receptor; SCID, severe combined immune deficiency.
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