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2 Chain, Matrix Metalloproteinase-2, and Membrane Type-1-Matrix/Metalloproteinase Are Required for Mimicry of Embryonic Vasculogenesis by Aggressive Melanoma1
Department of Anatomy and Cell Biology [R. E. B. S., E. A. S., L. M. G. G., M. J. C. H.] and The Holden Comprehensive Cancer Center [R. E. B. S., E. A. S., M. J. C. H.] at The University of Iowa, Iowa City, Iowa 52242; Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037 [N. K., M. B., V. Q.]; Cancer Genetics Branch, National Human Genome Research Institute [P. S. M.] and Extracellular Matrix Pathology, Laboratory Pathology [W. G. S-S.], National Cancer Institute, NIH, Bethesda, Maryland 20892
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP
(MMP-14) in aggressive compared with poorly aggressive melanoma cells.
These components colocalized with developing patterned networks and
antisense oligonucleotides to the Ln-5 2 chain (but not sense
oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9)
inhibited the formation of these networks. Cultures which did not
receive antibodies to either MMPs-2 or -14 contained the Ln-5 2
chain promigratory cleavage fragments. Poorly aggressive melanoma cells
seeded on collagen I matrices preconditioned by the aggressive cells
formed tubular networks along the Ln-5 2 chain-enriched tracks
deposited by the aggressive cells. These results suggest that increased
expression of MMP-2 and MT1-MMP, along with matrix deposition of the
Ln-5 2 chain and/or its cleavage fragments, are required for
vasculogenic mimicry by aggressive melanoma cells. Furthermore, the
apparent recapitulation of laminin-rich, patterned networks observed in
aggressive melanoma patients’ tissue sections by aggressive melanoma
tumor cells in three-dimensional culture may also serve as a model to
help identify specific molecular targets which could function as
templates for the coordinated migration of aggressive tumor cells and
their proteolytic remodeling of the ECM and may have profound
implications for the development of novel therapies directed at the ECM
to alter tumor progression. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Microarray analysis of matched sets of patient-derived cell lines representing highly aggressive versus poorly aggressive uveal melanoma has revealed candidate genes potentially important to melanoma vasculogenic mimicry (3) . Overall, the molecular profile associated with the aggressive melanoma tumor cells suggests a genetic reversion to a pluripotent, embryonic-like phenotype, illustrated by the expression of multiple molecular phenotypes. Thus, the focus of our current research is to decipher the biological relevance of the key genes required for the mimicry of embryonic vasculogenesis by aggressive melanoma cells.
The premise of this paper is that aggressive melanoma tumor cells,
capable of forming patterned tubular networks in three-dimensional
culture, interact with their
ECM3
environment differently than poorly aggressive tumor cells. Key to our
approach have been additional microarray gene chip analyses of
aggressive compared with poorly aggressive human cutaneous melanoma
cells. These analyses revealed that the aggressive cells express
significantly higher levels, compared with the poorly aggressive cells,
of the basement membrane ECM component Ln-5 (specifically, the 2
chain) and MMPs-1, -2, -9, and -14, (MT1-MMP) proteinases. Studies with
antibodies specific for these components show that the Ln-5 2 chain,
MMP-2, and MT1-MMP interact in a cooperative manner and are required
for the aggressive cells to form tubular networks in three-dimensional
cultures. Furthermore, we demonstrate the remarkable ability of
matrices conditioned by aggressive melanoma cells to induce poorly
aggressive melanoma cells to form tubular networks, suggesting that
instructional information is deposited into the ECM. On the basis of
these findings, these components may have diagnostic value and serve as
putative molecular targets for therapeutic intervention in aggressive
melanoma, as well as other aggressive cancers that demonstrate
vasculogenic mimicry during tumor progression.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Three-dimensional type I collagen gels were produced as follows: 25 µl of type I collagen (average 3 mg/ml; Collaborative Biomedical Products, Bedford, MA) were dropped onto 18-mm glass coverslips in 12-well culture dishes and polymerized with an application of 100% ethanol for 5 min at room temperature. After a wash with PBS, tumor cells were seeded onto the three-dimensional gel in complete medium. For experiments designed to analyze the matrix after it was preconditioned by the aggressive C8161 cells, the cells were removed after 2 days with 20 mM NH4OH followed by three quick washes with water.
Microarray Analysis.
cDNA microarrays detected altered gene expression in highly invasive
melanoma cells by the method described previously (3)
, and
the relative expression of selected genes critical for vascular
formation and maintenance is reported as highly invasive and metastatic
compared with poorly invasive melanoma cells (Table 1)
. Hybridization to cDNA microarrays followed the previously described
procedures.4
RNA extracted from the poorly invasive and highly invasive/metastatic
melanoma cells was converted to cDNA in the presence of fluorescent
nucleotides Cy3- or Cy5-dUTP. The labeled cDNA pools were mixed and
hybridized to microarrays containing cDNA elements selected from the
Unigene database. Fluorescence intensities for each gene were measured
with a custom instrument in the case of the NIHGRI array, and the
ratios were calculated as described (3)
.
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Immunohistochemical Detection of Laminin in Tissue Sections.
Formalin-fixed, paraffin-embedded uveal melanoma samples were sectioned
at 4 µm and stained with either H&E or PAS without hematoxylin.
Adjacent sections were stained for laminin using a rabbit (A105; Telios
Pharmaceuticals, Inc., San Diego, CA) or monoclonal antihuman laminin
antibody (clone LAM-89; Sigma Chemical Co.-Aldrich, St. Louis, MO) and
LSAB+ alkaline phosphatase detection system in a Dako Autostainer
(Universal Kit; Dako, Carpinteria, CA) as per the manufacturer’s
protocol. These samples were then treated with the Vector Red Substrate
(Vector Laboratories, Burlingame, CA) for 3–5 min, and the slides were
subsequently counterstained with Mayer’s Hematoxylin for 5–7 min.
Immunohistochemistry.
Cells were grown in three-dimensional cultures on coverslips coated
with a collagen I gel for 7 days. The Ln-5 2 chain, MMPs-1, -2, -9,
and MT1-MMP were localized in these cultures using antibodies
(anti-Ln-5 2 chain, clone D4B5, Chemicon, Temecula, CA; anti-MMP-1,
clone 36665.11, anti-MMP-2, clone 1A10, anti-MMP-9, clone 36020.111;
R&D Systems, Minneapolis, MN; and anti-MT1-MMP, clone 114–6G6,
Chemicon) and the Vectastain ABC and AEC chromogenic detection kits
(Vector Laboratories) as per the manufacturer’s protocol. Briefly, the
cultures were fixed in 3.7% formaldehyde in PBS for 10 min, then
treated with 3% H2O2 in
PBS for 5 min. After a PBS wash, the cultures were incubated with the
primary antibody (2 µg/ml in PBS) for 2 h, followed by detection
using the Vectastain ABC and AEC chromogenic assay kits (according to
the manufacturer’s protocol). After development of a brown-red color,
the samples were rinsed with water, then mounted on microscope slides
with an aqueous mounting medium (Aqua-Mount; Lerner Laboratories,
Pittsburgh, PA). Images were obtained using a Zeiss Axioskop 2
microscope (Carl Zeiss, Inc., Thornwood, NY), Spot 2 camera (Diagnostic
Instruments, Inc., Sterling Heights, MI) and Zeiss Axiovision 2.0.5
software.
For inhibition experiments, the collagen I matrix was treated for 30
min before seeding the cells with blocking antibodies to MMP-2 (2.7
µg/ml clone CA4001/CA719E3C; Chemicon), MMP-9 (4 µg/ml clone 6–6B;
Oncogene Research Products, Boston, MA), or MT1-MMP (4.5 µg/ml rabbit
anti-MT1-MMP catalytic domain; Chemicon) or an antibody to the Ln-5
2 chain (10 µg/ml clone D4B5; Chemicon), recombinant TIMP-1 (10
nM; Oncogene Research), or recombinant TIMP-2 (
100
nM; Chemicon). The matrices were then seeded with the
cells, and the antibodies or TIMPs were added daily thereafter.
Microinjection of Fluoresceinated Dyes.
Patterned tubular networks observed in 3-week-old cultures of C8161
human cutaneous melanoma cells grown on collagen I gels were injected
with 7% lucifer yellow dye in water (w/v, lithium salt; Molecular
Probes, Eugene, OR) using a Zeiss 135 Axiovert microscope and Eppendorf
5170 micromanipulator and 5242 microinjector (Brinkmann, Westbury, NY).
Briefly, the tubular patterns were located by phase-contrast microcopy
and injected with fluorescent dye using the "semiautomatic" setting
to permit varying the amount of time of injection to allow for proper
tracking of the dye. A video recording was made using an Optronics
camera (Goleta, CA) and video cassette recorder (AG6740; Panasonic),
and images were subsequently captured from the tape using a Matrox
Meteor frame grabber (Dorval, Canada) and Zeiss Axiovision 2.0.5
software on a personal computer.
Antisense Oligonucleotide Knockout of Ln-5 2 Chain.
The expression of Ln-5 2 chain by highly aggressive C8161 human
melanoma cells was transiently suppressed by treating the cells with
phosphorothioate-modified oligonucleotides (commercially prepared by
Integrated DNA Technologies, Coralville, IA) in the antisense (5'-3';
CCAGAGCGCAGGCATGGC) or sense (5'-3'; GCCATGCCTGCGCTCTGG;
oligonucleotide control) orientation or untransfected to serve as a
vehicle-only control. The cells were seeded into 60-mm culture dishes
and allowed to attach for 3 h, then washed once with serum-free
Opti-MEM (Life Technologies, Inc.), then incubated for 5 h with 2
µM sense or antisense oligonucleotides using the
Lipofectamine delivery system (Life Technologies, Inc.). After
transfection (24 h), the cells were harvested and seeded onto a
three-dimensional collagen I matrix in serum-containing medium. Fresh
oligonucleotides were added every other day. Cultures were analyzed by
phase contract microscopy daily for the presence of tubular network
formations.
Western Blot Analysis.
Extracellular levels of laminin were determined as described
(5)
. Briefly, serum-free conditioned medium was obtained
from the same number of cells in the same volume of medium and cultured
for the same period of time. After centrifugation, the medium was
concentrated 50-fold by freeze-drying, dissolved in sample buffer (plus
ß-mercaptoethanol), and incubated at >95°C for 5 min. Equal
volumes were loaded onto 4–12% SDS polyacrylamide gradient gels, and
the separated proteins were then transblotted onto an Immobilon-P
membrane (Millipore, Bedford, MA). Laminin was detected using antibody
D4B5 to the Ln-5 2 chain (Chemicon), a secondary antibody conjugated
to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West
Grove, PA) and enhanced chemiluminescence (Amersham, Piscataway, NJ).
Detection of the Ln-5 2 chain and its cleavage fragments in
three-dimensional cultures containing highly aggressive C8161 or MUM-2B
cells, plus and minus antibodies, was performed as follows: cells, plus
and minus inhibitory antibodies, were seeded as stated above, and after
2 days, the cells plus conditioned matrix were washed with PBS, then
scrapped into 50 µl of SDS polyacrylamide gel sample buffer (plus
ß-mercaptoethanol). After electrophoresis in either 4–12% SDS
polyacrylamide gradient gels (C8161 cultures) or 7.5% gels (MUM-2B
cultures), these samples were then analyzed by Western blotting as
described above. The blots were then digitized, and the average pixel
density associated with the band identified as Ln-5 2 chain was
determined using Scion Image for Windows (Beta 4.0.2; Scion
Corp.).5
The density for the MT1-MMP sample was normalized to 100, and
the values for the other treatments were calculated against this value.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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. In light
of our observations concerning the process of vasculogenic mimicry, and
its association with the aggressive phenotype of melanoma tumor cells
(2)
, we used these results to direct our examination of
the cellular and molecular mechanisms that contribute to vasculogenic
mimicry and melanoma tumor cell aggressiveness.
On the basis of the microarray data, and previous work which showed
that MMP inhibitors mitigate the ability of highly aggressive tumor
cells to form vascularized channels and tubular networks
(6)
, specific MMPs were examined to determine their
putative role in vasculogenic mimicry. Patterned, ECM-rich networks
formed by highly aggressive C8161 human melanoma cells grown on
collagen I gels were observed using PAS staining (Fig. 1A)
, and fluorescent dye injected into the network
demonstrated that they were tubular in nature and perfusable (Fig. 1, B and C)
. Furthermore, immunostaining with
specific anti-MMP antibodies showed that MMP-1 (Fig. 1D)
,
MMP-2 (Fig. 1E)
, MMP-9 (Fig. 1F)
, and MT1-MMP
(Fig. 1G)
colocalized with the patterned networks. Higher
concentrations of MMP-1 and MMP-2 were also measured in the conditioned
media from the highly aggressive (C8161 and MUM-2B) compared with the
poorly aggressive (C81-61 and MUM-2C) cells, and 10–20% of the MMP-2
was in its active form, whereas 20–35% could be subsequently
activated (i.e., via treatment with p-aminophenylmercuric
acetate; Table 2
). Although microarray data also identified an increase in MMP-9 gene
expression in the highly aggressive melanoma, the actual concentration
of MMP-9 in the conditioned media remained barely detectable (Table 2)
.
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, and TIMP-1 did not affect this process (Fig. 2B)
. TIMP-2 retarded the onset of the patterned network
formation (Fig. 2C)
, and although cells appear to lay along
some tracks in the matrix after 4 days, the tubular network was not as
well developed as the network in the untreated culture (Fig. 2A)
. Whereas antibodies to both MMP-2 (Fig. 2D)
and MT1-MMP (Fig. 2E)
inhibited the formation of the
patterned tubular networks, the antibody to MMP-9 did not (Fig. 2F)
.
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2 chain is more highly
expressed by the aggressive compared with the poorly aggressive
melanoma cells and prompted an examination of whether this ECM
component might play a role in vasculogenic mimicry. Western blot
analysis validated the microarray data and demonstrated that the
aggressive melanoma cell lines (MUM-2B and C8161) express the Ln-5 2
chain, whereas the poorly aggressive melanoma cell lines (MUM-2C and
C81-61) express little to no Ln-5 2 chain (Fig. 3A)
. Detection of the Ln-5 2 chain in three-dimensional
cultures of the highly aggressive C8161 cells on collagen I gels
revealed Ln-5 2 chain-rich, patterned networks (Fig. 3B)
,
similar to the networks detected by PAS staining (Fig. 1A)
.
Addition of antisense Ln-5 2 chain oligonucleotides (or an anti-Ln-5
2 chain antibody, D4B5; data not shown), but not sense Ln-5 2
chain oligonucleotides (which did not down-regulate the expression of
Ln-5 2 chain; data not shown), resulted in a loss of the Ln-5 2
chain-patterned networks (Fig. 3C)
. The Ln-5 2 chain-rich
patterned networks seen in the aggressive melanoma three-dimensional
cultures (Fig. 3B)
are reminiscent of laminin-rich,
patterned networks seen in patient tissue sections of aggressive uveal
melanoma (Fig. 3D)
.
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2 chain by MT1-MMP and MMP-2 proteolysis (5
, 7)
,
we examined whether some of these interactions could also be involved
in vasculogenic mimicry. Highly aggressive C8161 and MUM-2B cells were
grown on collagen I gels for 2 days with or without treatment with
antibodies to MMP-9, MMP-2, or MT1-MMP. The cells plus matrix were then
analyzed by Western blot for the presence of the Ln-5 2 chain and
its cleaved fragments (Fig. 3, E and F)
. The
samples which did not receive anti-MMP-2 or anti-MT1-MMP contained
mostly cleaved fragments of the Ln-5 2 chain and little to no
full-size (140 kDa) Ln-5 2 chain (Fig. 3, E and F
; Control). Whereas the addition of anti-MMP-9
did not appreciably alter this result, the addition of either
anti-MMP-2 or anti-MT1-MMP decreased the amount of cleaved Ln-5 2
chain fragments and increased the proportion of uncleaved Ln-5 2
chain in the samples (Fig. 3, E and F)
.
The observations that antisense oligonucleotides to the Ln-5 2 chain
(or anti-Ln-5 2 chain antibody) or antibodies to MMP-2 or MT1-MMP
could inhibit vasculogenic mimicry by highly aggressive tumor cells
grown on collagen I gels suggested that the highly aggressive cells
could be modifying their ECM in such a way as to initiate or promote
the vasculogenic mimicry process. To test this premise, highly
aggressive tumor cells capable of forming patterned tubular networks on
collagen I gels (Fig. 4A)
were removed from their matrix after 2 days and before the
visually apparent formation of networks (Fig. 4B)
. When the
acellular matrix was examined for laminin, a clear and discernable
pattern of laminin-positive networks is seen within the collagen I gel
(Fig. 4C)
. Whereas poorly aggressive tumor cells do not form
patterned tubular networks on collagen I gels (Fig. 4D)
,
they can be induced to form these patterned networks when seeded on
collagen I gels preconditioned by (and after the removal of) the highly
aggressive tumor cells (Fig. 4E)
.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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2 monomer) and MMPs-1, -2, -9, and MT1-MMP
in the highly aggressive cells. These observations suggested that the
aggressive cells have the potential to interact with and alter their
extracellular environment differently than the poorly aggressive cells
and provided the scientific framework for the study presented here.
The observations that the Ln-5 2 chain is more highly expressed by
the aggressive compared with the poorly aggressive melanoma cells, and
that it colocalized with the patterned tubular networks formed by the
aggressive cells, suggested that the Ln-5 2 chain may be a key
component and play a functional role in tumor cell vasculogenic
mimicry. Furthermore, there is a remarkable similarity between the Ln-5
2 chain-staining patterns formed by the aggressive melanoma cells in
three-dimensional culture with patterns of laminin-stained patient
tumor sections of aggressive melanoma and with patterns of matrix-rich
regions seen in human melanoma xenografts in nude mice
(9)
.
Laminins are major constituents of basement membrane ECM and play an active role in neurite outgrowth, tumor metastasis, cell attachment and migration, and angiogenesis (10, 11, 12) . It is now clear that the expression and distribution of laminin isoforms act to regulate the tissue-specific response of cells to basement membranes (10) . In addition, laminin expression also appears to be a prerequisite for embryogenesis and basement membrane formation in developing tissues, and it is thought to stabilize the organization of basement membranes (10) .
Laminins may undergo proteolytic cleavages attributable to
posttranslational (and possibly postsecretion) modifications that
result in laminin fragments, which may remain bound to the parent
molecule (10, 11, 12)
. In this context, laminin degradation
associated with basement membrane turnover and tissue remodeling can
expose and render active specific laminin sequences capable of
mediating cell interactions but were buried and not functionally
available before proteolytic degradation (10, 11, 12)
. In this
respect, Ln-5, which can be cleaved in the
NH2-terminal region of the 2 chain and the G
domain of the 
3 chain, can alter and regulate the integrin-mediated
migratory behavior of certain cells (5
, 7 , 10)
. Ln-5 is
found in many epithelial basement membranes and is an extracellular
substrate for both cell adhesion and migration. Whereas both
3ß1 and
6ß4 integrins can bind
to Ln-5, it is the interaction between Ln-5 and the
6ß4 integrin that can
lead to hemidesmosome assembly and the static adhesion of epithelial
cells to the basement membrane (5
, 7)
. However, in direct
opposition to this activity, Ln-5 can also promote vigorous cell
scattering when added to the medium of epithelial cell cultures
(5
, 7)
. This activity was found to be associated with the
proteolytic cleavage of the 2 chain of Ln-5 by activated MMP-2.
Whereas subsequent studies revealed that MT1-MMP appears to be the
primary MMP associated with the induction of epithelial cell migration
over proteolytically cleaved Ln-5, MMP-2, which can be activated by
MT1-MMP, may actually play an ancillary role by amplifying MT1-MMP
effects (5)
. An important aspect of this model is that
Ln-5 cleavage, either by MT1-MMP alone or in concert with
MT1-MMP-activated MMP-2, results in proteolytic components that are
anchored onto the cell surface because MT1-MMP is a transmembrane
protein. This could result in the spatially directed cleavage of Ln-5,
which may coincide with areas associated with high levels of migration
(5)
. Collectively, these studies reflect the complex
interactions that occur between MMPs, integrins, and the ECM in a
cell’s microenvironment (13)
that coordinately regulate
adhesion, migration, invasion, and, as suggested by the data presented
here, vasculogenic mimicry.
An interesting point is that we were not able to detect the
3 subunit of Ln-5 in the conditioned medium of
aggressive melanoma cells (data not shown). Furthermore,
immunoprecipitation with an antibody to the Ln-5 2 chain (data not
shown) indicated that the Ln-5 2 chain may be secreted by aggressive
melanoma cells as a monomer and not in the typical heterotrimeric
arrangement of laminins. More detailed studies on the monomeric Ln-5
2 chain will be presented elsewhere. Nonetheless, this finding is
supported by several reports indicating that the Ln-5 2 chain may be
detected at the leading edge of invasive tumor nests in the absence of
other laminin chains (14
, 15)
.
The observation that treating aggressive melanoma cells in
three-dimensional cultures with antibodies to MMP-2 or MT1-MMP (but not
MMP-9) blocked tubular network formation suggested that MMP-2 and
MT1-MMP were directly and/or coordinately involved in vasculogenic
mimicry, possibly resulting in a molecular signal and/or promigratory
cue being deposited in the ECM (5
, 7
, 10, 11, 12)
. This
concept is supported by the observation that treating aggressive
melanoma cells in three-dimensional cultures with antibodies to MMP-2
or MT1-MMP (but not MMP-9) resulted in a decrease in Ln-5 2 chain
cleavage fragments associated with promigratory cues embedded in a
matrix (5
, 7
, 10, 11, 12)
and an increase in the relative
amount of the uncleaved Ln-5 2 chain. Furthermore, although MMP-9
has been reported to be a trigger for the angiogenic switch during
carcinogenesis of pancreatic islets in transgenic mice
(16)
, it is MMP-2 activity that appears to be required for
the switch to the angiogenic phenotype in an in vivo
chondrosarcoma tumor development model (17)
.
Microarray analyses revealed that MMPs-1, -2, -9, and MT1-MMP are all more highly expressed in the aggressive compared with the poorly aggressive melanoma cell lines. Whereas immunohistochemical analyses demonstrated that these four MMPs also colocalize with the patterned networks formed by the aggressive cells on collagen I gels, development of these networks could only be inhibited by antibodies to either MT1-MMP or MMP-2 but not with the MMP-9 antibody. Although TIMP-1 did not inhibit the formation of patterned networks by the aggressive cells, this is not inconsistent with the microarray data which showed a significant increase in the expression of TIMP-1 by the aggressive cells compared with the poorly aggressive cells and could indicate that TIMP-1 is not acting strictly as an inhibitor of MMPs in this system. In this regard, previous work has shown that mRNA for TIMP-1 is more highly expressed in the colon cancer cell line SW620 (which expresses low levels of both the CC3 tumor suppressor gene and TIMP-3) compared with the SW480 cell line (which expresses higher levels of CC3 and TIMP-3; Ref. 18 ). SW620 cells also secrete a 10-fold increase of TIMP-1 protein compared with SW480 cells, and high TIMP-1 expression is associated with poor prognosis in colorectal cancer (18) . Taken together, these results, along with our observations, suggest that the association between TIMP-1 expression and tumor progression may be related to additional growth factor-like effects, which have been described for TIMP-1 in some systems (18 , 19) .
The inability of TIMP-2 to completely prevent the formation of
patterned networks by the aggressive cells is also not intuitively
clear because the inhibition of either MMP-2 or MT1-MMP activity with
antibodies is sufficient to prevent formation of these patterned
networks. However, this could be attributable to a couple of factors:
(a) the concentration of MMPs in the aggressive cell
cultures may be sufficiently large (specifically, MMP-1 and MMP-2;
Table 2
) that it may take a much higher concentration of TIMP-2 to
effectively inhibit all of the proteolytic activities associated with
MMP-2 and/or MT1-MMP (plus other MMPs in the culture that can bind
TIMP-2); or (b) because TIMP-2 can induce the activation of
pro-MMP-2 by MT1-MMP (via the formation of a MT1-MMP/TIMP-2/pro-MMP-2
ternary complex; Ref. 20
), it may require a higher
concentration of TIMP-2 to extend beyond the pro-MMP-2 activation point
and reach a level where it inhibits MMP activity.
A key point from this study is that poorly aggressive melanoma cells,
which do not express appreciable levels of the Ln-5 2 chain, can be
induced to form patterned tubular networks on collagen I gels
preconditioned by the aggressive cells. This result supports the
concept that molecular (promigratory) cues deposited in the matrix
(5
, 7
, 10, 11, 12)
and not soluble factors (as demonstrated in
Ref. 2
) can influence poorly aggressive cells to assume a
vasculogenic phenotype. Furthermore, treating the preconditioned matrix
with antibodies to the Ln-5 2 chain before seeding the poorly
aggressive cells abrogates their ability to engage in vasculogenic
mimicry. Together, these results indicate that the Ln-5 2 chain is
involved in the regulation of vasculogenic mimicry and appears to be
regulated itself through proteolytic cleavage by specific MMP
proteinases.
In conclusion, the ability of aggressive melanoma tumor cells to mimic
embryonic vasculogenesis when cultured on collagen I gels requires the
cooperative interaction between the Ln-5 2 chain, MT1-MMP, and
MMP-2. Furthermore, whereas blocking the function or activity of any
one of these three components inhibits tumor cell vasculogenic mimicry,
we also found that collagen gels preconditioned by aggressive melanoma
cells can induce poorly aggressive melanoma cells to assume a
vasculogenic phenotype and form endothelial-like networks. The apparent
recapitulation of laminin-rich, patterned networks observed in
aggressive melanoma patients’ tissue sections by aggressive melanoma
tumor cells in three-dimensional culture may also serve as a model to
help identify specific molecular targets, which could function as
templates for the coordinated migration of aggressive tumor cells and
their proteolytic remodeling of the ECM, and may have profound
implications for the development of novel therapies directed at the ECM
to alter tumor progression.
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FOOTNOTES |
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1 Supported by NIH/National Cancer Institute
Grants CA83137 (to R. E. B. S.), CA80318, CA88043-2S, and CA59702
(to M. J. C. H.), and Grants GM46902, CA47858-13S, and CA47858 (to
V. Q.). 
2 To whom requests for reprints should be
addressed, at Department of Anatomy and Cell Biology, The University of
Iowa, 51 Newton Road, 1-100 BSB, Iowa City, IA 52242-1109. Phone:
(319) 335-7755; Fax: (319) 335-7770; E-mail: mary-hendrix{at}uiowa.edu
3 The abbreviations used are: ECM, extracellular
matrix; MMP, matrix metalloproteinase; Ln-5, Laminin 5; TIMP, tissue
inhibitor of matrix metalloproteinase; PAS, periodic acid-Schiff.
4 Internet address:
http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/.
5 Internet address: http://www.scioncorp.com.
Received 4/11/01. Accepted 7/10/01.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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1 and ß1 chain peptides active for endothelial cell adhesion, tube formation, and aortic sprouting.. FASEB J., 13: 53-62, 1999. 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers.. Am. J. Pathol., 145: 782-791, 1994.[Abstract]
2 chain monomer in invading gastric carcinoma cells.. Cancer Res., 59: 5596-5601, 1999.This article has been cited by other articles:
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J. J. Partridge, M. A. Madsen, V. C. Ardi, T. Papagiannakopoulos, T. A. Kupriyanova, J. P. Quigley, and E. I. Deryugina Functional Analysis of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases Differentially Expressed by Variants of Human HT-1080 Fibrosarcoma Exhibiting High and Low Levels of Intravasation and Metastasis J. Biol. Chem., December 7, 2007; 282(49): 35964 - 35977. [Abstract] [Full Text] [PDF]
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A. P. Petty, K. L. Garman, V. D. Winn, C. M. Spidel, and J. S. Lindsey Overexpression of Carcinoma and Embryonic Cytotrophoblast Cell-Specific Mig-7 Induces Invasion and Vessel-Like Structure Formation Am. J. Pathol., May 1, 2007; 170(5): 1763 - 1780. [Abstract] [Full Text] [PDF]
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F. Spinella, L. Rosano, V. Di Castro, S. Decandia, M. R. Nicotra, P. G. Natali, and A. Bagnato Endothelin-1 and Endothelin-3 Promote Invasive Behavior via Hypoxia-Inducible Factor-1{alpha} in Human Melanoma Cells Cancer Res., February 15, 2007; 67(4): 1725 - 1734. [Abstract] [Full Text] [PDF]
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L.-M. Postovit, E. A. Seftor, R. E.B. Seftor, and M. J.C. Hendrix Influence of the Microenvironment on Melanoma Cell Fate Determination and Phenotype Cancer Res., August 15, 2006; 66(16): 7833 - 7836. [Abstract] [Full Text] [PDF]
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B. Wang, J. Sun, S. Kitamoto, M. Yang, A. Grubb, H. A. Chapman, R. Kalluri, and G.-P. Shi Cathepsin S Controls Angiogenesis and Tumor Growth via Matrix-derived Angiogenic Factors J. Biol. Chem., March 3, 2006; 281(9): 6020 - 6029. [Abstract] [Full Text] [PDF]
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R. A. Bartolome, I. Molina-Ortiz, R. Samaniego, P. Sanchez-Mateos, X. R. Bustelo, and J. Teixido Activation of Vav/Rho GTPase Signaling by CXCL12 Controls Membrane-Type Matrix Metalloproteinase-Dependent Melanoma Cell Invasion Cancer Res., January 1, 2006; 66(1): 248 - 258. [Abstract] [Full Text] [PDF]
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A. R. Hess, L.-M. Postovit, N. V. Margaryan, E. A. Seftor, G. B. Schneider, R. E.B. Seftor, B. J. Nickoloff, and M. J.C. Hendrix Focal Adhesion Kinase Promotes the Aggressive Melanoma Phenotype Cancer Res., November 1, 2005; 65(21): 9851 - 9860. [Abstract] [Full Text] [PDF]
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 E. C. Dietze, M. L. Bowie, K. Mrozek, L. E. Caldwell, C. Neal, R. J. Marjoram, M. M. Troch, G. R. Bean, K. K. Yokoyama, C. A. Ibarra, et al. CREB-binding protein regulates apoptosis and growth of HMECs grown in reconstituted ECM via laminin-5 J. Cell Sci., November 1, 2005; 118(21): 5005 - 5022. [Abstract] [Full Text] [PDF]
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P. Guo, Y. Imanishi, F. C. Cackowski, M. J. Jarzynka, H.-Q. Tao, R. Nishikawa, T. Hirose, B. Hu, and S.-Y. Cheng Up-Regulation of Angiopoietin-2, Matrix Metalloprotease-2, Membrane Type 1 Metalloprotease, and Laminin 5 {gamma} 2 Correlates with the Invasiveness of Human Glioma Am. J. Pathol., March 1, 2005; 166(3): 877 - 890. [Abstract] [Full Text] [PDF]
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N. Koshikawa, T. Minegishi, A. Sharabi, V. Quaranta, and M. Seiki Membrane-type Matrix Metalloproteinase-1 (MT1-MMP) Is a Processing Enzyme for Human Laminin {gamma}2 Chain J. Biol. Chem., January 7, 2005; 280(1): 88 - 93. [Abstract] [Full Text] [PDF]
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E.-M. Schnaeker, R. Ossig, T. Ludwig, R. Dreier, H. Oberleithner, M. Wilhelmi, and S. W. Schneider Microtubule-Dependent Matrix Metalloproteinase-2/Matrix Metalloproteinase-9 Exocytosis: Prerequisite in Human Melanoma Cell Invasion Cancer Res., December 15, 2004; 64(24): 8924 - 8931. [Abstract] [Full Text] [PDF]
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R. A. Bartolome, B. G. Galvez, N. Longo, F. Baleux, G. N. P. van Muijen, P. Sanchez-Mateos, A. G. Arroyo, and J. Teixido Stromal Cell-Derived Factor-1{alpha} Promotes Melanoma Cell Invasion across Basement Membranes Involving Stimulation of Membrane-Type 1 Matrix Metalloproteinase and Rho GTPase Activities Cancer Res., April 1, 2004; 64(7): 2534 - 2543. [Abstract] [Full Text] [PDF]
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Y. A. DeClerck, A. M. Mercurio, M. S. Stack, H. A. Chapman, M. M. Zutter, R. J. Muschel, A. Raz, L. M. Matrisian, B. F. Sloane, A. Noel, et al. Proteases, Extracellular Matrix, and Cancer: A Workshop of the Path B Study Section Am. J. Pathol., April 1, 2004; 164(4): 1131 - 1139. [Abstract] [Full Text] [PDF]
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W. Ruf, E. A. Seftor, R. J. Petrovan, R. M. Weiss, L. M. Gruman, N. V. Margaryan, R. E. B. Seftor, Y. Miyagi, and M. J. C. Hendrix Differential Role of Tissue Factor Pathway Inhibitors 1 and 2 in Melanoma Vasculogenic Mimicry Cancer Res., September 1, 2003; 63(17): 5381 - 5389. [Abstract] [Full Text] [PDF]
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