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From the * Department of Pharmacology; the
Department of Laboratory Medicine and
Pathology; the Department of Urologic Surgery;
the Masonic Comprehensive Cancer Center; and the ||
Department of Genetics, Cell Biology, and
Development, University of Minnesota; and the ¶
Minneapolis VA Medical Center, Minneapolis,
Minnesota.
| Correspondence to: Dr Michael J. Wilson, Research Service, Minneapolis VA Medical Center, One Veterans Drive, Minneapolis, MN 55417 (e-mail: Wilso042{at}umn.edu). |
| Received for publication August 20, 2008; accepted for publication January 2, 2009. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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C-MT1-MMP). Enhanced cell surface
MT1-MMP was determined by fluorescence-activated cell sorting analysis and
evidenced mechanistically by increased activation of proMMP-2 and invasion
into type-I collagen gels. PC-3 cells overexpressing MT1-MMP grew faster than
mock-transfected control cells subcutaneously in nude mice. MT1-MMP localized
in caveolae, as judged by immunofluorescence microscopy and sucrose-gradient,
detergent-resistant cell fractionation. C-MT1-MMP was strongly
associated with caveolae, whereas the WT form was present in both caveolae and
noncaveolae fractions. The role of plasma membrane MT1-MMP was supported by
localization of MT1-MMP by immunofluorescence microscopy at the cell surface
of tumor cells in primary prostate cancers. Increased plasma membrane
localization of MT1-MMP, either in caveolae or in other lipid raft structures,
is a mechanism to localize this proteinase in contact with extracellular
matrix and other pericellular proteins, the cleavage of which can facilitate
prostate cancer cell invasion and metastasis.
Key words: Cancer, matrix metalloproteinase type-1, MMP-14, tumor cell invasion, caveolae, type I collagen
Proteinases regulate functions that are critical for cancer cell growth, invasion, and metastasis. Proteolysis of extracellular matrix (ECM) proteins is considered fundamental to permit passage of cancer cells during invasion and metastasis. Matrix metalloproteinases (MMPs) comprise a family of about 25 zinc-dependent enzymes of similar structure that collectively can cleave all known ECM proteins (Basbaum and Werb, 1996; Powell and Matrisian, 1996; Ellenbroek and Stack, 1999; Stetler-Stevenson and Yu, 2001; Visse and Nagase, 2003). Although MMPs maintain the rate-limiting steps in ECM protein degradation, they work in concert or in cascades with plasminogen activators, proteinases of the coagulation system, plasma membrane–associated cathepsin B, and proteinases of diverse classes and families to process these proteins (Carmeiet and Collen, 1998). The discovery of the membrane-type MMPs (MT1-MMP through MT6-MMP), tethered to the plasma membrane by a transmembrane domain or glycosylphosphatidylinositol (GPI) anchor, brought on a new focus of MMP function at the cell surface (Zucker et al, 2003). It is now evident that in addition to MMP degradation of pericellular ECM proteins, MMPs have diverse functions in cancer, including processing of a variety of nonmatrix proteins (growth factors, growth factor receptors, cell adhesion molecules, and death domain factors), activation of other MMPs, release of growth factors from the cell surface and from ECM proteins, and exposure of cryptic sites in ECM proteins promoting cell migration (Egeblad and Werb, 2002; Lynch and Matrisian, 2002). Thus, degradation or activation of cell surface and ECM proteins by proteolysis can mediate rapid cellular responses that control cell growth, migration, differentiation, and death during tumor progression in the prostate (Egeblad and Werb, 2002).
There is considerable clinical data implicating a prominent role for MT1-MMP in cancer cell invasion and metastasis. Elevated expression of MT1-MMP correlated with poor prognosis is detected in different types of cancers originating in a variety of tissues (Seiki, 2003). There are several molecular characteristics of MT1-MMP that support the role of this MMP in cancer invasion and metastasis. Its transmembrane domain is a means by which it is inserted into the plasma membrane, particularly in caveolae in association with other proteinases, placing it on the cell surface in apposition to potential substrates. MT1-MMP is one of a select few proteinases (MMP-1, -8, and -13) that can cleave types I and II fibrillar collagens. It can also cleave a variety of other ECM protein substrates, including cell surface receptors, growth factors, cytokines, etc. In addition, MT1-MMP can activate proMMP-2 and proMMP-13, which in turn can cleave other protein substrates, amplifying the effect of MT1-MMP in the pericellular environment.
The expression of a number of MMPs, including MMPs-2, -7, -9, and -14 (MT1-MMP), has been reported in human prostate cancer tissues (Wilson and Sinha, 2008). MT1-MMP immunostains primarily in basal cells and basal-lateral membranes of secretory cells of benign prostatic glands and secretory cells in high-grade prostatic intraepithelial neoplasia (PIN) (Sroka et al, 2008). Although the level of MT1-MMP message has been reported to be lower in primary prostate cancer tissues and primary cell cultures derived from them than in nonmalignant prostate tissue (Jung et al, 2003), its localization in cancer cells has been described as varied, heterogeneous to diffuse (all cells in glandular structure positive; Upadhyay et al, 1999; Udayakumar et al, 2003), and with greater staining in invasive areas (Upadhyay et al, 1999). A role for MT1-MMP in prostate cancer is strongly suggested by a significant association of MT1-MMP with MMP-2 in the same specimens (Upadhyay et al, 1999), and an increased expression of the active form of MMP-2 with increased Gleason score (Stearns and Stearns, 1996). In our studies of MMPs in the human prostate cancer PC-3 cell line, we found that cell surface localization and not total expression of MT1-MMP was a limiting factor in the activation of proMMP-2 (Wilson et al, 2004). In this study we examined plasma membrane subdomain localization of MT1-MMP in more invasive PC-3 cells overexpressing MT1-MMP either by transfection or by selection for metastasis to lymph nodes by in vivo passage in nude mice. The presence of MT1-MMP in cholesterol-rich lipid raft compartments on the surface of PC-3 prostate cancer cells was associated with increased invasive behavior of these tumor cells.
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Materials and Methods |
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Antibodies and Reagents
Rabbit anti-MT1-MMP antibody (Lenti, Ab3) has been described
(Jiang et al, 2001).
Fluorescein isothiocyanate (FITC)-labeled mouse anti-MT1-MMP monoclonal
antibody was purchased from R&D Systems (Minneapolis, Minnesota). Rabbit
anti-caveolin-1 antibody was from BD Biosciences (Lexington, Kentucky).
Alexa-488 and -595 conjugated secondary antibodies were from Molecular Probes
(Eugene, Oregon). Nystatin, proteinase inhibitors, and secondary antibody
conjugates were from Sigma (St Louis, Missouri). BB-94 was a gift from British
Biotechnology (Oxford, United Kingdom).
Human Tissues
Prostate tissues were collected from radical prostatectomy specimens under
a protocol approved by the Internal Review Board of the Minneapolis VA Medical
Center and the University of Minnesota. These tissues were frozen immediately
on dry ice and stored at –70°C until prepared for sectioning with a
cryostat.
Nude Mice
Male nude mice (Nu–/Nu–) were purchased
from Charles River Laboratories (Worcester, Massachusetts) and were used
between 34 and 43 days of age for subcutaneous injection of tumor cells. Tumor
cells were washed 2 times with phosphate-buffered saline (PBS) and then
brought to a concentration so that 2 x 106 cells in 0.20 mL
PBS were injected subcutaneously in the right flank of each mouse. Mice were
monitored weekly for tumor growth, and tumors were measured in 2 dimensions
with calipers. Tumor volume was calculated as described previously
(Wilson and Sinha, 1997).
Tumors were removed at necropsy when they reached 1 cm3 in size, or
at the end of the experiment. Pieces of tumor were frozen in liquid nitrogen
or fixed in 10% buffered formalin in preparation for histology using
hematoxylin and eosin staining.
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C-MT1-MMP)
have been described (Jiang et al,
2001; Wang et al,
2004b). The DNA constructs were transfected into various cells by
Effectene (Qiagen, Valencia, California) according to the manufacturer's
protocol. Stable cell lines were selected with G418 in culture medium.
Transient transfected cells were used within 24 to 48 hours after
transfection. Select experiments were performed in the presence of the MMP
inhibitor BB-94 (Wang et al,
2004b), to prevent the autocatalytic decay of MT1-MMP.
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Fluorescence Flow Cytometry
The amounts of MT1-MMP on the plasma membrane were quantified by
determining the cell surface immunofluorescence of bound FITC-labeled mouse
anti-MT1-MMP using fluorescence-activated cell sorting (FACS) analysis. After
rapid rinsing of cells 2 times with serum-free Dulbecco modified Eagle medium
(DMEM) at 4°C, cells were incubated at 4°C for 60 minutes in
serum-free DMEM with the FITC-labeled mouse anti-MT1-MMP monoclonal antibody.
After washing of cells to remove excess secondary antibodies, cells were
detached and were then fixed with 3.7% formaldehyde prior to FACS analysis.
Cell surface MT1-MMP immunofluorescence was quantified by FACScan (Becton
Dickinson, Palo Alto, California). Fluorescence intensity of 10 000 cells was
collected for each sample. Cell Quest software (Becton Dickinson) was used to
calculate the mean fluorescence intensity of the cell populations.
Immunostaining and Confocal Microscopy
For internalization experiments, cells seeded on coverslips in 6-well
plates were washed 3 times with PBS and shifted to 4°C. Anti-MT1-MMP
antibodies were added to the cells at 0.2 µg/mL for 2 hours. Antibody was
subsequently removed and cells were washed before being shifted to 37°C
with prewarmed media for the indicated time. Cells were then fixed with 3.7%
paraformaldehyde in PBS (pH 7.4) for 30 minutes at room temperature and
blocked with PBS-diluent (0.3% Triton X-100, 1% normal donkey serum, 1% bovine
serum albumin, and 0.01% sodium azide, pH 7.2) for 60 minutes followed by
staining with secondary antibodies (Molecular Probes). The coverslips were
mounted by NO-FADE (10% glycerol in PBS and 0.1% p-phenylenediamine, pH 8.0).
For colocalization experiments, cells were labeled with anti-Caveolin-1
antibodies as the primary antibodies after fixation, followed by Alex-595
conjugated secondary antibody (Molecular Probes). Confocal microscopy was
carried out in the Biomedical Image Processing Laboratories at the University
of Minnesota using a Bio-Rad MRC 1024 system (Hercules, California) attached
to an Olympus (Melville, New York) microscope with a 60x oil objective.
The images were processed in Photoshop 7.0 (Adobe, San Jose, California).
Quantification was carried out in Openlab (Improvision, Coventry, United
Kingdom). The statistical analysis was done with GraphPad Prism software
program (San Diego, California).
Isolation of Triton X-100 insoluble complexes
Low-density Triton X-100 insoluble membrane lipid rafts of LN4 control and
MT1-MMP transfected variants were prepared as described by others
(Lisanti et al, 1993;
Stahl and Mueller, 1995). In
brief, cell lysates prepared in Mes-buffered saline (25 mM Mes, pH 6.5, 0.15 M
NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride) with 10 strokes of
a Dounce homogenizer were made 40% with sucrose and overlaid with a
30%–5% sucrose gradient. The gradients were centrifuged overnight at 190
000 x g and then fractionated. Individual fractions were probed
by Western blotting for caveolin-1 and MT1-MMP.
Cell Growth in Type I Collagen Gels
Cells (1.2 x 103) were mixed with 250 µL of type I
collagen (2 mg/mL in PBS; Collaborative, Waltham, Massachusetts) and allowed
to gel at 37°C for 30 minutes in 24-well plates, giving rise to
3-dimensional (3-D) collagen matrices. Fresh media containing 95% RPMI1640 and
5% fetal bovine serum were added to the wells with or without BB94 and changed
every 2 days. After 6 days, the growth of PC-3 and its derivatives was
photographed by a video camera attached to a Nikon microscope (Melville, New
York) at the University of Minnesota Bioimaging Processing Facility. Growth of
PC-3 cells in 3-D collagen gel was estimated by the diameter of the cell
clusters.
Cell Invasion on Type I Collagen Gel
500 µL of collagen (2 mg/mL in PBS; Collaborative) was prepared and
allowed to gel at 37°C for 30 minutes in 24-well plates giving rise to 3-D
collagen matrices. Cells (1.2 x 104) were plated on top of
the gel. Fresh media containing 95% RPMI1640 and 5% fetal bovine serum were
added to the wells with or without BB94 and changed every 2 days. After 4
days, the invasion of PC-3 and its derivatives were measured under a Nikon
microscope at the University of Minnesota Bioimaging Processing facility. The
invasion of PC-3 cells in 3-D collagen gel is estimated by the furthest depth
the cells invaded.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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C-MT1-MMP
(Figure 1B). The latter
construct is readily inserted and maintained in the cell surface without the
constraints regulating transport and insertion of WT-MT1-MMP
(Jiang et al, 2001). Stable
clones were picked up and characterized for MT1-MMP expression, proMMP-2
activation, and MT1-MMP subcellular localization. Although there was some
variability in the level of MT1-MMP cell surface localization of different
transfected substrains as determined by proMMP-2 activation, Pro5 cells
transfected with C-MT1-MMP consistently showed greater proMMP-2
activation than did WT-MT1-MMP transfected cells. However, proMMP-2 activation
by LN4 WT-MT1-MMP transfected cells was nearly equal to that of
C-MT1-MMP cells (Figure
1C). This pattern of cell surface MT1-MMP expression in Pro5 and
LN4 transfected cells was supported by FACS measurement of cell surface
immunofluorescence for MT1-MMP (Figure
1D). The level of C-MT1-MMP immunofluorescence on Pro5
cells was nearly 2 times that from WT-MT1-MMP, whereas the immunofluorescence
of C- and WT-MT1-MMP on LN4 cells was nearly equal. The MT1-MMP protein
expressed in WT- and C-MT1-MMP Pro5 and LN4 cells was examined by
immunofluorescence microscopy by first immunolabeling cell surface MT1-MMP
using a rabbit antibody and then permeabilizing the cells and using a mouse
antibody to detect intracellular and remaining MT1-MMP
(Figure 2). A greater level of
cell surface MT-MMP was detected in the LN4 cells for both WT- and C
constructs than in the Pro5 cells. This is consistent with the proMMP-2
activation and FACS measurements of a greater level of cell surface MT1-MMP in
LN4 than Pro5 cells.
The function of cell surface MT1-MMP on cell growth and invasion was tested
by plating vector control, WT-MT1-MMP, and C-MT1-MMP LN4 cells in 3-D
type I collagen gels (Figure
3). The size of colonies of WT- and C-MT1-MMP LN4 cells was
significantly larger at 4 days of culture than the mock transfected control
cells (Figure 3A). This growth
of overexpressing MT1-MMP LN4 cells was blocked by the MMP inhibitor BB-94,
indicating that enzymic activity of MT1-MMP is responsible for the enhanced
growth activity of the cells. BB-94 does not inhibit cell proliferation itself
in cells grown on plastic. The effect of MT1-MMP cell surface expression was
also examined with respect to tumor cell penetration into type I collagen
gels. The invasive potential of LN4 mock control cells was about 2-fold that
of Pro5 control cells 48 hours after plating cells on the collagen gel
surface, which substantiates the difference in cell surface MT1-MMP levels of
Pro5 and LN4 cells as evidenced by proMMP-2 activation and FACS analysis level
(Figure 1A and D). Pro5 cells
transfected with C-MT1-MMP invaded almost twice as far as those
transfected with WT-MT1-MMP and 3 times as far as those transfected with the
vector control. The LN4 WT- and C-MT1-MMP cells had a greater distance
of invasion than the Pro5 counterparts. The C-MT1-MMP LN4 cells invaded
farther than the WT-MT1-MMP cells, with the same trend as seen in the FACS
quantified cell surface MT1 (Figure
1D). BB-94 inhibited the invasiveness of all 3 groups, indicating
that the invasion is dependent upon MMP enzymatic activity and presumably
caused by MT1-MMP, which can cleave type I collagen
(Zucker et al, 2003). However,
these invasion assays were done in the presence of serum in the media, and
although MMP-2 cannot cleave type I collagen, it can degrade MT1-MMP
proteolyzed collagen fragments. Although we cannot determine the extent to
which activation of MMP-2 may contribute to cellular invasion of these cells,
invasion was clearly related to the extent of cell surface localization of
MT1-MMP.
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C-MT1-MMP in pro5 or LN4 cells
resulted in a marked increase in rate of tumor growth in nude mice as compared
with vector-only controls
(Table). In addition, it
appeared that the LN4 cells transfected with WT-MT1-MMP grew at a faster rate
than did C-MT1-MMP–expressing cells. This is evident in LN4 cells
in experiment 1, and also experiment 2, except that a large tumor had to be
removed early, reducing the average tumor size of the remaining tumors. A
reason for this is not clear, unless the absence of the cytoplasmic tail of
MT1-MMP alters substrate specificity of the proteinase and in some way growth
of the cells.
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Immunofluorescent Localization of MT1-MMP in Tumor Cells in Human Prostate Tissues
Because a greater level of MT1-MMP on the cell surface appeared related to
a greater MT1-MMP function and phenotype of PC-3 prostate cancer cells, we
examined MT1-MMP expression in human prostate tissues. Subcellular
localization of MT1-MMP in frozen sections of human prostate tissues was
examined using immunofluorescent microscopy. Expression of MT1-MMP was
demonstrated in benign hyperplastic (BPH) and cancerous prostate tissues by
Western blotting of RIPA buffer/protease inhibitor extracts of frozen sections
of human prostate tissue (Figure
4A). There was no difference in MT1-MMP levels between BPH samples
and those with 20% or less tumor. The level of MT1-MMP was significantly
higher in the Western blot of tumor tissue with 85% tumor (lane H) vs one with
20% tumor (lane F) from the same patient. The anti-MT1-MMP used in the Western
blot was the same antibody used for the immunofluorescent microscopy studies
and demonstrates remarkable specificity. We demonstrated MT1-MMP localization
in tumor cells of high Gleason grade tumors
(Figure 4Ba). Benign glands
were weak/negative in localization (not shown), but some more differentiated
cancerous glands showed a low level of cell boundary surface localization
(Figure 4Ba). The extent of
localization in Gleason 7 or 8 tumors appeared to be heterogeneous based on
the varied levels of fluorescent intensities amongst tumor cells
(Figure 4B), but there were
cells that showed distinct MT1-MMP localization (arrows) at the cell surface
and/or peripheral cytoplasm using immunofluorescent
(Figure 4B) and confocal
(Figure 4C) microscopy. Thus,
the cell surface localization of MT1-MMP in primary human prostate tumors
supports the contention that this factor is important in progression and
growth of prostate tumors.
The data above indicate an underlying molecular mechanism in the metastasis
of selected LN4 cells that positions and/or maintains greater levels of
MT1-MMP at the cell surface. The possible subdomain localization of MT1-MMP in
the plasma membrane of these prostate cancer cells was examined. Confocal
microscopy was used to examine the localization of caveolin-1 and MT1-MMP in
Pro5 and LN4 cells. The increased level of MT1-MMP on the cell surface of Pro5
cells transfected with the C construct is evident
(Figure 5A). There is evidence
of colocalization of MT1-MMP with caveolin-1 in the merged images
(Figure 5Ae), but at the same
time there appear to be caveolae without significant levels of MT1-MMP for
both Pro5 and LN4 cells (Figure 5 Ae and
Be). These data indicate that MT1-MMP is localized in caveolae,
which was further evaluated by separation of caveolae-enriched lipid raft
fractions of TritonX-100 resistant membrane components in sucrose gradients
(Figure 5C). Caveolae,
fractions enriched in caveolin-1, of LN4 control and WT- and C-MT1-MMP
transfected cells showed colocalization of MT1-MMP by Western blotting.
Control and WT-MT1-MMP cells also demonstrated MT1-MMP in fractions not
containing caveolin-1, indicating that MT1-MMP is found in other membrane
fractions that are not caveolae (Figure
5C). The C variant, on the other hand, is found almost
exclusively in the caveolae fractions, suggesting that C-MT1-MMP is
rapidly translocated to and retained in caveolae, and found only to a limited
extent in other membrane components of the cell.
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The possible localization of MT1-MMP in caveolae was also tested using
nystatin, a cholesterol-binding drug that disrupts caveolae but not
clathrin-coated pits or other submembranous structures
(Rothberg et al, 1992), on the
cell surface localization of MT1-MMP and caveolin-1. Control Pro5 cells
expressed little detectable MT1-MMP, but did show cell surface caveolin-1,
presumably in caveolae, as well as intracytoplasmic caveolin-1
(Figure 6). Nystatin treatment
increased the intracellular immunoreactivity to caveolin-1, indicating a
translocation of caveolin-1 from the cell surface. The C transfectant
showed greater levels of MT1-MMP on the cell surface than did WT, and this
localization was minimized by nystatin treatment. There appears to be some
colocalization of C-MT1-MMP with caveolin-1. Indication of cholesterol
in the cell surface localization of MT1-MMP is also evidenced by the
disruption of proMMP-2 activation by nystatin determined in zymograms
(Figure 7). Pro5 control cells
did not activate proMMP-2, whereas the WT- and C-MT1-MMP transfectants
did. The C-MT1-MMP activation of proMMP-2 appeared to be more resistant
to nystatin treatment, which may reflect a different plasma membrane domain
location for at least part of the C-MT1-MMP population.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In the human prostate, MT1-MMP has been localized by immunohistochemistry in basal cells and basal-lateral membranes of secretory cells of benign prostatic glands and in cells in high-grade PIN and cancer (Upadhyay et al, 1999; Udayakumar et al, 2003; Sroka et al, 2008), and although localization in cancer cells has been described as localized to apical cytoplasm (Sroka et al, 2008) or varied and heterogeneous to diffuse (Upadhyay et al, 1999), there was greater staining in invasive areas with a significant association of MT1-MMP in the same specimens (Upadhyay et al, 1999). We have now shown a cell surface localization as well as diffuse cellular distribution of MT1-MMP in some human prostate cancers. Although the level of MT1-MMP message has been reported to be lower in primary prostate cancer tissues and primary cell cultures derived from them than in nonmalignant prostate tissue (Jung et al, 2003), MT1-MMP, as well as MMP-2 and MMP-9, messages and proteins are detected in tumor and stromal cells of clinical specimens and are up-regulated in PC-3 cells growing on human bone in vivo in SCID mice (Nemeth et al, 2002).
MT1-MMP is anchored in the plasma membrane by a type I transmembrane domain. Cell surface activity of MT1-MMP is regulated in part by cellular trafficking, both in transporting the proteinase to and in removing it from the cell surface by internalization (Jiang et al, 2001; Zucker et al, 2002; Rozanov et al, 2004). Internalization of MT1-MMP can occur via clathrin-coated pits and caveolae (Ramacle et al, 2003). MT1-MMP and other proteinase system components such as MMP-2, cathepsin B, and urokinase receptor have also been localized in caveolae (Stahl and Mueller, 1995; Annabi et al, 2001; Puyraimond et al, 2001). Caveolae are flask-shaped invaginations of cholesterol-glycolipid–enriched raft microdomains of the plasma membrane (Anderson and Jacobson, 2002; Razani et al, 2002) that have functional roles in endocytosis and signal transduction and are commonly enriched in receptor tyrosine kinases, GPI anchored proteins, and caveolin-1 (Conner and Schmid, 2003). Caveolin-1 is a 22-kDa membrane-spanning protein in which both the amino and carboxyl ends are cytoplasmically oriented, and exists in a highly ordered oligomeric complex of about 14–16 monomers (Conner and Schmid, 2003). Caveolin-1 is highly expressed by PC-3 but not by LNCaP human prostate cancer cells (Thompson, 1998–1999; Lu et al, 2001), and by primary and metastatic prostate cancers, with highest levels found with increased Gleason grade and after androgen ablation therapy (Mouraviev et al, 2002). The control of caveolin-1 expression in prostate appears to be complex. Cui et al (2001) reported hypermethylation of CpG islands in the promoter region of the caveolin-1 gene determined in laser-capture microdissected tumor vs adjacent normal epithelium. They also noted substantial immunohistochemical staining of caveolin-1 in some of the tumor epithelium, whereas normal epithelium was consistently weak in expression. In contrast to prostate, caveolin-1 levels are down-regulated in a number of human cancers, including breast (Razani and Lisanti, 2001), in which caveolin-1 has been reported to have tumor suppressor activity (Sloan et al, 2004; Williams et al, 2004).
A plasma membrane caveolae localization for MT1-MMP function in PC-3 cells is indicated by the loss of cell surface MT1-MMP immunofluorescence and proMMP-2 activation upon disruption of caveolae by the cholesterol binding drug nystatin. In addition, there is colocalization of caveolin-1 with MT1-MMP in gradient-separated detergent-resistant membrane fractions. Male TRAMP mice heterozygous or homozygous for caveolin-1 knockout present with a much-reduced tumor burden and decreased lymph node and lung metastases, but no reduction in the number of tumors (Williams et al, 2005). Targeted down-regulation of caveolin-1 in TRAMP-C1 cells using small interfering RNA reduced their tumorigenic and metastatic potential in vivo and resulted in increased apoptosis (Williams et al, 2005). These data indicate that caveolae function in prostate tumor progression and that caveolae-localized MT1-MMP may have a critical role in mediating associated tumor growth and metastasis. However, there is evidence for functioning noncaveolar cholesterol-rich lipid rafts in prostate tumor cells. For example, epidermal growth factor receptor (EGFR) is in detergent-soluble membrane fractions of LNCaP cells, which do not have caveolae, but upon phosphorylation by Akt1, EGFR is localized in cholesterol-rich lipid rafts (Zhuang et al, 2002). It is possible that a portion of the membrane-associated MT1-MMP may be present in noncaveolar cholesterol-rich lipid rafts.
The data on caveolae in prostate cancer support our finding that
transfecting WT-MT1-MMP, or the mutated form lacking the cytoplasmic tail
(C), resulted in increased cell surface expression of MT1-MMP activity,
cell penetration of collagen gels, and growth in nude mice. Rozanov et al
(2004) have noted in MCF-7
breast tumor cells, as we have here in prostate PC-3 cells, that the
cytoplasmic tail–truncated MT1-MMP, in contrast to the wild-type form,
is preferentially localized in caveolae. It would appear that the absence of
the cytoplasmic tail affects the release mechanism of MT1-MMP from caveolae.
However, although they found that C MT1-MMP could activate proMMP-2 and
cause collagen gel contraction, it was not as able as WT-MT1-MMP to cleave
E-cadherin nor to generate the increased growth rate in vivo in nude mice as
were breast tumor cells with WT-MT1-MMP. MT1-MMP–stimulated growth of
U251 glioma (Deryugina et al,
2002) and MCF-7 breast tumor cells
(Sounni et al, 2002) is
attributable in part to up-regulation of vascular endothelial growth factor
(VEGF) and increased angiogenesis of the tumor in nude mice. This
up-regulation of VEGF transcription is dependent on the catalytic function of
MT1-MMP and its cytoplasmic tail (Sounni
et al, 2004). In contrast, our data showed that the growth rate of
both C- and WT-MT1-MMP–expressing prostate tumors was markedly
elevated and similar in nude mice. This may indicate that in prostate tumor
cells both MT1-MMP collagenase activity and the cleavage of cell surface
proteins can occur without regulation of these functions via the cytoplasmic
tail.
There is increasing evidence of regulation of MT1-MMP function via its
cytoplasmic tail domain. The cytoplasmic tail appears necessary for recycling
of MT1-MMP from the cell surface via clathrin-coated pits. There is also
evidence that the cytoplasmic domain regulates binding of hyaluronan at the
cell surface, because the wild-type but not the C form decreased the
amount of hyaluronan bound as well as levels of CD44, the hyaluronan receptor
(Annabi et al, 2004). The
cytoplasmic tail of MT1-MMP also appears to transduce hyaluronan cell surface
binding signaling through the mitogen-activated protein kinase (MAPK) pathway
because the MAPK extracellular signaling–regulated kinase inhibitor
PD98059 reduced hyaluronan cell surface binding in wild-type–expressing
but not in C-expressing U-87 glioma cells. Phosphorylation of tyrosine
14 of caveolin-1 by Src-family kinases upon VEGF stimulation of endothelial
cells increases its association with MT1-MMP via interaction of the
cytoplasmic domain with the phosphocaveolin-1
(Labrecque et al, 2004).
In conclusion, localization of MT1-MMP with MMP-2, urokinase, cathepsin B, and other proteinases in caveolae can serve as a mechanism to concentrate the proteolyzing power of the prostate cancer cell at the cell surface in apposition to extracellular proteins (Zucker et al, 2003). The plasma membrane–anchored MT1-MMP is the known main mediator of proteolytic events in the cancer cell surface that include proteolysis of ECM proteins including types I and II collagens, cleavage of cell surface receptors, and activation of MMP-2 and MMP-13 (Rozanov and Strongin, 2002). Activities of MT1-MMP and MMP-2 in turn are controlled by unique cleavage pathways, inhibition by tissue inhibitor of metalloproteinase trafficking to/from the plasma membrane, and molecular interactions that facilitate localization to the cell surface. Activation of MMP-2 introduces additional protein substrates to pericellular proteolysis in the tumor microenvironment. Our studies indicate that mechanisms to transport and/or support MT1-MMP in the prostate tumor cell plasma membrane, and activation of cell surface and pericellular MMP proenzyme forms, may be critical steps in progression of prostate tumor cells.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Annabi B, Lachambre M-P, Bousquet-Gagnon N, Page M. Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains. Biochem J. 2001; 353: 547 –553.[CrossRef][Medline]
Annabi B, Thibeault S, Moumdjian R, Beliveau R. Hyaluronan cell
surface binding is induced by type I collagen and regulated by caveolae in
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