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Regular Articles |
§
From the Departments of Dermatology,*
Obstetrics and
Gynecology,
and
Pathology,
Turku University Central Hospital;
MediCity Research Laboratory and the Department of Medical
Biochemistry,§
University of Turku, Turku,
Finland; the Department of Dermatology,¶
Helsinki University Central Hospital, Helsinki, Finland; and the
Department of Obstetrics and Gynecology,||
Ninewells
Hospital, University of Dundee, Dundee, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human collagenase-3 (MMP-13), originally cloned from breast carcinoma tissue,5 is characterized by exceptionally wide substrate specificity compared to other MMPs. MMP-13 cleaves fibrillar collagens, preferring type II collagen over types I and III, and displays over 40-fold stronger gelatinase activity than MMP-1 and MMP-8.6-8 In addition, MMP-13 degrades type IV, IX, X, and XIV collagens, tenascin, fibronectin, and aggrecan core protein.8,9 Apparently due to its ability to degrade a wide range of matrix components, the physiological expression of human MMP-13 is limited to situations in which rapid and effective remodeling of collagenous extracellular matrix is required, ie, fetal bone development and postnatal bone remodeling.10,11 On the other hand, MMP-13 apparently plays a role in excessive degradation of collagenous matrix in osteoarthritic cartilage,7,12,13 rheumatoid synovium,11,13,14 chronic cutaneous ulcers,15 intestinal ulcerations,16 and periodontitis.17 The wide substrate specificity of MMP-13 also makes it a potent proteolytic tool for invading tumor cells and the expression of MMP-13 has been documented in breast carcinomas,5,18,19 squamous cell carcinomas (SCCs) of the head and neck,20,21 cutaneous basal cell carcinomas,21 and chondrosarcomas.22 Our recent observations show that in SCCs of the skin, oral cavity, and larynx, MMP-13 is expressed mainly by tumor cells at the invading margin of the tumor (and also in some cases by stromal fibroblasts),20,21 whereas no MMP-13 expression is noted in intact or re-epithelializing epidermis, healthy oral mucosa, or normal keratinocytes in culture.15,20,21,23
To further elucidate the role of MMP-13 in tumor cell invasion, we have examined its expression in invasive malignant tumors of the female genital tract. Previous studies have demonstrated expression of gelatinase-A (MMP-2) and gelatinase-B (MMP-9) in endometrial carcinomas24-26 and expression of MMP-2, MMP-9, and MT1-MMP in cervical25,27-29 and ovarian30-34 carcinomas. At present, neither the level of expression of MMP-13 in any of these neoplastic tumors nor the role of MMPs in the invasion of vulvar carcinoma is known. In this study we show that MMP-13 mRNA is abundantly expressed by tumor cells of vulvar SCCs in vivo, whereas no expression of MMP-13 is detected in cervical SCCs or endometrial or ovarian adenocarcinomas in vivo. In addition, MMP-13 expression was noted in vulvar and cervical carcinoma cells in culture, especially by 5 of 6 vulvar and cervical SCC cell lines harboring human papilloma virus (HPV) 16 or 68 DNA, whereas no correlation was detected between MMP-13 expression and p53 gene mutations in vulvar SCC cell lines. These results provide evidence that MMP-13 is specifically expressed by malignantly transformed squamous epithelial cells, including vulvar SCC cells, and may serve as a marker for their invasive capacity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Formalin-fixed, paraffin-embedded specimens of vulvar SCCs
(n = 12, Table 1
),
cervical SCCs (n = 12; 1 in situ
carcinoma, 6 T1N0M0, 2 T2N0M0, 1 T3N1M0,1 T4N0M0 and 1 T4N0M1; 5 grade
II, 6 grade III), endometrial adenocarcinomas (n
= 11), and ovarian serous adenocarcinomas (n =
8) were obtained from the Department of Pathology, University of Turku,
Finland. In vitro transcribed antisense and sense RNA probes
were labeled with [
-35S]UTP as described
previously.35
A 491-bp ApaI fragment
from the 5' end of the human MMP-13 cDNA23
was
used to transcribe antisense and sense RNAs, respectively. The
specificity of these probes has been shown
previously.10,15-17,20,21
Generation and
specificities of human MMP-1 and stromelysin-1 (MMP-3) antisense and
sense RNA probes have been described
previously.36,37
Tissue sections 5 µm thick
were treated with proteinase K, washed in 0.1 mol/L triethanolamine
buffer containing 0.25% acetic anhydride, and covered with 50–100
µl of hybridization buffer containing 2.5–5 x
104
cpm/µl of
35S-labeled antisense or sense RNA probe. After
hybridization at 50–55°C for 18 hours in a humidified chamber, the
slides were washed under stringent conditions, including RNase A
treatment, to remove unhybridized probe.36
After
autoradiography for 25 to 35 days, the photographic emulsion was
developed and the slides were stained with hematoxylin and eosin.
Samples of tissues previously shown to express MMP-13 mRNA (breast
carcinoma)5
and MMP-1 and MMP-3 mRNA (chronic
dermal ulcers)15
were used as positive controls
and a labeled sense probe was used as a negative control in each
in situ hybridization.
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Immunostainings of sections parallel to those used for in situ hybridization were performed by the avidin-biotin-peroxidase complex technique37 using diaminobenzidine as chromogenic substrate. Monoclonal antibodies anti-MMP-9 (GE213, dilution 1:500; Diabor, Oulu, Finland),38 anti-MMP-13 (181–15A12, dilution 1:30) (Oncogene Research Products, Cambridge, MA), anti-MT1-MMP (114–6G6, dilution 1:10) (Oncogene Research)39 and anti-MMP-2 (42–5D11, dilution 1:200) (Oncogene Research) were used. Affinity-purified polyclonal antiserum against matrilysin (MMP-7)40 was diluted 1:800. For staining with MMP-9 and MMP-7 antibodies, sections were pretreated with 10 µg/ml trypsin. The tissues were counterstained with hematoxylin. Negative control stainings were performed with preimmune mouse ascites fluid for mouse monoclonal antibodies and with rabbit preimmune serum for polyclonal antibodies.
Cell Cultures
The establishment and characterization of the vulvar SCC cell
lines (Table 2)
,41-43
cervical
SCC cell lines (CaSki, Me-180, Hx-151, Hx-156, and
UT-CxC-1),44
cervical glassy cell carcinoma cell
line (UM-GCC-1),44
and endometrial carcinoma cell
lines (KLE, RL95-2, UM-EC-1, -2, UT-EC-2A, -2B, -2C, -3, -5, and
-6)45
have been described previously and their
p53 and HPV status has been documented.43-47
In
addition, ovarian carcinoma cell lines (n = 9)
(SK-OV-3, Ca-OV-3, UT-OV-2, -3, -4, -5, -6, -9, and -10) were used. All
cell lines were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 6 mmol/L glutamine, nonessential amino acids,
and 10% fetal calf serum (FCS). Cell lines UT-DEC-1 and UT-DEC-2,
established from premalignant vaginal intraepithelial
neoplasias48,49
were cultured in
Keratinocyte-Serum Free Medium (Gibco BRL, Gaithersburg, MD)
supplemented with bovine pituitary extract (50 µg/ml) and recombinant
epidermal growth factor (5 ng/ml) in early subcultures. UT-DEC-1 cells
in passage 89 were cultured in DMEM supplemented with 6 mmol/L
glutamine, nonessential amino acids, and 5% FCS.
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Human recombinant tumor necrosis factor- (TNF-) and
transforming growth factor-ß1 (TGF-ß1) were obtained from Sigma
Chemical (St. Louis, MO). Human recombinant transforming growth
factor- (TGF-) was obtained from PeproTech EC (Rocky Hill, NJ).
RNA Analysis
Total cellular RNA was isolated from cell cultures using the
single step method.50
Northern blot
hybridizations were performed as described
previously23
with cDNAs labeled with
[-32P]dCTP by random priming. Human MMP-13
cDNA fragments covering the coding region and part of the
3'-untranslated region, totaling 1931 bp, were used as
probes.23
In addition, a 2-kb human MMP-1
cDNA,51
a 1.5-kb human MMP-3
cDNA,52
a 2.7-kb MMP-2
cDNA,53
a 2-kb human MMP-9
cDNA,54
a 0.7-kb human MT1-MMP (MMP-14)
cDNA,55
a 1-kb human MMP-7
cDNA,56
and a 1.3-kb rat cDNA for
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)57
were used for Northern blot
hybridizations. [32P]-cDNA/mRNA hybrids were
visualized by autoradiography and the mRNA levels were quantitated by
densitometry and corrected for the levels of GAPDH mRNA in the same RNA
samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our recent observations show that MMP-13 is expressed by tumor
cells in SCCs of the head and neck, as well as by cell lines
established from corresponding tumors.20
In
contrast, MMP-13 is not expressed by normal human epidermal
keratinocytes in culture, in intact skin, or in re-epithelializing
cutaneous wounds, indicating that the ability to express MMP-13 is
specific to transformed keratinocytes.15,20,21,23
In the present study, we have further elucidated the role of MMP-13 in
tumor invasion by examining its expression in invasive malignant tumors
of the female genital tract with distinct histogenetic origin, ie, SCCs
of vulva and uterine cervix and adenocarcinomas of endometrium and
ovary. Initially, we examined the expression of MMP-13 mRNAs in these
tumors by in situ hybridization. Signal for MMP-13 mRNA was
detected in 9 of 12 vulvar SCC samples (5 of 6 primary SCCs and 4 of 6
recurrent tumors) (Table 1)
. In contrast, no MMP-13 mRNAs were detected
in any of the cervical SCCs (n = 12)
representing different stages of the tumor, nor in the endometrial
(n = 11) or ovarian (n =
8) adenocarcinomas examined (not shown). In comparison, expression of
MMP-1 mRNA was detected in 6 cervical SCCs, in tumor cells in 2
carcinomas, and in stromal cells in 4 tumors. In addition, MMP-1 was
detected in stromal cells in 2 endometrial carcinomas and 1 ovarian
carcinoma (not shown).
In the majority of MMP-13-positive vulvar SCCs, MMP-13 mRNA expression
was confined to tumor cells (Figures 1, A
-C, E, and F, and 2, A, B, D, and E).
However, in three tumors MMP-13 expression was also noted in stromal
cells (Table 1)
. No specific signal was detected in parallel tissue
sections hybridized with the sense probe (Figure 1D)
. No MMP-13 mRNA
could be detected in normal intact vulvar epithelium (Figure 1H)
.
|
; Table 1
).
Abundant MMP-13 expression was detected in both small (stage T1) and
larger (stages T2 and T3) tumors, indicating that expression of MMP-13
is activated early in vulvar SCC development (Table 1)
. In general,
expression of MMP-13 was detected in well differentiated (grade 1)
tumors, as well as in moderately (grade 2) and poorly (grade 3)
differentiated tumors (Table 1)
. However, differentiating tumor cells
were devoid of MMP-13 mRNA, whereas expression of MMP-13 mRNA was
detected in undifferentiated tumor cells (Figure 2, D and E)
.
Expression of MMP-13 was also examined by immunohistochemistry in five
vulvar SCCs expressing high levels of MMP-13 mRNA, ie, from patients 2,
3, 4, 7, and 9. Staining for MMP-13 was noted in all these vulvar SCCs,
primarily in tumor cells and occasionally in stromal fibroblasts
(Figure 2H)
. No staining was detected in negative controls using
preimmune mouse ascites fluid instead of primary antibody (Figure 2I)
.
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. Of these, in two tumors MMP-1 mRNA was detected only in
stromal cells. In many of the tumors examined, expression of MMP-1 in
tumor cells was lower than MMP-13 expression (Figure 1, E
-G). Of the 12
vulvar SCCs examined, two were entirely negative for both MMP-13 and
MMP-1 mRNA (Table 1)
. Intact vulvar epithelium was negative for MMP-1
mRNA (not shown). Coordinate Expression of MMP-13 and MT1-MMP by Tumor Cells in Vulvar SCCs
Latent MMP-13 is activated by stromelysin-1 (MMP-3), membrane type
1 MMP (MT1-MMP), and gelatinase-A (MMP-2).6,58
In
this context, we also examined the expression of these MMPs in vulvar
SCCs. Expression of MT1-MMP was detected by immunohistochemistry in all
10 vulvar SCCs examined (Table 1)
. Interestingly, the expression of
MT1-MMP was abundant in tumor cells, often noted in the regions with
MMP-13-positive tumor cells in parallel sections (Figure 2, A
-F and H).
In addition, in certain tumors, stromal fibroblasts stained positive
for MT1-MMP, although less intensely than tumor cells (Figure 2, C and F)
. Expression of MMP-2 was also detected by immunohistochemistry in
stromal cells of all 12 vulvar SCCs examined and in tumor cells in 9
SCCs (Table 1)
. In contrast to MT1-MMP, staining for MMP-2 was often
more pronounced in stromal fibroblasts than in tumor cells (Figure 2G)
.
MMP-2-positive cells were also detected in the vicinity of tumor cells
expressing MMP-13 in parallel sections (Figure 2, D, E, and G)
. These
results show that the expression of MMP-13 is associated with the
expression of MT1-MMP or MMP-2 in vulvar SCCs, providing an optimal
environment for pericellular activation of tumor cell-derived latent
MMP-13. Using in situ hybridization, expression of MMP-3
mRNA was detected in five tumors, all of which also expressed MMP-13
(Table 1)
. However, the signal for MMP-3 mRNA was generally observed in
isolated tumor cells and it did not seem to colocalize with that of
MMP-13 (not shown).
Expression of MMP-9 by Tumor and Inflammatory Cells in Vulvar SCCs
As MMP-13 has been show to activate latent
MMP-9,59
we also examined the expression of MMP-9
in vulvar SCCs by immunostaining. Interestingly, MMP-9 expression was
detected in mononuclear and polymorphonuclear inflammatory cells in
stromal infiltrate adjacent to tumor in all nine SCCs examined (Table 1)
. In addition, MMP-9-positive tumor cells were detected at the
invading edge of six tumors (Figure 3A
,
Table 1
). In certain vulvar SCCs, MMP-9-positive tumor cells or
inflammatory cells colocalized with MMP-13-expressing tumor cells
(Figure 3, A and B)
. However, MMP-9-positive tumor and inflammatory
cells were also detected in areas devoid of MMP-13 mRNA (not shown).
Expression of matrilysin (MMP-7) was also detected by immunostaining in
tumor cells of 3 of 5 vulvar SCCs examined (not shown).
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We also examined the expression of MMP-13 in 11 cell lines
established from primary or recurrent vulvar SCCs or their metastases
(Table 2)
. As shown in Figure 4A
and
Table 2
, basal expression of MMP-13 mRNAs was detected in 9 of 11
vulvar SCC cell lines examined (3 of 5 cell lines from primary
tumors and all 6 cell lines from recurrent tumors or metastases) (Table 2)
. Basal expression of MMP-1 mRNA was detected in 4 of 11 SCC cell
lines (Figure 4A
, Table 2
). Only 2 of 11 vulvar SCC cell lines were
negative for both MMP-13 and MMP-1 (Table 1)
. Interestingly, expression
of MT1-MMP was detected in all vulvar SCC cell lines examined (Figure 4A
, Table 2
). Expression of MMP-9 was detected at basal level in one
vulvar SCC cell line examined (Table 2)
, whereas expression of MMP-7
was detected in 7 cell lines (Figure 4A
, Table 2
). In contrast,
expression of MMP-3 or MMP-2 mRNA was not detected in any vulvar SCC
cell line examined (Table 2)
.
|
.43,46,47
Interestingly,
MMP-13 expression was detected in four vulvar SCC cell lines with p53
mutation and in one cell line that harbors HPV 16 DNA and has wild-type
p53 gene (Table 2)
. However, lack of MMP-13 expression in two cell
lines with mutated p53 shows that inactivation of p53 is not alone
sufficient for induction of MMP-13 expression (Table 2)
. Furthermore,
basal expression of MMP-13 mRNA was detected in all three cell lines
with wild-type p53 gene and no HPV DNA, indicating that inactivation of
p53 is not essential for induction of MMP-13 expression in vulvar SCC
cells (Table 2)
.
Enhancement of MMP-13 Expression in Vulvar SCC Cells by TNF- and
TGF-ß
The expression of MMP-13 in cell lines from SCCs of the head and
neck is enhanced by TNF-, TGF-ß, and
TGF-.20
In this context we examined the
regulation of MMP-13 gene expression in selected vulvar SCC cell lines
by Northern blot hybridizations. Expression of MMP-13 mRNAs was
enhanced by 2.5-fold and 17.5-fold by a 24-hour treatment with TNF-
(20 ng/ml) in cell lines UT-SCV-2 and UT-SCV-3, respectively (Figure 4B)
. TGF-ß1 (5 ng/ml), in turn, up-regulated expression of MMP-13
mRNAs in UM-SCV-7 and UT-SCV-2 cells by 32-fold and 6.3-fold,
respectively (Figure 4B)
. Interestingly, the expression of MMP-1 was
not stimulated by TNF- or TGF-ß in any of the cell lines, and was
even suppressed by 83% with TGF-ß1 in cell line UT-SCV-3. In
contrast, treatment of UM-SCV-7 and UT-SCV-2 cell lines by TGF- (10
ng/ml) enhanced MMP-1 mRNA expression threefold and 3.9-fold,
respectively (Figure 4B)
. In comparison, MMP-7 mRNA abundance was
up-regulated by TNF- in UM-SCV-7 (2.1-fold) and UT-SCV-2 (1.9-fold)
cells, and MMP-9 mRNA levels were stimulated by TGF-ß1 in UM-SCV-7
cells (5.8-fold) and by TNF- in UT-SCV-3 cells (4.6-fold) (Figure 4B)
. Interestingly, MT1-MMP mRNA abundance was enhanced by TGF-ß (up
to 4.6-fold), in all three cell lines and by TNF- (up to 3.7-fold)
and TGF- (up to 2.5-fold) in UT-SCV-2 and 3 cell lines (Figure 4B)
.
Expression of MMP-13 in Cervical Carcinoma Cells
The expression of MMP-13 was also examined in cell lines
established from cervical, endometrial, and ovarian carcinomas. Of the
six cervical carcinoma cell lines examined, one (UM-GCC-1) was derived
from glassy cell carcinoma and the remaining five from SCCs. None of
the cervical carcinoma cell lines examined has mutations in p53 gene,
but ME-180 cell line harbors HPV 68 DNA and other cell lines contain
HPV 16 DNA.43,44
Interestingly, 4 of 5 cervical
SCC cell lines expressed clearly detectable basal levels of MMP-13
mRNAs, suggesting an association between oncogenic HPV infection and
the ability to express MMP-13 (Figure 5)
.
However, no basal expression of MMP-13 gene was detected in cell lines
Hx-156 and UM-GCC-1 harboring HPV 16 DNA, indicating that
transformation by oncogenic HPV is not alone sufficient to turn on
MMP-13 expression in cervical carcinoma cells (Figure 5)
.
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, TGF-ß, or TGF- (not shown). Expression of MMP-13 by Cell Lines from Vaginal Premalignant Lesions
To further elucidate the role of oncogenic HPV in MMP-13
expression by transformed squamous epithelial cells, we examined two
immortalized cell lines established from premalignant vaginal
intraepithelial neoplasias. UT-DEC-1 and UT-DEC-2 express HPV 33 and
HPV 16 E6/E7 mRNA, respectively, and contain wild-type p53 and
retinoblastoma genes.48,49
Neither cell line
expressed MMP-13 mRNAs in passages 8 and 21 (UT-DEC-1) or in passages
10, 26, and 40 (UT-DEC-2), either at basal level or after treatment
with TNF- or TGF-ß (Figure 6
and
data not shown). These observations show that immortalization of
squamous epithelial cells by oncogenic HPV does not alone render them
capable of expressing MMP-13. UT-DEC-1 cell line was further examined
at late subculture (passage 89), at which stage abundant expression of
MMP-13 mRNAs was detected at basal level or after treatment with
TNF- (Figure 6)
. In contrast, treatment of cells with TGF-ß
suppressed MMP-13 mRNA expression by 60% (Figure 6)
. Interestingly,
UT-DEC-1 cells at subculture 89 grow anchorage-independently in the
presence of glucocorticoid and epidermal growth
factor,49
indicating that the ability to express
MMP-13 is associated with selection of a malignant phenotype of these
transformed vaginal epithelial cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression of MT1-MMP was detected by immunostaining in tumor cells of all vulvar SCCs examined and MT1-MMP mRNA was also detected in all cell lines derived from vulvar SCCs, suggesting that MT1-MMP plays an important role in their invasion capacity. However, less intense immunostaining for MT1-MMP was also observed in stromal fibroblasts in a subset of vulvar SCCs. Interestingly, MT1-MMP-positive tumor cells often colocalized with SCC cells expressing MMP-13 mRNA. Staining for MMP-2 was also detected in all vulvar carcinomas examined, but it appeared to be more pronounced in stromal fibroblasts often detected in the vicinity of MMP-13-expressing tumor cells. In SCCs of the head and neck, expression of MMP-2 and MT1-MMP mRNA has previously been reported in stromal cells60,61 and expression of MT1-MMP by tumor cells as well.62 It is likely that colocalization of the cells expressing MT1-MMP and MMP-2 with tumor cells producing MMP-13 creates optimal conditions for pericellular activation of tumor cell-derived latent MMP-13. In addition, expression of MMP-3 and MMP-7 by some SCC tumor cells provides further activity for a tumor-driven proteolytic cascade, in which latent MMP-13 can be activated by MT1-MMP or MMP-3, both of which are expressed by tumor cells. Furthermore, in analogy with cutaneous SCCs,60,63 expression of MMP-9 by invading tumor cells and adjacent tumor infiltrating inflammatory cells adds a further link to this MMP cascade, as MMP-13 can activate latent MMP-9.59 Together all these observations provide evidence that efficient breakdown of the extracellular matrix surrounding invasive SCCs involves interplay between tumor cells, stromal cells, and inflammatory cells. Because there are differences in the substrate specificity of the MMPs present in the peritumoral environment of vulvar SCCs, it can be proposed that each of these MMPs has a distinct role in SCC invasion. Therefore, it is possible that specific inhibition of the expression or activity of even one of these MMPs, eg, MMP-13, could disturb the balance in this complex network of proteases and markedly inhibit invasion by SCC tumor cells.
Expression of MMP-13 was also detected in 4 of 5 cell lines derived from cervical SCCs, although no expression of MMP-13 could be detected in vivo in any of the cervical carcinomas in our material, which contained moderately and poorly differentiated tumors representing different stages of invasion. It is therefore possible that MMP-13 does not play a crucial role in invasion of cervical SCCs in vivo, but that selection of an MMP-13-expressing cell population may occur during subculturing of cervical SCC cells as a consequence of genomic instability. This notion is also supported by our observation that UT-DEC-1, a cell line derived from premalignant vaginal dysplasia, expressed MMP-13 only after it had been selected by subculturing to grow anchorage-independently.
Transformation of vulvar or cervical epithelial cells is often associated with inactivation of p53 tumor suppressor gene, either by mutation or by oncogenic HPV E6 protein.43,46,47 In the present study, comparison of the p53 and HPV status of the vulvar SCC cell lines with their MMP-13 expression revealed detectable basal levels of MMP-13 mRNAs in cell lines with intact p53 gene and no HPV DNA, indicating that p53 inactivation is not essential for induction of MMP-13 expression in transformed vulvar squamous epithelial cells. In addition, no MMP-13 expression was detected in two vulvar SCC cell lines with mutated p53 gene, showing that inactivation of p53 does not alone render SCC cells capable of expressing MMP-13. Interestingly, one vulvar SCC cell line harboring HPV 16 DNA and wild-type p53 gene expressed abundant levels of MMP-13 mRNA. Furthermore, all cervical SCC cell lines expressing MMP-13 harbored HPV 16 or 68 DNA, suggesting an association between oncogenic HPV infection and induction of MMP-13 expression in vulvar and cervical SCC cells. However, no MMP-13 expression was detected either in two cervical carcinoma cell lines harboring HPV 16 DNA or in early subcultures of two HPV-positive cell lines from vaginal premalignant intraepithelial lesions, indicating that oncogenic HPV alone is not sufficient to induce MMP-13 expression. However, it is likely that loss of cellular growth control due to p53 inactivation and subsequent genomic instability plays an important role in the selection of an invasive, transformed squamous epithelial cell population capable of expressing MMP-13. In summary, these observations provide further evidence that MMP-13 expression is not simply an indicator for transformation of squamous epithelial cells, but may also serve as a marker for the invasive capacity of SCCs.
Expression of MMP-13 mRNAs in selected vulvar SCC cell lines, as in
SCCs of the head and neck, is stimulated by TNF- or TGF-ß, which
may play a role in stimulating vulvar SCC cells in vivo
during ulceration and inflammation of the tumors. However, the response
of the vulvar SCC cell lines to TNF- and TGF-ß appears variable,
indicating heterogeneity between these cell lines. Because TGF-ß is a
potent inhibitor of epithelial cell growth, it is not surprising that
malignant epithelial cells may lose their response to TGF-ß in the
process of malignant transformation. Nevertheless, these observations
show that MMP-13 expression by vulvar SCC cells is susceptible to
stimulation by TNF- and TGF-ß, both of which also enhance
expression of MMP-1 by normal
keratinocytes.1,2,23
However, expression of MMP-1
mRNA by vulvar SCC cells was not enhanced by TNF- or TGF-ß,
indicating differential regulation of MMP-13 and MMP-1 in vulvar SCC
cells. Interestingly, expression of MT1-MMP mRNA in these vulvar SCC
cells was stimulated by TGF-ß and, in two cell lines, also by TNF-
and TGF-, providing evidence that these inflammatory cell-derived
modulators may also play a role in up-regulation of MT1-MMP expression
in vulvar SCCs in vivo.
Invasion and metastasis of malignant cells is a multistep process involving detachment of cells from primary tumor, degradation of structural barriers such as basement membrane and collagenous extracellular matrix, and migration of cells through the degraded matrix.1-3 It is evident that invasion of SCC cells in vivo involves interplay between tumor cells, stromal cells, and inflammatory cells, all of which can express a variable set of MMPs and contribute to degradation of stromal matrix components of SCCs. In this context, it is interesting that MMP-13 is specifically expressed by transformed squamous epithelial cells. Furthermore, lack of MMP-13 expression in normal keratinocytes both in vitro23 and in vivo15,20,21 provides strong evidence that MMP-13 is specifically activated in SCC cells as the primary tumor progresses toward an invasive phenotype. This notion is supported by our observation that cell lines established from premalignant vaginal intraepithelial neoplasias do not express MMP-13 until selection of a late subculture cell population capable of anchorage-independent growth. This hypothesis is also supported by our recent findings showing lack of MMP-13 expression in cutaneous premalignant tumors or in SCCs in situ.21 These observations provide further evidence that MMP-13 expression is a marker for the invasion capacity of SCC cells in vivo. Finally, based on the observations of this study, it is conceivable that unveiling the regulatory mechanisms of MMP-13 gene expression in SCC cells may prove beneficial in developing novel therapeutic modalities to combat invasion and metastasis of these neoplastic cells.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Academy of Finland, Sigrid Jusélius Foundation, Cancer Research Foundation of Finland, Turku University Foundation, Paulo Foundation, and the Turku and Helsinki University Central Hospital Research Foundations.
Accepted for publication November 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and transforming growth factor-ß. Cell Growth Differ 1997, 8:243-250[Abstract]
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 G. Ghilardi, M. L. Biondi, M. Caputo, S. Leviti, M. DeMonti, E. Guagnellini, and R. Scorza A Single Nucleotide Polymorphism in the Matrix Metalloproteinase-3 Promoter Enhances Breast Cancer Susceptibility Clin. Cancer Res., December 1, 2002; 8(12): 3820 - 3823. [Abstract] [Full Text] [PDF]
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 M T Salmela, T T MacDonald, D Black, B Irvine, T Zhuma, U Saarialho-Kere, and S L F Pender Upregulation of matrix metalloproteinases in a model of T cell mediated tissue injury in the gut: analysis by gene array and in situ hybridisation Gut, October 1, 2002; 51(4): 540 - 547. [Abstract] [Full Text] [PDF]
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 M F Leeman, J A McKay, and G I Murray Matrix metalloproteinase 13 activity is associated with poor prognosis in colorectal cancer J. Clin. Pathol., October 1, 2002; 55(10): 758 - 762. [Abstract] [Full Text] [PDF]
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B. S. Nielsen, F. Rank, J. M. Lopez, M. Balbin, F. Vizoso, L. R. Lund, K. Dano, and C. Lopez-Otin Collagenase-3 Expression in Breast Myofibroblasts as a Molecular Marker of Transition of Ductal Carcinoma in Situ Lesions to Invasive Ductal Carcinomas Cancer Res., October 1, 2001; 61(19): 7091 - 7100. [Abstract] [Full Text] [PDF]
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