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Molecular Oncology, Markers, Clinical Correlates |
Department of Pathology, University of Oulu, 90014 Oulu, Finland
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of MT1-MMP mRNA had a tendency to be associated with a lower degree of differentiation in HCC, but such association was not noticed in pancreatic tumors. Correlation to the clinical data showed that MT1-MMP expression had a strong statistical association with a poor outcome of patients (P < 0.01). A similar tendency was also observed in pancreatic adenocarcinomas, but the association did not reach statistical significance. MMP-2 and MMP-9 mRNA expression did not have significant correlation with prognosis. The results of this study support the previous suggestions of the importance of MT1-MMP for malignant growth and indicate that increased MT1-MMP mRNA expression by tumor cells in HCCs and pancreatic adenocarcinomas may have prognostic significance.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In cancer research, much interest has been devoted recently to a gelatinase subgroup of MMPs that include MMP-2 (gelatinase A, Mr 72,000 type IV collagenase) and MMP-9 (gelatinase B, Mr 92,000 type IV collagenase). This is partly because of their ability to degrade type IV collagen, the major structural component of BMs and because of their assumed important role in cellular invasion (2) . MT1-MMP, the first member of a more recently established group of MMPs containing a membrane-spanning sequence, has been shown to have an important role in MMP-2 activation in cell membranes (3 , 4) . By immunohistochemistry, MMP-2 has been shown to be localized in carcinomas to the neoplastic epithelial cells (5, 6, 7) , but in situ hybridization, on the other hand, has shown that most of the expression of mRNA for MMP-2 occurs in fibroblasts and endothelial cells of the tumor stroma (6 , 7) . In numerous studies, expression of MMP-2 has been shown to be related to invasive phenotype and metastatic potential of tumor cells (8 , 9) .
HCCs are relatively uncommon in Western countries but common in parts of Africa and the Far East. The most important etiological factors implicated in HCC are hepatitis B and C, abuse of alcohol, and exposure to aflatoxin. Increased prevalence of HCC is also associated with cirrhosis, and generally the clinical prognosis of HCC is very poor.
Pancreatic adenocarcinoma is another aggressive tumor type that frequently develops early regional and distant metastases and therefore has an unfavorable prognosis. Previous studies have shown that many MMPs and TIMPs are overexpressed in these two tumor types (10, 11, 12, 13, 14) . However, results from these studies have not provided a systematic concept of how MMP-2, MMP-9, and MT1-MMP are expressed in mRNA as well as antigen levels, and moreover, what the clinical aspect of MMPs in these malignancies with poor prognosis is. Therefore, we collected a tumor series that comprised primary HCCs and pancreatic adenocarcinomas to study systematically MMP-2, MMP-9, and MT1-MMP expressions in these tumors. Because of some discrepancies in previous results, we wanted to clarify which cells are responsible for the production of these mRNAs and corresponding antigens and whether there are any differences in expression between tumors of high and poor differentiation. Because there are some previous reports with other malignancies that indicate the association of MMPs, especially that of MT1-MMP to poor prognosis of patients (15 , 16) , we also correlated our findings with the clinical outcome of the patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
.
The tumor material was fixed in 10% neutral formalin and embedded in
paraffin. The diagnoses of tumors were based on a light microscopic
examination of H&E-stained sections. Grading was done according to the
criteria of the WHO (17
, 18)
.
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In Situ Hybridization.
For in situ hybridization, the sections were treated with
0.2 M hydrochloric acid for 20 min at room
temperature and washed in diethylpyrocarbonate-treated water for 5 min.
Proteinase K (1 mg/ml; Roche Diagnostics, Indianapolis, IN) treatment
was carried out for 30 min at 37°C, and the reaction was terminated
with 0.2% glycine in PBS. Sections were fixed with 4%
paraformaldehyde in PBS for 20 min and washed in PBS. Acetylation was
done in 0.25–0.5% acetic anhydride in 0.1 M
triethanolamine for 10 min. Sections were washed in PBS and air-dried
for 1–2 h at room temperature. Incubation in prehybridization mixture
[10 mM DTT (Sigma, St. Louis, MO), 10
mM Tris-HCl, 10 mM
NaPO4, 5 mM EDTA (Sigma),
0.3 M NaCl, 1 mg/ml yeast tRNA, deionized
formamide 50%, and dextran sulfate 10% (w/v); 0.02% (w/v) Ficoll
(Amersham Pharmacia Biotech, Uppsala, Sweden), 0.02% (w/v)
polyvinylpyrrolidone, and 0.02 mg/ml BSA] was carried out for 2 h. Thereafter, the sections were washed in PBS and dehydrated. For
hybridization, the probes were first denatured by boiling them for 1
min and then placed on ice. Three x 106 cpm
of the 35S-labeled antisense or sense probe in 40
µl of prehybridization buffer was applied on each section, and the
hybridization was carried out at 50°C overnight. The
posthybridization washes were as follows: twice at 50°C for 1 h
in prehybridization mixture except for dextran sulfate and tRNA, 15 min
in 0.5 M NaCl in 10 mM
Tris-HCl, 1 mM EDTA at 37°C, 30 min in 0.5
M NaCl containing 40 µg/ml RNase A (Sigma) at
37°C, 15 min in 0.5 M NaCl at 37°C, 15 min
twice in 2x SSC, and 15 min twice in 1x SSC at 50°C. The sections
were then dehydrated in ethanol containing 300 mM
ammonium acetate and air-dried at room temperature for 1 h. For
autoradiography, the slides were dipped in NTB-2 film emulsion (Kodak,
New York, NY) and placed in light-tight boxes for 10–14 days.
The slides were developed in D-19 developer (Kodak) fixed in Agefix
(Kodak) and counterstained with H&E.
Evaluation of the in Situ Hybridization Results.
The results of the in situ hybridization experiment were
scored by using a semiquantitative scale as follows; negative (-), no
signal distinguishable from the background labeling; weak (+), signal
was slightly increased or only a few cells were observed to be labeled;
moderate (++), distinct labeling was observed in a moderate number of
cells; and strong (+++), distinct and abundant labeling observed in the
majority of the cells. The scoring was done independently by two
observers.
Immunohistochemistry.
Sections, 4 µm thick, were incubated overnight at 4°C with a
monoclonal antibody against MMP-2, MMP-9 (gifts from Dr. Matti
Höyhtyä, Biocenter Oulu, Finland), and MT1-MMP
(21)
. They were then incubated with a biotinylated
antimouse antibody (Dako, Glostrup A/S, Copenhagen, Denmark) for 30
min, followed by the avidin-biotin-peroxidase complex, and the
substrate solution (3,3diaminobenzidine tetrahydrochloride in
H2O2 in Tris buffer, pH
7.4; Sigma) for 10 min. For control stains, PBS was used instead of the
primary antibody.
Gelatin Zymography.
To assess the MMP-2 and MMP-9 activity in HCC tissues, gelatin
zymography modified from Heussen and Dowdle (22)
was
performed. Fifty µg of tumor tissue obtained from HCC and an equal
amount of adjacent nontumor tissue were manually homogenized.
Thirty-µl aliquots of both tumor and nontumor samples were dissolved
in the SDS sample buffer in the absence of reducing agents without
boiling. The samples were then run in SDS-PAGE in gels that also
contained copolymerized gelatin (1.5 mg/ml). After electrophoresis, the
gels were washed twice for 15 min in buffer containing 5 mM
calcium chloride, 1 µM zinc chloride, 50
mM-Tris-HCl (pH 7.6), supplemented with 2.5% Triton X-100.
Finally, the gel was incubated for 16 h in same buffer also
containing 0.2 M NaCl, 0.02% sodium trinitrate, and 10
mM calcium dichloride at 37°C. The enzyme activity was
visualized by staining the gels with a solution containing 50%
methanol, 10% acetic acid, and 0.1% Coomassie Brilliant Blue and
destaining in 10% methanol and 10% acetic acid.
Statistical Analysis.
Clinical data were collected from the patients’ clinical records, and
survival times were counted (months) from the date of partial
hepatectomy or pancreatectomy. Statistical analysis was performed by
the Kaplan-Meier method with log-rank analysis. HCCs were analyzed
comparing groups negative (-) for mRNA expression to positively
expressing (+ through +++) groups. Because there were only few cases of
pancreas adenocarcinomas that did not express any mRNAs investigated,
we combined negative (-) and low expression (+) groups and compared
those with moderate (++) and strong (+++) expressions. The results from
survival analysis were expressed by reporting Ps, and
values < 0.05 were regarded as significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and for pancreatic adenocarcinomas in Table 2
.
MT1-MMP mRNA Expression.
mRNA for MT1-MMP was detected in 12 of 36 (33%) HCCs. Of these, the
expression was seen exclusively in carcinoma cells in seven tumors. In
five cases, there was also expression in fibroblasts and, more weakly,
in endothelial cells. The stromal signal was generally weak and most
distinct in areas of abundant fibrosis and was often localized to cells
adjacent to islands of tumor cells. In some areas, positive labeling
was also seen in epithelial cells of proliferating, but not of normal,
bile ducts. In carcinoma cells of HCCs, the MT1-MMP mRNA expression was
independent from that in stroma, and no clear coexpression with
fibroblasts was noticed. The expression level in carcinoma cells varied
from negative to strong positivity and had a tendency to be associated
with a low degree of differentiation; 5 of 10 of grade III tumors
showed positive labeling (Fig. 1a
).
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). Normal
acinar or ductal structures were negative.
MMP-2 mRNA Expression.
MMP-2 mRNAs were detected in 14 of 36 (38%) of HCCs. The expression
occurred in stromal cells of 13 of 36 tumors. The mRNA level varied
from weak to moderate. In eight tumors, mRNAs were detected also in
carcinoma cells. Of stromal cells both fibroblasts and, to a lesser
extent, endothelial cells showed positive signals, usually in areas of
abundant fibrosis either around tumor islands or more diffusely in the
tumor stroma (Fig. 2a
).
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).
MMP-9 mRNA Expression.
MMP-9 mRNAs were observed in 12 of 36 (33%) HCCs. Twelve of them
showed expression in carcinoma cells and four also in the stromal
cells. The intensity of mRNA signal varied from weak to moderate and
was generally not associated with the amount of fibrous stroma. Within
tumor tissue, the expression was more pronounced in the more dysplastic
nodules. In stroma, both fibroblasts and endothelial cells contained
the mRNA. Occasionally, mRNAs were also seen in nontumorous hepatocytes
of cirrhotic areas and in cells of proliferating bile ducts (Fig. 3a
).
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).
Immunohistochemical Staining.
Immunohistochemical staining was performed to show the corresponding
MMP-2, MMP-9, and MT1-MMP protein expression in HCCs and pancreatic
adenocarcinomas. Because of the shortage of antibodies, four cases of
HCCs were stained. Carcinoma cells in all cases were positive for
MMP-2, and a clear staining reaction was also seen in stromal
fibroblasts and endothelial cells (Fig. 2c
).
MMP-9 staining reaction was seen in HCCs in individual carcinoma cells
scattered over the tumor area. Occasionally, stromal fibroblasts and
endothelial cells were seen to express MMP-9 antigen (Fig. 3c
). Because of a high content of endogenous peroxidases and
biotin in liver cells, resulting in increased possibilities for
nonspecific binding, the immunohistochemical stainings were not as
successful for MT1-MMP in HCCs. In some grade III carcinomas, there
was, however, intracytoplasmic or cell membrane staining of the
carcinoma cells for MT1-MMP.
Immunohistochemical staining for the presence of MMP-2 was performed on
four pancreatic adenocarcinomas. They all showed a clear positive
staining reaction in carcinoma cells but also in fibroblasts and
endothelial cells (Fig. 2d
). Occasional staining could also
be seen in cells of normal epithelial ducts of the adjacent pancreatic
tissue.
MMP-9 staining was performed for eight pancreatic adenocarcinomas. They
all showed a clear intracytoplasmic reaction in neoplastic epithelial
cells. Overlying tumor stroma remained, in most of the cases, negative,
and only occasional tumor fibroblasts and endothelial cells were seen
to express MMP-9 antigen (Fig. 3d
).
MT1-MMP staining was performed on eight pancreatic adenocarcinomas. In
all cases, intracellular staining was seen in both carcinomas and also
to a lesser extent in stromal cells. In some areas, a positive staining
reaction could also be seen in cell membranes of the neoplastic
epithelial cells (Fig. 1d
).
Gelatin Zymography.
Gelatinolytic activity has been previously well documented in
pancreatic adenocarcinomas (11
, 14)
. Therefore,
gelatin zymography was performed on HCCs (Fig. 4)
.
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Tumor and nontumor samples showed almost comparable bands for the latent Mr 92,000 MMP-9, but the active, Mr 82,000 form was seen only in HCC samples.
Statistical Analysis.
To analyze whether the expression of MMPs was associated with the
outcome of the patients, the semiquantitative results of in
situ hybridization were compared with the survival data of the
patients. A significant association was seen between the expression of
MT1-MMP mRNA by carcinoma cells of HCC and shortened survival of
patients (P = < 0.01, log rank; Fig. 5
). No such association was seen
in cases where the mRNA expression was seen present only in stromal
cells (P = 0.81, log rank). Generally, there was a
tendency of increased MT1-MMP mRNA expression in grade III tumors when
compared with grade I tumors, but the difference was not statistically
significant (P = 0.07).
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In pancreatic adenocarcinomas when compared with prognosis MT1-MMP mRNA expression by neoplastic cells did not associate with survival (P = 0.32, log rank), but the Breslow test gave a significant value (P = 0.04). No significant association was seen when correlated to the mRNA expression by the stroma (P = 0.12, log rank). Neither was there any difference in the mRNA expression between various differentiation grades (I–III) of pancreatic adenocarcinomas.
Because only one of the pancreatic tumors expressed moderately or strongly mRNA for MMP-2 by carcinoma cells, statistical analysis was not performed. The association of stromal expression to the patients’ outcome did not show statistical significance (P = 0.27, log rank). Nor was there any significant association between the patients’ prognosis and the expression of mRNA for MMP-9 by cancer cells (P = 0.65, log rank) or by stromal cells (P = 0.35, log rank). However, according to life tables, there was a tendency for a poorer prognosis of patients with increased MMP-9 mRNA expression by tumor cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There was a statistically significant association with a poor prognosis
of patients in HCCs when mRNA synthesis of MT1-MMP was present in
cancer cells themselves but not when stromal cells alone expressed the
mRNA. A similar tendency could also be observed in pancreatic
adenocarcinomas but with no statistical significance. The association
of increased MT1-MMP mRNA expression with shortened lifetime in HCCs is
easily understandable by its connection to the poor differentiation
grade of tumors, although it is generally difficult to assess the
prognosis of patients with HCC, because some patients with large and
progressive tumors have late recurrences and long survival periods. Our
result agrees also with Harada et al. (24)
, who
showed increased MT1-MMP mRNA expression in HCC to be associated with
capsular infiltration. Our results also support the previous reports of
the importance of MT1-MMP in malignant growth but also add evidence for
its association with poor prognosis of patients with a malignant
disease of the liver. MT1-MMP is one of the key enzymes among MMPs, and
its overexpression seems to have a significant effect on tumor growth.
To date, there is at least four mechanisms by which MT1-MMP can enhance
tumor progression: it can activate MMP-2 on tumor cell membranes
(3
, 4
, 24)
; it is a very effective degradative enzyme by
itself, having substrates such as fibronectin, tenascin, nidogen,
aggregan, and perlecan (25)
; MT1-MMP is a very potent
regulator for neovascularization (26)
, a phenomenon that
is critical for malignant growth (reviewed in Ref. 27
);
and recently MT1-MMP was shown to process directly laminin-5 
2
chain, which caused a strong migration effect by tumor cells over
laminin-5 surfaces (28)
. Previously, we have shown that
pancreatic adenocarcinomas strongly express laminin-5 2 chain by
tumor cells and also contain laminin-5-bearing BM structures around
tumor islands (29)
, and in a preliminary experiments, we
noticed that laminin-5 2 chain is abundantly expressed also by
HCCs.4
MT1-MMP and
laminin-5 can trigger an important migratory effect in carcinoma cells,
leading to their spreading. How these mechanisms together are operating
in malignant growth remains to be clarified. It seems, however, that
the association of MT1-MMP with poor prognosis of patients is rather
connected to tumor type and general growth pattern than differentiation
grade. Unraveling the regulatory mechanisms among the functions of
MT1-MMP, MMP-2, TIMP-2, and laminin-5 and possibly some other factors
may prove to be pivotal to our understanding of malignant behavior in
general.
In their study, Theret et al. (23) also showed a correlation between MT1-MMP mRNA levels and MMP-2 and TIMP-2 mRNA levels, as well as with MMP-2 activation in HCCs. In the present study, simultaneously increased MMP-2 and MT1-MMP expression by carcinoma cells was not observed. Gelatin zymography showed that although latent MMP-2 was present both in tumor and adjacent nontumor liver tissues, active MMP-2 was only seen in tumor tissue of HCC. This finding is in agreement with the result by Ogata et al. (11) , who also showed increased MMP-2 activation in tissue samples of HCC.
MMP-2 has been suggested to have a very important role in controlling tumor cell invasion because it is very commonly overexpressed in tumor masses and has a high ability to degrade BM macromolecules. In HCCs and pancreatic adenocarcinomas, MMP-2 mRNA synthesis occurred mainly by stromal fibroblasts and endothelial cells and only to a lesser extent by carcinoma cells. This result is similar to what has been reported previously in many tumor types such as breast, ovary, and colon carcinomas (5 , 6 , 7) . Bramhall et al. (12) also reported MMP-2 mRNA synthesis to be more abundantly present in the tumor stroma of pancreatic adenocarcinoma. It seems likely that the type or amount of tumor stroma is an important modulator for MMP-2 expression, because there was higher MMP-2 production in pancreatic tumors when compared with liver tumors, which are not usually accompanied by fibrosis so abundant as in pancreatic adenocarcinomas. Tumor stromal cells are also known to express many different types of regulatory factors that up-regulate MMP expression (30) . Although MMP-2 was abundantly synthesized by stromal cells, it immunolocalized mainly to tumor cells in HCCs and pancreatic adenocarcinoma.
In HCCs, MMP-9 mRNA was found to be expressed mainly by tumor cells. There was no clear association with the prognosis in mRNA expressions of MMP-9 or MMP-2. However, there was a tendency to have a strong expression for both mRNAs in tumors with a poorer survival of patients. In a previous study on HCCs, it was shown that increased MMP-9 mRNA expression, as studied by Northern hybridization, was associated with capsular infiltration (10) . Elevated plasma levels of MMP-9 in patients with HCC have also been observed, especially in patients having macroscopic portal vein invasion (31) . It is notable that in some cases of this study, MMP-9 mRNA expression varied from nodule to nodule, being stronger in the more dysplastic ones. Gelatin zymography showed the presence of Mr 92,000-sized MMP-9 in almost equal amounts in tumor samples of HCC and in adjacent nontumor liver tissue, but the active Mr 82,000 form was present only in tumor samples. All of this evidence indicates that MMP-9 production and functional activity are increased in HCCs. It is also an interesting notion that activated MMP-2 can activate proMMP-9 (32) . One could speculate that MT1-MMP/TIMP-2 complex in cell membranes starts the activation cascade by activating proMMP-2, which in turn is able to convert proMMP-9 to its active form.
In summary, our data show that mRNA synthesis of all MMPs investigated is up-regulated in HCCs and pancreatic adenocarcinomas but are differentially expressed by intensity and a cellular origin. Furthermore, MT1-MMP produced by carcinoma cells of HCCs was associated with a poor prognosis of patients, and a similar tendency was also present in pancreatic adenocarcinomas. The results support previous suggestions that MT1-MMP could be one of the key enzymes among MMPs in the process of invasion. Additionally, the uncontrolled production of MT1-MMP alone could be of significance for malignant growth and spread.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study is financially supported by the
Cancer Foundation of Northern Finland. 
2 To whom requests for reprints should be
addressed, at Department of Pathology, P. O. Box 5000 (Aapistie 5),
University of Oulu, 90014 Oulu, Finland. Fax: 358-8-537-5953.
3 The abbreviations used are: MMP, matrix
metalloproteinase; MT1, membrane type 1; HCC, hepatocellular carcinoma;
TIMP, tissue inhibitor of matrix metalloproteinase; BM, basement
membrane.
Received 8/30/99; revised 3/17/00; accepted 3/27/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 chain in pancreatic adenocarcinoma. J. Pathol., 180: 290-294, 1996.[CrossRef][Medline]
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