ARTICLE
Matrix metalloproteinases (MMPs) are a group of proteases involved in
the degradation of extracellular matrix (ECM). Degradation of basement
membranes or ECM surrounding cancer cells is an initial step for cancer
invasion and metastasis. Among the members of the MMPs, MMP-2 (72 kDa
type IV collagenase) is able to degrade basement membrane type IV collagen
and is therefore considered to be especially important in the malignant
behavior of cancer cells [1-3]. Since MMP-2 is secreted as an inactive
zymogen (pro MMP-2), an activation step is required for the tumor invasion.
ProMMP-2 is unique in that it is activated by membrane-associated metalloproteinases
(MT-MMPs) [4, 5] but not by serine proteinases [6]. MT-MMPs consist of
five members; MT1, MT2-, MT3-, MT4- and MT5-MMP, which have a transmembrane
domain in the hemopexin-like domain in the C-terminal end [2, 3]. It has
been demonstrated that MT1-MMP is expressed in many malignant tumors;
gastric cancers [7], ovarian tumors [8], breast cancers [9, 10], malignant
brain tumors [11] and urothelial carcinomas [12]. MT2-MMP and MT3-MMP
expressions in malignant tumors, however, have not been fully studied
yet.
Human skin melanoma is a highly malignant neoplasm. Breakdown of the
epidermal basement membrane is a prerequiste for the migration of the
melanocytic tumor cells into the dermis and distant organs. Recently it
has been demonstrated that the number of MMP-2-positive cells increases
with decreasing architectural organization and increasing atypia in the
melanocytic lesions and that expression MMP-2 and MT1-MMP colocalize in
the melanocytic lesions correlating with melanoma progression. Thus, activation
mechanism of MMP-2 appears to participate in the degradation of the basement
membrane of malignant melanocytic invasion and to be closely related to
the prognosis of the disease [13-16].
In this paper, in order to know the role of MT2-MMP and MT3-MMP in the
activation of MMP-2 in malignant melanoma, various clinical and histopathological
forms of melanoma were stained with monoclonal anti-MT2,3-MMP and anti-MMP-2
antibodies.
Materials and methods
Skin specimens
Skin specimens were obtained from the files of our department between
1994 and 1999. They include nevus cell nevus (n = 5), dysplastic nevus
(n = 2), juvenile melanoma (n = 3), superficial spreading melanoma (SSM)
(n = 3), acral lentiginous melanoma (ALM) (n = 3), nodular melanoma (NM)
(n = 2), and metastatic melanoma (n = 3).
Antibodies
All antibodies used here were purchased from Fuji Pharmaceutical Company
(Takaoka,Toyama, Japan). The monoclonal antibodies to MT1-MMP, MT2-MMP
and MT3-MMP were raised against the synthetic peptides REVPYAYIREGHEK
(corresponding to the amino acids at positions 160-173 in the human MT1-MMP)
(clone No 114-6G6) [10, 17], DTDNFQLPEDDLRG (corresponding to the amino
acids at positions 281-294 in the mouse MT2-MMP) (clone No 162-22G5) [10,
18] and EEVPYSELENGKRD (corresponding to the amino acids at positions
168-181 in the human MT3-MMP) (clone No 117-10C6) [19], respectively.
The monoclonal antibody to human MMP-2 was produced from the synthetic
peptides VTPRDKPMGPLLVATF (corresponding to the amino acids at positions
468-483 in the human MMP-2) (clone No 42-5D11) [20, 21]. Specificity of
each antibody has been described in each reference and found to show no
cross-reactivity each other.
Immunohistochemistry
Skin samples were fixed with 10% buffered formalin and embedded in paraffin.
Paraffin-embedded 4 µm sections were autoclaved to expose antigenic
sites at 120° C for 10 min in 10 mM citrate buffer, pH 6.0, then
blocked with 10% normal swine serum for 30 min. The sections were incubated
with anti-MT1,3-MMP antibodies and anti-MMP-2 antibody at 1:50 dilution
overnight at room temperature, then incubated with secondary antibody,
anti-mouse Ig antibody at 1:500 dilution for 1 hr. Antigen-antibody complex
was visualized with avidin-biotin complex.
For double immunofluorescence labeling, the sections were incubated
with anti-MMP-2 antibody (1:50 dilution) overnight, then with rhodamine-labeled
anti-mouse Ig (DAKO) for 2 hrs (1:20 dilution). After washing, the sections
were incubated for 2 hrs with anti-MT2-MMP or anti-MT3-MMP antibody (1:50
dilution) which had been labeled with FITC. Evaluation of fluorescence
was performed with confocal laser scan microscope (LSM410, Carl Zwiss,
Jena, Germany).
Results
Expression of MT1~3-MMPs and MMP-2 in nevus cell nevus, dysplastic nevus
and juvenile melanoma was not detected or detected only in a few cells
(not shown). Expression of MT2-MMP and MMP-2 in SSM and ALM was moderately
or strongly detected in melanoma cells, whereas MT1-MMP and MT3-MMP were
relatively weakly expressed by the tumor cells, especially at the edge
of the tumor. Normal skin including the epidermis and dermal tissue outside
the SSM was generally negative (Fig. 1a-d) except MMP-2 in which
dermal stromal fibroblasts were positive (Fig. 1d). Expression
of MT1,3-MMPs and MMP-2 in NM and metastatic melanoma cells was rather
intensely detected by the tumor cells, especially at the invasive edge
of the tumor nests (Fig. 2 a-d). Their expression was absent from
normal-appearing dermis distant from the tumor tissues. The summary of
the immunostaining patterns with the antibodies for MT-MMPs and MMP-2
was shown in Table I.
To know the correlation between MT-MMPs and MMP-2 expression in melanoma
cells, double immunofluorescence labeling was carried out. Colocalization
of MT2-MMP/MMP-2 (Fig. 3a) and MT3-MMP/MMP-2 in the periphery of
NM (Fig. 3b) was seen. MT2-MMP/MMP-2 and MT3-MMP/MMP-2 were also
colocalized in metastatic melanoma cells (not shown).
Article accepted on 10/4/01
CONCLUSION
Comment
It has been reported that the expression of MMP-2 in melanocytic lesions
correlates with later metastasis and is of prognostic value [13-16]. In
our studies, MMP-2 in NM and metastatic melanoma appears to be strongly
expressed (Table I). It is therefore of critical importance to
know the mechanism by which MMP-2 expression is activated in melanoma
cells. Regulatory mechanism of the activation of MMP-2 in melanoma cells
has been studied; Luca et al. [22] reported that interleukine-8
(IL-8) can directly activate MMP-2 gene transcription leading to the enhancement
of the invasion of melanoma cells in vivo. It has been also demonstrated
that TIMP-2 can regulate MMP-2 activation via modulation of MT1-MMP
in invasive melanoma cell lines [23]. In the present study, we have demonstrated
that the expression of MT2,3-MMP as well as MT1-MMP is generally activated
in primary and metastatic melanoma cells and activation of MT2,3-MMPs
and MMP-2 occurs in the same cells in vivo. Since it has been shown
that MT-MMPs are involved in the activation of inactive proMMP-2, the
active form of MMP-2 may be increased in melanoma cells which may result
in an increase in their tumorigenic and metastatic potential.
Recently MT1-MMP itself has been shown to degrade extracellular matrix
such as collagen [24] and aggrecan [25] in vitro. Therefore coordinate
expression of MT-MMPs and MMP-2 in melanoma cells in vivo may imply
synergic effect of MT-MMPs and MMP-2 on extracellular matrix degradation.
Taken together, expression of MT-MMPs in melanoma cells may support the
activity of MMP-2 by both activating proMMP-2 and by acting with a synergistic
effect on degradation of extracellular matrices, which will accelerate
pericellular matrix degradation in melanoma cell invasion and metastasis.
Expression of MT2-MMP appeared to be stronger than expression of MT1-
and MT3-MMPs, irrespective of the clinical and histopathological forms
of melanocytic tumors (Table I). It has been demonstrated that
expression of MT1,3-MMPs shows tissue or tumor specificity; MT3-MMP is
expressed preferentially in the brain, heart and placenta, while MT1-MMP
is widely expressed in various organs [19, 26, 27], MT1-MMP is detected
in the liver while MT2- and MT3-MMPs are not [19, 28], lung carcinoma
cells overexpress MT1-MMP but infrequently express MT3-MMP [18], prognosis
of breast carcinomas correlates with MT1-MMP expression levels but not
with MT2-MMP or MT3-MMP expression [10]. It is unclear whether rather
strong immunoreactivity of MT2-MMP may represent a phenomenon specific
to melanoma cells or skin tumors. Since expression of MT2- and MT3-MMPs
in malignant tumors from organs of various origins has not been fully
studied yet, further studies on MT2- and MT3-MMPs will answer the question.
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