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Expression of Matrix Metalloproteinases and Their Inhibitors Correlates With Invasion and Metastasis in Squamous Cell Carcinoma of the Head and Neck
Pornchai O-charoenrat, MD;
Peter H. Rhys-Evans, FRCS;
Suzanne A. Eccles, PhD
Arch Otolaryngol Head Neck Surg. 2001;127:813-820.
ABSTRACT
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Background Matrix metalloproteinases (MMPs) have been implicated in the invasion
and metastasis of head and neck squamous cell carcinoma (HNSCC). However,
a detailed analysis of MMPs and tissue inhibitors of MMPs (TIMPs) in relation
to the biological behavior of HNSCC has yet to be performed in clinical material.
Objectives To study a comprehensive profile of MMPs and their 2 main inhibitors
in HNSCC tissue samples and to correlate the patterns of expression with clinicopathological
characteristics, invasion, and metastasis.
Design This study included 54 consecutive patients with primary HNSCC, 27 of
which showed lymph node metastasis. Expression of MMP-1, MMP-2, MMP-3, MMP-7,
MMP-9, MMP-10, MMP-11, MMP-13, MMP-14, TIMP-1, and TIMP-2 was simultaneously
analyzed in tissue homogenates using semiquantitative reverse transcription–polymerase
chain reaction assay. Where feasible, levels of protein and enzyme activity
were confirmed by Western blot, enzyme-linked immunosorbent assay, and substrate
zymography. Conventional clinicopathological features, including mode of tumor
invasion, were also examined.
Results Significantly higher MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11,
MMP-13, and TIMP-1 levels were found in tumors vs specimens of matched normal
mucosa. No difference in the distribution of MMPs and TIMPs in relation to
age, sex, tumor site, or histological grade was observed. A significant correlation
was demonstrated between levels of MMP-1, MMP-9, and TIMP-1 and advanced T
stage and between MMP-9 expression and an infiltrative pattern of growth.
Enhanced expression of MMP-9 was strongly correlated (P<.001) and levels of MMP-2, MMP-7, and MMP-11 were weakly correlated
(P = .03-.05) with lymph node involvement.
Conclusions Overexpression of multiple MMPs and TIMPs is characteristic of HNSCC,
and analysis of specific MMPs, MMP-9 in particular, might be useful for evaluating
the malignant potential in individual HNSCC.
INTRODUCTION
SQUAMOUS CELL carcinoma of the head and neck (HNSCC) is a major problem
worldwide.1 One of the most characteristic
clinical features of HNSCC is its capacity to invade adjacent tissues and
metastasize locoregionally. Cancer cell invasion, metastasis, and angiogenesis
is a complex, multistep process involving the cooperation of multiple proteolytic
enzymes secreted by tumor or host cells and whose substrates include extracellular
matrix components.2 Evidence3-22
suggests that matrix metalloproteinases (MMPs) and their physiological tissue
inhibitors (TIMPs) might play a causal role in HNSCC progression.
The MMPs are a family of highly homologous extracellular zinc- and calcium-dependent
endopeptidases with enzymatic activity against almost all protein components
of the extracellular matrix. Based on the protein domain structure and substrate
specificity, the MMPs can be divided into 4 subclasses.2
The first group, which degrades types I, II, and III fibrillar collagens,
is composed of MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase),
and MMP-13 (collagenase-3). The second group, stromelysins, includes 4 members:
MMP-7 (matrilysin) contains the minimal number of domains, ie, a predomain,
a prodomain, and a catalytic domain, and MMP-3 (stromelysin-1), MMP-10 (stromelysin-2),
and MMP-11 (stromelysin-3) contain an additional carboxy-terminal hemopexinlike
domain. The stromelysins have a broad substrate specificity and are capable
of degrading many extracellular components, eg, laminin, fibronectin, and
proteoglycans. Matrix metalloproteinase 2 (gelatinase-A) and MMP-9 (gelatinase-B)
account for a separate class based on the presence of a fibronectinlike domain.
Gelatinases are able to cleave both the denatured forms of collagen and type
IV collagen found in basement membrane. Matrix metalloproteinase 2 and MMP-9
also contain a gelatin-binding domain that endows them with high affinity
for gelatin. The last group of MMPs contains the membrane-type MMPs (MT-MMPs),
which are composed of MMP-14 (MT1-MMP), MMP-15 (MT2-MMP), MMP-16 (MT3-MMP),
MMP-17 (MT4-MMP), and MMP-24 (MT5-MMP). Membrane-type MMPs have the unique
property of possessing a hydrophobic sequence at the C-terminus, which allows
insertion of the protein into the cell membrane. Some MMPs cannot be grouped
into any of these classes, including MMP-12 (metalloelastase), MMP-18, MMP-19,
MMP-20 (enamelysin), and MMP-23. These enzymes differ in substrate specificity,
regulation, tissue-specific expression, and potential interactions with additional
MMP and TIMP family members. Expression of MMP activity can be controlled
at the level of gene transcription, by proenzyme activation and by broad-spectrum
and specific inhibitors. Tumor cells might induce the host cells within the
surrounding stroma to secrete these enzymes or vice versa. Most MMPs are secreted
as latent proenzymes that undergo proteolytic cleavage of an amino-terminal
domain during activation. The net activity of MMPs is determined by the amount
of proenzyme expressed, the extent to which the proenzyme is activated, and
the local concentration of specific tissue inhibitors of MMPs, ie, TIMPs.
A multigene family of proteins named TIMPs has been demonstrated to
inhibit fully activated MMPs. Tissue inhibitors of MMPs comprise at least
4 members, and, together, they provide a tightly regulated mechanism for control
of MMP activation and function. Tissue inhibitor of MMP-1 and TIMP-2 have
molecular weights of 28.5 and 21.0 kd, respectively, and seem to act by forming
1:1 stoichiometric complexes with the active MMP.23
Tissue inhibitor of MMP-1 can inhibit the collagenases, MMP-3, and the gelatinases.24 Tissue inhibitor of MMP-2 binds preferentially to
MMP-2 but also inhibits the activities of MMP-1, MMP-3, MMP-7, and MMP-9.25 The local balance of these enzymes and inhibitors
seems to be a crucial factor in tumor invasion and metastasis.
We20 recently demonstrated the expression
of several members of the MMP and TIMP family in a large panel of HNSCC cell
lines and a strong correlation between some MMPs (MMP-9 in particular) and
their in vitro invasiveness. The aim of this investigation was to perform
a comprehensive analysis of MMPs previously identified in HNSCC, including
MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, and MMP-14 (MT1-MMP),
and 2 inhibitors, TIMP-1 and TIMP-2, in fresh tissue samples and to correlate
with the clinicopathological characteristics of HNSCC.
PATIENTS, MATERIALS, AND METHODS
PATIENTS AND FRESH TISSUE SAMPLES
The protocol for the following studies was approved by the Ethical Committee
of the Royal Marsden Hospital Trust, London, England. Fresh tissue samples
were obtained from 54 patients undergoing major surgical resection for HNSCC
at the Department of Head and Neck Surgery, the Royal Marsden Hospital, between
July 1, 1997, and October 31, 1999. The clinical and pathological characteristics
of patients are summarized in Table 1.
There were 44 men and 10 women (median age, 58.5 years; range, 27-83 years).
Patients had no detectable metastases in distant organs at the time of surgery.
None of the patients had previously received preoperative chemotherapy or
radiotherapy. Adjuvant treatment was given after radical surgery in appropriate
cases following the hospital's protocol. In each case, the portion of tumor
was resected near the advancing edge of the tumor, avoiding its necrotic center.
After excision, tissue samples were immediately snap-frozen and stored in
liquid nitrogen until use. Samples of the adjacent tissues were submitted
for histopathological study, which revealed that most cells were malignant.
Tumors were staged according to the TNM classification (5th edition)26 and were graded as well (G1), moderately (G2), and
poorly (G3) differentiated. T classification was evaluated according to tumor
size for tumors from the oral cavity or the oropharynx and tumor size and
extensiveness for tumors from the hypopharynx and the larynx. The mode of
cancer invasion was histologically classified as described previously27; grade 1 has a well-defined borderline, grade 2 has
a less well-defined borderline, grade 3 has groups of cells and no distinct
borderline, and grade 4 has diffuse invasion. In 27 patients, tissue samples
of metastatic lymph nodes (LNM) were also available for analysis. Matched
histologically normal mucosa of the upper aerodigestive tract, resected at
least 5 cm distant from the tumor area,3, 8
was obtained from 32 patients. As of June 30, 2000, median follow-up for living
patients was 20 months (range, 9-35 months); 35 patients (65%) were alive,
15 (28%) died of tumors, and 4 (7%) died of unrelated causes. Node-positive
cases in this study are patients in whom positive cervical nodes were identified
based on histological diagnosis after a neck dissection, and patients who
experienced no metastasis for at least 12 months after surgery are node-negative
cases.
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Table 1. Clinicopathological Features of 54 Patients With Head and
Neck Squamous Cell Carcinoma
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SEMIQUANTITATIVE REVERSE TRANSCRIPTION–POLYMERASE CHAIN REACTION
The semiquantitative reverse transcription–polymerase chain reaction
(RT-PCR) assay and the primer sequences have been described previously.20 The following genes were assayed using specific primer
pairs: MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 (MT1-MMP),
TIMP-1, and TIMP-2. ß-Actin was used to check RNA integrity and as an
internal control. Oligonucleotide primers were purchased from Genosys (Cambridge,
England). To control gel-to-gel variability, each PCR product from HT-1080
fibrosarcoma cells or MDA-MB 231 mammary carcinoma cells was also electrophoresed
as a control on every gel. The level of messenger RNA (mRNA) was calculated
as the ratio of tissue sample to control cell line on the same scan and was
then corrected as a ratio to the corresponding ß-actin level.
PREPARATION OF TISSUE HOMOGENATES
Tissues samples (wet weight, 300-400 mg) were homogenized in ice-cold
75-mM Tris-hydrochloride buffer, pH 7.4, containing 0.5% Triton X-100. After
removal of debris and nuclear pellets by centrifugation (10 000g for 10 minutes at 4°C), the protein concentration
was determined using a protein assay reagent kit (BCA; Pierce, Rockford, Ill).
The supernatants containing protein were stored at -70°C until required.
QUANTITATIVE ZYMOGRAPHY
Forty micrograms of the extract proteins was resolved under nonreducing
conditions in 10% sodium dodecyl sulfate–polyacrylamide gels copolymerized
with 0.1% (wt/vol) gelatin or 0.05% ß-casein. Gelatinolytic or caseinolytic
enzymes were detected as transparent bands on the blue background, and the
intensity of the bands was measured using image analysis software (Quantiscan,
Cambridge, England), as described previously.20
Results were expressed in arbitrary units per 40 µg of total protein.
Conditioned medium from 12-O-tetradecanoyl-phorbol-13-acetate
(TPA)–treated HT-1080 fibrosarcoma cell line and TPA-treated MDA-MB
231 mammary carcinoma cells served as a positive control and a standard for
intergel variations for gelatin zymography and casein zymography, respectively.
QUANTITATION OF MMP-1, TIMP-1, AND TIMP-2 BY ENZYME-LINKED IMMUNOSORBENT
ASSAY
Concentrations of MMP-1, TIMP-1, and TIMP-2 in the same tissue homogenates
(500 µg per sample) as used in zymography were measured using commercially
available enzyme-linked immunosorbent assay (ELISA) kits (Chemicon International
Inc, Temecula, Calif). The values measured represent pro–MMP-1, TIMP-1,
and TIMP-2 concentrations, with a range of detection at 0.16 to 10.0, 1.2
to 49.0, and 20.0 to 320.0 ng/mL, respectively. Results were calculated as
nanograms per 1 mg of total protein tissue extracts. Two independent experiments
were performed. In each experiment, tissue lysates were prepared from 2 separate
pieces of the same tissue specimen, and ELISA values were measured in duplicate
for each sample.
WESTERN BLOT ANALYSIS
Equal amounts of protein (100 µg) from the same tissue homogenates
as used in zymography were resolved under reducing condition in 10% and 15%
sodium dodecyl sulfate–polyacrylamide gel electrophoresis for MMP and
TIMP detection, respectively, and transferred onto a nitrocellulose membrane
(Highbond-C extra; Amersham International, Buckinghamshire, England), then
probed with the appropriate primary antibody. Antibodies to MMP-2, MMP-3,
MMP-7, MMP-9, and MT1-MMP were provided by British Biotech (Oxford, England).
Antibodies to TIMP-1 and TIMP-2 were purchased from Chemicon (Harrow, England).
Blots were washed, incubated with a secondary antibody coupled to horseradish
peroxidase (Serotec, Oxford), and developed using the luminol reagent (Santa
Cruz Inc, Santa Cruz, Calif) and Kodak X-OMAT AR film (Eastman Kodak, New
York, NY) with an intensifying screen. Levels of proteins were determined
by image analysis using Quantiscan software. As a negative control, the primary
antibody, which was preabsorbed with corresponding proteins overnight at a
ratio of 1:10, or normal serum was reacted with the membrane filter. The specific
bands were absent when the preabsorbed antibody or normal serum was used.
Purified human MMP-2/MMP-9, MMP-3/MMP-7, TIMP-1 (28 kd), and TIMP-2 (24 kd)
were obtained from CalBiochem (Nottingham, England).
STATISTICAL ANALYSIS
All statistical analyses were performed using statistical software (GraphPad
Prism version 2.01; GraphPad Software Inc, San Diego, Calif). To compare levels
of mRNA expression between tumor tissues and control (histologically normal)
tissues and to determine the significance of increased mRNA expression of
MMPs and TIMPs with various clinicopathological variables, the Mann-Whitney
test and the Kruskal-Wallis test with the Dunn multiple comparison test were
used when comparing 2 groups and 3 or more groups, respectively. The 2-tailed
Fisher exact test was used to analyze the contingency table. Levels of mRNA
and protein were measured from 2 parts of the same specimens in duplicate
where feasible. Correlations between the mRNA and protein levels were computed
using the 2-tailed Spearman nonparametric correlation. Results are expressed
as mean ± SEM. P<.05 was considered statistically
significant. Unless otherwise stated, each experiment was performed twice
with virtually identical results.
RESULTS
EXPRESSION OF MMPs AND TIMPs IN HNSCC
Lesions from primary HNSCC (n = 54) and LNM (n = 27) and histologically
normal adjacent mucosa (n = 32) were examined for mRNA levels of multiple
MMPs and TIMPs. Using semiquantitative RT-PCR assay, mRNA expression of MMP-1,
MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-13, and TIMP-1 was significantly greater
in malignant tissues (primary tumors and/or LNM) compared with mRNA levels
in histologically normal mucosa (P = .02 to <.001)
(Figure 1). No significant differences
were found between mRNA levels of MMP-14 (MT1-MMP) and TIMP-2 in tumors and
control tissues. In addition, no differences were found between expression
of most MMPs tested in primary HNSCC vs LNM, except for the MMP-3 gene, where
the levels in primary tumors (14.55 ± 3.91) were significantly higher
than those in LNM (1.09 ± 0.32) (P = .03).
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Figure 1. Semiquantitative reverse transcription–polymerase
chain reaction analysis of matrix metalloproteinases (MMPs) and tissue inhibitors
of MMPs (TIMPs). Each value is the ratio of MMP or TIMP to ß-actin messenger
RNA level and is the mean of 3 independent RNA samples. The MMPs and TIMPs
for which no messenger RNA was detected by 50 or more polymerase chain reaction
cycles are indicated by zeros in the graphs. Horizontal lines indicate mean
values; LNM, lymph node metastasis; and MT1, membrane-type 1.
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Levels of MMP-1, TIMP-1, and TIMP-2 proteins were also measured from
tissue homogenates using the ELISA kit (Figure
2). Compared with control tissues, (1) MMP-1 protein levels were
4.1-fold greater in primary tumors and 3.8-fold greater in LNM (P<.001 for both) and (2) TIMP-1 protein levels were 1.6-fold higher
in primary tumors (P = .009) and 1.9-fold in LNM
(P = .007); TIMP-2 protein levels were detected in
tumors and in control tissues, but no significant differences were observed,
corresponding to the RT-PCR results.
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Figure 2. Quantitation of matrix metalloproteinase
(MMP) 1 (pro form) (A), tissue inhibitor of MMP (TIMP) 1 (B), and TIMP-2 (C)
proteins in tissue homogenates. Error bars represent SEM. LNM indicates lymph
node metastasis.
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We used substrate zymography to determine the activity of MMP-2/MMP-9,
MMP-3/MMP-7, and TIMP-1/TIMP-2. Gelatin zymography revealed a varied profile
of MMPs: lysis zones corresponding to molecular weights of 92, 84, 72, and
62 kd and high-molecular-weight (approximately 120-kd) gelatinases were seen
(Figure 3A). Gelatinases of 92 and
84 kd correspond to pro–MMP-9 and an active form of MMP-9, respectively.
Those of 72 and 62 kd are considered to be pro–MMP-2 and its active
form, respectively. These results are supported by the profile of TPA-treated
HT-1080 cells, which express all of these enzyme species and whose gelatinolytic
zones matched those of the HNSCC samples. The high-molecular-weight gelatinase
detected in some samples might be due to a complex of MMP and TIMP. Indeed,
2 inhibitors tested, TIMP-1 and TIMP-2, were detected in most HNSCC tissues
by ELISA (Figure 2) and gelatin
reverse zymography (data not shown). Casein zymography demonstrated lytic
zones corresponding to molecular weights of of approximately 57, 45, 29, and
21 kd and several small bands between 57 and 45 kd and 29 and 21 kd (Figure 3B). The 57- and 45-kd bands correspond
to latent and active forms of MMP-3, respectively. The 29- and 21-kd bands
are considered to be pro–MMP-7 and its active form, respectively. The
nature of gelatinolytic (MMP-2, MMP-9, and MMP-14) and caseinolytic (MMP-3
and MMP-7) enzymes or inhibitors (TIMP-1 and TIMP-2) was confirmed by Western
blotting of the same tissue homogenates with specific antibodies (only data
of MMP-14 are shown in Figure 3C).
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Figure 3. A, Gelatin zymography; B, casein
zymography; and C, Western blot analysis of matrix metalloproteinase 14 protein.
Tissue homogenates from 5 representative matched pairs of primary tumors (T),
metastatic lymph nodes (LN), and histologically normal tissues (N) were subjected
to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Conditioned
medium from 12-O-tetradecanoyl-phorbol-13-acetate–treated
HT-1080 fibrosarcoma cell line and MDA-MB 231 mammary carcinoma cells served
as a positive control and as a standard for intergel variations for gelatin
zymography and casein zymography, respectively. Molecular mass markers, in
kilodaltons, are indicated on the left (Mr x 103).
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Using computerized image analysis of transparent bands, we quantitated
the gelatinolytic and caseinolytic activities in tissue homogenates. The results
(Figure 4) showed that the MMP-9
(92 and 84 kd, respectively) and MMP-2 (72 and 62 kd, respectively) activities
were significantly greater in primary tumors and LNM compared with the levels
in control tissues. Compared with control tissues, the levels of high-molecular-weight
gelatinase (approximately 120 kd), MMP-3 (45-51 kd), and MMP-7 (21-29 kd)
were also greater in the primary HNSCC, although the levels in LNM did not
reach statistical significance (Figure 4).
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Figure 4. Gelatinolytic and caseinolytic
activities in tissue homogenates. Values are obtained from computerized image
analysis of transparent bands in substrate zymography. Error bars represent
SEM.
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Our results imply that several members of the MMP and TIMP family are
present in primary and secondary HNSCC. Apart from MMP-14 and TIMP-2, all
the molecules tested (including TIMP-1) were up-regulated in primary HNSCC
and/or LNM compared with adjacent histologically normal mucosa.
LEVELS OF MMPs AND TIMPs IN RELATION TO CLINICOPATHOLOGICAL VARIABLES
Relationships between the mRNA expression of MMPs and TIMPs in 54 primary
tumors and their clinicopathological variables were analyzed. As shown in Table 2, MMP-1, MMP-9, and TIMP-1 mRNA
expression in primary HNSCC showed a statistically significant relationship
with a higher T classification (T3-T4) (P = .004,
.001, and .003, respectively). A significant correlation was found between
MMP-9 expression and an infiltrating pattern of growth (P = .002). In addition, enhanced mRNA expression of MMP-9 was strongly
correlated with the presence of lymph node involvement (P<.001), whereas MMP-2, MMP-7, and MMP-11 levels were weakly correlated
(P = .04, .03, and .049, respectively). Comparing
primary tumors with early lesions (pathological stages I and II) and advanced
diseases (stages III and IV), higher expression levels of MMP-2 and MMP-9
were observed in the latter group (P = .01 and <.001,
respectively). On the other hand, when patients were separated into 4 groups
according to the cutoff values for MMP-2 and MMP-9 expression obtained from
the mean mRNA levels determined by RT-PCR in the primary tumors, groups with
high MMP-2 and high MMP-9 expression showed the highest incidence of LNM (100%; P = .002; odds ratio, 28.24) and advanced pathological
stages (100%; P = .02; odds ratio, 12.78), whereas
groups with low MMP-2 and low MMP-9 expression showed the lowest incidence
of nodal metastasis (23%; P<.001) and advanced
stage (38%; P<.001) compared with other groups
(Figure 5). In contrast, there was
no association between MMP-1, MMP-2, MMP-9, MMP-11, or TIMP-1 levels and age,
sex, site of primary tumors, or histological grade (Table 2). Furthermore, no association between expression of MMP-3,
MMP-10, MMP-13, MMP-14, TIMP-2, ratio of MMP-9 to TIMP-1, or ratio of MMP-2
to TIMP-2 and any clinicopathological variables was observed (data not shown).
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Table 2. Correlation Between Clinicopathological Variables and Messenger
RNA Levels of MMPs and TIMPs in Primary HNSCC Tissues*
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Figure 5. Correlation between lymph node
metastasis (A) or advanced pathological stages (B) and the messenger RNA expression
of matrix metalloproteinase (MMP) 2 and MMP-9 in primary head and neck squamous
cell carcinoma tissues. Node – and Node + are primary tumors without
and with neck node metastasis, respectively. The cutoff values obtained from
the mean values of MMP messenger RNAs determined by reverse transcription–polymerase
chain reaction in primary tumors were used to separate primary head and neck
squamous cell carcinoma into groups with low and high expression.
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COMMENT
A number of studies have attempted to delineate which, if any, of the
MMPs and TIMPs are required for HNSCC to grow and spread.28
Until now, the predictive value of the MMPs and TIMPs in invasion and metastasis
of HNSCC has been controversial, partly because of the varying methods used
to detect MMP expression. Because the components of the extracellular matrix
are complex, the combined action of various MMPs is essential for the efficient
degradation of the structure. Thus, a comprehensive study of the expression
of multiple MMPs and their inhibitors (TIMPs) is important for understanding
the complex processes by which tumors acquire their invasive and metastatic
potential. In the present study, we quantified the expression of a comprehensive
set of MMPs and TIMPs previously identified in HNSCC and studied their relationship
with clinicopathological variables in an attempt to determine whether overexpression
of certain specific proteases could be particularly relevant to progression
in this disease.
Using the highly sensitive RT-PCR assay, we studied the expression of
all genes of interest within the same tissue samples. Where available, we
also confirmed the presence of proteins and enzyme activities using immunoblot,
enzyme immunoassay, and substrate zymography. We used tumor margins in the
present studies based on the hypothesis that the cellular events in the tumor-stromal
interface might be more closely related to the metastatic potential of the
tumor than the (often necrotic) center. Coexpression of several members of
the MMP family seems to be a general characteristic of human HNSCC. The simultaneous
expression of several MMPs is consistent with the concept that extracellular
matrix remodeling during tumor progression requires the synergistic action
of several proteolytic enzymes produced by tumor cells or by stromal cells.2 Evidence suggests that each of these MMP genes might
have a distinct role in tumor progression. Some members of the MMP family,
such as MMP-2 and MMP-11, are expressed mainly in stromal fibroblasts and
might be regarded as paracrine, stroma-derived factors necessary for the progression
of HNSCC.8, 11 Both MMP-7 and MMP-9
were expressed exclusively in epithelial cells3, 10-11;
MMP-1 and MMP-10 were found principally within fibroblasts surrounding tumor,
in endothelial cells, and also in neoplastic cells.3-5,8
Muller et al8 reported that increased MMP-11
gene expression might be a useful marker for defining subpopulations of aggressive
HNSCC. Increased expression of MMP-14 was detected at the tumor cell surface,
especially at the invasive edge of tumor cell nests, in most HNSCC tissues
assayed.13
Although inhibition of in vitro and in vivo tumor invasion by TIMPs
has been demonstrated,20, 29 increased,
rather than decreased, TIMP levels have been shown to be related to poor outcome
in several malignant tumors, such as bladder cancer.30
The present finding of increased TIMP-1 expression in HNSCC might be explained
by the growth-promoting activity of TIMPs on a variety of cell types31 or the induction of TIMPs by secreted MMPs (or vice
versa) from tumor-host interaction in the extracellular milieu. The correlation
between increased TIMP-1 and TIMP-2 levels with less aggressive tumors was
found in some studies,7, 11, 17
although the opposite pattern was also reported16, 18
(and confirmed in the present study).
Several studies have examined relationships between the expression of
gelatinases and malignant potential in HNSCC, but the results are still inconclusive.
In oral cancers, MMP-2, but not MMP-9, was found to correlate with LNM and
poor clinical outcome.6, 10 One
study17 found that high levels of MMP-2 and
MMP-9 were related to the invasiveness of oral SCC, whereas another11 showed no difference in MMP-2 and MMP-9 levels between
primary HNSCC and LNM. Most recently, several studies19-22
have shown that MMP-9 might play a more important role than MMP-2 in the invasive
and metastatic potential of HNSCC. Among 9 MMPs and 2 TIMPs tested in the
present study, MMP-9 overexpression showed the strongest correlation with
the presence of neck nodal metastases and advanced pathological stages. The
underlying roles that some MMP family members play in the process of lymphatic
metastasis remain to be elucidated. In addition, it is possible that some
MMPs and TIMPs did not show statistical significance because of a type II
error (relatively small sample size with high variability), and another study
with a larger sample size might be required.
The up-regulation of several MMPs in lymph node–positive patients
suggests that the evaluation of MMPs, MMP-9 in particular, in HNSCC tissues
at the time of presentation might allow identification of a subset of patients
with HNSCC who are more susceptible to metastatic spread via lymphatic pathways
and permit therapy to be offered accordingly. With the application of highly
sensitive RT-PCR analysis, preoperative assessment from small tissue biopsies
or even needle aspirates will become more useful in assessing the malignant
potential of HNSCC. Aggressive neck management in tumors showing multiple
MMPs positive, MMP-9 in particular, might be considered to avoid later lymphatic
spread. Because of the relatively short follow-up, we are unable to demonstrate
yet whether MMP/TIMP expression is related to survival. This awaits confirmation
in a longer follow-up period.
In conclusion, the results of these studies suggest that expression
of multiple MMPs and TIMPs is characteristic of HNSCC and that no specific
member of the MMP family is solely responsible for HNSCC progression. The
correlative studies of MMP/TIMP expression in human head and neck tumor tissues
suggest the potential role of MMP-2, MMP-7, MMP-9, and MMP-11 in progression
and metastasis of human HNSCC. Combined analysis of these MMPs, MMP-9 in particular,
might be useful in evaluating the malignant potential in individual HNSCC.
AUTHOR INFORMATION
Accepted for publication March 27, 2001.
This project was supported by a Fellowship from the Faculty of Medicine,
Siriraj Hospital Medical School (Dr O-charoenrat), and by the Siriraj Hospital
Fund, Bangkok, Thailand.
Presented at the annual meeting of the American Head and Neck Society,
Fifth International Conference on Head and Neck Cancer, San Francisco, Calif,
August 1, 2000.
We are grateful to all staff in the Department of Head and Neck Surgery
and operating theaters at the Royal Marsden Hospital for help with collecting
the clinical specimens. We also thank Gary M. Box, BSc, and William J. Court,
MSc, for excellent technical assistance.
Corresponding author and reprints: Pornchai O-charoenrat, MD, Division
of Head and Neck Surgery, Department of Surgery, Siriraj Hospital Medical
School, Bangkok 10700, Thailand (e-mail: pornchaio{at}hotmail.com).
From the Division of Head and Neck Surgery, Department of Surgery,
Siriraj Hospital Medical School, Bangkok, Thailand (Dr O-charoenrat); the
Head and Neck Unit, Royal Marsden Hospital, London, England (Dr Rhys-Evans);
and the Section of Cancer Therapeutics, Institute of Cancer Research, Sutton,
England (Dr Eccles).
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