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Short Communication |
From the Department of Pathology, University of Erlangen-Nürnberg, Erlangen, Germany
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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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Autonomous growth is found in both benign colorectal adenomas and malignant colorectal carcinomas. The essential step in the progression from adenomas to carcinomas is invasive growth, whereby basal membranes and extracellular matrix are degraded. One class of proteins participating in this process are matrix matalloproteinases (MMPs). However, recent evidence suggests that MMPs play a more complex role in tumor progression. They are necessary to create a microenvironment supporting the initiation and maintenance of growth of primary tumor and metastasis.9 Their mechanisms are not yet fully understood; however, they could also participate in processes like tumor angiogenesis. In contrast to other MMPs such as gelatinase A (MMP-2), stromelysin 1 (MMP-3), and stromelysin 3 (MMP-11), which are produced by stromal cells, MMP-7 (also named matrilysin) is expressed in the tumor cells of colorectal carcinomas and other epithelial tumors.10 Overexpression of MMP-7 has been demonstrated for about 80% of colorectal carcinomas.10,11 MMP-7 was shown to be important for the growth of colon adenoma12 and the tumorigenicity of colon cancer cells.13 Additionally it was shown that MMP-7 plays an important role for the invasive and metastatic potential of cancer cells.14,15
In APC gene mutated colorectal cancers overexpression of ß-catenin is often found predominantly at the invasion front.16 This led us to the hypothesis that ß-catenin/TCF are directly involved in invasion processes by activating invasion associated genes. A GenBank search for the TCF consensus binding sequence (5'-A/T A/T CAAAG-3') in the promoters of such genes identified several MMPs. Here we demonstrate that the promoter of the human MMP-7 gene contains two TCF binding sites and is activated by ß-catenin/TCF. Moreover, MMP-7 expression is correlated with ß-catenin expression in hereditary and sporadic human colon carcinomas. We conclude that overexpression of the tumor progression factor MMP-7 in up to 80% of colorectal carcinomas can be caused by mutations in the APC gene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following double stranded oligonucleotides were used as probes or for competition: TCF(1.): 5'-ACATACTTTCAAAGTTCTGTA; TCF(1.m): 5'-ACATACTGCCAAAGTTCTGTA; TCF(2.): 5'-AAAAATCCTTTGAAAGACA-AA; TCF(2.m): 5'-AAAAATCCTTTGGCAGACAAA; TBE1: 5'-CGCACCTTTGATTTCTGCACCTTTGATTTCT. Probes were end-labeled to 3 x 108 dpm/µg 0.5 ng probe was incubated with 0.5 µg of bacterially expressed GST-TCF-4 (codons 265–496) as described.5 For specific competition 500x amounts (250 ng) of unlabeled oligonucleotides were used.
Cells and Cell Culture
HeLa and HT29 cells were obtained from ATCC. All cell lines were cultivated under standard conditions in DMEM + 10% fetal bovine serum.
DNA Clones
phMMP-7prLuc was constructed by cloning the XhoI/HinDIII fragment (encoding nucleotides -933 to +35 of the human MMP-7 promoter) of the plasmid p-933HPCAT (gift from L. Matrisian, Vanderbilt University, Nashville, TN) into the XhoI/HinDIII opened vector pGL3-Basic (Promega, Mannheim, Germany). For mutation of phMMP-7prLuc (wild type; WT) with the QuickChange mutagenesis kit (Stratagene, Heidelberg, Germany) we used the following primers: pTCF(1.m): 5'-GTTAATGAAAAATAACACATACTGCCAAAGTTCTGTAGACTC; pTCF(2.m): 5'-GACAGAAAAAAAAATCCTTTGGCAGACAAATACATTGTG.To construct the four times tandem repeat clones p4xTCF(1.)-Luc, p4xTCF(1.m)-Luc, p4xTCF(2.)-Luc and p4xTCF(2.m)-Luc, we polymerized the corresponding double-stranded oligonucleotides used for EMSAs and cloned them into the HinDIII cut and blunted vector ptkLuc (gift from E. Serfling, Department of Pathology, Wuerzburg, Germany), containing a thymidine kinase minimal promoter. Following plasmids were gifts from other researchers: pGST-TCF4(DBD), pcDNA/myc-hTCF4 and pcDNA/myc-DN-TCF4 from K. Kinzler and B. Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD), pcDNAhß-catenin was a gift from H. C. Clevers (University Hospital, Utrecht, The Netherlands), pSV40Ets1 from J. Ghysdael (Institute Curie, Orsay, France), pMSc-fos from M. Schuermann (Marburg, Germany) and pRSVc-jun from P. Angel (Karlsruhe, Germany).
Transfections and Reporter Assays
HeLa and HT29 cells were grown in 60-mm tissue culture dishes to a confluency of 50% and transfected with 2 µg of reporter construct, 0.5 µg of pCMVßGal-control, and 3 µg of expression vectors or empty vector pCNA3 as indicated, using Lipofectamine Plus (Life Technologies, Karlsruhe, Germany). Cells were harvested after 40 hours, luciferase assays were carried out and normalized for transfection efficiency through ß-galactosidase activity using Dual Light Kit (Perkin Elmer, Weiterstadt, Germany). Assays were performed in triplicate.
Immunohistochemistry
Formalin-fixed, paraffin-embedded tissue blocks of colorectal
cancers with variable expression of ß-catenin were used. Three tumors
were from genetically defined familial adenomatosis polyposis (FAP)
patients and two from patients with hereditary non-polyposis colorectal
cancers (HNPCC). APC gene status of the HNPCC cases was considered as
normal, based on loss of heterozygosity analyses (marker D5S107).
However, exact sequencing data of the APC gene were not available for
these cases. The other cases were sporadic carcinomas. 3-µm serial
sections were deparaffinized, rehydrated, and incubated with the
different mouse monoclonal antibodies for 2 hours. Antigen retrieval
was used for 
-MMP-7, mib-1 (microwave treatment), and CEA (protease
XIV). Dilutions were 1:300 for -MMP-7 (Chemicon, Hofheim, Germany),
1:200 for -ß-catenin (Transduction, Lexington, KY), 1:30
for -Mib-1 and -CEA (Dako, Hamburg, Germany). Biotinylated rabbit
-mouse Ig antiserum diluted 1:50 was used as the secondary antibody.
After washing, slides were incubated for 30 minutes at room temperature
with streptavidin-coupled alkaline phosphatase (Dako, Hamburg, Germany)
and developed for 12 minutes at 37°C using Fast Red (Sigma,
Deisenhofen, Germany) as substrate. After rinsing in water, sections
were counterstained with hemalum (Merck, Darmstadt, Germany),
dehydrated, and coverslipped.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By searching the GenBank, we identified potential TCF binding
sites in the promoters of several human invasion associated genes
including MMP-1, MMP-3, and MMP-7. We focused on MMP-7 because of its
overexpression in most colorectal cancers. Three transforming growth
factor (TGF)-ß inhibitory elements (TIEs), two binding sites for
ets transcription factors (PEA3), and one AP-1 binding site were
already described in the human MMP-7 promoter.17
We
identified two potential TCF binding elements: TCF(1.) from -109 to
-103 and TCF(2.) from -194 to -188, which matched the consensus
sequence 5'-A/T A/T CAAAG-3' (Figure 1a)
.
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. GST protein alone did not form a
complex. Specificity was confirmed by using the two mutated TCF sites
as probes, which then were not bound by TCF-4. Moreover, we used
wild-type or mutated TCF(1.), TCF(2.) and TBE1, a TCF site of the
c-myc promoter,5
as specific competitors. ß-Catenin and TCF4 Activate the Human MMP-7 Promoter
To determine whether TCF-4 binding to the defined elements has a
functional consequence, we transiently transfected luciferase reporter
constructs controlled by the human MMP-7 promoter together with
different expression constructs into HeLa cells (Figure 2b)
. Cotransfections of TCF-4 or
ß-catenin expression clones led to a 3.2-fold or 1.88-fold
activation, respectively, compared to the empty vector pcDNA3. This was
similar to the known stimulatory effect of TPA on the MMP-7 promoter
(2.36- fold).17
A synergistic activation (9.4-fold) was
found after cotransfection of TCF-4 and ß-catenin expression
constructs. We further tested the effect of overexpressed Ets1 and
c-Jun/c-Fos, binding to the PEA3 and the AP-1 site in the MMP-7
promoter. Isolated stimulation of the promoter through only one of
these elements had only a weak effect in HeLa cells. However combined
expression of both TCF4/ß-catenin and Ets1, c-Jun/c-Fos led to a
maximum activation of the MMP-7 promoter. This was similar to a
combined activation by TCF4/ß-catenin and TPA, which mimics AP-1
activation. The empty luciferase vector did not respond to either
activator (not shown). Thus TCF/ß-catenin contributed to a high
extent (~60%) to the maximum activation of the human MMP-7 promoter
achieved through all defined elements.
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. Mutation in TCF(1.) reduced the stimulatory effect of
TCF-4 plus ß-catenin to about 30% compared to the wild-type promoter
(Figure 2c)
. The promoter mutated only in TCF(2.) could be activated by
up to 44% compared to the wild-type promoter by both activators.
Interestingly, mutations in both sites could not fully suppress
stimulation by ß-catenin/TCF-4 (1.81-fold). Recent data demonstrating
that ß-catenin/TCF also activates c-jun gene
transcription18
indicate that this residual stimulation
could be due to indirect stimulation through the AP-1 site (-67 to
-61) of the human MMP-7 promoter.17
We next tested isolated TCF(1.) and TCF(2.) by cloning four copies of
each wild-type and mutated site upstream of a thymidine kinase-minimal
promoter luciferase construct (Figure 2d)
. Both polymerized TCF sites
were responsive to ß-catenin/TCF-4. TCF(2.) was activated stronger,
which is consistent with our observation of a higher binding affinity
of TCF-4 to this site (Figure 1b)
. Nucleotide substitutions, which
abolished TCF-4 binding, prevented activation.
Dominant Negative TCF Suppresses the Activity of the MMP-7 Promoter in Colon Cancer Cells
The HT29 colon cancer cell line has no functional APC protein due
to mutations on both alleles. Thus the MMP-7 promoter should be
constitutively activated by endogenous ß-catenin/TCF-4. Accordingly,
cotransfection of expression vectors for both factors had only a weak
additional stimulatory effect on the wild-type promoter (Figure 2e)
.
Similar results were obtained with the APC gene deficient colon cancer
cell lines SW480 and SW620, which showed an even weaker MMP-7 promoter
activation by a combination of TCF4 and ß-catenin expression vectors
(data not shown). To further confirm the importance of
ß-catenin/TCF-4 for the activation of the human MMP-7 promoter, we
tried to inhibit its endogenous activity in HT29 by a dominant-negative
TCF-4 mutant (DN-TCF-4).4
DN-TCF4 lacks the ß-catenin
binding domain, thereby binding to the TCF elements without activating
transcription. Transfection of the DN-TCF-4 construct suppressed the
promoter activity to 18%. In contrast DN-TCF-4 had only a weak
inhibitory effect on the promoter construct with mutations in both TCF
sites (endogenous activity of this construct was 40% compared to WT
promoter).
Correlation of Nuclear ß-Catenin and MMP-7 Expression in Human Colorectal Carcinomas
ß-Catenin is overexpressed in a high percentage of sporadic
colorectal carcinomas. If MMP-7 activation is highly dependent on
ß-catenin, one would expect a correlated expression of both proteins.
Serial sections of 16 representative colorectal cancers with strong,
weak, or no immunohistochemically detectable ß-catenin were stained
with -MMP-7 antibodies. Included were both sporadic and hereditary
forms of colon carcinomas. All cases showed a correlation in the
expression pattern of both proteins. Especially, all three included
tumors from FAP patients with defects in the APC gene expressed both
ß-catenin and MMP-7. Two investigated tumors from HNPCC patients with
high grade microsatellite instability did not express nuclear
ß-catenin and MMP-7. However, we had not included HNPCC tumors with
mutations in the APC or ß-catenin gene, which occur in >50% of
these tumors. Representative results for a ß-catenin high and low
expressing tumor are shown (Figure 3)
.
Immunohistochemistry for the colon tumor marker CEA and the
proliferation marker mib-1 were used to control the staining efficiency
of the different tumors. Interestingly, in all investigated cases we
found no correlation of ß-catenin expression and the proliferation
activity, measured by the percentage of mib-1 stained cells (compare
Figure 3, d and h
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data can explain important previous observations in intestinal tumors of mice heterozygous for the ApcMin allele (Min/+), the animal model mimicking the human FAP syndrome.12 There, Wilson et al showed that the majority of these adenomas (88%) overexpressed MMP-7 mRNA. Furthermore they demonstrated that intestinal tumorigenesis in these mice was suppressed by an additional targeted disruption of the MMP-7 gene. The authors suggested a cooperative mechanism of the APC gene defects and MMP-7 expression for tumorigenesis. Our data, defining MMP-7 as a target gene of ß-catenin/TCF, support these findings and can also explain the high frequency of MMP-7 overexpression (up to 80%) in human colorectal cancers,10,20 which is similar to the frequency of APC gene mutations.1
In contrast to other MMPs, MMP-7 was shown to be an important factor already for the growth of early colon adenomas.12 Accordingly, low amounts of MMP-7 are also found in earlier adenomas.21 However there is an increase in MMP-7 expression, correlated with tumor progression. Late, highly dysplastic colon adenomas, the first progression stage with invasive potential, show a high MMP-7 expression level.20-22 This is consistent with a postulated role of MMP-7 also for later tumor progression steps.14,15 We also detected potential TCF binding sites in the promoters of other MMPs (data not shown), one of which (MMP-1) is also overexpressed in human colorectal cancer and associated with poor prognosis.23 Recent work also demonstrated an indirect effect of ß-catenin on the expression of uPAR,18 another molecule involved in invasion. These data extend the effect of APC gene mutations from autonomous proliferation, which is already a characteristic of benign colon adenomas, to later steps in colon cancer development.
An important question to be discussed is why high MMP-7 expression is
restricted to later progression steps, whereas APC gene mutations are
probably the earliest genetic defect in colorectal carcinogenesis.
Previous analysis of the MMP-7 promoter also defined binding sites for
the transcription factors AP-1 and PEA3.17
Both factors
are downstream effectors of the ras signaling
pathway24-26
and activated K-ras was shown to enhance
MMP-7 expression in colon cancer cell lines.27
Activating
mutations in the K-ras oncogene are accumulated in a high percentage of
late, highly dysplastic colon adenomas and colorectal
carcinomas.1,28
Thus, APC and K-ras mutations could have
an additive effect in activating MMP-7 gene transcription in colorectal
tumors. This is supported by our results that maximum activation of the
MMP-7 promoter is achieved by a combination of TCF4, ß-catenin,
ets-1, c-jun, and c-fos expression vectors (Figure 2b)
. Further work
will investigate the relative significance of the different
transcriptional activators. In common with other MMP genes, the
regulatory elements of MMP-7 possess TGF-ß inhibitory elements,
negatively regulating its transcription.17
It is now clear
that inactivating mutations in molecules of the TGF-ß signaling
pathway are additional important genetic alterations during colon tumor
progression, and also often occur during the step from benign to
malignant growth.29
Moreover, mice with a combined defect
in the APC gene and the TGF-ß pathway develop more aggressive
malignant tumors compared to mice with an isolated APC gene
defect.30
This indicates functional cooperation of both
pathways. One could speculate that both constitutive activation of
ß-catenin/TCF and inactivating mutations in the TGF-ß pathway are
necessary for efficient activation of MMP-7 transcription. Thus MMP-7
could be a model for an effector gene simultaneously affected by
mutations in the two main oncogenic pathways in colorectal
carcinogenesis, the WNT and the TGF-ß pathway. Further work will
address this point.
Our results, defining MMP-7 as a target gene of ß-catenin, explain the high percentage of MMP-7 overexpressing colorectal cancers. The possible functions of MMP-7, not only in early but also in late tumorigenesis,14,15 extend the effects of APC gene defects in colon tumors.
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Acknowledgements |
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Footnotes |
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Accepted for publication June 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Also indicated are
the previously defined TGF-ß inhibitory element-like sequences
(TIE), the ets
(PEA3) and the AP-1
binding sites (
).
Numbers indicate nucleotide positions relative to the transcription
initiation site (+1). The
diagram is not to scale. Also shown are the nucleotide sequences of
both sites and the mutated sequences used in the following experiments
(TCF(1.m)
and
TCF(2.m)).
b: TCF-4 binds specifically to both TCF sites. EMSA with
TCF(1.) and
TCF(2.) sequences
(wt) or their mutants
(mt) as probes, incubated
with GST-TCF-4 (TCF-4) or
GST alone. For specific competition the unlabeled oligonucleotides used
as probes or TBE2 (TCF binding site of the c-myc
promoter) were used.
