Yonago Acta medica 2009;52:47–56

Functional Polymorphisms in the Promoter Regions ofMatrix Metalloproteinase-2, -3, -7, -9 and TNF-alphaGenes, and the Risk of Colorectal Neoplasm in Japanese
Hideyuki Ohtani, Naoto Maeda and Yoshikazu Murawaki
Division of Medicine and Clinical Science, Department of Multidisciplinary Internal Medicine,
School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504 Japan

Colorectal carcinogenesis involves environmental factors and genetic predispositions. Recent studies have suggested the associations between colorectal neoplasm and functional
polymorphism of matrix metalloproteinases (MMPs) and cytokine genes. In this study, we
analyzed polymorphisms of MMPs and tumor necrosis factor (TNF)-alpha genes, focusing
on the susceptibility to colorectal neoplasm and the tumor progression. The subjects were
186 patients (95 men and 91 women) who underwent total colonoscopy, and were classified
into cancer, adenoma and non-neoplasm (control) groups of 47, 72 and 67 patients, respectively. The polymorphisms at the MMP-2 –1306C/T, MMP-3 –1171 5A/6A, MMP-7 –181A/
G, MMP-9 –1562C/T and TNF-alpha –308G/A loci were analyzed. Regarding backgroundfactors, significant differences were found in the age, sex ratio and alcohol-drinking andcigarette-smoking histories in the adenoma and cancer groups, compared to those in thecontrol group. On these factors-adjusted logistic regression analysis of polymorphisms
and disease susceptibility, no significant difference was noted in the frequency of any polymorphism in the adenoma and cancer groups, compared to those in the control group.
The analysis of the involvement of polymorphisms in tumor progression in the adenoma
and cancer groups revealed that the odds ratio for the MMP-3 5A allele was significantly
higher in the cancer group (2.74; 95% confidence interval = 1.11–6.74, P = 0.02). The polymorphisms of MMP genes and TNF-alpha genes were not associated with the susceptibility to colorectal neoplasm, but the involvement of the MMP-3 5A allele in the progressionof adenoma to cancer was suggested.
Key words: colorectal neoplasm; gene analysis; matrix metalloproteinase; polymorphism; tumor
necrosis factor-alpha

Recently, the rate of colorectal cancer has been
increasing rapidly in Japan, and it is now themain cause of death from malignant disease, as inmany other countries (Yoshimi and Sobue, 2004).
In fact, age-standardized rates are similar to thosein Caucasian populations of the United States(Yiu et al., 2004). The reason has generally been
ascribed to the Westernized diet, characterized bya high intake of fat and meat, popular after WorldWar II (Kono, 2004), whereas the relevance ofgenetic predispositions has not been sufficiently
analyzed in Japanese. Colorectal neoplasm isknown to be a multifactorial disease, with dietaryfactors, lifestyle habits and genetic predispositions
contributing to its development.
Abbreviations: MMP, matrix metalloproteinase; RFLP,
restriction fragment length polymorphism; TNF, tumor
necrosis factor47

H. Ohtani et al.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that play key roles not only inextracellular matrix degradation but also in all
stages of cancer initiation, invasion and metastasis (Curran and Murray, 1999; Zhu et al., 2001;
Behrens et al., 2003). Furthermore, recent studies
have suggested that MMPs are involved in tumor
initiation and development, including the regulation of cell proliferation, apoptosis, angiogenesis,
loss of cell adhesion and immune responses tocancer (Egeblad and Werb, 2002). In fact, theover-expression of MMPs has been demonstrated
in various cancers (Adachi et al., 1999; Ohashi
et al., 2000; Aglund et al., 2004; Jordan et al.,2004). Polymorphisms of MMPs in the promoter
region, naturally occurring sequence variations,
may result in the differential expression of MMPs
in individuals (Ye, 2000). To date, the promotersof MMP-2, -3, -7 and -9 genes have been reported
to contain polymorphisms, exhibit allele-specificeffects on the regulation of MMP gene transcription, and have been associated with changes in thesusceptibility to or development of some cancers(Liang et al., 2002; Ghilardi et al., 2002, 2003;
Matsumura et al., 2005).
On the other hand, previous studies haveshown that tumor necrosis factor (TNF)-alphaexpression may act as a high-risk factor or a poor
prognostic factor in some cancers (Warzocha etal., 1997; El-Omar et al., 2003; Machado et al.,
2003; Sharma et al., 2008). However, a few previous reports have suggested that there is no signifi-

cant association between the TNF-alpha –308A/G
polymorphism allele and colorectal cancer development (Park et al., 1998; Landi et al., 2003).
To explore the possible association between
these polymorphisms and the risk of colorectal
neoplasms, we analyzed promoter genes in MMP2, -3, -7 and -9 and TNF-alpha in a Japanese sample of colorectal neoplasm patients and controls
who had no finding of colonoscopy.

Materials and Methods
Subjects
A total of 186 Japanese subjects (91 women and
95 men, with a mean age of 64 ± 13 years) werestudied between August 2003 and March 2007.
All subjects had undergone total colonoscopy
just prior to enrollment. One hundred nineteen
patients with histologically confirmed colorectal
neoplasm (47 cancers and 72 adenomas) and 67
non-neoplasm subjects (controls) were interviewed
regarding their medical history, family history ofcolorectal cancer, anamnesis of diabetes, and habits such as alcohol intake and cigarette smoking.
Before enrollment and blood sampling, written
informed consent was obtained from all recruitedsubjects. This study was approved by the Ethics
Committee of Tottori University Faculty of Medicine.

Table 1. Sequences of PCR primers
Gene
Polymorphism
Forward primer and reverse primer (5´–3´)
			

Tm
(˚C)

Restriction
enzymeMMP-2–1306 C/T
			
MMP-3
–1171 5A/6A
			
MMP-7
–181 A/G
			
MMP-9–1562 C/T			
TNF-alpha–308 G/A
			

60

Xsp I

65

Tth111 I

65

EcoR I

60

Sph I

55

Nco I

CTT CCT AGG CTG GTC CTT ACT GA
CTG AGA CCT GAA GAG CTA AAG AGC T
CTT CCT GGA ATT CAC ATC ACT GCC ACC ACTGGT TCT CCA TTC CTT TGA TGG GGG GAA AGATGG TAC CAT AAT GTC CTG AAT GAT ACC TAT G
TCG TTA TTG GCA GGA AGC ACA CAA TGA ATTGCC TGG CAC ATA GTA GGC CC
CTT CCT AGC CAG CCG GCA TC
GAG GCA ATA GGT TTT GAG GGC CAT
GGG ACA CAC AAG CAT CAA G

48

Polymorphisms and colorectal neoplasm

A

DNA extraction

MMP-2 (–1306 C/T): Xsp I
M
1
2
3
(bp)

Genomic DNA samples were extracted from peripheral white blood cells using a DNA extracting
kit (DnaQuickII: Dainipponseiyaku, Osaka, Japan) according to the manufacturer’s instructions.

4

5
(bp)

194 —

— 193
— 162

Determination of genotypes
C/T

Using the extracted DNA as a template, the promoter region of each MMP was amplified by PCR
using commercially available kits (HotStarTaq:
QIAGEN, GmbH, Germany), following the manufacturer’s instructions. The sequences of primers
are summarized in Table 1. The methods used
to type the MMP-2 –1306 C/T, MMP-3 –1171
5A/6A, MMP-7 –181A/G, MMP-9 –1562 C/T and
TNF-alpha –308 G/A polymorphisms have been
described previously (Jormsjö et al., 2001; Vasku
et al., 2004; Zhang et al., 2004; Perri et al., 2005;
Tuet al., 2007). Briefly, reactions were carried out
under the following conditions: 5 min at 95˚C,
and then amplification for 35 cycles consisting of
30 s at 95˚C, 30 s at an appropriate temperatureand 30 s at 72˚C. A final extension step at 72˚C
for 5 to 10 min was added to terminate the amplification. Subsequently, the PCR products weredigested with appropriate restriction enzymes thatcleave from 1 to 2 fragments. The digests werethen electrophoresed on a 1 to 2% agarose gel using TBE buffer (45 mM Tris-borate, pH 8.3, and2 mM EDTA) to confirm cleavage using molecular size marker IV (Nippon Gene, Tokyo, Japan).
Gels were stained with ethidium bromide andvisualized under UV light. As examples for gel
documentation, the results of MMPs genotyping
were shown in Fig. 1.

B

C/C

MMP-3 (–1171 5A/6A): Tth111 I
M
1
2
3
4

5

— 129
— 97

118 —

6A/6A 5A/5G

C

5A/6A

MMP-7 (–181 A/G): ExoR I
M
1
2
3

4

5

194 —

— 150
— 120

118 —

A/A

D

A/G

MMP-9 (–1562 C/T): Sph I
M
1
2
3

4

5

603 —

— 435

310 —
234 —
194 —

— 247
— 188

C/C

C/T

T/T

Fig. 1. Genotyping of MMP-2 C/T, MMP-3 5A/6A, MMP-7 A/G and MMP-9 C/T by PCR-restriction fragment
length polymorphism.
A: MMP-2 C/T. The PCR products were digested with XspI restriction enzyme and subjected to electrophoresis on a 2.5%
agarose gel. M, molecular marker; 1 and 2, C/T genotype; 3, 4 and 5, C/C genotype.
B: MMP-3 5A/6A. The PCR products were digested with Tth111I restriction enzyme and subjected to electrophoresis
on a 2.5% agarose gel. M, molecular marker; 1, 3 and 4, 6A/6A genotype; 2, 5A/5A genotype; 5, 5A/6A genotype.
C: MMP-7 A/G. The PCR products were digested with EcoRI restriction enzyme and subjected to electrophoresis on a
2.5% agarose gel. M, molecular marker; 1, 2 and 4, A/A genotype; 3 and 5, A/G genotype.
D: MMP-9 C/T. The PCR products were digested with Sph I restriction enzyme and subjected to electrophoresis on a
2.5% agarose gel. M, molecular marker; 1 and 4, C/C genotype; 2 and 3, C/T genotype; 5, C/T genotype.

49

H. Ohtani et al.

Table 2. Characteristics of subjects
Control [67]Ratio		Age (year)		
Sex
Male/female
Family history§Yes/noSmoking status
Smoker/non-smoker
Alcohol status
Drinker/non-drinker
DiabetesYes/noPatient with
Adenoma [72]
Ratio
P†

61.3 ± 2.0
25/42
8/59
19/48
21/46
5/62

66.3 ± 1.1
42/30
3/69
31/41
36/36
5/67

0.04
0.02
NS
NS
0.03
NS

Ratio

Cancer [47]P†P‡

67.3 ± 1.7
28/19
10/37
26/21
25/22
9/38

0.04
0.03
NS
0.006
0.03
NS

NS
NS
0.008
NS
NS
NS

[ ], number of subjects.
Ratio, except age (mean ± SD).
NS, not significant.
† Compared with the control subjects.
‡ Compared with patients with adenoma.
§ Patients with family history of colorectal cancer in the 1st degree relatives.

colon. The backgrounds of groups are shownin Table 2. The mean age was 67.3 ± 1.7 years(range: 27–90) in the colorectal cancer group and66.3 ± 1.1 years (range: 36–88) in the adenoma
group. Compared to the control group, significant
differences were noted in the age, sex ratio andalcohol-drinking history in the adenoma and cancer groups. No significant difference was notedin the cigarette-smoking history between the adenoma and control groups (P = 0.10), but one waspresent between the cancer and control groups (P= 0.01). There were no significant differences in
the familial medical history of colorectal cancer
or anamnesis of diabetes, compared to the control
group.
On comparison of the adenoma and cancergroups, no significant differences were present in
the age, sex ratio, or alcohol-drinking or cigarettesmoking history, but the frequency of familial
medical history of colorectal cancer was significantly higher in the cancer group (P = 0.007).
The frequency of anamnesis of diabetes was
slightly higher in the cancer group, but the difference was not significant (P = 0.08).

Statistical analysis
The significance of differences in means or proportions was measured using analysis of variance or
the chi-square test. Comparison of the genotypes of
MMP-2, -3, -7 and -9 and TNF-alpha and the allelotype distribution in the study groups was performedby means of a 2-sided contingency table using thechi-square test. Relationships between the genotypes and clinicopathological characteristics of thepatients were evaluated by Fisher’s exact test. To
evaluate the increased risk of colorectal neoplasm
associated with the presence of polymorphic alleles, odds ratios and 95% confidence intervals werecomputed, adjusted by logistic regression for several
covariates potentially associated with the colorectal
neoplastic risk such as age, sex, family history ofcolorectal cancer, alcohol intake, smoking status
and diabetes mellitus. All statistical analyses were
performed using the software package SPSS II for
Windows (version 11.0 J, SPSS Japan, Tokyo)

Results
Characteristics of subjects

Genotype distributions and the susceptibility for adenoma and cancer

Subjects who had undergone colonoscopy weredivided into a control group with no finding andpatient groups with adenoma or cancer of the

The frequencies and odds ratios of polymorphisms of the MMP-2, -3, -7 and -9 and TNF50

Polymorphisms and colorectal neoplasm

Table 3. Genotype and allele frequencies of polymorphisms of the MMP-2, -3, -7 and -9 and TNFalpha gene in controls and patients
Genotype
and allele

Control [67]
Number(%)

Patient with adenoma [72]Number
Odds ratio†
P(%)(95% CI)		

Patient with cancer [47]Number
Odds ratio†(%)(95% CI)

MMP-2 –1306 C/TCC
64 (95)66 (92)1		
41 (87)CT
3 ( 5)
6 ( 8)			
5 (11)TT0 ( 0)
0 ( 0)			
1 ( 2)CT + TT3 ( 5)
6 ( 8)
2.39
0.26
6 (13)
					(0.51–11.7)				
T
0.02		
0.04				0.07MMP-3 –1171 5A/6A
6A6A
50 (75)58 (81)1		
31 (66)
6A5A
17 (25)13 (18)			
14 (30)
5A5A
0 ( 0)
1 ( 1)			
2 ( 4)
6A5A + 5A5A
17 (25)14 (19)0.79
0.59
16 (34)					(0.34–1.84)				
5A
0.13		
0.1				0.19MMP-7 –181 A/G
AA
55 (82)65 (90)1		
45 (96)AG
12 (18)7 (10)			
2 ( 4)GG
0 ( 0)
0 ( 0)			
0 ( 0)AG + GG
12 (18)7 (10)
0.61
0.35
2 ( 4)
					(0.21–1.75)				
G
0.09		
0.05				0.02MMP-9 –1562 C/TCC
47 (70)54 (75)1		
30 (64)CT
19 (28)17 (24)			
16 (34)TT
1 ( 2)
1 ( 1)			
1 ( 2)CT + TT
20 (30)18 (25)0.78
0.53
17 (36)					
(0.35–1.71)				
T
0.16		
0.19				0.13TNF-alpha –308 G/A 1
GG
65 (97)71 (99)1		
46 (98)GA
2 ( 3)
1 ( 1)			
1 ( 2)AA0 ( 0)
0 ( 0)			
0 ( 0)GA + AA2 ( 3)
1 ( 1)
0.62
0.71
1 ( 2)
					(0.04–8.02)				
A
0.01		
0.01				
0.01

P

1
2.98
(0.62–14.29)

0.17

1
1.56
(0.64–3.81)

0.32

1
0.21
(0.40–1.11)

0.06

1
1.41
(0.61–3.28)

0.41

1
0.77
(0.06–9.31)

0.83[ ], number of subjects.
CI, confidence interval.
† Odds ratio adjusted on age, sex, smoking and alcohol status.

alpha gene are shown in Table 3. Regarding the
distribution of alleles in our study, most patients
had C/C alleles in MMP-2, 6A/6A in MMP-3, A/
A in MMP-7, C/C in MMP-9 and G/G in TNFalpha, and, in reverse, none had G/G alleles in
MMP-7, nor A/A alleles in TNF-alpha.
Logistic regression analysis adjusted for age,sex ratio, and alcohol-drinking and cigarettesmoking histories, which were background factors

with significant differences, was performed in thecontrol, adenoma and cancer groups.
There were no significant differences ofthese genotypes between controls and adenoma
patients, and between controls and cancer patients,suggesting that colorectal neoplasm susceptibility
was not found in any polymorphism of the MMP2, -3, -7 and -9 or TNF-alpha gene.
51

H. Ohtani et al.

Table 4. Genotype and allele frequencies of polymorphisms of the MMP-2, -3, -7 and -9 and TNFalpha gene in patients with adenoma and cancer

Association with tumor progression
To investigate the association with tumor progression from adenoma to cancer, the frequencies andodds ratios of polymorphisms of the MMP-2, -3,-7 and -9 and TNF-alpha gene in the adenoma andcancer groups were calculated (Table 4). Logistic
regression analysis adjusted for a familial historyof colorectal cancer and anamnesis of diabetes
was performed. We found that patients with the
MMP-3 5A allele had significantly higher odds
ratio (2.74) of cancer (P = 0.02). However, othergenotypes showed no significant difference between the adenoma and cancer groups.

Genotype
andallelePatient with
AdenomaCancer[72][47]
Number Number Odds ratio†(%)(%) (95% CI)

P

MMP-2 –1306 C/TCC
66 (92) 41 (87)1
CT + TT
6 ( 8)
6 (13) 1.54
0.49					 (0.43–5.488)
MMP-3 –1171 5A/6A
6A6A
58 (81) 31 (66)1
6A5A + 5A5A 14 (19) 16 (34) 2.74
0.02					 (1.11–6.74)
MMP-7 –181 A/G
AA
65 (90) 45 (96)1
AG + GG
7 (10)
2 ( 4) 0.58
0.52					 (0.11–3.00)
MMP-9 –1562 C/TCC
54 (75) 30 (64)1
CT + TT
18 (25) 17 (36) 1.43
0.41					 (0.61–3.37)
TNF-alpha –308 G/A
GG
71 (99) 46 (98)1
GA + AA1 ( 1)
1 ( 2) 0.58
0.58					 (0.13–36.13)

Discussion
It has been reported that MMPs play an importantrole in various cancer metastases and invasion
including colorectal cancer (Asano et al., 2007),
but no consistent finding has been obtained withregard to the association of any MMP gene polymorphism with colorectal cancer progression.
Similarly, no association between TNF-alpha andcolorectal cancer has been identified. We investigated polymorphisms of the MMP-2, -3, -7 and-9 and TNF-alpha gene and the susceptibility
to colorectal neoplasm. In addition, considering that most colorectal cancer cases develop via
the adenoma-carcinoma sequence (Fearon andVogelstein, 1990), we studied the association oftumor progression with each gene polymorphism.
MMP-2 expression in colorectal cancer tissue has
been reported to be correlated with the diseasestage and prognosis, and considered to play animportant role in colorectal cancer invasion andmetastasis (Turpeenniemi-Hujanen, 2005). In the
MMP-2 gene, C/T polymorphism is present at–1306, and the transcription activity is higher in
the C than in the T allele (Price et al., 2001). In
studies reported by Heittaratchi et al. (2007) andElander et al. (2006), –1306 C/T was not associated with the sensitivity of colorectal cancer, 5-year
survival rate or tumor characteristics, whereas Xu

CI, confidence interval.
† Odds ratio adjusted on family history and diabetes.

et al. (2004) reported that the risk of colorectal
cancer development was higher in CC than in CT
and TT types, showing that no consistent findings
have been obtained with regard to the associationof MMP-2 gene polymorphisms with colorectal
cancer. In our study, no association of MMP-2
gene polymorphisms with the susceptibility to colorectal neoplasm or tumor progression was noted.
In colorectal cancer tissues, MMP-3 ismainly expressed in stromal cells (Newell et al.,1994). In the MMP-3 gene, 5A/6A polymorphism
is present at –1171, and the transcription activity
is higher in the 5A than in the 6A allele (Ye et al.,1996). Hinoda et al. (2002) reported that the 6A
allele with low transcription activity indirectly
contributed to colorectal cancer development, but
no other study has supported this finding (Biondi
et al., 2000; Ghilardi et al., 2001; Xu et al., 2006;
52

Polymorphisms and colorectal neoplasm

Hettiaratchi et al., 2007). The clinicopathological significance of the MMP-3 expression in colorectal cancer remains unclear. No association
with colorectal neoplasm susceptibility was notedin the present study, but the frequency of the 5A
allele with high transcription activity during the
progression of adenoma to cancer was high in thecancer group. Sternlicht et al. (1999) investegatedthe MMP-3 in the breast cancer, and reported
that MMP-3 promoted spontaneous premalignantchanges and malignant conversion in the mammary glands of transgenic mice. Furthermore, regarding breast cancer, Nelson et al. (2008) reported that the overproduction of MMP-3 in mammary gland tissue triggered surrounding cells toincreasingly produce reactive oxygen species, andinduced DNA injury and genetic instability, finally leading to malignant conversion. Colorectal
cancer may develop via a similar course.
While MMP-7 has a broad substrate specificity, it also exhibits “sheddase” activity mediating cell surface protein release, and its influenceon the growth and progression of colorectal,
esophageal, stomach, and lung cancers has been
investigated (Leeman et al., 2003; Zhang et al.,2005). A strong correlation of MMP-7 with colorectal cancer malignancy has been reported, inwhich the expression frequency of MMP-7 wasparticularly high in cancer tissues, and the MMP7 expression level was higher in metastatic lesions than in the primary lesion (Yoshimoto et al.,1993). In the MMP-7 gene, A/G polymorphism
is present at –181 of the promoter lesion, and thetranscription activity is higher in the G than in
the A allele (Ghilardi et al., 2003). Ghilardi etal. (2003) reported that the MMP-7 –181 G/Ggenotype was involved in colorectal cancer andtumor progression in Italians, but we detected noassociation of MMP-7 gene polymorphisms withcolorectal neoplasm susceptibility or tumor progression. One reason may have been the biased
distribution of MMP-7 polymorphisms. The frequency of MMP-7 G/G was about 0.5% in healthy
subjects in a study performed in China (Lu et al.,2006), but about 20% in another Asian country,

India (Singh et al., 2008), suggesting regionalvariation. Actually, none of the subjects analyzed
showed MMP-7 G/G in our study.
Regarding MMP-9, Zucker and Varcrca(2004) reported that the MMP-9 level rose in an
early stage after the progression of adenoma tocolon cancer, and Roeb et al. (2001) reported that
MMP-9 transcription activity was 5 times higher
in colorectal cancer than in the normal mucosa.
In the MMP-9 gene, T/C polymorphism is present
at –1562, and the transcription activity is higher
in the C than in the T allele (Zhang et al., 1999).
Matsumura et al. (2005) reported a relationshipbetween stomach cancer invasion and the MMP-9
T allele position, and Grieu et al. (2004) reported
that C homo improved the prognosis of breast
cancer. Regarding colorectal cancer, Elander etal. (2006) investigated the relationship between
C/T polymorphism at –1562 and colorectal cancer, and found no correlation, as observed in ourstudy.
In the TNF-alpha gene, G/A polymorphism
is present at –308, and the transcription activity
is higher in the A than in the G allele (Abraham
and Kroeger, 1999). Previous reports, however,support that there is no significant association between the TNF-alpha –308 A/G allele and colorectal cancer susceptibility (Park et al., 1998; Landi etal., 2003). The findings of our study are similar to
those reports.
The present study clarified that the susceptibility to colorectal cancer is more strongly affected by age, gender, and cigarette-smoking andalcohol-drinking histories in Japanese, as reported
by Otani et al. (2003), than gene polymorphismsstudied. Colorectal cancer is a multifactorial disease involving environmental factors as well asa genetic predisposition, and properly cannot be
explained by genetic predisposition alone. However, detailed clarification of the involvement of agenetic predisposition may contribute to the prediction and early discovery of cancer development
as well as elucidation of the molecular mechanism
involved in the carcinogenic factors and earlystage of carcinogenesis.
53

H. Ohtani et al.

In conclusion, no association with the susceptibility to colorectal neoplasm was noted inany polymorphism of the MMP-2, -3, -7 and -9
and TNF-alpha genes, but the involvement of the
MMP-3 5A allele in tumor progression was suggested.

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Received December 8, 2008; accepted January 5, 2009
Corresponding author: Hideyuki Ohtani

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