Strong association of interleukin-6 -174 G>C promoter polymorphism
with increased risk for oral cancer
Eleftherios Vairaktaris1, Athanasios Yiannopoulos2, Antonis Vylliotis1, Christos
Yapijakis1, Spyridoula Derka1, Stavros Vassiliou1, Emeka Nkenke3, Zoi Serefoglou1,
Vasilis Ragos1, Giorgos Papageorgiou1, Eleni Vorris1, Elena Critseli4, Dimitris
Avgoustidis1, Friedrich Neukam3, Efstratios Patsouris5
1. Department of Maxillofacial Surgery, University of Athens Medical School, Vas.
Sofias 93 & Dim. Soutsou 1, Athens 11521, Greece
2. Department of A’ Surgical Clinic, University of Athens Medical School, Mikras
Asias 75, Athens 11527, Greece.
3. Department of Oral and Maxillofacial Surgery, Universität Erlangen, Klinik und
Poliklinik fűr Mund-, Kiefer-, Gesischtschirurgie, Glueckstrasse 11, Erlangen D91054, Nűrnberg, Germany.
4.
5. Department of Pathology, University of Athens Medical School, Mikras Asias 75,
Athens 11527, Greece.
Short running title: Association of interleukin-6 with oral cancer
Correspondence to:
Dr. Eleftherios Vairaktaris, MD,DDS,PhD,PhD
Department of Oral and Maxillofacial Surgery, University of Athens Medical School,
Vas. Sofias 93 & Dim. Soutsou 1, Athens 11521, Greece.
Tel: +30-210-6443035; Fax: +30-210-6443803; Email: lvairakt@med.uoa.gr
Source of support: This work was co-funded by the European Social Fund and National
Resources (EPEAEK II “Pythagoras” 70/3/7391) grant to E.V.
1
Abstract
In light to recently found contribution of factors associated with thrombosis and
inflammation to carcinogenesis, we investigated the possible association of interleukin-6
(IL-6) with increased risk for oral cancer. In DNA samples of 162 patients with oral
squamous cell carcinoma and 156 healthy controls of comparable ethnicity, age and sex,
we studied the -174 G>C polymorphism in the IL-6 gene, which affects its transcription.
The C allele frequencies were significantly increased in patients compared to controls,
42.6% versus 23.1% (P<0.001), respectively. The CC homozygotes had a seven-fold
greater risk for developing oral cancer (odds ratio 7.39, 95% C.I. 2.61-20.92), while the
GC heterozygotes had a four-fold greater risk (odds ratio 3.74, 95% C.I. 2.29-6.11). A
significant increase of C alleles was observed in patients regardless their smoking or
alcohol consumption habits, early or advanced stage of cancer, presence or absence of a
family history for cancer or thrombophilia (Fischer values P<0.001). These findings
suggest that the -174 G>C polymorphism, by affecting the IL-6 gene expression, is
associated with aggressive progress of oral oncogenesis.
2
Introduction
Oral squamous cell carcinoma (OSCC) is one of the most common human
malignancies, characterised by low survival rate, which makes this disease a serious
public health problem (1). Oral carcinogenesis is a multistep process occurring due to
environmental factors such as tobacco and alcohol abuse and gene alterations in
oncogenes and tumor suppressor genes (2). Recently, common polymorphisms in
angiogenesis, inflammation and thrombosis-related genes have been associated with
increased risk for oral cancer (3-7). One such factor related with both thrombosis and
malignancies is interleukin 6 (IL-6) (8-11).
IL-6, a phosphorylated glycoprotein containing 185 amino acids, is a pleiotropic
cytokine involved in many physiological processes, including inflammation, bone
metabolism, synthesis of C-reactive protein (CRP) and hematopoiesis (12). IL-6 and
related cytokines have also been found to induce cachexia in cancer patients by altering
metabolism of lipids and proteins. Furthermore, IL-6 functions as a growth-regulating
factor in several human tumors, including plasmacytoma, melanoma, and gastric, ovarian
and renal cell carcinoma (12). This particular role of IL-6 in neoplastic disease is
supported by the observation that circulating levels of the cytokine increase markedly
during development and progression of tumors, including those of the breast, pancreas,
gastrointestinal tract, lung, ovary, prostate, and others (9-11).
A single nucleotide polymorphism, upstream of the transcription start site of the
IL-6 gene, involving substitution of cytosine (C) for guanine (G) (henceforth referred to
as IL-6 -174G>C) affects gene expression and is associated with differences in plasma
IL-6 levels (13). Other polymorphisms at adjacent sites -597, -572 and -373 also seem to
3
affect the IL-6 promoter activity, but they are in linkage disequilibrium with the
polymorphism at position -174 (13). The frequency of the less common C allele ranges
between 23-42 %, in various populations (14,16).
The majority of studies indicate that IL-6 gene expression is constitutively greater
when the more common G allele is present (14,16). Nevertheless, the IL-6 gene
expression increases after an inflammatory stimulus when the C allele is present, such as
in individuals with chronic inflammations or older age (17). It has been suggested that the
important determinant of plasma IL-6 concentration is simply not the peak value after an
inflammatory stimulus, but rather the time taken for activity to return to basal levels after
stimulation (13). In subjects with chronic inflammatory activity such as smokers, patients
with cardiovascular disease or elderly populations, the association between IL-6 genotype
and circulating IL-6 may be the converse of that observed in young healthy populations
or middle-aged populations without inflammation (13). Consistent with this, the IL-6
production of peripheral blood mononuclear cells from C allele carriers increased with
age whereas this phenomenon was not observed in the GG genotype (13). In sum, while it
seems clear that the -174G>C polymorphism has clearly an impact on IL-6 gene
expression, the exact mechanisms remain to be delineated.
Interestingly, several studies report elevated IL-6 in serum, saliva and tumor
biopsies obtained from patients with oral cancer (18-19). In light of the above, we studied
the IL-6 -174 G>C polymorphism in patients with oral cancer and healthy controls in
order to determine whether this polymorphism is associated with increased risk for this
type of cancer.
4
Materials and Methods
The individuals under study were 318 unrelated Greeks and Germans, consisting
of 162 patients with oral squamous cell carcinoma and 156 healthy blood donors of
similar age, ethnicity and sex. The patients were mostly men (N=130) and their age
ranged between 40-84 years (58.5±10.1 years, median 58.5 years). The sex ratio of the
controls (N=120 men) and their age (ranged 31-83 years; 55.5±11.9 years, median 55.5
years) were comparable to those of the patients.
The patients, who were included in this study, had developed oral cancer and were
operated recently or up to a decade ago. In addition to clinical presentation, a biopsy with
pathological diagnosis of tumor stages I-IV and a family history regarding cancer and
thrombophilia were available. Sixty of them (37%) had one or two first degree relatives
with cancer and their age range (median=58.7 years) did not differ significantly from the
whole group of patients. Furthermore, thirty two patients (19.8 %) had one or two firstdegree relatives with idiopathic thrombosis and an earlier age range (median =58 years)
but again with no statistical difference compared to the whole group. Sixteen patients
(9.9%) had a positive family history for both cancer and thrombophilia (median age=56.3
years).
Most of the participants in the two groups generally, worked in a low-risk
environment (with the exception of one patient and three controls who worked in
chemical factories). No data were available on controls regarding their family history or
smoking and alcohol consumption habits.
Blood samples were collected from patients and controls under study after
informed consent. DNA was isolated from blood with the use of Nucleon
TM
kit
5
(Amersham). Molecular detection of the (-174G/C) polymorphism in the IL-6 gene was
performed by restriction fragment length polymorphism typing. This involved a
combination of PCR amplification and digestion with restriction endonuclease Nla III
followed by gel electrophoretic analysis. The PCR conditions consisted of an initial
denaturation step at 95 oC, followed by 35 cycles of 94 oC for 55 sec, 61 oC for 1 min, and
72 oC for 50 sec, as well as a final elongation step at 72 oC for 5 min. The primers used
were
Forward:
5’-TGACGACCTAAGCTGCACTTTTC-3’
and
Reverse:
5’-
GGGCTGATTGGAAACCTTATTAAGA-3’. The generated PCR product of 93 bp was
cleaved by restriction enzyme Nla III into two fragments of 52bp and 41bp only if the C
allele was present (Fig 1).
The frequencies of alleles and genotypes of the whole group or subgroups of
patients were compared to the respective frequencies of the control group using the chi
square or Fisher’s exact test and odds ratios. The significance level was set at P<0.05 and
the results are presented as hazard ratios with 95% confidence intervals (CI). For the
purpose of statistical analysis all unknown variables of controls were assumed to be nil,
thus, odds ratios obtained for some subgroups of patients may overestimate the true
likehood of IL-6 genotypes and those variables.
Results
The prevalence of detected IL-6 genotypes in healthy controls and patients with
oral cancer are shown in Tables 1-3. The data for the two tested populations (Greek and
German healthy controls) were analyzed together, since there were no significant
6
differences of allele frequencies of the (-174G/C) polymorphism among the two
populations.
For -174G>C IL-6 promoter polymorphism, genotype distributions and allele
frequencies in the control group, in patients group and subgroups were as expected for a
sample in Hardy-Weinberg equilibrium. Compared to the control group, the
heterozygotes and the homozygotes for the C allele were significantly increased in the
group of patients and in every subgroup (P<0.001).
The observed C allele frequency in the control group was 23.1%, therefore similar
to other European populations. The detected C allele frequencies, in the patients’ group
and in every subgroup were significantly elevated in comparison to the control group
(value P<0.001), with the only exception being the patients with alcohol abuse where no
significant difference was observed (Table 3). The same significant difference was
observed in the subgroup of patients with nicotine abuse (P<0.001), while the minimal
number of non-smoking patients (N=10) does not permit any safe conclusions to be
drawn. Finally, there were no major differences in the frequencies of the two IL-6 alleles
due to categorizations of sex, age, and age at onset of oral cancer.
Interestingly, compared to individuals with the GG genotype, the relative risk for
oral squamous cell carcinoma for GC heterozygotes was 3.74 (2.29-6.11), while for CC
homozygotes was 7.39 (2.61-20.62). Additionally, GC heterozygotes have a relative risk
of 2.81 (1.56-4.96) for developing oral cancer in stages I&II and 5.6 (2.82-11.13) in
stages III&IV. For CC homozygotes the respective relative risks are even greater, 5.94
(1.89-18.64) for stages I&II and 10.82 (2.93-39.94) for stages III&IV.
7
Discussion
IL-6 is a multifunctional molecule produced by a variety of cells (12). The
cytokine exerts its effects through activating pathways mediating cell proliferation and by
inducing proteins that inhibit apoptosis (12). In neoplastic disease, IL-6 circulating levels
increase markedly during development and progression of tumors (12). Indeed, high IL-6
levels have been shown to correlate with poor prognosis and high mortality in prostate
and colorectal cancer patients (9-11).
A single nucleotide polymorphism was identified at position -174 within the
promoter of the IL-6 gene with two alleles, G and C. IL-6 gene expression is
constitutively greater when the more common G allele is present, while it is elevated by
chronic inflammation in the presence of the C allele (14,16-17). Furthermore, several
studies in oral cancer, report elevated IL-6 in serum, saliva and tumor biopsies obtained
from patients with oral cancer (18-19).
In light of the above, in the present study the genotypes and allele frequencies of
the -174G>C polymorphism were investigated in a cohort of 162 patients with oral
cancer in comparison to 156 healthy controls of equivalent age, sex and ethnicity. Despite
the relatively small sample of studied individuals, the overall obtained data revealed a
strong association of the -174G>C IL-6 polymorphism with an increased risk for oral
squamous cell carcinoma. Compared to the controls, a significant increase of GC
heterozygotes and CC homozygotes was observed not only in the whole group of
patients, but in all subgroups of patients regardless their alcohol consumption habits,
early or advanced stage of cancer, presence or absence of a family history for cancer or
8
thrombophilia. Accordingly, the G allele frequency was decreased in the whole group of
patients and in every subgroup.
IL-6 gene expression is constitutively greater when the more common G allele is
present, and this allele has been incriminated for increased risk of thrombotic events
(14,16). In light of the above, the fact that the G allele frequency is decreased in the
whole group of patients with oral cancer, as well as in every subgroup of them, might
indicate that C allele carriers are protected from immediate risk for life-threatening
thrombotic events, but not from cancer, which is a more prolonged process of multiple
steps. This notion is in accordance not only to the findings of the present study, but with
reports of association of the C allele with increased risk for breast and colorectal cancer
(14,15).
Furthermore, the fact that the C allele frequency is increased in the whole group
and in every subgroup of our patients might suggest that it is actually the presence of this
allele that causes the elevation in IL-6 circulating levels reported in several studies
investigating serum, saliva and biopsies of patients with oral cancer (18,19). This notion
is consistent with the C allele-related increase of IL-6 gene transcription observed in
elderly individuals (over 40 years-old), such as those studied in the present work (17). It
follows that, in C allele carriers age-related inflammation may increase risk for
oncogenesis in certain tissues (including those in the oral region), while the actual target
tissue may depend on accidental mutational events or environmental factors such as
tobacco abuse. Unfortunately, the small amount of available non-smoking patients who
were included in this study did not allow us to further test this hypothesis.
9
Additionally, the statistical analysis showed that CC homozygotes have twice as
much greater relative risk for developing oral cancer than GC heterozygotes.
Furthermore, C allele carriers have twice as much greater relative risk for developing oral
cancer in stages III&IV than in I&II. Therefore, it can be assumed that -174G>C IL-6
promoter polymorphism is associated with a more aggressive type of oral cancer.
In conclusion, the studied IL-6 polymorphism is strongly associated with
increased risk for oral cancer in certain individuals. Similar associations have been
reported for polymorphisms in other interleukin genes, as well (20). As a consequence, it
is of great importance to perform further genetic association studies regarding the
contribution of additional cytokines or other factors related to angiogenesis, inflammation
and thrombosis to oncogenesis in the oral region. Any positive findings could ultimately
result in the undertaking of preventive measures safeguarding the health status and lives
of certain at risk individuals in the general population.
10
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11
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The IL-6 -174 G>C polymorphism is associated with cardiovascular diseases and
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14. DeMichele A, Martin AM, Mick R et al. Interleukin-6 -174 G>C polymorphism is
associated with improved outcome in high-risk breast cancer. Cancer Res
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15. Landi S, Moreno V, Gioia-Patricola L et al. Association of common polymorphisms
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peroxisome proliferators-activated receptor γ with colorectal cancer. Cancer Res
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13
Table 1. Prevalence of IL-6 (-174G/C) polymorphism in healthy controls and patients with oral cancer (total group of patients and
subgroups in regard to cancer stage). aFischer’s P-value; bχ2 P-value; cage-adjusted odds ratios.
Genotypes
C/C
G/G
G/C
Total
Prevalence
of C allele
C allele
frequency
Carrier
frequency
of C allele
Controls
(%)
Patients
(%)
P-value
ORc
(CI)
6
(3.8%)
90
(57.7%)
60
(38.5%)
156
(100%)
18
(11.1%)
42
(25.9%)
102
(63.0%)
162
(100%)
P<0.001a
7.39
(2.61 - 20.92)
72
(23.1%)
138
(42.6%)
P<0.001b
72
(40%)
P<0.001b
66
(45.8%)
P<0.001b
66
(42.3%)
120
(74.1%)
P<0.001b
62
(68.9%)
P<0.001b
58
(80.6%)
P<0.001b
1 (referent)
P<0.001a
3.74
(2.29 – 6.11)
Patients with
cancer stages I&II
(%)
10
(11.1%)
28
(31.1%)
52
(57.8%)
90
(100%)
P-value
ORc
(CI)
P<0.001a
5.94
(1.89-18.64)
1 (referent)
P<0.001a
2.81
(1.56-4.96)
Patients with
cancer stages III&IV
(%)
8
(11.1%)
14
(19.4%)
50
(69.5%)
72
(100%)
P-value
ORc
(CI)
P<0.001a
10.82
(2.93-39.94)
1 (referent)
P<0.001a
5.6
(2.82-11.13)
14
Table 2. Prevalence of IL-6 (-174G/C) polymorphism in healthy controls and patients with oral cancer in regard to family history of
either cancer or trombophilia. aFischer’s P-value; bχ2 P-value; cage-adjusted odds ratios.
Genotypes
C/C
G/G
G/C
Total
Prevalence
of C allele
C allele
frequency
Carrier
frequency
of C allele
P-value
ORc
(CI)
6
(3.9%)
90
(57.7%)
60
(38.5%)
156
(100%)
Patients
with
family
history
of cancer
(%)
6
(10%)
12
(20%)
42
(70%)
60
(100%)
P<0.001a
7.19
(2.03-25.47
72
(23.1%)
54
(45%)
66
(42.3%)
48
(80%)
Controls
(%)
Patients
without
family
history
of cancer
(%)
12
(11.8%)
30
(29.4%)
60
(58.8%)
102
(100%)
P-value
ORc
(CI)
P<0.001a
7.47
(2.4-23.2)
P<0.001b
84
(41.2%)
P<0.001b
72
(70.6%)
1 (referent)
P<0.001a
5.23
(2.54-10.74)
Patients
with
family
history
of thr
(%)
8
(25%)
8
(25%)
16
(50%)
32
(100%)
P-value
ORc
(CI)
P<0.001a
13.02
(3.74-45.34)
P<0.001b
32
(50%)
P<0.001b
24
(75%)
1 (referent)
P<0.001a
3.11
(1.78-5.43)
Patients
without
family
history
of thr
(%)
10
(7.7%)
34
(26.2%)
86
(66.1%)
130
(100%)
P-value
ORc
(CI)
P<0.001a
5.49
(1.74-17.31)
P<0.001b
106
(40.8%)
P<0.001b
P<0.001b
96
(73.8%)
P<0.001b
1 (referent)
P<0.001a
2.95
(1.2-7.29)
1 (referent)
P<0.001a
3.98
(2.36-6.71)
15
Table 3. Prevalence of IL-6 (-174G/C) polymorphism in healthy controls and patients with oral cancer in regard to either alcohol
consumption or smoking habits. aFischer’s P-value; bχ2 P-value; cage-adjusted odds ratios; N.S.: not significant P-value.
Genotypes
C/C
G/G
G/C
Total
Prevalence
of C allele
C allele
frequency
Carrier
frequency
of C allele
P-value
ORc
(CI)
Patients
without
tobacco
abuse
(%)
18
(11.8%)
38
(25%)
96
(63.2%)
152
(100%)
P<0.001a
8.87
(3.06-25.68)
0
(0%)
4
(40%)
6
(60%)
10
(100%)
P<0.001a
72
(23.1%)
132
(43.4%)
P<0.001b
6
(30%)
P<0.001b
44
(42.3%)
N.S.
94
(42.7%)
P<0.001b
66
(42.3%)
114
(75%)
P<0.001b
6
(60%)
N.S.
38
(73.1%)
P<0.001b
82
(74.5%)
P<0.001b
Controls
(%)
Patients
with
tobacco
abuse
(%)
6
(3.9%)
90
(57.7%)
60
(38.5%)
156
(100%)
1 (referent)
P<0.001a
3.98
(2.4-6.6)
P-value
ORc
(CI)
1 (referent)
P<0.001a
2.14
(0.6-7.65)
Patients
with
alcohol
abuse
(%)
6
(11.5%)
14
(26.9%)
32
(61.6%)
52
(100%)
P-value
ORc
(CI)
P<0.001a
9.31
(2.27-38.11)
1 (referent)
P<0.001a
3.68
(1.78-7.59)
Patients
without
alcohol
abuse
(%)
12
(10.9%)
28
(25.5%)
70
(63.6%)
110
(100%)
P-value
ORc
(CI)
P<0.001a
6.75
(2.23-20.45)
1 (referent)
P<0.001a
3.76
(2.17-6.5)
16
Figure 1: Nla III restriction pattern of the IL-6 -174 G>C polymorphism, observed in three
patients (CC homozygote , GC heterozygote, GG homozygote, M: molecular weight marker).
M
GC
GG GG CC
93 bp
52/41 bp
17