Download in the MKK4 promoter is associated with decreased risk of acute

Functional variant ()1304T>G) in the MKK4 promoter
is associated with decreased risk of acute myeloid
leukemia in a southern Chinese population
Lan Jiang,1,2,6 Ping Zhou,3,6 Aining Sun,1,2,6 Jian Zheng,1,2 Bin Liu,4 Yonghe You,1 Chun Zhang,3 Depei Wu1,2 and
Yifeng Zhou1,2,5
1Soochow
University Laboratory of Cancer Molecular Genetics, Cyrus Tang Hematology Center, Department of Hematology, Jiangsu Institute of
Hematology, The First Affiliated Hospital of Soochow University, Suzhou; 2Thrombosis and Hemostasis Key Laboratory of the Ministry of Health, Soochow
University, Suzhou; 3The Third Hospital Affiliated to Nantong University, Wuxi; 4The No. 12 Hospital of Guangzhou, Guangzhou, China
(Received February 8, 2011 ⁄ Revised April 7, 2011 ⁄ Accepted April 13, 2011 ⁄ Accepted manuscript online April 25, 2011)
As a member of the MAPK kinase family, mitogen-activated protein kinase kinase (MKK) 4 (NM__003010.2) is know to be involved
in the regulation of apoptosis, inflammation, and tumorigenesis.
Several polymorphisms have been identified in the promoter
region of the MKK4 gene and we hypothesized that genetic variations in this region may alter gene expression, and thus cancer
risk. In the present study, we genotyped two polymorphisms in
the promoter of the MKK4 gene, namely )1304T>G (rs3826392)
and )1044A>T (rs3809728), in 433 patients with AML and 600 controls, and assessed the association between those polymorphisms
and the risk of AML. Compared with the )1304TT genotype,
patients with the )1304TG genotype had a significantly decreased
risk of AML (adjusted odds ratio (OR) 0.67; 95% confidence interval
(CI) 0.51)0.87), with the risk decreased even further in those carrying )1304GG (OR 0.56; 95% CI 0.31–0.97). Additional experiments,
which focused on reporter gene expression driven by MKK4 promoters, demonstrated that the presence of a )1304G allele led to
greater transcriptional activity than the presence of a )1304T
allele. However, no significant association was observed between
the MKK4 )1044A>T polymorphism and the risk of AML. These
findings suggest that the functional )1304G>T variant may contribute to the risk of AML by enhancing the transcriptional activity
of MKK4. Thus, this polymorphism may be a genetic modifier
for the development of AML. (Cancer Sci, doi: 10.1111/j.13497006.2011.01965.x, 2011)
A
s the name suggests, AML is a cancer of the myeloid line
of blood cells. It is characterized by the rapid growth of
abnormal white blood cells, which accumulate in the bone marrow, thus interfering with the production of normal blood cells,
and is the most common acute leukemia affecting adults. The
incidence of AML increases with age: the median age at diagnosis is 63 years and its incidence is approximately 10-fold greater
in people >65 years of age than in those <65 years of age.(1)
The incidence of AML is slightly greater in men than in women,
with a male:female ratio of 1.3:1.(2) Epidemiological studies
have established many etiologic factors for AML, including
other blood disorders,(3) chemical exposure,(4,5) ionizing radiation,(6) and genetics.(7) Of these, chemical exposure and ionizing
radiation are both cell stressors and are capable of activating
MAPK pathways, which are known to participate in the regulation of apoptosis, inflammation, and tumorigenesis.(8)
At least three distinct MAPK signaling pathways have been
identified, namely the ERK, p38 kinase, and JNK pathways.(9)
All MAPK family members are activated by the phosphorylation
of threonine and tyrosine residues within a TXY motif, which is
catalysed by the single dual-specificity kinase MAPK kinase
(MKK). Two upstream MKK, namely MKK4 and MKK7, are
responsible for the phosphorylation and activation of JNK.(9,10)
doi: 10.1111/j.1349-7006.2011.01965.x
ª 2011 Japanese Cancer Association
When it is overexpressed in mammalian cells, as well as under
in vitro conditions, MKK4 can also phosphorylate and activate
different p38 MAPKs, such as p38a, p38b, SAPK3 (p38g) and
SAPK4 (p38d).(9)
The MKK4 gene, also known as JNKK1, has been mapped to
chromosome 17p11.2 (NM_003010.2). The gene is over 120 kb
in size, contains 11 exons, and encodes a 399-amino acid protein
in humans.(9,11) It has been found that MKK4 is of vital importance in tumor formation and development because it may act as
a tumor suppressor gene.(12–14) It is known that the promoter
region determines the transcriptional activity of a gene, so
genetic variations in the promoter region may affect gene function. Furthermore, environmental factors that possible contribute
to the induction of different cancers, including AML, may cause
gene mutations in the promoter region.(8) Therefore, in the present study, we evaluated the hypothesis that polymorphisms in
the promoter region of the MMK4 gene are associated with the
risk of AML. Four common single nucleotide polymorphisms
(SNP; those with a minor allele frequency [MAF] >5%) have
been found in the 1.6-kb promoter region of MKK4 according
to the GenBank dbSNP database: )1304T>G (rs3826392),
)1044A>T (rs3809728), )641C>G (rs2190853), )84T>C
(rs9892151). Because Wei et al.(8) had proved, in a Chinese
population, that )1044A>T, )641C>G, and )84T>C were in
complete linkage disequilibrium (LD) with each other. If )1044
A>T is associated with AML risk, so will the other two sites,
conversely, if there is no association between )1044A>T and
AML risk, )641C>G and )84T>C will not have association
with AML either. So, the selection of )1044A>T is enough for
)641C>G and )84T>C.
In the present hospital-based case-control study, we investigated the association between two MKK4 polymorphisms,
namely )1304T>G (dbSNP ID: rs3826392) and )1044A>T
(dbSNP ID: rs3809728), and the risk of AML. Moreover, we analyzed the correlation between the )1304T>G polymorphism and
MKK4 mRNA levels in AML bone marrow. Recombinant reporter plasmids containing )1304T>G and )1044A>T were also
constructed to determine their effect on MKK4 gene expression.
Materials and Methods
Study subjects. The present study included 433 AML patients
and 600 healthy controls. All subjects were ethnically homogeneous Han Chinese. Patients with newly diagnosed AML were
recruited consecutively from March 2001 to May 2010 at the
First Affiliate Hospital of Soochow University (Suzhou, China).
5To whom correspondence should be addressed.
E-mail: [email protected]
6These authors contributed equally to this work.
Cancer Sci | 2011
All eligible patients diagnosed at the hospital during the study
period were invited to participate in the study, with 91%
agreeing to do so. There were no restrictions in terms of age,
stage of disease, or histology preventing people from participating in the study. The population controls consisted of cancerfree people living in the Suzhou region. Control subjects were
selected from a nutritional survey conducted over the same period as the cases were collected.(15,16) The selection criteria for
control subjects included no history of cancer and the control
population was matched in terms of age and sex with the AML
patient group. At the time of recruitment, informed consent was
obtained from each subject. This study was approved by the
Medical Ethics Committee of The First Affiliate Hospital of
Soochow University.
Genotyping analysis. Genomic DNA was extracted from a 5mL bone marrow sample, which was obtained from patients
before they were given chemotherapy or radiotherapy to avoid
any influence of these treatments on outcomes. Genotypes were
analyzed using PCR-based methods as described below. Genotyping was performed without knowledge of the subject’s case
or control status. A 30% masked random sample of cases and
controls was tested twice by different people, and the results
were concordant for all masked duplicate sets.
We developed a PCR-RFLP method to determine AML-associated MKK4 polymorphisms. The primer pair designed to
amplify the target DNA fragment containing the )1304T>G
(rs3826392) polymorphism was 5¢-CTT GTT CCA AAC CCA ATT TC-3¢ (forward) and 5¢-GGG CTA C TG ATT
TCC AGA TG-3¢ (reverse), which produced a 232-bp fragment.
Similarly, the primer pair designed for )1044A>T (rs3809728)
was 5¢-CTA CGA TTT GTA AGC CAA CCA-3¢(forward) and
5¢-CCA ACA TGC TGT GAA GAA CTC-3¢ (reverse), which
produced a 235-bp fragment. The PCR was performed in a 25lL reaction system containing 5 mM MgCl2, 0.1 mM dNTPs,
3.0 units Taq polymerase and the manufacturer’s buffer (Fermentas, Burlington, VT, Canada). The PCR procedure consisted
of an initial melting step at 94C for 5 min, followed by 35
cycles of 94C for 45 s, annealing at 58C for )1304T>G and
60C for )1044A ⁄ T for 45 s and 72C for 45 s, with a final
extension step at 72C for 7 min. A native endonuclease AflII
(Fermentas) site was present in the amplified fragment containing the )1304G>T (rs3826392) polymorphism. After digestion
by AflII at 37C for at least 3 h, the major G allele produced a
single 232-bp band, whereas the minor C allele produced two
bands (111 and 121 bp). The two bands could be separated easily by 3% agarose gel electrophoresis. The amplified fragment
containing the )1044A>T (rs3809728) polymorphism could be
cut using Tsp509I (Fermentas) at 65C for at least 3 h. After
digestion, the major A allele produced two bands (129
and106 bp), whereas the minor T allele produced a single 235bp band. The genotype identification by PCR-RFLP was confirmed by DNA sequencing (Figs 1,2).
Construction of reporter plasmids. Because the MKK4
)1304T>G polymorphism was found to be associated with a
significantly decreased risk of AML, we then determined
whether this polymorphism had an effect on MKK4 gene expression in vitro. The T allelic reporter constructs were prepared by
amplifying the 1612-bp MKK4 promoter region (from )1528 to
+84 bp relative to the translation start site) from subjects homozygous for the T allele ()1304TT), including the artificial KpnI
and HindIII enzyme restriction sites with a forward primer of
5¢-gccggtacctaatctgtagtgctgcttcta-3¢ and a reverse primer of
5¢-tggaagcttcgccggggaccctacggggc-3¢. The amplified fragments
were then cleaved with the KpnI and HindIII enzymes (New
England BioLabs, Ipswich, MA, USA). The pGL3 basic vector
(Promega, Madison, WI, USA) was also cleaved with the KpnI
and HindIII enzymes, and the fragments and pGL3 basic vector
were then ligated by T4 DNA ligase (New England BioLabs).
The p1304T–1044T, p1304G–1044A, and p1304G–1044T reporter constructs were obtained from the p1304T–1044A constructs by site-directed mutagenesis using the QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA, USA). All
constructs were sequenced to confirm the allele, orientation,
and integrity of each insert.
Transient transfections and luciferase assays. Three different
cell lines, namely HL-60 (AML-M2 origin), NB4 (AML-M3
origin) and SHI-1 (AML-M5b origin) (China Center for Type
Culture Collection, Wuhan University, Hubei, China), were
grown in RPMI 1640, supplemented with 10% (v ⁄ v) heat-inactivated FCS, 2 mM L-glutamine, 100 units ⁄ mL penicillin, and
100 units ⁄ mL streptomycin at 37C and 5% CO2 in a humidified
incubator for 2 days. For transient transfection experiments,
5 · 104 cells were plated in 10-mm 24-multiwell plates and
grown to 60–70% confluence. Transfection was performed using
Fig. 1. (a) Representative PCR-RFLP for different genotypes containing the )1304G>T polymorphism site. M, DNA size marker; lanes 1, 3, 4, 5,
TT genotype; lanes 2, 7, TG genotype; lane 6, GG genotype. (b) DNA sequencing analysis. The PCR products with different PCR-RFLP profiles
were sequenced to confirm the genotypes.
2
doi: 10.1111/j.1349-7006.2011.01965.x
ª 2011 Japanese Cancer Association
Fig. 2. (a) Representative PCR-RFLP for different genotypes containing the )1044A>T polymorphism site. M, DNA size marker; lane 1, TT
genotype; lanes 2, 4, 5, 6, AA genotype; lanes 3, 7, AT genotype. (b) DNA sequencing analysis. The PCR products with different PCR-RFLP profiles
were sequenced to confirm the genotypes.
Lipofectamine Reagent (Life Technologies, Rockville, MD,
USA) according to the manufacturer’s instructions. Cells were
cotransfected with 0.5 lg reporter plasmid and 0.1 lg pRLSV40 (Luciferase Assay System; Promega); the latter was used
to standardize transfection efficiency.
Luciferase activity was determined using a luciferase assay
system (Promega) according to the manufacturer’s instructions.
Briefly, cells were scraped into lysis reagent, transferred to
microfuge tubes and centrifuged for 30 s at 12 000g. Luciferase
activity was measured using a manual luminometer (TD20 ⁄ 20;
Turner Designs, Sunnyvale, CA, USA) after mixing 100 lL
luciferase assay reagent with 20 lL of 1:10 diluted cell lysate.
Measurements for each sample were recorded three times at 10s intervals. Three independent transfection experiments were
performed for each plasmid construct, and each experiment
was performed in triplicate. Results are expressed as luciferase
activity as a ratio of pRL-SV40.
Real-time analysis of MKK4 mRNA. Bone marrow samples
were obtained from 41 AML patients recruited consecutively
over the period February 2008–April 2010 at the First Affiliate
Hospital of Soochow University (Suzhou, China). Samples were
placed immediately in liquid nitrogen and stored at )80C until
analysis. Total RNA was isolated from bone marrow using TRIzol reagent (Molecular Research Center, Cincinnati, OH, USA).
A 2-lg aliquot of total RNA from each specimen was reverse
transcribed into single-strand cDNA using oligo primer and SuperscriptII (Invitrogen, Carlsbad, CA, USA). Relative gene
expression of MKK4 was determined using b-actin as an internal
standard and the ABI Prism 7000 sequence detection system
(Applied Biosystems, Foster City, CA, USA) based on the
SYBR green method. The primers pairs used in the present
study were as follows: for MKK4, 5¢-aac aac act ggg att tca ct3¢ (forward) and 5¢-tca cta ctc cgc att act aca-3¢ (reverse); and
for b-actin, 5¢-ggc ggc acc acc atg tac cct-3¢ (forward) and
5¢-agg ggc cgg act cgt cat act-3¢ (reverse). The PCR reaction
mixture (final volume 20 lL) contained 0.1 lM each primer,
10 ll 1· SYBR Premix EX Taq premix reagent (Perfect Real
Time, Takara, Dalian, China), and 50 ng cDNA. The cycling
conditions consisted of 95C for 2 min, followed by 40 cycles
of 95C for 15 s and 60C for 1 min. Expression of MKK4 in
Jiang et al.
individual samples was normalized against that of b-actin using
a modification of the method described by Lehmann and
Kreip.(17) All analyses were performed in a blinded fashion, with
laboratory personnel unaware of the genotyping results.
2
Statistical analysis. Two-sided v tests were used to assess
differences in the distribution of age and sex between AML
patients and controls, as well as between alleles and genotypes.
Hardy–Weinberg equilibrium (HWE) was assessed using a
Table 1. Characteristics of the AML patients and control subjects
from the Chinese populations used for the association study
No. of men
No. of women
Age at diagnosis (years)
20
21–40
41–60
‡61
Lineage
Myeloid
Myeloid and lymphoid
Myeloid and monocytic
Classification of diagnosis
M0
M1
M2
M3
M4
M5
Unknown
Karyotype
Aberrant
Normal
Unknown
AML patients
(n = 433)
Control subjects
(n = 600)
240 (55.4)
193 (44.6)
336 (56.0)
264 (44.0)
47
149
155
82
60
223
220
97
(10.9)
(34.4)
(35.8)
(18.9)
(10.0)
(37.2)
(36.7)
(16.2)
308 (71.1)
118 (27.3)
7 (1.6)
1
82
118
73
37
68
54
(0.2)
(18.9)
(27.3)
(16.9)
(8.5)
(15.7)
(12.5)
207 (47.8)
176 (40.6)
50 (11.5)
Data show the number of subjects in each group, with percentages
given in parentheses.
Cancer Sci | 2011 | 3
ª 2011 Japanese Cancer Association
Table 2. Genotype frequencies of the two single nucleotide
polymorphisms in the MKK4 gene in patients and controls and their
associations with AML
Control subjects
(n = 600)
)1304G>T
Genotype
TT
GT
GG
Allele
T
G
)1044A>T
Genotype
AA
TA
TT
Allele
A
T
AML
(n = 433)
OR† (95% CI)
318 (53.0)
244 (40.7)
38 (6.3)
274 (63.3)
141 (32.6)
18 (4.1)
1.00 (Reference)
0.67 (0.51–0.87)
0.56 (0.31–0.97)
0.00097
880 (73.3)
320 (26.7)
689 (79.6)
177 (20.4)
1.00 (Reference)
0.71 (0.57–0.87)
0.001
402 (67.0)
173 (28.8)
25 (4.2)
285 (65.8)
123 (28.4)
25 (5.8)
1.00 (Reference)
1.01 (0.76–1.32)
1.41 (0.79–2.49)
0.444
977 (81.4)
223 (18.6)
693 (80.0)
173 (20.0)
1.00 (Reference)
0.91 (0.73–1.14)
0.427
P value‡
Results
Unless indicated otherwise, data show the number of subjects in each
group, with percentages given in parentheses. OR, odds ratio; CI,
confidence interval. †Data were calculated by unconditional logistic
regression and adjusted for sex and age status. ‡P values for v2
analysis or Fisher’s exact test.
goodness-of-fit v2 test to compare expected genotype frequencies with the observed genotype frequency (p2 + 2pq + q2 = 1).
Associations between the status of the control subjects and each
SNP were estimated using an unconditional logistic regression
model, with adjustment for age and sex. Logistic regression
modeling was also used for the trend test.
Data were further stratified by age, sex, karyotype, and lineage
to evaluate the variable-related odds ratios (OR) among the
various MKK4 genotypes. Homogeneity within different variable-related ORs levels was tested. Potential multiplicative and
additive interactions among gene–gene and gene–environmental
factors were also evaluated using logistic regression analysis.
The 2LD program and the PROC ALLELE statistical procedure
in SAS ⁄ Genetics (SAS Institute, Cary, NC, USA) were used to
detect the LD of two SNPs. Statistical power was calculated
Table 3.
using PS Software (http://biostat.mc.vanderbilt.edu/twiki/bin/
view/Main/PowerSampleSize, accessed Dec 14, 2010). All tests
were two sided and analyses were performed using SAS (version
9.1; SAS Institute). P < 0.05 was considered significant.
Characteristics of the study population. Selected characteristics of AML patients and controls are summarized in Table 1.
The average age of patients and controls was 43 and 42 years,
respectively (P = 0.347). Similarly, there were no significant
difference in the proportion of men and women within each of
the two groups (P = 0.855).
MKK4 genotypes and risk of AML. The genotyping results are
given Table 2. The allele frequencies for rs3826392G and
rs3809728A were 0.267 and 0.814, respectively, in the control
group and 0.204 and 0.800, respectively, in AML patients. The
observed genotype frequencies of rs3826392 and rs3809728
polymorphisms in both controls and patients did not deviate
from those expected based on HWE (v2 = 1.003, d.f. = 1,
P = 0.316 for rs3826392; and v2 = 1.247, d.f. = 1, P = 0.264
for rs3809728).
The frequencies for the )1304GG, GT, and TT genotypes in
AML patients differed significantly from those in the control
group (Ptrend = 0.00097). Relative to the )1304GG genotype,
)1304GT and )1304TT were both associated with a significantly decreased risk of AML, with an OR of 0.56 (95%
confidence interval [CI] = 0.31–0.97) and 0.67 (95% CI = 0.67–
0.87), respectively. However, the difference in genotype frequencies at the rs3809728A>T site between AML patients and
the control group was not significant (Ptrend = 0.444).
In the control group, LD analysis revealed that the linkage
between two loci was relatively weak (D¢ = 0.296; r2 = 0.065),
suggesting that each may have an independent effect on the risk
of AML.
Stratification analysis of MKK4 )1304 T>G genotypes and risk
of AML. The risk of AML related to MKK4 genotype was fur-
ther examined with stratification according to age, sex, lineage,
and karyotype. However, as indicated in Table 3, there was no
significant association between age, sex, lineage, and karyotype
and these two polymorphisms.
Effects of the MKK4 )1304T>G polymorphism on transcriptional activity in AML cell lines. To determine the transcrip-
tional activity of the native MKK4 promoter in AML cells, two
Stratification analysis of the MKK4 gene )1304G>T (rs3826392) genotypes by selected variables in AML patients and control subjects
Patients (n = 433)
TT
Age (years)
£40
>40
Sex
Male
Female
Karyotype
Aberrant
Normal
Unknown
Lineage
Myeloid
Myeloid and lymphoid
Myeloid and monocytic
GG + GT
Controls (n = 600)
Adjusted OR (95% CI)†
TT
GG + GT
CG + CC vs GG
P value‡
125 (28.9)
149 (34.4)
71 (16.4)
88 (20.3)
156 (26.0)
162 (27.0)
127 (21.2)
155 (25.8)
0.71 (0.47–1.05)
0.62 (0.41–0.86)
0.64
150 (34.7)
124 (28.6)
90 (20.8)
69 (15.9)
171 (28.5)
147 (24.5)
165 (27.5)
117 (19.5)
0.60 (0.44–0.89)
0.73 (0.45–1.04)
0.65
138 (31.9)
103 (23.8)
33 (7.6)
69 (15.9)
73 (16.9)
17 (3.9)
318 (53.0)
318 (53.0)
318 (53.0)
282 (47.0)
282 (47.0)
282 (47.0)
0.51 (0.37–0.78)
0.81 (0.55–1.12)
0.59 (0.30–1.11)
0.32
190 (43.9)
77 (17.8)
7 (1.6)
118 (27.2)
41(9.5)
0 (0.0)
318 (53.0)
318 (53.0)
318 (53.0)
282 (47.0)
282 (47.0)
282 (47.0)
0.72 (0.52–0.93)
0.60 (0.38–0.92)
0.08 (0.01–1.32)
0.27
Unless indicated otherwise, data show the number of subjects in each group, with percentages given in parentheses. † Odds ratios (OR) were
adjusted for sex and age in a logistic regression model. ‡P values are to test for homogeneity for ORs of MKK4-1304T>G SNP among different
strata. CI, confidence interval.
4
doi: 10.1111/j.1349-7006.2011.01965.x
ª 2011 Japanese Cancer Association
luciferase reporter gene constructs were generated by PCR,
spanning )1528 to +84 bp of the MKK4 promoter region, with a
T ⁄ G or A ⁄ T at the )1304 or )1044 polymorphic sites. These
constructs were used to transiently transfect three AML cell
lines (i.e. HL-60, NB4, and SHI-1 cells). As shown in Fig. 3, we
found that )1304G-containing MKK4 promoter resulted in an
approximate 1.8–3-fold increase in reporter expression compared with the )1304T-containing promoter in the HL-60, NB4,
and SHI-1 cell lines.
Effects of the MKK4 )1304T>G SNP on MKK4 mRNA levels.
The effects of the )1304T>G SNP on MKK4 expression were
examined by real-time PCR evaluation of MKK4 mRNA in individual samples of AML bone marrow. The results revealed that
MKK4 mRNA levels (normalized against b-actin) were significantly greater in patients with the )1304TG and )1304GG
genotypes compared with levels in patients with the )1304TT
genotype (mean [±SD] expression 0.104 ± 0.043, 0.205 ±
0.133, and 0.064 ± 0.022, respectively; P < 0.001; Fig. 4.
Discussion
In the present study investigating 433 AML patients and
600 cancer-free controls, we found that the )1304G>T polymorphism in the promoter region of MKK4 was associated with the
risk of developing AML. The risk of developing AML decreased
as the number of )1304G alleles increased. Moreover, we found
(a)
that the )1304G variant allele significantly increased the transcriptional activity of the MKK4 gene compared with the
)1304T allele, both in vitro and in vivo. However, there was no
significant difference in the susceptibility to AML between different genotypes of the )1044A>T locus.
All the findings in the present study of the genotyping, realtime PCR, and transient transfection experiments suggest a
tumor suppressor role for MKK4. This suppressor function is
supported by results from other studies. For example, homozygous deletion of MKK4 that eliminates its coding portions has
been identified in pancreatic carcinoma cell lines and lung
carcinoma cell lines.(10) Ganiatsas et al.(18) have reported that
loss-of-function mutations in the MKK4 gene are present in
approximately 5% of tumors from various human tissues. Moreover, MKK4 has been identified as a suppressor of the metastasis
of prostate and ovarian cancers,(19,20) and the lack of expression
of MKK4 in resected gastric adenocarcinoma was found to be
highly associated with poor survival.(21) However, the role of
MKK4 in cancer is complex, because several studies have also
suggested a pro-oncogenic role for MKK4.(22–25)
In recent years, it has been established that the MAPK signaling pathways play crucial roles in the pathogenesis of various
hematologic malignancies.(26) In a recent study, biochemical
analysis of 67 primary adult AML patients demonstrated a correlation between the constitutive activity of JNK in leukemic
blasts and treatment failure in AML.(27) Importantly, a relationship between JNK activity and increased multidrug resistanceassociated protein efflux was also observed.(27) So far, no report
has directly demonstrated a role for constitutive activation of the
p38 MAPK pathway in the pathophysiology of AML. However,
a recent study has shown that p38 and its downstream effector
MAPK-activated protein kinase 2 (MAPKAPK2) were activated
during treatment of the NB-4 acute promyelocytic leukemia cell
line with all-trans retinoic acid (ATRA).(28) Conversely, the
(a)
(b)
(b)
Fig. 3. Transient reporter gene expression assays with constructs
containing a full-length MKK4 promoter. (a) Schematic of the
reporter gene constructs having a full-length MKK4 promoter. The
only difference between the four constructs is a T>G or A>T at the
)1304 and )1044 polymorphic sites. (b) Luciferase expression of four
constructs in HL-60, NB4, and SHI-1 cells cotransfected with pRL-SV40
to standardize transfection efficiency. Luciferase levels of pGL-3 Basic
and pRL-SV40 were determined in triplicate and standardized for
transfection efficiency. Fold increases were determined by defining
the activity of the empty pGL-3 Basic vector as 1. Data shown are the
mean ± SD fold increase from three independent transfection
experiments, each performed in triplicate. (h), p1304T–1044T; ( ),
p1304G–1044A; ( ), p1304T–1044A; ( ), p1304G–1044T.
Jiang et al.
Fig. 4. Expression of MKK4 mRNA in bone marrow samples of
individuals who carry different genotypes for the (a) )1304T>G and
(b) )1044A>T polymorphisms.
Cancer Sci | 2011 | 5
ª 2011 Japanese Cancer Association
MEK inhibitor PD98059 was found to block the induction of
differentiation of NB-4 cells(29) and HL-60 cells(30) in response
to ATRA. Other studies have shown that, under certain circumstances, the p38 pathway can cooperate with the ERK pathway
to mediate cytokine-induced proliferation of AML cells.(31)
In summary, the JNK and p38 MAPK pathways both play a
functional role in the pathogenesis and pathophysiology of
AML. Because MKK4 is a direct activator of both JNK and p38,
we hypothesized that dysfunction caused by MKK4 mutations
may possibly affect susceptibility to the development of AML.
Our hypothesis was supported by the results of our genotyping,
real-time PCR, and transient transfection experiments. The present study is the only study thus far to investigate the association
between MKK4 mutations and ⁄ or expression and susceptibility
to AML.
Few studies have investigated the MKK4 )1304T>G polymorphism, so only three studies, including our present study,
can provide a comparison for the tested frequencies of the allele
and genotypes. Wei et al.(8) found that, 723 Chinese control subjects, the frequency of the TT, TG, and GG alleles was 53.8%,
40.8%, and 5.4%, respectively. Similarly, in another study of
lung cancer in 1056 controls, the frequency of the TT, TG, and
GG alleles was 57.3%, 35.9%, and 6.8%, respectively.(32) These
values are similar to the frequency of 53.0%, 40.7%, and 6.3%
for the TT, TG, and GG alleles, respectively, determined in the
600 control subjects in the present study. The corresponding figures for these genotypes in the HapMap database (http://hapmap.ncbi.nlm.nih.gov/, accessed Jan 2, 2011; HapMap Genome
Browser #27 (Phase 1, 2 and 3–merged genotypes and frequencies) are 63.1%, 33.3%, and 3.6% in 84 Chinese; 74.4%, 23.3%,
and 2.3% in 86 Japanese; 54.0%, 41.6%, and 4.4% in 113 European descendents; and 17.0%, 42.9%, and 40.2% in 112 Africans. These data suggest that the role of the MKK4 )1304T>G
polymorphism in cancer risk may vary with ethnicity, a possibility that warrants further investigation.
Although we found that the MKK4 )1304GG ⁄ GT genotypes
were associated with a decreased risk of AML, our study may
have certain limitations because of its design. Selection bias
and ⁄ or systematic errors may have occurred because the AML
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6
patients recruited to the study were recruited from those attending hospital, whereas the control subjects were recruited from
the community. Furthermore, some factors that may interact
with genotype or act as potential confounders in the analysis,
such as information regarding minimal residual disease, were
not available in the present case control study. Other limitations may be related to the fact that the present study was a
hospital-based case control study that was restricted to a Chinese Han population. However, the genotype frequencies
observed in the control group were in agreement with the
Hardy–Weinberg disequilibrium law, suggesting that our subject sampling was sufficiently random. We also achieved over
90% study power (two-sided test, a = 0.05) in detecting an OR
of 0.65 for the )1304GG + GT genotypes (which occurred at a
frequency of 47.0% in the control group), relative to the
)1304TT genotype. This evidence suggests that our findings
are sound and of note.
In conclusion, the present study indicates that, in the Chinese
population, carriers of the )1304GG and GT genotypes have a
decreased risk of AML compared with carriers of the MKK4
)1304TT genotype. To the best of our knowledge, the present
study is the first to demonstrate a significant association between
the MKK4 )1304G>T polymorphism and the risk of developing
AML. Larger, and preferably population-based, case control
studies, as well as well-designed mechanistic studies, are warranted to validate our findings.
Acknowledgments
This study was supported in part by the National Natural Scientific Foundation of China (81001278 and 81072366) and the Suzhou Science and
Technology Agency (SYS201052). The authors thank Drs Jianying
Liang and Mengxing Xue (Department of Hematology, First Affiliated
Hospital of Soochow University, Suzhou, Jiangsu, China) for their assistance in recruiting the subjects.
Disclosure Statement
The authors have no conflicts of interest to declare.
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ª 2011 Japanese Cancer Association