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Association of calcium/calmodulin-dependent protein kinase kinase1 rs7214723 polymorphism with lung cancer risk in a
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Chinese population Da Chen1, Fangming Zhong1, Ye Chen2* 1
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Huanchengdong Road, Hangzhou, Zhejiang, China. 2
Department of Pneumology, The Second Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou, Zhejiang, China
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*Correspondence
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+86-0571-85632119; Fax: +86-0571-86574316
to:
Ye
Chen;
E-mail:
[email protected];
Tel:
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ACCEPTED MANUSCRIPT
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The Cardiothoracic Surgery Department, Hang Zhou Red Cross Hospital, 208
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Abstract
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Calcium/calmodulin-dependent protein kinase kinase1 (CAMKK1) could specially
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recognize and activate Calcium/calmodulin-dependent protein kinase (CAMK) I and
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IV. Furthermore, The activation of CAMK showed positively correlation with
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proliferation of lung cancer (LC). In addition, A genome-wide association study has
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identified rs7214723 (E375G) in the CAMKK1 gene as a susceptibility locus for LC
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in the UK population. Therefore, we conducted a case-control study involving 320 LC
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patients and 320 controls to validate this conclusion in a Chinese population.
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Genotyping was performed using a custom-by-design 48-Plex single nucleotide
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polymorphism (SNP) Scan™ Kit. Our results indicate that the individuals with CC
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genotype of rs7214723 polymorphism had the higher risk of LC than those who
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carried TT genotype. Moreover, CAMKK1 rs7214723 polymorphism showed
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positively correlation with the elevated risk of LC in the allelic model and recessive
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model, but not in the dominant model. Stratified analysis further confirmed this
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significant association in male groups and smokers. In conclusion, CAMKK1
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rs7214723 polymorphism may be associated with the increased risk of LC. However,
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larger studies with more diverse ethnic populations are needed to confirm these
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results.
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Keywords: CAMKK1, polymorphism, lung cancer, molecular epidemiology
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2
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Introduction
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Lung cancer (LC) is the leading cause of cancer death with high incidence rate
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[1]. Due to complex biological characteristics, lung cancer has extremely difficulty in
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diagnosis and treatment at early stage [2]. In 2007, new 222,500 LC cases and
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155,870 LC deaths are expected to occur in United States [3]. Tobacco using, air
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pollution, exposure to carcinogens, genetic factors and other factors can cause LC [4,
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5]. In addition, genetic factors play an important role in modifying an individual’s risk
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for LC.
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The calcium/calmodulin dependent protein kinase kinase (CAMKK) gene is
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located on chromosome 17p13.2 and has 19 exons. CAMKKs phosphorylate and
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activate specific downstream protein kinases, including calcium/calmodulin
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dependent protein kinase (CAMK) I, CAMK IV, and 5'-AMP-activated protein kinase
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(AMPK), which mediates a variety of Ca2+ signaling cascades [6]. CAMK I was
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demonstrated to be associated with the proliferation of LC cell lines [7]. Moreover,
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Williams et al. demonstrated that the activation of CAMK (CAMK Ⅱ and CAMK IV)
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inhibits cell cycle progression in small cell lung carcinoma (SCLC) cells [8].
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Furthermore, Shao et al. revealed that the activation of MAPK contributes to growth
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inhibition and apoptosis in human LC cell [9]. There is a wide consensus that
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CAMKK have been shown to undergo autophosphorylation and contains two
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isoforms (CAMKK1 and CAMKK2) [10]. Therefore, we guessed that CAMMK1, a
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member of CAMKK, may play a role in the development of LC indirectly.
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Rs7214723, a T to C transition, leads to a glutamate (E) to glycine (G)
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substitution at the amino acid position 375. A genome-wide association study (GWAS)
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has identified rs7214723 (E375G) polymorphism of CAMKK1 gene as a
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susceptibility locus for LC in UK Caucasians [11]. Subsequently, Truong et al. tended 3
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to replicate this finding, but failed to find significant association between this single
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nuclear polymorphism (SNP) and LC risk among Caucasians and Asians [12]. In 2013,
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Zhang et al. found CAMKK1 rs7214723 polymorphism contribute to LC risk in a
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Chinese population and the T allele of rs7214723 could be viewed as a risk allele for
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LC [13]. Notably, there were two contradictory findings in the Asian populations.
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Therefore, we conducted a hospital-based study with 320 cases and 320 controls to
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validate the role of CAMKK1 rs7214723 polymorphism in modifying the risk of LC.
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Patients and methods
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Study subjects
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A total of 320 patients diagnosed with lung cancer were consecutively recruited
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from the Second Affiliated Hospital of Zhejiang Chinese Medical University, between
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September 2014 and October 2016. The subjects with family history of cancer and
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biochemical abnormalities did not conform to our inclusion criteria. The healthy
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controls were free of LC and recruited from the same institutions during the same
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time period. They were frequency matched (1:1) to the LC cases based on sex and age
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(± 5 years), A detailed questionnaire related to smoking habits was completed for each
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patient and control by a trained interviewer. Informed consent was obtained from all
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patients and controls prior to their participation. The protocol for this study was
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approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang
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Chinese Medical University (Hangzhou, Zhejiang, China).
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DNA extraction and Genotyping To investigate the polymorphism of CAMKK1, all study participants provided 2 4
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mL of peripheral blood in ethylenediaminetetraacetic acid (EDTA) tubes and stored at
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-80 ℃ until use. DNA was extracted by using the QIAamp DNA Blood Mini Kit
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(Qiagen,
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custom-by-design 48-Plex SNP scanTM Kit (Genesky Biotechnologies Inc., Shanghai,
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China), which was described in previous case-control studies [14, 15]. This kit was
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developed according to patented SNP genotyping technology by Genesky
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Biotechnologies Inc., which was based on double ligation and multiplex fluorescence
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PCR. For quality control, repeated analyses were done for 4% of randomly selected
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samples with high DNA quality.
Hilden,
Germany).
SNP
genotyping
was
performed
using
a
95 96
Statistical analysis
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Hardy–Weinberg equilibrium (HWE) for CAMKK1 genotype distributions in
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controls was tested by a goodness-of-fit chi-squared test. The demographic and
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clinical characteristics of study participants were evaluated by using the chi-squared
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test. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the
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association between CAMKK1 gene polymorphisms and risk of LC by logistic
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regression analyses. The most common homozygote was seen as a reference group.
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All statistical analyses were performed using the SPSS ver22.0 software package. P
A polymorphism was associated with a decreased risk of
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esophageal cancer in a Chinese population. Clin Biochem. 46, 1469-1473.
247
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Witczak, C.A., N. Fujii, M.F. Hirshman, and L.J. Goodyear. (2007)
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Ca2+/calmodulin-dependent protein kinase kinase-alpha regulates skeletal
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muscle glucose uptake independent of AMP-activated protein kinase and Akt
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activation. Diabetes. 56, 1403-1409.
251
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Mizuno, K., L. Ris, A. Sanchez-Capelo, E. Godaux, and K.P. Giese. (2006)
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Ca2+/calmodulin kinase kinase alpha is dispensable for brain development but
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is required for distinct memories in male, though not in female, mice. Mol Cell
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Biol. 26, 9094-9104.
255 256
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Okuno, S., T. Kitani, and H. Fujisawa. (1997) Studies on the substrate specificity of Ca2+/calmodulin-dependent protein kinase kinase alpha. J 11
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Biochem. 122, 337-343.
258 259
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Table 1 Patient demographics and risk factors in lung cancer Variable Cases (n=320) Age (years) ≤50 122 >50 198 Sex Female 70(21.9%) Male 250(78.1%) Smoking status Nonsmoker 65(20.3%) Smoke 255(79.7%) Histology Squamous cell carcinoma 180(56.3%) Adenocarcinoma 94(29.4%) others 46(14.4%)
Controls (n=320)
P
102 218
0.369
75(23.4%) 245(76.6%)
0.495
78(24.4%) 242(75.6%)
0.217
— — —
Table 2 Logistic regression analysis of associations between CAMKK1 rs7214723 polymorphism and risk of lung cancer Genotype Cases*(n=320) Controls*(n=320) OR (95% CI) n % n % TC vs. TT 122/133 38.1/41.6 130/149 40.6/46.6 1.05 (0.75–1.48) CC vs. TT 61/133 19.1/41.6 38/149 11.9/46.6 1.80 (1.13-2.87) CC vs. TC vs. TT TC+CC vs. TT 183/133 57.2/41.6 168/149 52.5/46.6 1.22 (0.89-1.67) CC vs. TC+TT 61/255 19.1/79.9 38/279 11.9/87.2 1.76 (1.13-2.73) C vs. T 244/388 38.1/60.6 206/428 32.2/66.9 1.31(1.04-1.65) *The genotyping was successful in 316 cases and 317 controls. Bold values are statistically significant (P
