| Background:Lung cancer is one of the leading causes of death in the world. It was estimated that a death rate of 53.80/105 would have occurred in the USA in 2009. The data from the municipal death registry system in Guangzhou city of China have showed that the death rate of lung cancer was 48.79/105 between 2000 and 2002, the rate have increased almost three times in recent 30 years. In addition, the data from Shanghai and Tianjin have indicated that lung cancer is also a growing health problem in China. Epidemiological studies of lung cancer have established many etiologic factors; including smoking, air pollution, radiation, tuberculosis, nickel compounds and high body mass index (BMI).These factors are all stressors to the cells, which activate many protein kinase pathways, such as the mitogen-activated protein kinase (MAPK), protein kinase C (PKC), protein kinase A (PKA), and calcium/calmodulin dependent protein kinase (CAMK). Among of them, MAPK pathways are the best known, which typically consist of MAPKKK (MAPKK kinase), MAPKK (MAPK kinase) and MAPK. MAPKs molecules are a group of Ser/Thr protein kinases, responsible for the phosphorylation and activation of the proteins with"Ser/Thr-X-Pro"motif, which evolutionary conserved exists in all eukaryotes. Mammalian MAPK signaling pathways can be divided into four groups based on their structure and function: ERKs (Extracellular signal-Regulated Kinases), p38 MAPKs, JNKs (c-Jun NH2-terminal Kinases) and ERK5.There are comparative spectifity of substrates and effectors in different MAPK pathways. However, MAPK pathways do not function in isolation, and these groups are integrated and are impacted by each other. MAPK pathways also interate with other signaling pathways, such as JAK-STAT pathway, PKA pathway, hormone signaling (especially the Estrogen Receptor pathway), phos-phatidylinositol (PI)-3 kinase, and Ca2+ pathway. Thus, the key gather points of signaling cross-talk and the critical mediator of MAPK pathways may play pivotal roles in tumorigenesis.Mitogen-activated protein kinase kinase 4, also known as MKK4, MAP2K4, MEK4, JNKK1 and SEK1, is a critical mediator of stress-activated protein kinase signal that regulate apoptosis, inflammations, and tumorigenesis, eliciting a variety of biological responses to extracellular signals that include growth factors, hormones, pro-inflammatory cytokines and various stress stimuli. MKK4 can be activated through Ras-dependent or non Ras-dependent manner by many up-stream kinase of stress-activated protein kinase signal, such as MEKK (MEK kinase) 14, TAK1 (TGF-activated kinase 1), MUK (MAPK-upstream protein kinase), Tpl-2 (Tumor progression locus 2), MLK-3 (Mixed lineage kinase 3), ASK1 (Apoptosis signal-regulating kinase 1), MST (mammalian STE20-like kinase), suggesting MKK4 as a pivotal cross-talk gather point of stress-activated protein kinase signals. MKK4 in turn activates c-Jun NH2-terminal kinases (JNKs) and p38 MAPKs. Both JNK and p38 activate many transcriptional factors in nuclei, such as c-Jun,activating transcription factor-2 (ATF-2), Ets-like protein 1 (Elk-1), p53, deletion target in pancreatic carcinoma 4 (DPC4), NF-AT4, C/EBP homology protein (Chop), and Max. However, p38 MAPKs, unsimilar to JNKs, have a role in the regulation of phosphorylation of cytoplasm subtrates, such as MK2/MK3 (MAPK activated protein kinase 2/3) and HSP25/HSP27 (heat shock protein 25/27).Through regulating these substrates directly or indirectly, MKK4 plays a wide and complex role in carcinogenesis. It has been found that MKK4 plays an important role in tumor formation and development. Homozygous deletion of MKK4, which eliminates its coding portions, has been identified in pancreatic carcinoma cell lines and lung carcinoma cell lines. It is also reported that loss-of-function mutations in the MKK4 gene have been found in about 5% of tumors from various human tissues. Furthermore, MKK4 has been identified as a suppressor of metastasis of prostate and ovarian cancers, and lack of expression of the MKK4 gene in resected gastric adenocarcinoma was found to be highly associated with poor survival. Taken together, some researchers have defined MKK4 as a candidate tumor suppressor. However, the role of MKK4 in cancer appears to be complex because a pro-oncogenic role of MKK4 was also reported. These reports suggest that further study of MKK4 gene may help for cancer prevention and treatment.However, lung cancer just occurres in 15% of heavy smokers. Only a small part of individuals who exposured to the risk environments of lung cancer developed lung cancer disease. It suggestes that genetic susceptibility plays a role in the development of lung cancer. Because mutations and deletions of MKK4 gene are usually reported in cancer, we should evaluate the association between the genetic variants of MKK4 gene and cancer risk.Human MKK4 gene, (Genomic accession No: NM003010.2), spans >120 kb on chromosome 17p11.2, has a promoter region of 1.6 kb, contains 11 exons and encodes the 399-amino acid protein. Several polymorphisms have been identified in the MKK4 gene. One of our previous studies investigated the role of polymorphisms in the MKK4 promoter and found that the polymorphism rs3826392 (-1304T>G) [-1304 nt to initiation transcription code ATG (A in bold indicate the boundary)], but not rs3809728 (-1044A>T), was associated with a decreased risk of sporadic colorectal cancer. Because various environmental factors of lung cancer can also activate MKK4 and MAPK pathways, and MKK4 is usually poorly expressed in lung cancer, we hypothesized that genetic variants in the MKK4 promoter may alter its expression and thus cancer risk. Hypotheses:Environmental carcinogens usually activate MAPK pathways, which regulate apoptosis, inflammations and tumorigenesis. MKK4 is a critical mediator of MAPK pathways. Several putative functional polymorphisms have been identified in the MKK4 gene. We hypothesized that genetic variants in the MKK4 promoter may alter its expression and thus cancer risk.Objectives:In this hospital-based case–control study conducted in a Southern Chinese population, we genotyped two common polymorphisms (-1304T>G and -1044A>T) of the MKK4 promoter in 1056 patients with newly diagnosed lung cancer and 1056 cancer-free controls, and used SAS 9.13 software to analyze the genetic variant and lung cancer risk through non-conditional logistic regression, with and without adjustment for age, sex, smoking status, alcohol drinking status, BMI, and family history of cancer. In the stratification analysis, we assessed the main effect of MKK4 polymorphisms in each subgroup and the possible interaction between MKK4 polymorphisms and selected variables on cancer risk. We also performed western blotting analysis and luciferase assays to identify whether genetic variants in MKK4 alter its gene expressions and functions, and thus cancer risk.Methods:In this study, we used 1056 patients with newly diagnosed primary lung cancer and 1056 sex and age (±1 year) frequency matched cancer-free controls genetically-unrelated ethnic Han Chinese from Guangzhou City and surrounding regions in Southern China. Patients with histopathologically confirmed diagnosis were consecutively recruited between March 2007 and March 2009 at the five urban or suburb hospitals of Guangzhou city. The 1056 cancer-free controls, frequency matched to the cases by sex and age (±1 years) were randomly selected from a subject pool with 10 000 individuals who participated in the healthy checkup programs in the community health stations in Guangzhou City during the same time period when the cases were recruited. All subjects have lived in Guangzhou at least 10 years.Each participant was scheduled for an interview after a written informed consent was obtained, and the trained interviewers collected the subjects'data on smoking status, alcohol use and other factors including family history of cancer with a structured questionnaire. Each subject was asked to donate 5 mL of blood after having given their informed consent. The genomic DNA of each subject was extracted by a routine method for further SNP genotyping.Based on our resequencing data of 30 normal Han Chinese subjects, we found that four common SNPs (i.e. minor allele frequency > 5%) in the MKK4 promoter region in Southern Chinese: -1304T>G (rs3826392), -1044A>T (rs3809728), -641C>G (rs2190853) and -284T>C (rs9892151). Further linkage disequilibrium (LD) analysis showed that the pairs of three SNPs [i.e. -1044A>T (rs3809728), -641C>G (rs2190853) and -284T>C (rs9892151)] were in complete linkage disequilibrium (LD) (r2 = 1.00,D'= 1.00, for each pairs), but these three SNPs were not in complete LD (r2 < 0.80, D'< 0.80) with the other SNP -1304T>G (rs3826392). Therefore, we genotyped only two SNPs (-1304T>G and -1044A>T) that covered genetic information of the above four loci in the MKK4 promoter region.In 1056 lung cancer patients and 1056 cancer-free controls, two common polymorphisms, -1304T>G and -1044A>T, were genotyped by an allelic discrimination method using allele-specific fluorogenic probes (Taqman assay, 5' nuclease assay). MGB probes but not TARMA probes were chosen for genotyping, because of their higher Tm values and higher genotyping resolution. SAS 9.13 software was used to analyze the genetic variant and lung cancer risk through non-conditional logistic regression, with and without adjustment for age, sex, and smoking status, alcohol drinking status, BMI, and family history of cancer. In the stratification analysis, we assessed the main effect of MKK4 polymorphisms in each subgroup and the possible interaction between MKK4 polymorphisms and selected variables on cancer risk. A multiplicative interaction was suggested when OR 11 > OR 10×OR 01, in which OR 11 = the OR when both factors were present, OR 01 = the OR when only genetic factor was present, OR 10 = the OR when only environmental factor was present.To analyze the correlation between MKK4 polymorphisms and MKK4 protein expression, we tested MKK4 protein expression levels in 30 lung cancer tissues by western blotting analysis. All tumor samples were histologically confirmed. The MKK4 protein expression levels were normalized to that ofβ-actin by calculating the relative expression levels. One-way ANOVA tests were used for analyzing the results of in vivo MKK4 protein expression in tumor tissues by the genotypes. We used the linear regression model to evaluate the main effect of MKK4 genotypes on the MKK4 protein expression with adjustment for age, sex, smoking and alcohol usage.To determine the allele-specific effect of the MKK4 variants on the promoter activity, we generated a series of luciferase reporter gene constructs that contained 1612 bp of the MKK4 promoter region which covered both studied polymorphisms [from -1528 to +84 bp relative to the translation start site ATG (A in bold indicate the boundary)]. The three human cell lines, 16HBE (a human bronchial epithelial cell line), A549 (a human lung adenocarcinoma cell line) and L78 (a human lung squamous carcinoma cell line), were transfected with the vector DNA with the MKK4 promoter region. As an internal standard, all cells were cotransfected with pRL-TK plasmids, which contained the Renilla luciferase gene. The pGL3-basic vector without an insert was used as a negative control. Luciferase activity was normalized against the activity of the Renilla luciferase gene. Differences in the expression levels of different constructs were determined by Student's t test.Results:This study included 1056 patients with newly diagnosed primary lung cancer and 1056 sex and age frequency matched cancer-free controls. Of the 1056 cases included in this study, there were 384 (36.4%) cases of adenocarcinoma, 369 (34.9%) of squamous cell carcinoma, 43 (4.1%) of large cell carcinoma, 128 (12.1%) of small cell lung cancer, and 132 (12.5%) of mixed-cell or undifferentiated carcinoma. According to the 2009 IASLC (the International Staging Committee of the International Association for the Study of Lung Cancer) staging classifications, there were 154 (14.5%) cases of stage I, 94 (8.9%) of stage II, 333 (31.5%) of stage III, and 475 (45.0%) of stage IV. Two-sided chi-square tests were used to assess differences in the distributions of age, sex, smoking status, alcohol use, BMI, and family history of cancer between cases and controls. Overall, the differences in distributions of age, sex, alcoholic drinking and the family history of cancer between the cases and controls were not statistically significant (P =0.931, 1.000, 0.916 and 0.942, respectively). However, the cases were more likely to have a history of current or ever smoking, and have a low or a normal body mass index (BMI) than were controls (P = 0.028, and <0.001, respectively). Therefore, these variables were further adjusted for in the multivariate logistic regression model to control possible confounding on the main effects of the studied polymorphisms. Additionally, they were used in the later stratifications and gene-environment interaction analysis.The genotype and allele distributions of the MKK4 polymorphisms -1304T>G (rs3826392) and -1044A>T (rs3809728) among the cases and controls were genotyped using TAQMAN method. The Hardy-Weinberg equilibrium (HWE) was tested by a goodness-of-fit chi-square test to compare the expected genotype frequencies with observed genotype frequencies (p2+2pq+q2=1) in cancer-free controls. The observed genotype frequencies of these two polymorphisms were all in agreement with the Hardy-Weinberg equilibrium in the control subjects (P= 0.704, and 0.688, respectively), suggesting the subjects'selection are in random. The linkage disequilibrium (LD) analysis in controls showed that the linkage between the -1304T>G locus and -1044A>T locus was very weak (D'= 0.072, and r2 = 0.004), suggesting that each may have an independent effect on risk of lung cancer. Therefore, we did not perform the association of the HAPLOTYPE of above two polymorphisms with lung cancer risk.The frequencies of -1304TT, -1304TG and -1304GG in patients were 65.5%, 30.2%, and 4.3%, respectively. And the corresponding figures in controls were 57.3%, 35.9%, and 6.8%, respectively. Genotype frequencies of -1304T>G locus between the cases and the controls were significant different (χ2 = 15.079, P<0.001). Logistic regression analysis showed that compared with the most common -1304TT genotype, carriers of the -1304TG heterozygote had a decreased risk of lung cancer [adjusted Odds Ratio (OR) = 0.74; 95% confidence interval (CI) = 0.61-0.90; P= 0.002], and -1304GG homozygote had a further decreased risk of lung cancer (adjusted OR=0.62, 95% CI=0.408-0.936; P = 0.024). There was a significant trend for an allele dose-effect on the decreased risk of lung cancer (adjusted Ptrend<0.001). The association between -1304G variant genotypes (-1304TG+GG) and decreased lung cancer risk was statistically significant at a 5% type I error level (adjusted OR=0.72, 95%CI = 0.60-0.87; P<0.001). However, for the -1044A>T polymorphism, both genotype and allele frequencies did not differ significantly between the cases and the controls (P = 0.703 and 0.426, respectively), and consistently, there was no significant association between this polymorphism and risk of lung cancer.We further performed a stratification analysis of the associations between MKK4 variant genotypes and risk of lung cancer in subgroups by age, sex, smoking, drinking, BMI, and family history of cancer. We combined -1304TG and -1304GG in a group to increase the statistical power. The decreased risks of lung cancer associated with the -1304G variant genotypes (-1304TG+GG) did not differ by age (younger or older than 60 years), sex, smoking and drinking status, the family history of cancer and tumor histological types (P >0.05 for all subgroups). However, the protection of -1304G variant genotypes against lung cancer risk was observed in the subjects with a low BMI (BMI<18.0kg/m2, adjusted OR = 0.27; 95% CI = 0.13-0.53; P <0.001) and a normal BMI (adjusted OR = 0.73; 95% CI = 0.59-0.90; P = 0.0028), but not in those with overweight (BMI >25.0, adjusted OR = 1.00; 95% CI = 0.63-1.59; P= 0.997).Furthermore, we assessed possible interactions (or effect modifications) between MKK4 polymorphisms and age, sex, smoking, drinking, family history of cancer and BMI groups on the risk of lung cancer. While fitting multiplicative models, we found that the -1304G variant genotypes interacted with low or normal BMI status (adjusted P value for interaction was 0.003).To test the relation between the genotypes and phenotypes (expressions) of MKK4 gene, we tested the MKK4 protein expression levels in 30 lung cancer tissues. We found that MKK4 protein expression levels of the G variant carriers were significantly higher in tumor tissues than those of the -1304TT genotype (0.78±0.09 ofβ-actin level for -1304GG, 0.71±0.12 for -1304TG, 0.50±0.26 for -1304TT; ANOVA test: adjusted F = 4.728, P = 0.017, Ptrend = 0.009). However, no significant association was observed between the -1044A>T polymorphism and risk of lung cancer.To further evaluate the effect of the important polymorphism -1304T>G on transcript activity of the MKK4 promoter, we generated two luciferase reporter gene constructs that contained 1612 bp of the MKK4 promoter region (from -1528 to +84 bp relative to the translation start site ATG) with a T or G allele at the -1304 polymorphic site, and with A allele at the -1044 polymorphic site. We transfected the vector DNA with either -1304T or -1304G allele into three lung cell lines, 16HBE, A549 and L78. The three lung cell lines were also co-transfected with pRL-TK plasmids (as an internal standard) and with pGL3-basic vectors without an insert (as a negative control). The luciferase assay showed that the G allele in the promoter significantly increased about 2-3 folds of the transcription activity of the MKK4 gene in vitro in the three types of cell lines: compared with the transcription level with the -1304T allele, the vectors with the -1304G allele had a 243% increase in 16HBE, P < 0.001; those with -1304G allele had a 301% increase in A549, P <0.001; those with -1304G allele had a 206% increase in L78, P = 0.001. These results suggested that the -1304G allele in the promoter region increases transcriptional activity of the MKK4 gene.Innovation:To the best of our knowledge, this study provided the first evidence that functional genetic variant MKK4 -1304T>G is associated with lung cancer risk, and that the T>G transition of -1304 locus increase the promoter activity. Our data suggested that the -1304G variant may be a susceptibility biomarker for lung cancer. Moreover, we first found that BMI interacted with genetic variants in lung cancer risk. Limitation:Our study was limitated to the Southern Chinese population. Larger population-based studies to replicate our findings, and further functional studies, such as electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP), are warranted.Conclusions:In this hospital-based case–control study, we found that the -1304T>G polymorphism in the MKK4 promoter was associated with lung cancer risk, and the risk decreased as the number of -1304G allele increased in an allele dose–response manner; however, the protective role of the -1304G allele was absent in the overweighters. Furthermore, we found that levels of MKK4 protein expression were significantly higher in tumor tissues from patients carrying the -1304G variant genotypes than those carrying the -1304TT genotype. In the in vitro assays, we found that the -1304G variant allele significantly increased the transcription activity of the MKK4 gene compared with the -1304T allele, suggesting that the -1304T>G polymophism is potentially functional. However, for the -1044A>T polymorphism, no statistical evidence was found for any association between this polymorphism and lung cancer risk.Our data suggest that the functional -1304G variant in the MKK4 promoter contributes to a decreased risk of lung cancer by increasing the promoter activity and that the G variant may be a marker for susceptibility to lung cancer. |
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