New insights into the association between AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G variants and cancer risk

Background Many epidemiological studies have investigated association of AXIN2 variants on overall cancer risks; however, the available results remain inconsistent. Methods An updated analysis was conducted to ascertain a more accurate estimation of the correlation between AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G polymorphisms and cancer risk. We also used in silico tools to assess the effect of AXIN2 expression on cancer susceptibility and overall survival time. Results A total of 4281 cases and 3955 control participants were studied. The overall results indicated that AXIN2 148 C/T variant was associated with cancer risk (allelic contrast: OR = 0.88, 95% CI 0.77–0.99, Pheterogeneity = 0.004; dominant model: OR = 0.82, 95% CI 0.69–0.96, Pheterogeneity = 0.022), especially for lung and prostate adenocarcinoma. Similar results were observed in 1365 C/T polymorphism (OR = 0.71, 95% CI 0.61–0.98, Pheterogeneity = 0.873; dominant model: OR = 0.66, 95% CI 0.47–0.94, Pheterogeneity = 0.775). Moreover, in subgroup analysis by ethnicity, similar findings were obtained for Asian and Caucasian populations. Results from in silico tools suggested that AXIN2 expressions in lung adenocarcinoma were lower than that in normal group. Conclusions Our findings indicated that AXIN2 148 C/T and 1365 C/T variants may be associated with decreased cancer susceptibility.


Background
The continuing changes in global population and epidemiology indicate that the burden of cancer will continue to increase in the coming decades. Cancer is considered as a multifactorial disease and its occurrence is associated with several factors such as lifestyle, environment and single nucleotide polymorphism (SNP) [1][2][3]. With the remarkable development of a series of genotyping technologies including genome-wide association studies (GWAS), our understanding of genetic factors related to carcinogenesis has substantially expanded [4][5][6]. Wnt/βcatenin signaling pathway is known to play a central role in the process of embryogenesis, and abnormalities of this pathway are associated with numerous human malignant tumors [7,8]. Axin2 protein acts as a negative regulator of Wnt pathway and plays a crucial role in cell differentiation, migration, cytometaplasia, and apoptosis [9][10][11]. Axin2 protein is also involved in down-regulation of β-catenin translocation ito the nucleus. In this process, Axin2 binds to transcription factors and subsequently inhibits the expression of numerous target genes including vascular matrix metalloproteinases (MMP), cox 2, and endothelial growth factor (VEGF) [12,13].

Literature retrieval strategy
PubMed, Web of Science, Google Scholar, and China Wanfang Databases were systematically searched to identify all eligible published articles on AXIN2 variants and cancer susceptibility. The following terms were utilized for searching abstracts and titles: "Axin OR AXIN2", "polymorphism OR SNP OR variant", and "cancer OR adenocarcinoma OR carcinoma OR tumor". The latest search was conducted on Jan 31, 2019 with no language restrictions. Furthermore, we also carefully screened and manually searched the review or original publications for more eligible studies.

Study selection
Two authors independently chose the eligible studies based on the inclusion criteria: (a) case-control studies that evaluated the association between AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G variants and cancer risk; (b) studies that involved available information for measuring odds ratio (OR) with 95% confidence intervals (CIs); (c) genotype distribution in controls must be conformed to Hardy-Weinberg equilibrium (HWE).

Data extraction
All related information was independently screened by two investigators (L Shi and B Xu) from each enrolled study, including the name of first author, year of publication, country of origin, ethnicity, source of control, genotyping method, cancer type, total number of participants, P value for HWE, age range, genotyping data of AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G variants in cases and controls. Disagreement should be resolved by discussion with a third author (W Zhang). If the controversial content still existed, it should be addressed by all investigators to reach a consensus.

Statistical analysis
The strength of the relationship between AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G polymorphisms and cancer susceptibility was measured by calculating OR with 95% CI. A total of four genetic models were adopted in the current analysis, including allelic comparison model (M-allele vs. W-allele), homozygote contrast model (MM vs. WW), heterozygote model (MW vs. WW), and dominant model (MM + MW vs. WW). The χ 2 -test-based Q test was performed to investigate P value for heterogeneity among eligible researches. If P < 0.05, indicating that a significant heterogeneity was found, we employed the random-effects model (DerSimonian-Laird method) [34]. On the other hand, the fixed-effects model (Mantel-Haenszel method) was carried out [35]. We adopted qualitative funnel plot to assess possible publication bias by calculating the standard error of log(OR) for each research plotted against its log(OR). We further conducted quantitative Egger's test to evaluate funnel plot asymmetry [36]. The web-based program was applied to check for deviations from the Hardy-Weinberg equilibrium (HWE) of distribution frequencies (http://ihg2. helmh oltz-muenc hen.de/cgibi n/hw/hwa1.pl) [37]. The P value more than 0.05 suggested an HWE balance. Moreover, we applied leave-one-out sensitivity analyses to calculate the stability of pooled results [38]. All of the above analyses were conducted by STATA software v11.0 (Stata Corporation, TX).

In silico analysis of AXIN2 expression
An online gene expression database was adopted to investigate the AXIN2 expression in lung and prostate adenocarcinoma tissues and the paracancerous tissues. (http://gemin i.cance r-pku.cn/) [39]. RNA expression profiles of 446 pathologically diagnosed lung adenocarcinoma (including 387 Caucasians, 51 African-Americans, and 8 Asians) and 153 prostate adenocarcinoma tissues (containing 147 Caucasians and 6 African-Americans) were evaluated by this database. The Cancer Genome Atlas (TCGA) samples were also utilized to investigate the high and low expression of AXIN2 on cancer susceptibility and overall survival time. Moreover, the String online server was applied to assess the gene-gene correlation of AXIN2 (http://strin g-db.org/).

Characteristics of studies
As was shown in Table 1, 15 articles were finally retrieved in the present analysis, which contains 22 case-control studies for AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G variants. There were 2909 cancer subjects and 2907 control volunteers for 148 C/T polymorphism, 587 cancer subjects and 605 controls for 1365 C/T variant, 785 cases and 443 controls for rs4791171 A/G variant. Furthermore, we checked the minor allele frequencies (MAF) of three AXIN2 variants by Trans-Omics for Precision Medicine (TOPMed) online (https ://www.ncbi.nlm.nih. gov/snp/) ( Fig. 1 Global, 0.547. In stratified analysis by ethnicity, seven studies were performed in Caucasian populations, twelve studies were in Asian descendants, and two were done in Arabians and one was in Latin descendants. Eight studies were conducted using population based controls and the rest 14 studies were utilizing hospital based controls. The classical genotyping method, PCR-restriction fragment length polymorphism (RFLP) was adopted in nine of these studies.

In silico analysis of AXIN2 expression
Results from in silico tools suggested that AXIN2 expression in normal group was higher than that in lung adenocarcinoma tissue (Fig. 4a). However, no obvious difference was indicated for prostate adenocarcinoma (Fig. 4b). Moreover, we explored whether the AXIN2 expression had an effect on the overall survival time of lung adenocarcinoma patients. However, Kaplan-Meier estimate showed no vital difference of overall survival time between high and low AXIN2 expression groups (P = 0.40, Fig. 5).  Fig. 6, results from funnel plots appeared symmetrical in the overall analysis under dominant model, which indicated a lack of publication bias. Sensitivity analyses were also utilized to assess the pooled OR by omission of any one study. The  results suggested that the current data from pooled ORs were relatively stable. No single study can substantially change the overall OR (Fig. 7).

Discussion
To date, large quantities of studies have been conducted to explore whether the variants confer individual's susceptibility to carcinoma. However, results from the previous publications have yielded controversial results [21,22]. A previous study based on Indian descendants found a strong protective effect in participants having heterozygous genotype for 1365 C/T variant [30], while another study group did not observe such positive correlation in Turkish population [27]. In 2005, Wu et al. performed a meta-analysis and found that AXIN2 rs2240308 variant may increase the risk of cancer, especially lung cancer in Asian descendants [40]. Two years later, another metaanalysis indicated no obvious correlation between this variant and cancer risk in the overall analysis. Moreover, researches of this article observed that rs2240308 polymorphism was significantly associated with a decreased cancer risk in Asian population [41]. The overall goal of the present study was to evaluate all eligible data based on the inclusion criteria to enhance the statistical powers and draw more accurate conclusions. In the current study, a total of 4281 cases and 3955 control participants were investigated. The overall results showed evidence that AXIN2 148 C/T variant was associated with decreased cancer risk, especially for lung and prostate adenocarcinoma, which is in line with conclusions identified by Kanzaki et al. Liu et al. and Gune et al. [19,26,28]. Similar results were observed in AXIN2 1365 C/T polymorphism (under allelic contrast, heterozygote comparison, and dominant genetic model). Moreover, in subgroup analysis by ethnicity, positive findings were obtained for Asian and Caucasian populations. In the stratified analysis by source of control, similar findings were identified in population-based studies for AXIN2 148 C/T variant, which is consistent with the findings reported by Yu et al. [41]. Moreover, results from in silico tools showed that AXIN2 expressions in lung cancer and prostate cancer are lower than that in normal counterpart. High expression of AXIN2 may have longer OS time than low expression group for lung cancer participants, which were consistent with results derived from the present meta-analysis. Nevertheless, we indicated no significant difference between the high expression and low/ medium expression of AXIN2 in prostate cancer patients.
Some limitations of the above analysis should be mentioned. Firstly, the numbers of enrolled articles in the current analysis were still not large enough for the comprehensive analysis, especially for AXIN2 1365 C/T and rs4791171 A/G variants. Four articles towards AXIN2 1365 C/T and three articles for rs4791171 A/G polymorphism were eligible based on the selection criteria. Secondly, insufficient original data from the raw articles limited further evaluation of potential interactions, including relationship between the AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G variants and different tumor grade and stage. Thirdly, meta-analysis was based on unadjusted estimates, which may lead to serious confounding bias. Furthermore, gene-gene interaction would also participate in etiological mechanism of carcinoma. As shown in Fig. 8, at least 20 related genes may be involved in such interaction, which are required to be further investigated in future studies. On the other hand, core advantages in current analysis should also be acknowledged. Firstly, a comprehensive study of the correlation of the AXIN2 148 C/T, 1365 C/T, and rs4791171 A/G variants with overall cancer susceptibility  is statistically more powerful than single case-control study. All the studies according to the inclusion criteria were accumulated in our analysis. Secondly, genotype distribution of controls is conformed to Hardy-Weinberg equilibrium (HWE) in any of the enrolled studies and no significant publication bias was found, which indicated that conclusions of the present analysis are relatively trustworthy.

Conclusions
Taken together, the current study showed evidence that AXIN2 148 C/T and 1365 C/T variants may be associated with decreased cancer susceptibility, especially for lung and prostate adenocarcinoma. Future large scale studies with standardized unbiased cases and well-matched