Pin1 Promoter rs2233678 and rs2233679 Polymorphisms in Cancer: A Meta-analysis

Cancer is a multi-step process resulting from complex interactions between genetic and environmental factors. Host genetic susceptibility plays an important role in developing cancer. Such various susceptibilities could be explained, in part, by single nucleotide polymorphisms (SNPs) of susceptible genes (Xue et al., 2010; Chung et al., 2011; Perez-Losada et al., 2011). PIN1 (peptidylprolyl cis/trans isomerase, NIMAinteracting 1), which is one member of the parvulin peptidyl-prolyl isomerase (PPIases) families, is a 18 kDa protein containing a carboxy-terminal catalytic domain and a WW amino-terminal protein–protein interaction domain which can change conformation of phosphoproteins by recognizing and binding to specific phospho-Ser/Thr-Pro motifs (Lu et al., 2007; Liou et al., 2011; Theuerkorn et al., 2011). Its high expression was correlated with tumor progression and prognosis of patients in several types


Introduction
Cancer is a multi-step process resulting from complex interactions between genetic and environmental factors. Host genetic susceptibility plays an important role in developing cancer. Such various susceptibilities could be explained, in part, by single nucleotide polymorphisms (SNPs) of susceptible genes (Xue et al., 2010;Chung et al., 2011;Perez-Losada et al., 2011). PIN1 (peptidylprolyl cis/trans isomerase, NIMAinteracting 1), which is one member of the parvulin peptidyl-prolyl isomerase (PPIases) families, is a 18 kDa protein containing a carboxy-terminal catalytic domain and a WW amino-terminal protein-protein interaction domain which can change conformation of phosphoproteins by recognizing and binding to specific phospho-Ser/Thr-Pro motifs (Lu et al., 2007;Liou et al., 2011;Theuerkorn et al., 2011). Its high expression was correlated with tumor progression and prognosis of patients in several types Yan-Mei Zhu 1,2 , Jing-Wei Liu 1 , Qian Xu 1 , Yuan Yuan 1 * of cancer (Ayala et al., 2003;Miyashita et al., 2003;Fukuchi, et al., 2006;He et al., 2007). The PIN1 gene (NC_000019.8) spans over ~14 kb on chromosome 19p13, contains four exons, encodes a 163-amino acid protein, and has a promoter region of 1.5 kb. Two putative functional single nucleotide polymorphisms (SNPs) in the PIN1 promoter (rs2233678G>C: c.-842G>C [842nt upstream to initiation transcription code ATG] and rs2233679C>T: c.-667C>T) have been submitted to the PIN1 locus-specific database (www.LOVD.nl/PIN1). Dysregulation of PIN1 protein function and expression owing to SNPs in PIN1 promoter may alter the PIN1 signaling pathway, thereby modulate the risk of cancer.
Despite a series of molecular epidemiological studies aiming to examine the association between these two polymorphisms and the susceptibility of different cancer types, the available results remain conflicting. For the -842G>C polymorphism, Han et al. (2010) found that -842C variant alleles (GC+CC) were associated with decreased risk in breast cancer; Lu at al (Lu et al., 2013) found that -842CG heterozygote but not -842CC homozygote had a significantly decreased risk of nasopharyngeal carcinoma; In another two studies, -842G>C polymorphism had no influence on breast cancer (Naidu et al., 2011) and hepatocellular carcinoma risk (Segat et al., 2007). For -667C>T polymorphism, some studies found that -667T allele was associated with increased risk of nasopharyngeal carcinoma (Lu et al., 2013) and hepatocellular carcinoma (Segat et al., 2007), but others found that this polymorphism had no association with esophageal carcinoma (You et al., 2013), lung cancer , breast cancer (Han et al., 2010;Naidu et al., 2011) and squamous cell carcinoma of the head and neck (Lu et al., 2009). Therefore, it is highly necessary to perform a quantitative and systematic investigation with rigorous methods. To further evaluate the association between Pin1 polymorphisms (-842G>C, rs2233678 and -667C>T, rs2233679) and the risk of cancer, a metaanalysis was conducted on all eligible published studies in current study.

Identification and eligibility of relevant studies
The MEDLINE, EMBASE databases, Chinese National Knowledge Infrastructure (CNKI), Web of Science, and BIOSIS databases were used simultaneously using the following query: ('peptidylprolyl cis/trans isomerase, NIMA-interacting' or 'Pin1') and ('cancer' or 'tumor' or 'neoplasm' or 'malignancy' or 'carcinoma') and 'polymorphism' by two independent investigators (YM Zhu and JW Liu). Last search update was June 30, 2013. All published papers matching the eligible criteria were retrieved. Additional studies were identified by a manual search of references of original or review articles on this topic. Studies included in our meta-analysis have to meet the following criteria: (i) evaluated the relationship of the Pin1 polymorphisms Pin1-842G/C (rs2233678) or Pin1-667C/T (rs2233679) and cancer risk; (ii) in a casecontrol study design; (iii) contained available genotype frequency; (iv) excluded benign tumors, precancerous lesions. Major reasons for exclusion of studies were (i) only case population; (ii) the study did not have the outcomes of comparison reported or it was not possible to determine them; (iii) duplicate of previous publication.

Data extraction
Two of the authors (YM Zhu and JW Liu) extracted all data independently using a standardized extraction form and reached a consensus on all items. In the present study, the following information was extracted: first author, year of publication, country, ethnicity, cancer types, genotype frequencies for cases and controls, source of control groups (population-or hospital-based controls) and evidence of Hardy-Weinberg equilibrium (HWE). Meanwhile, we categorized larneal squamous cell carcinoma, nasopharyngeal carcinoma, esophageal carcinoma, squamous cell carcinoma of the head and neck into ' squamous cell carcinoma of the head and neck' for the stratified analysis.

Statistical analysis
Firstly, the strength of the association between the Pin1 polymorphisms (rs2233678 and (rs2233679) and cancer risk was measured by ORs with 95% confidence intervals (CIs). The statistical significance of the OR was determined using the Z test. Statistical heterogeneity between studies was assessed with the χ 2 -based Q test and Ι 2 (Higgins et al., 2003), heterogeneity was considered significant when P<0.05, and Ι 2 was used to qualify variation in OR attributable to heterogeneity. When heterogeneity was not an issue, fixed effect model with Mantel-Haenszel method was used (Ramsberg et al., 2012). Otherwise, a random effect model with Inverse variance method was used. Generally, we first evaluated the risks of the variant homozygote and heterozygote compared with the wild-type homozygote (CC vs GG and CG vs GG for rs2233678/TT vs CC and TC vs CC for 2233679). As to allele comparison, the risks of variant allele versus wildtype allele (C allele vs G allele for rs2233678/T allele vs C allele for 2233679) were estimated respectively. Then we evaluated the risks of the dominant and recessive effects of the variant allele (CC+CG vs GG and GG vs CG+GG for rs2233678/TT+TC vs CC and TT vs TC+CC for 2233679), respectively. In addition, we also performed stratification analyses on cancer type (divided into squamous cell carcinoma of the head and neck, lung cancer, breast cancer and hepatocellular carcinoma), ethnicity and source of control. We tested significance of deviation of genotype distribution at the polymorphic site from that expected from Hardy-Weinberg equilibrium (HWE) in the control sample for each of the selected case-control data sets. A χ 2 test was performed to examine HWE when genotype data was available. If HWE disequilibrium existed (P<0.05), or it was impossible to evaluate this equilibrium, sensitivity analysis was performed. Begg and Mazumdar (Begg et al., 1994) adjusted rank correlation test and the Egger regression asymmetry test (Egger, 1997) were utilized to provide a diagnosis of publication bias. All analyses were performed using Stata version 11.0 software (Stata,

Characteristics of studies
According to the searching strategy, 105 papers were found. We reviewed the titles, abstracts and the full texts of all retrieved articles through defined criteria. Finally, 9 studies including a total of 5427 cancer cases and 5469 controls were selected in our meta-analysis (Segat et al., 2007;Lu et al., 2009;Zhao, 2009;Han et al., 2010;Lu et al., 2011;Naidu et al., 2011;Cao, 2012;Lu et al., 2013;You et al., 2013) (Figure 1). The characteristics of the selected studies are listed in Table 1. The rs2233678 and rs2233679 polymorphisms were both investigated in 9 studies with the same cases and controls as mentioned above. The distribution of genotypes in the controls was consistent with the Hardy-Weinberg equilibrium for all selected studies, except for tow studies (Segat, et al., 2007;Zhao, 2009) for rs2233678 polymorphism.

Meta-analysis
The overall evaluation of the association between these two polymorphisms and cancer risk is presented in Table 2 Table 3, specific data for PIN1 -842G>C SNP was stratified, on the basis of sample size, into two subgroups: large sample (the total number of controls and cases not less than 500) and small-and-moderate sample (the total number of controls and cases less than 500) subgroups. In the overall rs2233679 polymorphism analysis, the

Sensitivity analyses and publication bias
Every one single study involved in the meta-analysis was deleted each time to reflect the influence of the individual data set to the pooled ORs. This procedure did not change the pooled ORs supporting the robustness of our findings. Furthermore, when two studies that did not reach HWE in controls were excluded, the results were in agreement with the findings from foregoing analysis for all populations. Begg's and Egger's test were conducted to evaluate publication bias. These different test methods have come to the same conclusion. Both of them revealed statistical significance for publication bias in homozygous comparison models for -842G>C (rs2233678) and -667C>T (rs2233679). The results were shown in Table  5.

Discussion
It is well known that individual susceptibility plays important role in the development of most cancers. Polymorphisms of genes involved in carcinogenesis may have accounted for the susceptibility. Therefore, genetic susceptibility, especially single nucleotide polymorphism (SNP), to cancer has been a research focus in scientific community.
Many studies have been done to figure out the impact of Pin1 promoter SNPs on multiple types of cancer. The most intensively concerned ones are -842G>C (rs2233678) and -667C>T (rs2233679). However, the existing data were contradictory. To better understanding of the association between these polymorphisms and cancer risk, a metaanalysis with larger sample and subgroup analysis is necessary. The current study is the first meta-analysis associating Pin1 two promoter polymorphisms (-842G>C [rs2233678] and -667C>T [rs2233679]) with cancer risk.
Our study showed that -842C is a protect factor for cancer risk in total analysis. Considering the number of studies included in this article, we performed the stratified analyses by cancer types from different organs, by ethnicity and by control selection bias. Our results suggested that -842C tends to be a protective factor on squamous cell carcinoma of the head and neck, lung cancer and breast cancer. Contrary to other cancer types, in one hepatocellular carcinoma study, the variant -842C allele is associated with increased cancer risk, contrary to others. The reason may be that the -842G>C (rs2233678) polymorphism may have different effect on carcinogenesis in different organs, reflecting the diversities of the susceptible factors for different tumor types. In addition, this observed different effect could be likely due to chance because it is a small sample size with only 228 cases and 250 controls may have generated a fluctuated risk estimate or may have insufficient statistical power to detect a slight effect. So studies with larger sample size in hepatocellular carcinoma are necessary to fully understand the relationship between the polymorphism and the risk of hepatocellular carcinoma. -842C is also a protect factor both in Asian and Caucasian. When stratifying the source of control, significant associations were observed in hospital-based and population-based controls. This may result from most of the included studies matching age, sex and residential area to control selection bias. In stratified analysis by sample size, -842C is a protect factor in large sample size subgroup but not in small-and-moderate subgroup, which may due to insufficient statistical power for small size study.
Previous functional analyses of the PIN1 -842G>C polymorphism found that -842C variant allele had a lower transcription activity in luciferase assay which indicated that PIN1 gene expression driven by the variant -842C allele was much lower than those driven by the -842G allele (Lu et al., 2009;Lu et al., 2011). Furthermore, the deficiency in binding of nuclear protein by the -842C allele probe was also observed compared to the -842G allele probe in electrophoretic mobility shift assay (EMSA). All of above indicated that -842 C variant genotypes might decreased PIN1 protein expression, lead to reduced oncogenic phosphorylation signals and thus reduce the cancer risk.
Our meta-analysis results showed that -667C>T polymorphism decreased cancer risk in total analysis. When stratified analysis by cancer type, no association was found in any cancer type. In the stratified analyses by ethnicity, significantly decreased cancer risks were found for -667C>T among Asians but not in Caucasians. Different ethnicities may have different genetic backgrounds, which influence the association between polymorphism and cancer susceptibility. Inconsistency between the two ethnicities can be explained by the possibility that different ethnic groups live with multiple life styles and environmental factors and thus yield diverse geneenvironment interactions (Molina et al., 2009;Dick, 2011;Carpenter et al., 2013). And different populations carry different genotype and/or allele frequencies of this locus polymorphism: the rs2233679 C allele among controls between Asian (0.445) and Caucasian (0.662) is highly significantly different, and it may lead to various degrees of cancer susceptibility (Gao et al., 2010;De et al., 2012;Euhus et al., 2013). When stratified analysis by sample size, the results was inconsistent between two subgroups. In large sample subgroup, -667T allele is a protect factor, which is opposite to that in small-and-moderate sample subgroup. This difference may be caused by the small sample size. There are only 3 studies in small-andmoderate sample subgroup, the total case number is 501 and control number is 506. Compared with large sample size, small sample size may have limited statistical power to result a real risk estimate. Furthermore, functional study should be performed to validate these results. Despite our efforts in performing a comprehensive analysis, some limitations exist in our meta-analysis. First, we pooled the data using unadjusted information, whereas a more precise analysis could to be conducted if detailed information of original data is available. Second, a lack of original data of the reviewed studies limited our further evaluation of potential interactions, including the interactions between different genes and between gene and environment factors. Third, only English and Chinese documents were included in this meta analysis, while reports that were written in other languages and other unpublished data or ongoing studies were not available, which may cause certain publication bias in our metaanalysis. The last but not the least, the pooled sample size was relatively limited in this meta-analysis. Therefore, this meta-analysis could only preliminarily appraise the association of rs2233678, rs2233679 polymorphisms with currently-reported cancers. More studies are still required to get a more reliable result.
In conclusion, our meta-analysis suggested that the -842G>C (rs2233678) polymorphism may contribute to genetic susceptibility for overall cancers risks especially in squamous cell carcinoma of the head and neck, lung cancer and breast cancer, as well as in Asian and Caucasian. The -667C>T (rs2233679) polymorphism might be associated with genetic susceptibility for overall cancers risks especially in Asian. Future well-designed and larger population studies are of great value to confirm these findings. Moreover, combination of genetic factors together with environmental exposures should also be considered.