Background
Breast cancer is one of the most common malignant tumors contributing to the high mortality of females worldwide. The etiology of breast cancer is a complex combination of both environmental and genetic factors, so the determination of genetic polymorphism provided a new way to investigate the etiology of such complex genetic disease. Accordingly, significant associations have been demonstrated on some gene polymorphisms with breast cancer risk [
1]. So far, accumulating evidence convincingly emphasizes that the host immune system is involved in the regulation of cancer development and progression. T lymphocyte, whose function is central to the adaptive immune response, plays a critical role in immune surveillance of cancer cells [
2,
3]. Therefore, the molecules, in particular the costimulatory ones, mediating regulation of T-cell activity may influence cancer susceptibility [
4].
Inducible costimulator (
ICOS) molecule is a member of the
CD28 family that generates indispensable secondary signals to determine the activation and development of the immune response.
The gene encoding
ICOS is located on chromosome 2q33, which contains
CD28 and
CTLA-4, another two numbers of
CD28 family. Not expressed by naive Th cells,
ICOS is induced following T cell activation [
5]. Interaction between
ICOS and its ligand (
ICOS-L;
CD275), a molecule highly expressed on B cells and dendritic cells, provides costimulatory signal to induce T cell proliferation, secretion of various cytokines and up-regulation of cell surface molecules [
6]. Blocking of
ICOS with Abs or an ICOS-Ig fusion protein results in the inhibition of immune responses mediated by Th1 and Th2 [
7,
8]. Furthermore, a recent research revealed that impaired function of CD4
+ and CD8
+ T cells were observed in
ICOS-deficient patients [
9].
Although the polymorphisms in
ICOS gene have been extensively studied in various diseases, including cancers [
10,
11], the association between
ICOS gene polymorphisms and the risk of breast cancer remains unclear. To determine the key roles of
ICOS in tumor immunity, we genotyped five potentially functional SNPs, including rs11889031, rs10932029 (IVS1+173), rs4675374, rs10183087 (c.602) and 10932037 (c.1624), and investigate their associations with both the risk and clinicopathologic features of breast cancer in Chinese women from Heilongjiang Province, northeast of China.
Discussion
The etiology of breast cancer is complicated, in which the genetic factor plays an important role. Although high-penetrant susceptibility genes such as
BRCA1 and
BRCA2 demonstrate potent associations with the familiar breast cancer, many low-penetrant susceptibility genes predisposing to breast cancer remain to be elucidated. Previous studies showed that SNPs of
Cytotoxic T lymphocyte antigen -4 (
CTLA-4) and
B and T lymphocyte attenuator (
BTLA) might confer susceptibility to breast cancer, which suggests the critical role of costimulatory molecules in the development of breast cancer [
12,
13]. In addition,
CTLA-4 and
ICOS are located within a stretch of 100 kb on chromosome 2q33. Treated with CTLA-4 blockade, ICOS expressed higher on CD4
+ T cells from peripheral blood and tumor tissues of bladder cancer patients and this CD4
+ICOS
hi T cell population produced higher IFN-γ, indicating that ICOS interacts with CTLA-4 and plays an important role in tumor immunity [
14,
15]. Thus, we predicted that
ICOS gene polymorphisms might also be related to the risk of breast cancer. In this study, we analyzed five potentially functional SNPs in
ICOS gene, including rs11889031C/T in promotor, rs10932029 C/T and rs4675374C/T in the intron1, rs10183087A/C and rs10932037 C/T in the 3'-untranslating region (UTR), and determine their associations with breast cancer. Here, we first report that some of the alleles, genotypes and haplotypes of the above SNPs are associated with the risk and clinicopathological features of breast cancer.
This case-control study on the variants in the
ICOS gene revealed that only rs10932029 may confer susceptibility to breast cancer. The association between rs10932029 C/T and breast cancer risk found in the study cohort was also observed in the validation cohort. Patients carrying rs10932029 CT genotype and C allele had an increased risk of breast cancer, which indicates that CT genotype and C allele may play risk roles in breast cancer. Rs10932029 C/T and rs4675374 C/T are both located in intron1 of
ICOS. Introns are important for mRNA processing and transporting. Many studies have demonstrated the presence of regulatory elements and splicing control elements in mammalian introns, particularly in the first intron. Moreover, mutations occurring in introns can induce the aberrant splicing due to the disruption of splicing enhancers and alteration of the pre-mRNA, as a result, impair the translation efficiency [
16‐
18]. Rs10932029 is a well studied variant in some diseases. Interestingly, one study demonstrated that this SNP could influence the expression of
CTLA4 isoforms [
19], but later study could not confirm this conclusion [
20]. Although a correlation between rs10932029 and breast cancer risk was ascertained in our study, the mechanism underlying this result is not immediately evident. The potential explanation might be concluded as follows. Firstly, this polymorphism might regulate
ICOS mRNA processing and translation. Secondly, CT genotype and C allele might down-regulate the ICOS expression and then increase the breast cancer risk. However, rs4675374 located in the same intron did not show any association with the risk of breast cancer.
Allelic variants located in the promoter region may change the motif of functional DNA binding sites and thereby affect their affinities for the relevant transcription factors. Notably, with a location in the promoter region, rs11889031 is situated in the NF-κB binding site [
21]. However, although this potentially functional variant may affect the function of the gene, our data did not show any association between this SNP and the risk of breast cancer.
Rs10183087 A/C and rs10932037 C/T are both located in the 3'-UTR of the
ICOS. Multiple regulatory elements in 3'-UTR can affect mRNA stability and degradation as well as nuclear export [
22]. Using the PupaSuite software [
23], rs10183087 is speculated to be located in the exonic splicing enhancers(ESE), so it may influence mRNA splicing, eventually, affect protein function. One previous study showed that rs10183087 was associated with delayed graft function [
24], but no relation was found in malignant melanoma susceptibility [
11]. Our study also did not show any statistical significant association between this SNP and the risk of breast cancer. Therefore, whether this SNP affect
ICOS function needs to be further studied. Additionally, rs10932037A/C was located in a MicroRNA-binding site. Altering the strength of miRNA binding, SNPs in 3'UTR targeted by miRNAs were associated with the risk of breast cancer [
25,
26]. In line with this hypothesis, one study had verified that rs10932037 can regulate the expression of
ICOS mRNA [
19]. However, we failed to find the association between rs10932037 and the susceptibility to breast cancer. The correlation between rs10932037 and genetic susceptibility to a variety of diseases has been widely studied, but the results were inconsistent. Rs10932037 was proposed to be associated with kidney graft survival and the outcome of hematopoietic stem cell transplantation (HSCT) [
24,
27], but another study about gastric mucosa-associated lymphoid tissue lymphoma did not show any significant association [
10]. Considering the minor allele frequency of rs10932037 was 2.53% in controls, further study using a large sample size is needed to confirm this association.
We further analyzed the association between haplotypes and the risk of breast cancer. We found that CTCAC haplotype containing rs10932029 T allele appeared to be protective against breast cancer, whereas CCCAC haplotype containing rs10932029 C allele may be a risk factor in breast cancer. This result also highlighted the important role for rs10932029.
In addition, we found that ICOS gene polymorphisms were associated with clinicopathologic features of breast cancer patients. For rs10932029, a correlation was found between it and C-erbB-2 status in the study cohort, but no relation was found in the validation cohort. As it is known, the over-expression of C-erbB-2 can lead to insensitive endocrine treatment, the recurrence and metastasis of tumor, resulting in poor prognosis and overall survival [
28,
29]. Nevertheless, in consideration of the converse results in two independent cohorts, whether rs10932029 was associated with C-erbB-2 status needs to be further investigated. We also found that rs11889031 was associated with PR status and lymph node metastasis in both study and validation cohorts. The expression of ER or PR has long been regarded as predictive markers of breast cancer endocrine therapy [
30,
31]. Moreover, cases of positive lymph node metastasis have higher mortality [
32]. So our data indicate that genotypes and alleles in rs11889031 may be important in forecasting the prognosis of breast cancer.
In the analysis of the association between haplotypes and clinicopathlogical features, we observed that CTTAC and CTCCC haplotypes were associated with ER expression, and CTTAC haplotype showed a correlation with the status of PR. Therefore, these two haplotypes may provide valuable prognostic information for the survival of breast cancer patients.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FYX designed the primers and wrote the drafts. DLL collected patients and controls blood samples. QJZ, ZKF, WGY and SC performed the PCR-RFLP experiments. JZ contributed to statistical analysis. DP and DJL conceived of the study, and participated in its design and coordination and helped draft the manuscript. All authors read and approved the final manuscript.