Association of CTLA-4 gene 49 G/A polymorphism in breast cancer patients with invasive ductal carcinoma

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1 International Journal of advances in health sciences (IJHS) ISSN Vol2, Issue1, 2015, pp Research Article Association of CTLA-4 gene 49 G/A polymorphism in breast cancer patients with invasive ductal carcinoma 1 Sushma Chinnapaka, 2 Sudha Murthy, 3 Shiva Prasad, 4 C Naidu, 5 K Rudrama Devi and 4 T Subramanyeshwar Rao 1 Department of Genetics, Osmania University, Hyderabad, Telangana, India 2 Department of Lab Medicine, Hyderabad, India 3 Asian Institute Of Gastroenterology, Hyderabad, Telangana, India 4 Basavatarakam Indo American Cancer Hospital and Research Institute, Hyderabad, Telangana, India 5 Department of Zoology, University College of Science, Osmania University, Hyderabad, Telangana, India Corresponding Author: rudramadevi.genetics2011@gmail.com [Received-25/01/2015, Accepted-02/02/2015] ABSTRACT Background: Weak immunity surveillance has been the main reason for progression and rapid development of various cancers reported. Though the association between cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene 49 G/A polymorphism and breast cancer has been widely assessed, a definitive conclusion remains elusive and still focused by scientific community. The present study aims to evaluate the potential influences of CTLA-4 gene polymorphisms on breast cancer risk in invasive ductal carcinoma of tumor and healthy s. Results: Tumor of forty confirmed breast cancer patients were included as cases. The same forty patients adjacent non-tumor samples were considered as controls. The prevalence of AA, GG and AG genotypes was %, % and 42.8% in the tumor and non-tumor s respectively. Individuals containing tumor and non-tumor samples showed significance in breast cancer, in which homozygous A/A and heterozygote G/A found to be more significant than G/G genotype. Conclusion: The study confirmed that the presence of at least one A allele may increase the risk of breast cancer when compared to the presence of G allele in same patient. Key words: Invasive Ductal carcinoma, CTL4, Breast Cancer INTRODUCTION It is estimated that about 12.7 million multiple cancer cases and 7.6 million cancer deaths have occurred in 2008 worldwide, with more than half of the cases and about two-thirds of the deaths in the developing countries [1]. Out of 13 known solid tumors, breast cancer is the most common

2 malignancy seen in women worldwide and its rate is increasing in both developed and developing countries resulting in higher female morbidity. The exact etiology of breast cancer disease is not known. Genetic predisposition alone may not be responsible for causing breast cancer but a combination of susceptible g e n e s and exposure t o environmental factors can contribute to the development of b o t h nonfamilial and sporadic cancers. Other reason as mentioned earlier for rapid outcome of cancer is the weak immune surveillance [2]. Genetic alterations which up regulate the immune system activity will decrease the risk of developing cancer, but at the same time, increase the risk of developing certain autoimmune diseases [3]. Conversely genetic alterations which down regulate the immune system activity increase cancer susceptibility but decrease the risk of developing autoimmune diseases. Experimental evidence of Dunn and Aggarwal [4,5] findings in the field of tumor immunology demonstrates the interactions between immune system and tumor cells. It has become clear that the immune system can facilitate tumor progression through three phases: elimination, equilibrium, and escape. Both innate and adaptive responses are well coordinated through soluble and membrane bound regulators in the progression of breast cancer carcinoma leading to the disposal of the most suitable effectors for controlling the growth of tumor. Yet the biological significance of these responses is uncertain. T lymphocytes and natural killer (NK) cells are believed to be significant antitumor response in cell-mediated immunity. Hence variants of these genes that regulate the activation and proliferation of T lymphocytes and NK cells may affect the risk of breast cancer. In recent years Cytotoxic T-lymphocyte Antigen-4 (CTLA-4) gene has been in research focus, and several epidemiological studies attempted to assess the possible interaction between the CTLA- 4 gene polymorphism and cancer susceptibility, including breast cancer, cervical cancer, lung cancer, glioma etc. CTLA-4 is a member of the superfamily of immunoglobulin that are mainly expressed by activated T cells. This antigen is encoded by a gene on chromosome 2q33 and is established that blockade of CTLA-4 receptors lead to the enhancement of immune response. CTLA-4 is a negative regulator of T-cell proliferation and activation, numerous studies revealed that it plays an important role in cancer immune surveillance and may also be involved in tumor development and progression. In the later year Vandenborre reported that blocking TLA-4 function and enhancing T cell activation has resulted in having several different types of malignant neoplasms in tumor-transplanted mice inhibited or cured, and owned long-lasting antitumor immunity. This clearly suggests that CTLA-4 plays an important role in carcinogenesis. Similar results were found by Egen [6] in tumor where CTLA-4 is up regulated on the T cells with the help of TGF-b (a suppressive cytokine secreted by the tumor cells), and during the early stage of tumorigenesis, CTLA-4 may elevate the T-cell activation threshold, thereby attenuating the antitumor response and increasing cancer susceptibility. It is a receptor expressed by activated T lymphocytes. It interacts with the B-7 cell surface molecule on antigen-presenting cells and inhibits T cell activation and clonal expansion. Recent report [7] showed that mice deficient of CTLA-4 gene were born healthy but died early due to severe lymphoproliferative disorders and autoimmune diseases. Of late, a number of studies stated that genetic variants of the genes that regulate the activation and proliferation of T lymphocytes and nature killer (NK) cells may influence cancer risk [8,9]. CTLA-4 gene composed of four exons that encode several functional domains of the CTLA-4 protein and possess several vital SNPs, more than 100 singlenucleotide polymorphisms have been identified in the CTLA-4 gene region. But important SNP s are +49A/G (rs231775), -318C/T (rs ), CT60G/A (rs ), 1661A/G (rs ), and K Rudrama Devi, et al. 45

3 -1722T/C (rs733618) SNPs, etc [10, 11]. The role of these polymorphisms on different types of cancers has been evaluated and validated, telling that different polymorphisms of CTLA-4 are associated with susceptible to various cancers. However, the results of the different studies are conflicting. Hadinia [12]. reported that, there is no significant association between CTLA A/G polymorphism and colorectal cancer but, Hou [13] found that CTLA A/ G is associated with significantly increased risk of gastric cancer. Thus, the association between CTLA-4 gene polymorphisms and cancer susceptibility requires considerable investigation. Similarly, conflicting results were found with other variant +49 G/A. Among all the CTLA-4 genetic variants, +49 G/A polymorphism is the only genetic variation in CTLA-4 gene that causes an amino acid change in the resulting protein. It causes a 17Thr to 17Ala substitution in the leading peptide of CTLA-4 receptor. The role of this protein in increasing cancer susceptibility is well known and affirmed in various populations across the world, especially Asians. Conventional collective wisdom through research in all these years is questioned by Sachin Minhas,2014 [14]. Hence, the present paper investigated the association of CTLA-4 gene +49 G/A variant polymorphism in breast cancer. Materials and Methods The Project was approved by the Institutional Ethics Committee of Basavatarakam Indo American Cancer Institute and Research Centre, (Hyderabad) in accordance with the Helsinki Declaration. Enrolment of study subjects: In this study a total of 40 patients, diagnosed with breast cancer (pathologically, histologically/cytologically) between June 2009 to November 2012 were enrolled. The disease was determined clinically through FNAC, malignant cytology by Pathologists. With the informed consent from the subjects, tumor sample and non-tumor adjacent sample were collected. Only those confirmed with invasive ductal carcinoma were signed up for this study. Genotyping of CTLA-4 gene +49 G/A polymorphism Genomic DNA was isolated from blood samples of lung cancer cases and controls by salting out method. A polymerase chain reaction followed by restriction fragment length polymorphism (PCR- RFLP) assay was used to detect dimorphism of +49 G/A. The polymorphic region was amplified by PCR in a 25 µl reaction solution containing 0.5 µg genomic DNA, 2.5 µl of 10X NEB buffer, 1.5 µl of 25 mm MgCl2,0.5 µl 10 mm dntps, 1 U Taq DNA polymerase (Bioserve, Hyderabad, India) and 1 µl of 10 pm/ µl of each primer (Bioserve, Hyderabad, India), 12.3 µl of PCR water and 5.0 µl of enhancer were used. Presence of 311 bp PCR product confirmed the standardization of gene for PCR. PCR product was subjected to restriction digestion with Bbv I (NEB, cat no: R0173S). Since the G to A transition in promoter region of CTLA-4 abolishes the recognition site for Bbv I enzyme, the G/G genotype yielded digested bands of bp, A/A resulted a single fragment of 311 bp and 311 and bp A/G genotype with three fragments. Statistical analysis Genotype frequencies for CTLA -4 polymorphisms were found to be in Hardy- Weinberg equilibrium (HWE). HWE was tested for cases and controls separately. The allele and genotype frequencies were obtained by direct counting. The p values of <0.05 were considered statistically significant. MEDCALC software was used to estimate frequencies in the tumor and healthy. Comparisons of the distributions of the allele, genotype and haplotype frequencies were performed using the chi-square test. The relative risk associated with alleles was estimated as an odds ratio (OR) with a 95% confidence interval (CI). K Rudrama Devi, et al. 46

4 RESULTS Table 1: Genotype frequencies of CTLA-4 polymorphisms in breast cancer s and healthy Genotype Control (n=38) Tumor Tissue (n=38) p value A/A 16 (42. 1%) 16 (42. 1%) 1.0 G/G 06 (15.8%) 06 (15.8%) 1.0 G/A 16 (42.1%) 16 (42.1%) 1.0 Genotype distribution results of CTLA-4 gene (49A/G) are presented in Table 1. To enhance statistical power of the genotypes for detection of their association with the risk of the disease, we compared the three genotypes (AA, GG and AG) in tumor and non-tumor of same patient. The prevalence of AA, GG and AG genotypes at position 49 of exon-1 by PCR-RFLP method was found to be %,15.8.2% and 42.8% in the tumor and non-tumor s respectively. Table 2: Allelic Frequencies of CTLA-4 polymorphisms in breast cancer s and healthy Group A Frequency G Frequency Control Tumor Total p value >0.05 Statistical analysis using odds ratio showed no statistically significant difference between the tumor and non-tumor groups (P>0.05). There is no significant relationship between the genotypes of these two sampling s i.e., tumour and non-tumour s and the presence of breast cancer (Table 1). There was also no significant difference between the prevalence of any allele, either A allele or G allele at this position in both cases (tumour and nontumour) due to the similar results (table 2). Table 3: Association of CTL G/A gene polymorphism and breast cancer risk Control Chi- Contingency Tumor Square Coefficient A/A G/G G/A A/A G/G G/A P Value <0.05 But when we have analyzed the individual genotypes (tumour and non-tumour samples) by the Cochran-Armitage test, it showed significance in breast cancer samples, where presence of homozygous A/A and G/A showed significance with G/G genotype (table 3). We found that presence of at least one A allele may increase the risk of breast cancer when compared with the presence of G allele in same patient sample (p<0.05) DISCUSSION: The +49 G/A polymorphism at position 49 in CTLA-4 exon 1 (rs231775) has been reported, which causes an amino acid change (threonine to alanine) in the peptide leader sequence of the CTLA-4 protein [15]. Previous studies have found that the G allele has lower mrna efficiency and decreased CTLA-4 protein production than the A allele [16]. Since CTLA-4 downregulates T-cell production, individuals with GG genotype (less CTLA-4 production) have higher T-cell proliferation than those with the AA genotype [17]. Other studies [11,18] found that G/A polymorphism may influence the ability of CTLA- 4 to bind with B7.1 and subsequently, may affect T-cell activation. Therefore CTLA-4 inhibits excessive expansion of activated CD28+ T-cells, serving as a negative regulator or feedback inhibitor of the clonal expansion process. There K Rudrama Devi, et al. 47

5 are accumulating data suggesting that CTLA-4 deficiency will induce or exacerbates autoimmunity, enhances tumor-immunity or prevents induction of immunologic tolerance [10]. Due to this reason, the risk allele for cancer susceptibility (allele A) is the opposite of that allele found for susceptibility to autoimmune diseases (allele G) [19]. Therefore, it can be postulated that factors such as different genetic polymorphisms that lead to suppression or decreased function of T-cells can play a role in tumor genesis in breast cancer. The present study found that the presence of A/A or presence of atleast one allele of A genotype may increase the risk of breast cancer when compared with G/G genotype in tumor. Similar results were reported [11,18] in Chines [20] and in Iranian breast cancer patients. Sun et al. reported the associationn of cancer of oesophagus with the lung among the Chinese [11] and some of the urinary system malignancies such as RCC is well established [21]. An association with gastric cancer was found by Hou [22]. Metaanalysis [23] also reported evidence for an association between CTLA-4 +49A/G polymorphisms and multiple cancers in the general population, particularly solid tumors. A recent study [15] stating a negative association between North Indian breast cancer patients and CTLA-4 +49A/G polymorphisms may be due to variation in sample selection i.e. healthy subjects as controls for comparison unlike the existent study which considered the adjacent non-tumor of the same patient. Other reports are also inconclusive in other cancers like colorectal neoplasm among Italian Caucasians [24] found to be not associated. This is due to ethnic disparities, which exist with respect to the prevalence of a polymorphism in a particular ethnic population and its association with a disease. For example, CTLA G/A polymorphism is known to be associated with the risk of colorectal cancer in Chinese but no such association was seen in a study on Turkish patients [25,26]. Also a metaanalysis report of CTLA4 +49A/G polymorphism showed an association with an increased risk of Hashimoto s thyroiditis in Asian but not Caucasian populations [27]. By virtue of unique genetic make-up, one particular ethnic population may not hold the same for some other population of different ethnic background [28, 29]. The reported discrepancies among studies can be due to population specific differences as well as sample size and multiple subgroup analysis The efficacy of the current study lies in including both tumor and adjacent non-tumor from the same breast cancer confirmed patient which endowed a direct evaluation of the +49 G/A polymorphism of CTLA-4 gene. Thus further studies should involve more number of subjects for the better evaluation of significance of the +49 G/A polymorphism in breast cancer assessment. CONCLUSION From the results, it is clear that +49 G/A polymorphism of CTLA-4 gene may play a role in the etiology of breast cancer. Further studies are required to confirm these findings. Competing interests: None REFERENCES: 1. Jemal A, Bray F, Center MM, et al: Global cancer statistics. CA Cancer J Clin 2011, 61: Reiman JM, Kmieciak M, Manjili MH, et al: Tumor immunoediting and immunosculpting pathways to cancer progression. Semin Cancer Biol 2007, 17: Ghaderi A: CTLA4 gene variants in autoimmunity and cancer: a comparative review. Iran J Immunol 2011, 8: Dunn GP, Old LJ, Schreiber RD: The three Es of cancer immunoediting. Annu Rev Immunol 2004, 22: K Rudrama Devi, et al. 48

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