Functional Polymorphisms of FAS and FASL Gene and Risk of Breast Cancer – Pilot Study of 134 Cases

Fas/Fas ligand (FasL) system is one of the key apoptotic signaling entities in the extrinsic apoptotic pathway. De-regulation of this pathway, i.e. by mutations may prevent the immune system from the removal of newly-formed tumor cells, and thus lead to tumor formation. The present study investigated the association between −1377 G/A (rs2234767) and −670 A/G (rs1800682) polymorphisms in Fas as well as single nucleotide polymorphisms INV2nt −124 A/G (rs5030772) and −844 C/T (rs763110) in FasL in a sample of Iranian patients with breast cancer. This case-control study was done on 134 breast cancer patients and 152 normal women. Genomic DNA was extracted from whole blood samples. The polymorphisms were determined by using tetra-ARMS-PCR method. There was no significant difference in the genotype distribution of FAS rs2234767 polymorphism between cases and controls. FAS rs1800682, FASL rs5030772, and FASL rs763110 genotypes showed significant associations with an increasing risk of breast cancer (odds ratio OR = 3.18, P = 0.019; OR = 5.08, P = 0.012; OR = 2.40, P = 0.024, respectively). In conclusion, FAS rs2234767 was not associated with breast cancer risk. Though, FAS rs1800682, FASL rs5030772, and FASL rs763110 polymorphisms were associated with the risk of breast cancer in the examined population.


Introduction
Breast cancer is the main cause of cancer-related death among women in western countries, with the sporadic form of the disease constituting more than 90% of all breast cancers [1,2]. Breast cancer is also one of the most frequent malignancies among Iranian women [3,4]. In addition to environmental factors and body weight, genetic invariability affects not only the chance of cancer development but also its aggressiveness, therapy response and overall prognosis [5,6,7]. Apoptosis is one of physiological forms of cell death that plays an important role in maintaining tissue homeostasis. Abnormal regulation of apoptosis contributes to the pathogenesis of cancer [8,9]. The acquired capability to oppose apoptotic stimuli is one of the main characteristics of a malignant cell. Alterations of apoptotic pathways are key events in the development of variety of human diseases including cancer [10,11].
The FAS/FASL signaling system activates a major extrinsic cell death pathway, that is of particular importance for the regulation of acquired immune response, maintaining immuno-privileged sites and fulfils other regulatory functions [12,13]. Fas (TNFRSF6/CD95/APO-1) is a cell surface receptor, expressed in a variety of tissues [14]. FasL, also known as TNFSF6 or CD95L, is a member of the tumor necrosis factor superfamily and the natural ligand to Fas [15]. The Fas interaction with the FasL may trigger the death signal cascade, and eventually the cell expressing FAS would die. Studies have shown that decreased expression of Fas and/or elevated expression of FasL could be detected in many kinds of human tumors [16,17].
The FAS/FASL system may have two opposite effects on cancer. The expression of FAS on tumor cells, if downstream signaling pathways are functional, may assist FAS-triggered killing of tumors by immune-effector cells [15,18]. The expression of FASL on tumor cells may repel specific antitumor immune response, thus turning tumor into an immuno-privileged site [19,20]. The role anticancer-drug-induced FAS/FASL system in tumor-cell killing has been championed by some [21], it is currently however often disregarded as experimental artifact [22,23].
Two polymorphisms have been identified in the FAS promoter region: one in the silencer region, G to A substitution at nucleotide position 21377 (rs2234767), and the other in the enhancer region, A to G substitution at nucleotide position 2670 (rs1800682). These two polymorphisms are located within the stimulatory protein-1 (Sp1) and the signal transducers and activators of transcription 1 (STAT1) transcription factor binding sites, respectively [24,25]. Because these sequence variations in the FAS gene promoter region may influence Fas expression and deregulate cell death signaling, they could contribute to carcinogenesis [25]. The human FASL gene is located on chromosome 1q23, consists of four exons spanning ,8 kb, and encodes 281amino acids [26]. There are two reported polymorphisms: C to T changes at nucleotide position 2844 (FASL-844 C/T, rs763110) in the promoter region [27] and A to G change at nucleotide position 2124 of intron 2 (FASL INV2nt 2124 A/G, rs5030772). FASL 2844 C/T is located in a putative binding motif for a transcription factor, CAAT/enhancer-binding protein h, and the 2844 C allele may increase basal expression of FASL compared with the 2844 T allele [27], suggesting that the FASL 2844 C/T polymorphism may influence FASL expression and FasL mediated signaling, and ultimately, the susceptibility to cancer. So far, the functional relevance of the FASL IVS2nt 2124 A/G polymorphism has not been reported.
Numerous studies investigated the role of FAS and FASL gene polymorphisms in the etiology of various cancers including cervix, breast, bladder, lung, prostate, head and neck and esophagus [25,28,29]. However, the role of FAS and FASL gene polymor-phisms in breast cancer has not been conclusively established. Thus, the present study, that assesses the association between 21377 G/A and 2670 A/G polymorphisms in FAS as well as single nucleotide polymorphisms 2844 C/T and INV2nt 2124 A/G in FASL adds an important information about the role of this death pathway in breast cancer.

Cancer patients and controls
This case control study was performed in 134 patients with histologically confirmed breast cancer and in 152 population based healthy women who participate in a screening project for metabolic syndrome with no history of cancer. The local Ethical Committee of Zahedan University of Medical Sciences approved the study protocol and written informed consent was obtained from all subjects. Two ml of venous blood drawn from each subject and genomic DNA was extracted from peripheral blood as described previously [30].

T-ARMS-PCR assay
Tetra-amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) is a rapid and simple technique for detection of single nucleotide polymorphism [30,31,32]. In the present study we designed T-ARMS-PCR assay for the detection FAS and FASL polymorphisms. The primers used are listed in Table 1. Two allele-specific (inner) primers have been designed in opposed directions and, in combination with the common primers, can simultaneously amplify both the wild-type and the mutant alleles in a single-tube PCR (see result section for schematic depiction). PCR was performed using commercially available PCR premix (AccuPower PCR PreMix, BIONEER, Daejeon, Korea) according to the manufacturer's instructions. For detection of polymorphisms of FAS 2670 A/G (rs1800682) and 21377 A/G (rs2234767), 1 mL template DNA (,100 ng/mL), 1 mL of each primer (10 pmol/mL), and 15 mL DNase-free water were added into a 20 mL PCR tube containing the AccuPower PCR PreMix. The same PCR condition was used for detection of FASL (rs763110), except that 0.6 mL of each primer and 16.6 mL water were added. For FASL (rs5030772), 1 mL template DNA, 1 mL of outer primer, 0.5 mL of inner primer, 0.5 mL of DMSO and 15.5 mL was added.
PCR cycling was performed at 95uC for 5 min, followed by 30 cycles of a denaturation for 30 s at 95uC, annealing for 30 s at different temperatures (Table 1), extension s at 72uC (30 s for rs1800682 and rs2234767; 40 s for rs5030772 and rs763110) and a final extension for 10 min at 72uC in a thermocycler (Corbett research, Australia). The amplified products were separated by electrophoresis on a 2% agarose gel containing 0.5 mg/ml ethidium bromide.
The genotyping analysis was randomly repeated for 10% of samples for confirmation for quality control and the results were 100% concordant. To confirm the genotyping results, selected PCR-amplified DNA samples (n = 3, respectively, for each genotype) were examined by DNA sequencing and the results determined by T-ARMS-PCR were concordant with those determined by sequencing (see the result section for more details).

Real-time RT-PCR (qRT-PCR) assay
We investigated the expression levels of FAS in 10 tumor samples and 16 adjacent normal tissue breast normal tissues. Briefly, total RNA was extracted from the tissues using TRIzol reagent. Then complementary DNA (cDNA) synthesis from total RNA was catalyzed in a final volume of 25 mL by cDNA synthesis kit (Bioneer, K-2045, Korea) according to the manufacturer's protocol. Expressions for the Fas and the housekeeping GAPDH genes were investigated using real-time quantitative PCR (qRT-PCR) (Applied Biosystems). FAS (F: 59-TGAAGGACATGGCT-TAGAAGTG-39, R: 59-GGTGCAAGGGTCACAGTGTT-39) and GAPDH primers (F: 59-GAAGGTGAAGGTCGGAGTC-39, R: 59-GAAGATGGTGATGGGATTTC-39) were used [33]. Samples were assayed in a 25 mL reaction mixture including 2 mL of cDNA, 12.5 mL of 26 Master Mix (Fermentas, Cat No. K0221), 1 mL of each specific primer (10 mM), and 8.5 mL of RNase-free water. qRT-PCR reactions were performed with an initial incubation at 95uC for 10 minutes followed by 40 cycles at 95uC for 15 seconds and 60uC for 60 seconds.

Statistical analysis
The statistical analysis of the data was performed using the SPSS 18.0 software. Genotypes and alleles were compared between groups by use of x2 test. The strength of the association between the polymorphisms and cancer risk was measured by odds ratios (ORs) with 95% confidence intervals (CIs). Binary logistic regression was used for all analysis variables to estimate risk as odds ratio (OR) with 95% confidence intervals (CIs) using age as covariate. According to our findings, sample power was calculated for FAS and FASL polymorphisms by comparison of each genotype with the sum of other related genotypes ( Table 2) at each polymorphic region by using STATA 10 software and is shown in table 3.

Results
The study group consists of 134 histopathologically confirmed female cases with breast cancer (age; 47.97613.27 years), and 152 healthy females (age; 43.53613.52 years). The clinicopathologic characteristics of patients are summarized in Table 2. The present study examined the association between 21377 G/A and 2670 A/G polymorphisms in FAS as well as single nucleotide polymorphisms 2844 C/T and INV2nt 2124 A/G in FASL (Fig. 1). Should any of the studied polymorphisms be significantly associated with ant of the type of breast cancer, it would provide important indirect information about the role of death pathway in breast cancer. The study was performed applying Tetra-amplification refractory mutation system-polymerase chain reaction. The stretches of genes targeted for amplification are depicted in figure 2 and the corresponding primers used for the T-ARMS-PCR are shown in Table 1. The amplified products were separated by electrophoresis on a 2% agarose gel containing 0.5 mg/ml ethidium bromide (please see figure 3 as an example). For quality-control purposes, the genotyping analysis was randomly repeated for 10% of samples for confirmation for quality control and the results were 100% concordant. To confirm the genotyping results, selected PCR-amplified DNA samples (n = 3, respectively, for each genotype) were examined by DNA sequencing and the results determined by T-ARMS-PCR (Fig. 3) were concordant with those determined by sequencing (Fig. 4). Table 3 summarizes the genotype and allele frequencies of the examined polymorphisms in the FAS gene promoter region (21377 G/A, rs2234767; and 2670 A/G, rs1800682), as well as two in the FASL gene (2844 C/T in the promoter region, rs763110; and 2124A/G in the second intron, rs5030772). Significances of associations (pvalues) of those alleles with breast cancer risk are also shown in Table 3 (second-from the right column). The current study does not demonstrate a significant difference in FAS 21377A/G genotype frequencies between patients and controls (x 2 = 0.49, p = 0.78). Hence no association was observed between 21377A/G polymorphism of FAS and risk of breast cancer (Table 3). On the other hand the genotype distribution of the FAS-670A/G polymorphism was significantly different between patients and control groups (x2 = 8.25, p = 0.016). There was a significant association between the FAS 2670 A/G polymorphism and breast cancer risk and the 2670 GG genotype is a risk factor for susceptibility to breast cancer (OR = 3.181; 95% CI = 1.21-8.33; p = 0.019).
There was a significant difference between breast cancer subjects and controls in the distribution of FASL Ivs2nt 124 A/ G (rs5030772) genotypes (x2 = 9.15, p = 0.010) and the GG genotype exhibited significant association with the risk of breast cancer (OR = 5.08; 95% CI = 1.04-18.022; p = 0.012). A significant difference was identified in the distribution of FASL 2844 C/ T genotype between patients and controls (x2 = 7.57, p = 0.022) and the TT genotype showed an increased risk for breast cancer (OR = 2.40; 95% CI = 1.12-5.14; p = 0.024).
Genotype distribution of FAS-1377A/G polymorphism in case and control are not consistent with Hardy-Weinberg equilibrium (HWE). The distribution of FAS 2670 A/G genotype did not deviate from the HWE in case patients or control subjects. While, genotype distribution of FASL Ivs2nt 124 A/G and 2844 C/T deviate from the HWE in case group. Furthermore, haplotypes analysis was performed (Tables 3 and 4). The results showed that rs2234767A/rs1800682A/rs5030772A/rs763110T, rs2234767G/ rs1800682G/rs5030772A/rs763110T and rs2234767A/ rs1800682A/rs5030772G/rs763110T haplotypes decreased the The expression level of Fas was determined in breast cancer and normal tissues by qRT-PCR using the 2 2DDCT method. The results showed that the expression levels of FAS mRNA were not significantly different between breast cancer and normal tissues (P = 0.588).

Discussion
Breast tumor cells frequently down-regulate FAS and/or upregulate FASL expression [34,35,36,37]. Death receptor activation initiates extrinsic apoptotic pathways, which in some cell types may be sufficient to carry apoptosis, whereas in others it also branches towards the intrinsic/mitochondrial pathway, via caspase-8-dependent Bid-cleavage [38,39,40]. Numerous polymorphism sites have been identified to influence the development of breast cancer [2,7]. In the present study we found lack of association between FAS expression and breast cancer. In normal breast epithelium, Fas protein is expressed constitutively, while in primary breast cancer, its expression was found to be less uniform [41]. It has been reported that the lack of Fas in the primary tumor was associated with perilymphatic fat infiltration and metastasis either to the regional lymph nodes or to the bones [42,43].
Several previous researches have focused on the association between different gene polymorphisms within cell death pathways and the risk of developing malignancies and developing cancers. FAS/FASL polymorphisms commands major attention as it may play different, context-dependent roles in cancer [44,45]; 1) apoptosis promotion by FasL on T-lymphocytes in Fas-expressing cancer cells, this mechanism could play an important responsibility in cell mediated cytotoxic reactions against malignant cells. 2) malignant cells could escape immune system expression of FasL, thus repelling Fas-expressing immune-effector cells, hence preventing the immune system from recognizing mutated tumor cells (tumor as immuno-privileged site). 3) FasL expressed on T cells, may not only aids them to kill target (cancer) cells, but it was also associated with an enhanced rate of activation-induced cell death in T cells [46]. Various correlations have been drawn from genetic polymorphisms in the death pathway genes FAS and FASL and the risk of cancer development [47,48,49,50,51,52].
In the present study, we investigate whether the FAS-1377 G/A, FAS-670 A/G, FASL-844 T/C and FASL Ivs2nt 124 A/G polymorphism in cell death pathway genes were associated with the risk of the development of breast cancer in a sample of Iranian population (south east of Iran). We found that the FAS-1377 G/A did not affect the risk of breast cancer, while FAS 2670 G/A, FASL Ivs2nt 124 A/G and FASL 2844 C/T gene polymorphisms are risk factors for this disease in our study population. Previous researches have also addressed FAS/FASL polymorphisms in  [53]. On the other hand, Zhang et al have found a significant association between FAS 21377G/A and FASL-844T/C gene polymorphisms and risk of breast cancer, but they reported no association between FAS 2670 G/A and breast cancer risk [46]. We observed a significant association between breast cancer risk and FAS 2670 G/A genotype, thus our findings show partly-different association pattern that the above mentioned ones. Moreover, they observed a significantly increased breast cancer risk associated with the FAS 21377G/A genotype, but we report lack of such association. They reported decreased risk associated with FASL 2844TT (OR, 0.66; 95% CI, 0.43-1.00) genotype, but we found increased risk with mentioned genotype (OR, 2.40; 95% CI, 1.12-5.14). Contrary to our results, Krippl et al., in a study of 500 breast cancer patients and 500 controls in a Caucasian population in Austria, reported a significant association between FAS 21377G/A and increased risk of breast cancer, but they found no associations with FAS 2670G/ A and FASL 2844C/T [54]. The FASL 2844 C/T has a considerable impact on the promoter activity of the FASL gene in an in vitro assay system, because the polymorphism affects a binding affinity for the transcription factor C/EBP. A significantly higher basal expression of FasL is associated with the FasL 2844 C allele compared with the T allele [27]. Hence, the FASL 2844 C allele, which drives a higher expression of FASL in T cells, was associated with an enhanced rate of activation-induced cell death in T cells [46]. It has been reported that FASL 2844 T allele has a possible protective effect on cancer risk [55]. Although the exact mechanism for this inverse association in our study was not clear, other polymorphisms of FASL may also play a role. There is no clear explanation for deviation from HWE in our population. The possible reason may be due to genetic drift.
We recognize some limitations of the present study: (i) we have no data on some known risk factors (e.g., family history, previous benign conditions, oral contraceptive or hormone therapy use, etc.), (ii) a relatively small sample size. Nevertheless, we believe that the presented here data provides an important input into the debate regarding the clinical relevance of investigated polymorphisms.
Our work is a valuable addition to the ongoing discussion on the influence of various polymorphisms within the signaling molecules pertaining to cell death pathways, and cancer formation. The so far only partial overlapping of conclusions reached from our data analysis as compared to previously published studies (please see above) may reflect differences in genetic background between the studied populations, and variations in external (i.e. environmental), or other (i.e. obesity) factors that influence studied populations [5,8,56]. As cell death pathways are ubiquitous, active in most cells in eucaryotes, our manuscript provides additional voice in the discussion on the role of Fas/FasL system on the clinical outcome of breast-and other cancers. One has to keep in mind that although apoptosis is the dominant, fastest cell death program, autophagy appears to play increasingly important role as an accessory cell death mechanism [57,58,59]. . Two forward and two reverse specific primers are used to produce three potential products. Product sizes were 158 bp for A allele, 207 bp for G allele, and 309 bp for two outer primers (control band) for Fas rs1800682 (A). Product sizes were 216 bp for G allele, 340 bp for A allele, and 507 bp for control band for Fas rs2234727 (B). Product sizes were 145 bp for T allele, 192 bp for C allele, and 284 bp for control band for FasL rs763110 (C). Product sizes were 197 bp for G allele, 300 bp for A allele, and 438 bp for control band for FasL rs5030772 (D). doi:10.1371/journal.pone.0053075.g002