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Carcinogenesis Advance Access originally published online on December 20, 2006
Carcinogenesis 2007 28(5):1067-1073; doi:10.1093/carcin/bgl250
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Functional polymorphisms in FAS and FASL contribute to increased apoptosis of tumor infiltration lymphocytes and risk of breast cancer

Bailin Zhang{dagger}, Tong Sun1,{dagger}, Liyan Xue2, Xiaohong Han3, Baoning Zhang, Ning Lu2, Yuankai Shi3, Wen Tan1, Yifeng Zhou1, Dan Zhao1, Xuemei Zhang1, Yongli Guo1 and Dongxin Lin1,*

Center of Breast Diseases and Department of Abdominal Surgery
1 Department of Etiology and Carcinogenesis
2 Department of Pathology
3 Department of Medical Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China

* To whom correspondence should be addressed. Tel: +86 1087788491; Fax: +86 1067722460; Email: dlin{at}public.bta.net.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The FAS–FASL system plays crucial role in counterattack of cancer cell against immune system. This study examined the effects of FAS (–1377G/A and –670A/G) and FASL (–844T/C and 7896G/C) polymorphisms on breast cancer risk and apoptosis of T lymphocytes. The effect on breast cancer risk was determined by case–control analysis of 840 patients and 840 controls. The effects on T-lymphocyte apoptosis were determined by activation-induced cell death (AICD) of T cells ex vivo and by analyzing apoptotic tumor-infiltrating lymphocytes (TILs) in breast cancer tissue. We found moderately increased risk associated with FAS –1377AG [odds ratio (OR), 1.29; 95% confidence interval (CI), 1.05–1.59] and –1377AA (OR, 1.36; 95% CI, 1.01–1.82) genotypes compared with the –1377GG genotype and decreased risk associated with FASL –844CT (OR, 0.76; 95% CI, 0.62–0.94) and –844TT (OR, 0.66; 95% CI, 0.43–1.00) genotypes compared with the –844CC genotype. T lymphocytes with the FASL –844CC genotype had heightened FASL expression that is associated with increased AICD of the T cells stimulated by MCF-7 cells or phytohemagglutinin compared with the FASL –844TT genotype (10.38 ± 4.09% and 24.29 ± 1.50% versus 6.03 ± 0.41% and 17.96 ± 3.66%; P < 0.05 and 0.001). Breast cancer patients with the FASL –844CC genotype had higher apoptotic TILs in their cancer tissues than those with the FASL –844TT genotype (33.7 ± 1.2% versus 19.1 ± 2.0%; P = 0.007). These findings indicate that functional polymorphisms in FAS and FASL contribute to increased apoptosis of tumor infiltration lymphocytes and risk of breast cancer.

Abbreviations: AICD, activation-induced cell death; CI, confidence interval; FASL, FAS ligand; MMC, mitomycin C; OR, odds ratio; PBMC, peripheral blood mononuclear cell; SNP, Single nucleotide polymorphism; TIL, tumor-infiltrating lymphocyte; TUNEL, transferase-mediated dUTP nick end labeling


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Breast cancer is one of the most common cancers and leading causes of cancer death among women all over the world (1). It has been well known that the increased risk of developing breast cancer is associated with hormone and reproductive events such as early menarche, late menopause and nulliparity (2). Lifestyle factors including alcohol consumption, physical inactivity and perhaps smoking also contribute to the risk of breast cancer (2,3). On the other hand, women with a family history of breast cancer are at high risk for the cancer, suggesting that genetic makeup may play an important role. The family aggregation of breast cancer has led to the identification of germ line mutations in some genes such as BRCA1/2, P53 and ATM as inherited susceptibility genes (4–7). However, it is estimated that only a small fraction of breast cancer cases result from these inherited mutations of susceptibility genes (8). Accumulating evidence has suggested that other more common but low-penetrance genetic variants may exist to contribute to breast cancer formation (9,10).

Apoptosis is known to participate in various biological processes such as development, maintenance of tissue homeostasis and elimination of cancer cells. The aberrant regulation of apoptosis contributes to many types of human diseases including cancer (11,12). During carcinogenesis, tumor cells develop many mechanisms to subvert the immune system and suppress the antitumor immune response, among which FAS and FAS ligand (FASL) molecules play an important role in immune escape (13). Decreased FAS but elevated FASL expression has been found in many types of cancer including breast cancer (14–17). It has been shown that tumor cells may counterattack FAS-sensitive tumor-infiltrating lymphocytes (TILs) using heightened expressed FASL and this mechanism may lead to tumor cell immune privilege that sequentially contributes to cancer formation and progression (14,16,18–20).

Activation-induced cell death (AICD), referred to the induction of apoptosis of previously activated T cells on subsequent encounter with antigen (21), is another important mechanism responsible for the increased apoptosis rate among tumor infiltration lymphocytes that may protect transformed cells from elimination by antitumor immune responses and therefore contribute to carcinogenesis and cancer progression (22,23). AICD is a FASL-dependent process (24). Cytotoxic T cells increase expression of both FAS and FASL upon activation by antigen or other stimulation and subsequently undergo ‘suicide’ or ‘fratricide’ by FASL liganding to FAS (25,26). Antigenic stimulation within the tumor microenvironment might also enhance the expression and function of FASL on T cells, resulting in activation of autocrine or paracrine mechanisms of apoptosis (27). FASL-mediated AICD is directly regulated by the level of this death ligand (28), whereas FASL transcriptional regulation happens in both T cells and non-lymphoid cells, such as a variety of tumor cells.

Single nucleotide polymorphisms (SNPs) in the promoter regions of FASL and FAS genes have been linked to the differential expression of these two genes. The FAS –1377G->A and FAS –670A->G polymorphisms are situated within the consensus sequences of binding sites for transcription factors Sp1 and STAT1, respectively. These SNPs have been associated with decreased FAS expression probably due to destroying binding elements for the transcription factors (29,30). The FASL –844T->C SNP is located in a binding motif for transcription factor CAAT/enhancer-binding protein ß, and a considerably higher basal expression of FASL is associated with the FASL –844C allele compared with the –844T allele (31). Another FASL SNP (7896G->C) with a relatively high frequency for the variant allele in Chinese is located in the 3' untranslational region (http://www.ncbi.nlm.nih.gov/SNP), but the effect of this SNP on FASL function is unknown yet. The functional significance of FAS and FASL SNPs may be associated with alterations in FAS- and FASL-mediated apoptosis and thus individual susceptibility to cancer.

We and other investigators have shown that the aforementioned FAS and FASL SNPs are associated with increased risk of the development of certain cancers including esophageal cancer (32), lung cancer (33,34) and cervical cancer (35,36), suggesting that genetic variation in the death pathway may confer susceptibility to common cancers. In the present study, we investigated the association between the FAS and FASL SNPs and the risk of developing breast cancer in a large case–control study. In addition, we also examined the effects of FAS and FASL polymorphisms on AICD of T lymphocytes ex vivo and apoptosis of TILs in vivo in breast cancer tissues. We found that FAS and FASL SNPs are associated with significantly increased T-lymphocyte apoptosis and moderately increased risk of developing breast cancer.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Study subjects
To determine the association between FAS or FASL genotype and risk of developing breast cancer, we designed a case–control study consisting of 840 patients with breast cancer and 840 cancer-free controls. All subjects were ethnic Han women and residents of Beijing and its surrounding regions. A large portion of patients (n = 520) and controls (n = 520) have been characterized in our previous study (37). In the current study, we added 320 more patients and 320 more controls to extend the sample size to increase the statistical power. Briefly, the eligible patients diagnosed histologically as primary breast carcinoma were recruited between June 1997 and March 2004 at Cancer Hospital, Chinese Academy of Medical Sciences (Beijing), with a response rate of 90%. Tumors were staged according to American Joint Committee on Cancer staging criteria (38) and histologically classified according to the World Health Organization classification (39). The control subjects were from a nutritional survey conducted in the same region during the period of case collection. The control subjects were selected from a database consisting of 2500 individuals based on a physical examination including chest radiography and abdominal ultrasonography. The selection criteria included no history of cancer and benign breast diseases and frequency matching to patients by age (±5 years). For FAS and FASL expression and AICD assays ex vivo, vein blood samples were obtained from a group of 40 healthy volunteers (22 males and 18 females) working in Cancer Hospital, Chinese Academy of Medical Sciences, aged 19–49 years (mean age = 31.8 years). All these volunteers were HLA-A2 positive with no history of malignant diseases. At recruitment, informed consent was obtained from each subject and this study was approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Hospital and Institute.

MCF-7 cell culture, antigen preparation and MMC treatment
Human breast cancer cells MCF-7 were cultured in RPMI 1640 medium with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. For production of MCF-7 cell antigen, portions of logarithmic growth MCF-7 cells were collected and washed with cold phosphate-buffered saline. These cells were then shattered by freezing in liquid nitrogen and melted in a 37°C water bath alternately five times, followed by centrifugation at 22 000g for 15 min at 4°C. The supernatant was collected and the total amount of protein was determined. Another portion of 106 MCF-7 cells/ml in RPMI 1640 medium was treated with 100 mg/ml mitomycin C (MMC) (Sigma-Aldrich, St Louis, MO) for 1 h at 37°C with occasional shaking to inhibit proliferation. These cells were then washed four times with the medium before use in the activation of peripheral blood mononuclear cells (PBMCs) as described below.

PBMC isolation and culture
PBMCs were isolated by Ficoll-Paque (Sigma-Aldrich) density-gradient centrifugation and suspended in RPMI 1640 medium (5 x 106 cells/ml) as used for MCF-7 cells. PBMCs were distributed into 24 well tissue culture plates at 1 ml per well and cultured in the presence of 25 µg/ml PHA (Sigma-Aldrich) for 6 h or cultured with 50 µg/ml MCF-7 cell antigen for the first 4 h, followed by the addition of MMC-treated MCF-7 cells (1:10 PBMCs) and cultured for the next 68 h. PBMCs cultured without PHA or MCF-7 antigen/cells served as respective controls (35). Cultured PBMCs were used to assay expression of FAS, FASL and CD25 and the apoptotic index among CD3+ or CD4+ cells, and the cell culture supernatants were collected to determine IL-2 production as described below.

Flow cytometry analysis
A flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA) was used to determine the cell surface expression of FAS, FASL, CD25, CD3 and CD4 and cell apoptotic index indicated by Annexin V and propidium iodide staining as described in our previous report (35).

Determination of IL-2 and soluble FASL
PBMC culture media were collected and IL-2 concentrations were determined by a human IL-2 ELISA kit (R&D systems, Minneapolis, MN), according to the manufacturer's protocol. Optical density was measured at 450 nm with a multilabel counter (Wallac Victor2 1420; PerkinElmer Life and Analytical Sciences, Boston, MA). The levels of soluble FASL (sFASL) in PBMC culture supernatants were measured by ELISA with the human FAS Ligand/TNFSF6 immunoassay kit (R&D systems). Assay diluent RD1S (200 µl) was added to each well followed by the addition of 50 µl standard or sample, then incubated for 2 h in room temperature. After washing four times with wash buffer, 200 µl FASL conjugate was added to each well and incubated for 2 h. After washing, 200 µl substrate solution was added to each well and incubated for 30 min in the dark. Finally, the colorable reaction was terminated by adding 50 µl stop solution (2N sulfuric acid). Optical density of each well was read at 450 nm with the multilabel counter within 30 min.

Determination of apoptotic TILs in breast cancer specimens
Tissue array of 29 individual breast tumor specimens was constructed to determine the relationship between FAS and FASL genotype and the apoptotic rate of TILs by immunohistochemical staining. All tumors were invasive ductal carcinoma (stage II) except for one intraductal papillary carcinoma. Samples were selected according to FAS and FASL genotypes to obtain a similar number among different genotype groups. Three tissue cores from each cancer tissue were sliced (5 µm) and mounted onto adhesive-coated slides. The apoptotic TILs were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using Klenow FragELTM DNA Fragmentation Detection Kit (EMD Biosciences, Darmstadt, Germany) according to the manufacturer's protocol. To identify specifically apoptotic TILs, CD3+ cells in the same tissue were stained after TUNEL staining by using mouse antihuman CD3 IgG (DakoCytomation Denmark A/S, Glostrup, Denmark). The slides were then lightly counterstained in hematoxylin and mounted with aqueous permanent mounting medium (Ultramount, Dako) for a long-term preservation. Under our experimental conditions, the CD3+ cells were in red, whereas CD3+/TUNEL+ cells represented brown nuclei and red cytoplasm and surface. The apoptotic index of TILs was calculated as the percentage of CD3/TUNEL dual-positive cells among total number of CD3+ cells counted in tumor region. Cell counting was performed under the microscope by using a grid ocular (Olympus WHK x10, Tokyo, Japan) independently by two investigators (T.S. and B.Z.), who were unaware of genotyping data at that time.

Genotyping
Genotypes of FAS –1377G/A, FAS –670A/G and FASL –844T/C polymorphisms were determined by PCR-based restriction fragment length polymorphism assays as described previously (32). FASL 7896G/C genotypes were also determined by PCR-based restriction fragment length polymorphism. The primers were 5'-TGAGAAGGTGAGGAATGAGGA-3' and 5'-CCAGGGACAATAGCCTACCA-3' that produce a 241 bp PCR product under the conditions of 2 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 63°C and 30 s at 72°C and a final elongation step of 7 min at 72°C. Genotype was distinguished by digestion with restriction endonuclease AccI (New England Biolabs, Beverly, MA). A 10% masked random sample of subjects was tested twice by different persons and the results were concordant for all the masked duplicate sets.

Statistical analysis
One-way analysis of variance was used to examine the differences in the levels of cell surface molecules, secreted IL-2, sFASL, apoptotic T cells ex vivo, and apoptotic TILs in vivo among different genotype. Arcsine transformation of square root was performed before the differences of apoptotic TIL rates were tested. Because the expression levels of FAS and FASL on T cells might be influenced by the activation status of T cells (40), an analysis of covariance was performed when analyzing FAS/FASL expression by using the expression level of CD25+/CD4+ cells and the concentration of IL-2 in cell culture media as covariates. The association between FAS and FASL genotypes and the risk of breast cancer was estimated using odds ratios (ORs) and their 95% confidence intervals (CIs) that were computed by unconditional logistic regression. All the ORs were adjusted for age. We tested the null hypotheses of additive and multiplicative gene–gene interactions between the FAS and FASL polymorphisms and evaluated departures from additive and multiplicative interaction models (41) by including main effect variables and their product terms in the logistic regression model. A P value of <0.05 was used as the criterion of statistical significance, and all statistical tests were two-sided tests. All analyses were performed with computer programs from Statistical Analysis System (SAS Institute, Cary, NC). Haplotype frequencies and linkage disequilibrium coefficient were estimated using PHASE (42) and 2LD software (43).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
FAS and FASL genotypes and their expression on T cells
To determine the effects of FAS and FASL SNPs on the expression of these genes, PBMCs isolated from 40 healthy donors carrying different genotypes were incubated with PHA- or MMC-treated MCF-7 cells. The proportion of CD25+/CD4+ cells to CD3+ cells was determined as an index of T-cell activation. In addition, the levels of IL-2 in the supernatants of culture media of PBMCs were also determined as an indicator of T-cell activation. It was found that the proportion of CD25+/CD4+ cells (mean ± SD, n = 40) in the PBMC cultures with PHA was 50.83 ± 14.08% that was significantly higher than that in the cultures without PHA (2.27 ± 0.88%; P < 0.001). In parallel, the IL-2 level (mean ± SD, n = 40) in the PBMC culture media with PHA was significantly higher than that in the PBMC culture media without PHA (191.25 ± 66. 41 pg/ml versus 28.82 ± 15.39 pg/ml; P < 0.001). Similarly, the proportion of CD25+/CD4+ cells was significantly elevated when PBMCs were incubated with MMC-treated MCF-7 cells compared with that without MMC-treated MCF-7 cells (9.50 ± 2.88% versus 6.83 ± 2.61%; P < 0.001). The activation of T cells by MCF-7 antigen was also evidenced by the fact that the mean IL-2 level in the PBMC culture media incubated with MCF-7 cells was significantly higher than that in the PBMC culture media without MCF-7 cells (140.74 ± 55.20 pg/ml versus 98.93 ± 53.47 pg/ml; P < 0.001). These results demonstrated that both PHA and MCF-7 cell antigens activated PBMCs under our study conditions. Moreover, neither the elevated population of CD25+/CD4+ cells nor the IL-2 concentration in the media was correlated to investigated FAS or FASL genotypes (data not shown).

We then examined whether FAS or FASL genotypes have impact on FAS or FASL expression on T cells in PHA- or MCF-7 cell-stimulated PBMCs. We observed a significant difference in the FAS expression on T cells after incubation with PHA as a function of FAS genotype. The FAS –1377AA genotype had a significantly lower FAS expression levels (mean ± SD) compared with the GG or GA genotype [46.31 ± 5.02% (n = 6) versus 66.63 ± 3.53% (n = 12) and 57.92 ± 2.61% (n = 22); P = 0.002 and P = 0.048, respectively], whereas the levels were not significantly different between the GG and GA genotype (P = 0.355). Similar results were obtained for the FAS –670 SNP that is in high linkage disequilibrium with the FAS –1377 SNP. For FASL expression after stimulation by PHA, the FASL –844CC genotype had significantly higher levels than the CT or TT genotype [1.72 ± 0.10% (n = 18) versus 1.22 ± 0.10% (n = 18) or 0.32 ± 0.25% (n = 4); P = 0.002 and P < 0.001, respectively], and the difference between the heterozygous CT genotype and the variant homozygous TT genotype was also statistically significant (P = 0.002). After stimulating by MCF-7 cells, the FASL expression in subjects with the TT genotype was 0.22 ± 0.10% that was significantly lower than that in subjects with the CC (0.39 ± 0.06%; P < 0.001) or CT (0.36 ± 0.03%; P < 0.001) genotype. However, the levels between the CC and CT genotypes were not statistically different probably due to the limited statistical power (Figure 1A). These findings clearly demonstrated that the investigated SNPs in the promoter region of FAS or FASL have a substantial impact on the activation-induced expression of FAS and FASL on T cells.


Figure 1
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  		Fig. 1. The levels of FASL expression in PHA- or MCF-7 cell-stimulated PBMCs from individuals carrying different FASL genotype. PBMCs from individuals were cultured with or without MCF-7 cell antigen and MMC-treated MCF-7 cells for 72 h or cultured with or without PHA for 6 h. FASL expression levels were adjusted for CD25 and IL-2 levels. (A) FASL expression on CD3+ cells as a function of FASL genotypes. Columns, mean; bars, ±SD. Expression level among the –844CC genotype was significantly different from that among the CT or TT genotype; *P < 0.01, **P < 0.001. (B) Soluble FASL expression in culture supernatants as a function of FASL genotypes. Columns, mean; bars, ±SD. Expression level among the –844CC or CT genotype was significantly different from that among the TT genotype; *P < 0.05.

 
FASL genotype and the expression of sFASL
ELISA showed that the sFASL concentrations in PBMC cultured without PHA were very low and no significant difference in the levels was found among different genotypes. However, sFASL expression levels were greatly heightened in PBMCs stimulated with PHA and the levels were significantly different among three FSAL genotypes (Figure 1B): The mean level (±SD) in the culture of PBMCs having the FASL –844TT genotype was 1310.24 ± 359.89 pg/ml, that was significantly lower than that in the culture of PBMCs having the –844CC (2826.82 ± 1390.77; P = 0.022) or CT (2426.59 ± 1362.25; pg/ml; P = 0.017) genotype, whereas the levels were not significantly different between the CC and CT genotypes (P = 0.351).

FAS or FASL genotypes and AICD of T cells ex vivo
To examine the effects of FAS and FASL genotypes on AICD of T cells, PBMCs incubated with PHA or MCF-7 cells were labeled with FITC-Annexin V, propidium iodide and PerCP-Cy5.5-CD3, and the proportion of Annexin V+ cells in the CD3+ gate was determined by flow cytometry (Figure 2A). We found that neither FAS nor FASL genotype had effect on the spontaneous apoptosis of T cells during culture. However, when T cells were incubated with either PHA or MCF-7 cells, the differences in AICD among three different FASL –844 genotypes were striking (Figure 2B). After incubation with MCF-7 cells, the mean proportion (±SD) of Annexin V+ cells was significantly higher in T cells carrying the FASL –844CC genotype (10.38 ± 4.09%) compared with that in T cells carrying the FASL –844TT (6.03 ± 0.41%; P = 0.049) genotype. Similarly, after incubation with PHA, the differences in AICD of T cells among three FASL genotypes were also significant, with the proportion of Annexin V+ cells being higher in T cells carrying the FASL –844CC genotype (24.29 ± 1.50%) than that in T cells carrying the FASL –844CT (19.84 ± 1.46; P < 0.001) or TT genotype (17.96 ± 3.66%; P < 0.001). However, the differences in AICD of T cells stimulated either with MCF-7 cells or with PHA were not significant among different FAS genotypes (data not shown).


Figure 2
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  		Fig. 2. AICD of T cells in PBMCs from healthy individuals carrying different FASL genotypes determined by flow cytometry. Annexin V assay was performed to detect the apoptosis index by flow cytometry in CD3+ cells. Apoptosis index was defined as the ratio of Annexin V+/CD3+ cells. (A) Representative flow cytometry showing apoptotic cells in PBMCs from two individuals with different FASL genotypes after stimulation by PHA. (B) The relationship between FASL genotypes and rates of AICD of T cells. The FASL –844CC genotype had a significantly higher rate of AICD compared with the –844CT or TT genotype when PBMCs were incubated with PHA or MCF-7 cell antigens; *P < 0.05, **P < 0.001.

 
FAS and FASL SNPs and apoptotic TILs in breast cancer tissues
We further examined the association between FAS and FASL SNPs and apoptosis of TILs in breast tumor tissues using CD3- and TUNEL-dual staining in tissue array slides (Figure 3). We found that the mean percentage (±SD) of apoptotic TILs among total CD3+ lymphocytes (apoptotic index) in breast tumors with the FASL –844CC genotype was significantly higher than that in breast tumors with the FASL –844TT genotype [33.7 ± 1.2% (n = 10) versus 19.1% ± 2.0% (n = 12); P = 0.007], whereas the values were not significantly different between the –844CC and –844CT (24.7 ± 1.4%, n = 7) or –844CT and –844TT genotypes. Similar results were obtained when the data were analyzed in the context of FASL haplotypes; the apoptotic index of TILs in the –844C/7896G haplotype carriers (n = 7) was 35.5 ± 1.6% compared with 19.1 ± 2.0% in non-carriers (n = 12) (P = 0.011). However, no such significant difference in apoptotic index of TILs in breast tumor tissues was found among patients carrying different FAS genotype or haplotype.


Figure 3
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  		Fig. 3. Identification of apoptotic TILs in breast tumor tissues using CD3-TUNEL dual staining in a tissue array slide. (A) CD3+ lymphocytes in tumor area, exhibiting red cytoplasmic/cell surface staining. (B) Apoptotic TILs in tumor area identified by CD3-TUNEL dual staining, showing brown TUNEL-positive nuclei staining with red CD3+ cytoplasmic/cell surface staining.

 
FAS and FASL SNPs and breast cancer risk
Baseline clinical characteristics of patients and controls are summarized in Table I. The age distribution in patients was not significantly different from that in controls (P = 0.651). For cancer staging, 23.0% of patients had stage I breast cancer, whereas 61.1, 6.7 and 1.5% of patients had stage II, III and IV breast cancer, respectively. Tumor staging information was unavailable among 7.7% patients.


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  		Table I. Baseline clinical characteristics of breast cancer patients and controls

 
Genotyping data are shown in Table II. Among subjects, 12 patients and 18 controls failed to be genotyped in more than one polymorphic site because of PCR amplification problems with their DNA samples. All observed genotype frequencies in both controls and patients were consistent with Hardy–Weinberg equilibrium. In our study population, the FAS –1377 and FAS –670 and FASL –844 and FASL 7896 polymorphisms were in linkage disequilibrium (D' = 0.85 and 0.87, respectively). It was observed that genotype frequencies for FAS –1377, FASL –844 and FASL 7896 polymorphisms among patients were significantly different from those among controls ({chi}2 = 7.06, 9.22 and 6.77, P = 0.029, 0.010 and 0.034, respectively). However, the distributions of FAS –670A->G genotypes were not significantly different between patients and controls ({chi}2 = 0.01, P = 0.995).


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  		Table II. Allelic and genotypic frequencies of FAS and FASL among controls and cases and their association with breast cancer risk

 
Logistic regression analysis (Table II) revealed that subjects carrying the FAS –1377AA genotype had a moderately excessive risk of developing breast cancer (adjusted OR, 1.36; 95% CI, 1.01–1.82; P = 0.045) compared with those carrying the GG genotype. FAS –1377GA heterozygous genotype was also associated with higher risk (OR, 1.29; 95% CI, 1.05–1.59; P = 0.017). When the AA and GA genotypes were grouped for analysis, the OR compared with the –1377GG genotype was 1.30 (95% CI, 1.07–1.59; P = 0.009). Subjects with the FASL –844TT genotype had a decreased risk for breast cancer compared with those with the CC genotype (OR, 0.66; 95% CI, 0.43–1.00; P = 0.045) and a similar association was also presented for the heterozygous CT genotype (OR, 0.76; 95% CI, 0.62–0.94; P = 0.008). For the FASL 7896 polymorphism, the GC genotype was associated with a moderately decreased risk (OR, 0.80; 95% CI, 0.65–0.98; P = 0.031) and the CC genotype associated with a marginally deceased risk (OR, 0.66; 95% CI, 0.41–1.08; P = 0.081) compared with the GG genotype. When the CC and GC genotypes were grouped for analysis, they were associated with significantly decreased risk (OR, 0.78; 95% CI, 0.64–0.95; P = 0.013) compared with the GG genotype. The increased risk of breast cancer associated with the FAS and FASL genotypes was not influenced by menarche age, menstrual periods before cancer diagnosis, menopause status and ER or PR status (data not shown).

We then analyzed the association between risk of breast cancer and FAS –1377 and –670 and FASL –844 and 7896 polymorphisms in the context of haplotypes (Table III). It was found that the FAS –1377A/–670A haplotype had a higher (OR, 1.45; 95% CI, 1.06–2.00; P = 0.021) but the FAS –1377G/–670G haplotype had a lower (OR, 0.68; 95% CI, 0.49–0.93; P = 0.016) risk compared with the FAS –1377G/–670A haplotype. For the FASL polymorphisms, the –844T/7896C haplotype was associated with a significantly lower risk compared with the –844C/7896G haplotype (OR, 0.79; 95% CI, 0.67–0.94; P = 0.009).


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  		Table III. Haplotype frequencies of FAS and FASL among controls and cases and their association with breast cancer risk

 
Because FAS and FASL work together in apoptotic cell death, we also examined whether there was a statistical interaction between the FAS and FASL polymorphisms in risk of breast cancer. However, the data did not show any significant interaction between the polymorphisms in these two genes, although the highest risk (OR, 3.00; 95% CI, 1.45–6.24) was seen among subjects carrying the FAS –1377AA and FASL –844CC genotypes.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Apoptosis plays a key role in cancer development. By constitutive expression of death receptor ligands such as FASL, tumor cells may adopt AICD mechanism from cytotoxic lymphocytes to delete the attacking antitumor T cells through the induction of apoptosis via death receptor and death receptor ligand interaction (44). In the previous studies, we have shown that polymorphisms in FAS/FASL pathway confer host's susceptibility to cancers of the esophagus, lung and cervix (32,33,35) and the mechanism might be implicated in AICD of TILs (35). In the present study, we extended our findings to breast cancer. On the basis of genotyping 840 patients and 840 controls in Chinese women, we observed a moderate modification of the risk for developing breast cancer by FAS and FASL polymorphisms. In addition, we also examined the genotype–phenotype correlation between the FAS and the FASL polymorphisms and AICD of T cells stimulated ex vivo by human breast cancer cells or apoptotic TILs in vivo in breast cancer tissues. The results are positive and generally in agreement with our previous findings (32,33,35). Taken together, these data further support our notion that the FAS- and the FASL-triggered apoptosis pathway may play a role in human carcinogenesis.

We have previously established an ex vivo system to examine the expression of FAS and FASL and AICD of T cells and shown that the FASL –844T->C change strongly influences the expression of FASL and AICD of T cells stimulated with PHA or cervical cancer cells (35). In the current study, by using this experimental model, we also observed that the FASL –844T->C change is associated with elevated expression of both soluble and membrane types of FASL and AICD of T cells cocultured with human breast cancer cell line MCF-7 cells. This genotype and phenotype correlation between the FASL polymorphism and AICD of T cells stimulated by MCF-7 cells encouraged us to look at the difference in apoptotic TILs in vivo in breast tumor microenvironment in patients carrying different FAS and FASL genotype. As a result, using immunohistochemical CD3- and TUNEL-dual staining method, we found that the mean percentage of apoptotic TILs among total CD3+ lymphocytes in breast tumors with the FASL –844CC genotype was significantly higher than that in breast tumors with the FASL –844TT genotype, although no such difference was observed for the FAS polymorphisms. These findings are consistent with the results obtained from ex vivo model, indicating that the functional FASL polymorphism is associated with AICD of TILs in breast tumor microenvironment in vivo. Because FAS–FASL-mediated AICD of TILs is believed to play a role in tumor development and progression (22,23), individuals carrying the FASL –844CC genotype and, thus, having higher FASL expression and apoptotic TILs upon tumor antigenic stimulation would be anticipated to be susceptible to cancer development. On the other hand, it has been shown that breast tumor cells frequently downregulate FAS and upregulate FASL expression (14–17). Because of the functional consequence of the FASL –844C->T polymorphism, individuals carrying the –844CC genotype may also have higher FASL expression on breast tumor cells than those carrying the –844TT genotype. The heightened expression of FASL may assist transformed cells to counterattack the FAS-expressing TILs, resulting in immune evasion of these malignant cells. This mechanism may also contribute to increased risk of the development of breast cancer in subjects with the FASL –844TT genotype.

We did not observe any significant association between the investigated functional polymorphisms in FAS and apoptotic rate of TILs in vivo in breast cancer tissues and similar results were also obtained when PBMCs were incubated with PHA or MCF-7 cells in the ex vivo experimental model, which is consistent with our previous study (35). These results indicate that, unlike FASL, a subtle change of widely expressed FAS might not be sensitive enough to detect a subtle change of AICD of T cells. However, we did observe a moderate but significant risk of breast cancer associated with the FAS –1377A allele that are in agreement with our previous results from large molecular epidemiological studies showing that the FAS –1377A allele is associated with increased risk for lung cancer and esophageal cancer (32,33).

To the best of our knowledge, only one study on FAS and FASL polymorphisms and susceptibility to breast cancer has been reported so far. Krippl et al. (45) described a case–control study of 500 breast cancer patients and 500 controls in a Caucasian population in Austria and reported a significant association between the FAS –1377A but not –670G variant allele and increased risk of breast cancer (OR, 1.42; 95% CI, 1.08–1.88; P = 0.013). They also showed a positive association between the FAS –1377A/–670G haplotype and increased breast cancer risk (OR, 1.39; 95% CI, 1.02–1.90; P = 0.038) but this association was less pronounced than that of the –1377A allele alone. Their results are similar to ours showing that the –1377A allele but not the –670G allele is risk allele and the –1377G/–670G haplotype seemed to be associated with lower risk of the cancer. Taken together, these results suggest that the –670A->G SNP may not have functional effect on risk of breast cancer in both Caucasian and Chinese populations. For the FASL –844C->T polymorphism, however, the results are not consistent between our study and the study by Krippl et al. We observed a significantly increased breast cancer risk associated with the FASL –844CC genotype, but they reported a null association (45). The reason for this discrepancy is not clear but may reflect the difference in genetic background between the two study populations, such as other unknown functional SNPs in FASL and/or other breast cancer-related genes. On the other hand, the inconsistent results may also reflect different gene–environment interaction in breast carcinogenesis in different populations, which did not address in both studies. We noticed that patients in our study are much younger than those in the study of Kripple et al. (50 years versus 57 years), and such early onset of breast cancer among Chinese women compared with Caucasian women has already been documented (46,47), suggesting that given a carcinogenic exposure, Chinese women who developed breast cancer might be more susceptible than Caucasian women due to genetic makeup.

In summary, our study demonstrates an association between genetic polymorphisms in death pathway genes FAS and FASL and susceptibility to breast cancer in Chinese women. Functional analyses showed that the FASL –844CC variant is associated with heightened AICD of T cells stimulated ex vivo by human breast cancer cells and heightened apoptotic rate of TILs in vivo in breast cancer tissues. These results indicate that natural occurring genetic variation in death pathway may underlie phenotypic variation in susceptibility to cancer.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
This work was supported by National Natural Science Foundation grant 30530710 and State Key Basic Research Program grant 2004CB518701.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 17, 2006; revised November 14, 2006; accepted December 10, 2006.


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