Polymorphisms of FAS and FAS Ligand Genes Involved in the Death Pathway and Risk and Progression of Squamous Cell Carcinoma of the Head and Neck

Squamous cell carcinoma of the head and neck (SCCHN), including those of the oral cavity, pharynx, and larynx, is the sixth most frequently occurring cancer and the seventh leading cause of cancer-related deaths worldwide (1). In the U.S., there will be an estimated 40,500 new cases and 11,170 deaths from SCCHN in 2006 (2). Although tobacco smoking and alcohol use are the major risk factors in the etiology of SCCHN (3), only a proportion of exposed individuals develops SCCHN in their life span, suggesting individual susceptibility to exposure-related carcinogenesis.

Apoptosis is a biological process that regulates physiologic cell death, varies in different tissues and organisms (4), and plays an important role in maintaining homeostasis (5). Accumulating evidence suggests that abnormal regulation of apoptosis is likely to contribute to the pathogenesis of a variety of human diseases, including cancer (6).

FAS, also known as TNFRSF6/CD95/APO-1, is a cell surface receptor that is involved in apoptotic signaling in many cell types (7). FAS ligand (FASLG), also known as TNFSF6/CD95LG, is a member of the tumor necrosis factor superfamily that can trigger the apoptotic cell–death cascade by cross-linking with its receptor, FAS (8). Therefore, the FAS/FASLG pathway plays a crucial role in regulating apoptosis and maintaining cellular homeostasis. Studies have shown that dysregulation of this pathway leads not only to reduced FAS expression but also to aberrant FASLG expression in a variety of tumors, including SCCHN (9–12). The FAS/FASLG pathway may also participate in the immunosuppression process observed in head and neck cancer (12). These data collectively suggest that the FAS/FASLG pathway may play an important role in the development and progression of SCCHN.

The human FAS gene (GenBank accession no. AY450925), located on chromosome 10q24.1, consists of nine exons and eight introns, and encodes 334 amino acids (13). Two polymorphisms have been identified in the FAS promoter region: one in the silencer region, G to A substitution at nucleotide position −1377 (FAS −1377 G>A, rs2234767), and the other in the enhancer region, A to G substitution at nucleotide position −670 (FAS −670 A>G, rs1800682). These two polymorphisms are located within the Sp1 and the signal transducers and activators of transcription 1 transcription factor binding sites, respectively (13, 14). Because these sequence variations in the FAS gene promoter region may influence FAS expression and dysregulate cell death signaling, they could contribute to carcinogenesis (14).

The human FASLG gene (GenBank accession no: Z96050) is located on chromosome 1q23, consists of four exons spanning ∼8 kb, and encodes 281 amino acids (15). There are two reported polymorphisms: C to T changes at nucleotide position −844 (FASLG −844 C>T, rs763110) in the promoter region (16) and A to G change at nucleotide position −124 of intron 2 (FASLG INV2nt −124 A>G, rs5030772; ref. 17). FASLG −844 C>T is located in a putative binding motif for a transcription factor, CAAT/enhancer-binding protein β, and the −844 C allele may increase basal expression of FASLG compared with the −844 T allele (16), suggesting that the FASLG −844 C>T polymorphism may influence FASLG expression and FASLG-mediated signaling, and ultimately, the susceptibility to cancer. To date, the functional relevance of the FASLG IVS2nt −124 A>G polymorphism has not been reported.

The FAS −1377 G>A, FAS −670 A>G, and FASLG −844 C>T polymorphisms have been reported to be associated with increased risk of developing esophageal squamous cell carcinoma in a northern Chinese population (18), and the FAS −1377 G>A and FASLG −844 C>T polymorphisms were found to be associated with increased risk of lung cancer (19). Our previous study showed that the FAS −670 A>G polymorphism was associated with an increased risk of lung cancer (20), and a similar association was reported with cervical cancer in a Japanese population (21). Both the FAS −1377 G>A and FAS −1377 A/−670 A haplotype were also found to be associated with risk of acute myeloid leukemia (14). No association was found, however, between the FAS −670 A>G polymorphism and risk of non–melanoma skin cancer (22), although mutations in the FAS gene were reportedly involved in skin carcinogenesis (23, 24). To the best of our knowledge, there is no report on the association between the FAS and FASLG polymorphisms and risk of SCCHN.

Because of the role of the FAS and FASLG genes in regulating cell death, and because abnormal expression of FAS and/or FASLG has been observed in a variety of tumors, including SCCHN, we hypothesized that the FAS and FASLG polymorphisms contribute to genetic susceptibility to SCCHN. To test this hypothesis, we genotyped four polymorphisms of FAS and FASLG genes in our ongoing hospital-based case-control study of SCCHN and evaluated their associations with the risk of developing SCCHN.

Study subjects and data collection. The recruitment of study subjects has been described previously (25). Because this analysis was built on an ongoing DNA phenotype study, only those patients who did not have any treatment at the time of recruitment were eligible. Briefly, the study population included 721 patients with newly diagnosed and pathohistologically confirmed SCCHN and 1,234 cancer-free control subjects recruited between May 1995 and September 2003. Approximately 95% of the eligible patients contacted (among those registered with the M.D. Anderson Cancer Center) chose to participate in this study. The response rate among the eligible patients with SCCHN was 95%. Among the 721 patients with primary SCCHN who were included in the analysis, 222 (30.8%) had cancers of the oral cavity, 362 (50%) had cancers of the pharynx, and 131 (19%) had cancers of the larynx. Patients with second SCCHN primary tumors, primary tumors of the nasopharynx or sinonasal tract, primary tumors outside the upper aerodigestive tract, cervical metastases of unknown origin, or histopathologic diagnoses other than SCC were excluded.

The regional lymph node (N) involvement of pharyngeal cancer was defined from N₀ to N₃ as follows (26): N₀, no regional node metastasis; N₁, metastasis in a single ipsilateral lymph node, ≤3 cm in the greatest dimension; N₂, metastasis in a single ipsilateral lymph node, >3 cm but <6 cm in the greatest dimension; or in multiple ipsilateral lymph nodes, none >6 cm in the greatest dimension; or in bilateral or contralateral lymph nodes, <6 cm in the greatest dimension; N₃, metastasis in a lymph node >6 cm in the greatest dimension. The extent of the primary pharyngeal cancer was defined from T₁ to T₄ as follows (26): T₁, tumor ≤2 cm at the greatest dimension (oropharynx) or tumor limited to one subsite of hypopharynx (hypopharynx); T₂, tumor >2 cm but <4 cm in the greatest dimension (oropharynx) or tumor invades more than one subsite of the hypopharynx or an adjacent site, without fixation of hemilarynx (hypopharynx); T₃, tumor >4 cm in the greatest dimension (oropharynx) or the tumor invades more than one subsite of the hypopharynx or an adjacent site, with fixation of hemilarynx (hypopharynx); and T₄, tumor invades adjacent structures.

Cancer-free control subjects were recruited from persons who were not hospital patients or seeking health care, and who accompanied the case patients to the clinics. We first surveyed potential control subjects at the clinics by using a screening sheet to determine their willingness to participate in research studies and to obtain information on demographics, smoking status, and personal history of cancer. We then selected the frequency-matched eligible subjects to the cases by age (±5 years) and sex. Among the respondents for the screening, ∼90% were eligible (no personal cancer history) and the response rate was ∼90% in those we further contacted for recruitment. We interviewed each eligible subject to obtain data on tobacco smoking and alcohol use. After signing informed consent forms, each subject donated 30 mL of blood, of which, 1 mL was used for genomic DNA extraction with a DNA blood Mini Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's instructions. The research protocol was approved by the M.D. Anderson Institutional Review Board. It should be noted that in such a hospital-based case-control study, these cases and controls were not a random sample of a defined population and that the cases were not a random sample of all patients seen at M.D. Anderson. However, it is unlikely that the genotype of the subjects may have determined the selection of the subjects.

Genotyping.FAS and FASLG polymorphisms were identified by using the PCR-RFLP method, as previously described (27). The following primers were used to amplify the target fragments containing these four polymorphisms (mismatch bases are underlined): 5′-TGTGTGCACAAGGCTGGCGC-3′ (forward) and 5′-TGCATCTGTCACTGCACTTACCACCA-3′ (reverse) for FAS −1377 G>A, 5′-ATAGCTGGGGCTATGCGATT-3′ (forward) and 5′-CATTTGACTGGGCTGTCCAT-3′ (reverse) for the FAS −670 A>G (18), 5′-CAATGAAAATGAACACATTG-3′ (forward) and 5′-CCCACTTTAGAAATTAGATC-3′ (reverse) for FASLG −844 C>T, and 5′-GCAGTTCAGACCTACATGATTAGGAT-3′ (forward) and 5′-CCAGATACAGACCTGTTAAATGGGC-3′ for FASLG IVS2nt −124 A>G (28). The amplified PCR products were 122, 193, 85, and 230 bp for −1377 G>A, −670 A>G, −844 C>T, and IVS2nt −124 A>G polymorphisms, respectively. The BstUI, ScrFI, DraIII, and FokI restriction enzymes (New England Biolabs, Beverly, MA) were used to distinguish the −1377 G>A, −670 A>G, −844 C>T, and IVS2nt −124 A>G polymorphisms, respectively, which resulted in 104 and 18 bp fragments in the case of the −1377 G allele; 136 and 57 bp fragments in the case of the −670 G allele; 66 and 19 bp fragments in the case of the −844 T allele; and 180 and 50 bp fragments in the case of the IVS2nt −124 G allele. More than 10% of the samples were randomly selected for confirmation, and the results were 100% concordant.

Statistical analysis. We used the χ² test to evaluate differences in the frequency distributions of selected demographic variables, smoking status, alcohol use, and each allele and genotype of the four polymorphisms of the FAS and FASLG genes between the cases and controls. Unconditional univariate and multivariate logistic regression analyses were done to obtain the crude and adjusted odds ratios (OR) for risk of SCCHN and their 95% confidence intervals (CI). Multivariable adjustment was conditional on effects of age, sex, smoking status, and alcohol use. Furthermore, the 2LD software was used to calculate the D′ value among the two FAS polymorphisms (−1377 G>A and −670 A>G) and among the two FASLG polymorphisms (−844 C>T and IVS2nt −124 A>G; refs. 29, 30). Considering the potential interaction of the FAS and FASLG genes on risk of SCCHN, the associations between the combined genotypes of the FAS or FASLG polymorphisms and risk of SCCHN were evaluated. The combined genotype data were further stratified by subgroups of age, sex, smoking status, alcohol use, and primary tumor site. To increase the statistical power, a priori genetic models (that is, dominant, codominant, or recessive) were assumed to collapse the genotype data. We tested the null hypotheses of additive and multiplicative gene-gene interactions and assessed departures from additive and multiplicative interaction models (31). A greater-than-multiplicative interaction was suggested when OR₁₁ > OR₀₁ × OR₁₀, in which OR₁₁ is the OR when both factors were present, OR₀₁ is the OR when only factor 1 was present, OR₁₀ is the OR when only factor 2 was present (31). To assess evidence for departure from a multiplicative model, we modeled interaction terms between variables using standard unconditional logistic regression. We were specifically interested in searching for interactions indicating a greater-than-multiplicative relationship (i.e., interaction terms from the logistic regression with positive coefficients) because these interactions identify subgroups of individuals who may be at particularly high risk for developing SCCHN. We were also interested in identifying departures from additive models. Empirically, a greater-than-additive interaction was indicated if OR₁₁ > OR₁₀ + OR₀₁ − 1. When the test for multiplicative interaction was not rejected, further tests for additive interaction were done by a bootstrapping test of goodness of fit of the null hypothesis of an additive model with no interaction against an alternative hypothesis that allows an additive interaction. To perform the hypothesis test for additive models, we implemented bootstrapping using Stata 8.2 (Stata Corp. LP, College Station, TX). All statistical tests were two-sided, and P < 0.05 was considered statistically significant. We analyzed all data, except for additive models, using SAS software (version 8e; SAS Institute, Cary, NC).

Characteristics of the study population. The frequency distributions of selected characteristics of the case patients and control subjects are presented in Table 1. The cases and controls seemed to be well matched on age and sex: the mean age was 57.0 years for the cases (±11.9 years; range, 18-90 years) and 57.2 years for the controls (±11.6 years; range, 20-87 years; P = 0.277), and 74.9% and 25.1% of the cases and 74.1% and 25.9% of the controls were men and women, respectively (P = 0.686). As expected, however, there were more current smokers (34.8%) and drinkers (51.2%) among the cases compared with controls (25.4% and 43.7%, respectively), and these differences were statistically significant (P < 0.001 for both exposures). Therefore, these variables were further adjusted for in the multivariate logistic regression analysis.

Association between the FAS and FASLG polymorphisms and risk of SCCHN. As shown in Table 2, the frequencies of minor −1377 A and −670 G alleles were higher in the cases (0.122 and 0.483, respectively) than in the controls (0.118 and 0.452, respectively) but the difference was not statistically significant (P = 0.748 and P = 0.065, respectively). The −1377 AA genotype was more frequent in the cases (2.4%) than in the controls (1.0%) and was associated with a significantly increased risk of SCCHN compared with the GG genotype (adjusted OR, 2.23; 95% CI, 1.07-4.64) compared with the −1377 GG genotype, and the risk did not change substantially under the assumption of a recessive genetic model (OR, 2.27; 95% CI, 1.09-4.72), compared with the variant genotypes (i.e., GG + AG). The −670 GG was also more frequent in the cases (23.2%) than in the controls (20.9%) and was associated with a borderline significantly increased risk of SCCHN (OR, 1.29; 95% CI, 0.99-1.68), and the risk associated with the variant genotypes (i.e., GG + AG) under the assumption of a dominant genetic model became statistically significant (OR, 1.24; 95% CI, 1.01-1.52) than in the −670 AA genotype. For the two FASLG polymorphisms, the frequencies of minor −844 T and IVS2nt −124 G alleles were lower in the cases (0.333 and 0.141, respectively) compared with the controls (0.354 and 0.147, respectively) but the differences were not statistically significant (P = 0.195 and P = 0.640, respectively). Although there were no statistically significant associations between these two polymorphisms and risk of SCCHN, the frequencies of the variant (i.e., −844 T and IVS2nt −124 G) genotypes were lower in the cases than in the controls. All distributions of the frequencies of observed genotypes in the controls were consistent with the Hardy-Weinberg equilibrium model (Table 2).

Association between the combined genotypes of the FAS or FASLG polymorphisms and risk of SCCHN. Further analysis suggested that there was linkage disequilibrium between the two FAS polymorphisms (D′ = 0.875, P < 0.001 for −1377 G>A and −670 A>G) and the two FASLG polymorphisms (D′ = 0.892, P < 0.001 for −844 C>T and IVS2nt −124 A>G). Such an incomplete linkage disequilibrium suggests, however, that a joint effect between the two FAS polymorphisms or the two FASLG polymorphisms may exist. To evaluate the genotype-genotype interaction, we dichotomized the FAS genotypes as either −1377 (GG + AG) or −1377 AA and −670 AA or −670 (GG + GA), and the FASLG genotypes as either −844 CC or −844 CT + TT, and −IVS2nt −124 AA or −IVS2nt −124 (AG + GG; Table 3). When we used the combined genotype of the −1377 (GG + AG)/−670 AA as the reference, we found that only the −1377 AA/−670 (GG + AG) genotype was associated with a significantly higher risk of SCCHN (OR, 2.69; 95% CI, 1.24-5.83). In contrast, compared with the −844 CC and -IVS2nt −124 AA genotype, none of the other genotype combinations was associated with risk of SCCHN, although they tended to be associated with a lower risk (Table 3). No statistical evidence was found for any interaction from either multiplicative or additive models between the two FAS polymorphisms or the two FASLG polymorphisms.

Association and stratification analysis of the combined genotypes of the FAS polymorphisms associated with risk of SCCHN. We then focused on the stratification analysis of the FAS polymorphisms because we found some association of the FAS, but not FASLG, polymorphisms with risk of SCCHN. To facilitate further analysis, we dichotomized combined genotypes of the two FAS polymorphisms into two groups, i.e., with −1377 (GG + AG)/−670 AA and without −1377 (GG + AG)/−670 AA [i.e., the −1377 (GG + AG)/−670 (GG + AG), −1377 AA/−670 AA, and −1377 AA/−670 (GG + AG) all combined]. As shown in Table 4, compared with the −1377 (GG + AG)/−670 AA genotype, we found that individuals carrying the combined genotypes without −1377 (GG + AG)/−670 AA had a higher risk of SCCHN (adjusted OR, 1.24; 95% CI, 1.01-1.52), and this increased risk was more pronounced among the subgroups of patients ages ≥50 years (OR, 1.32; 95% CI, 1.04-1.68), women (OR, 1.67; 95% CI, 1.09-2.56), and patients with pharyngeal cancer (OR, 1.32; 95% CI, 1.01-1.73). However, these ORs between subgroups were not statistically different, and the P value for the additive interaction was 0.213 and 0.066 for the age and sex subgroups, respectively.

Association between the combined genotypes of the FAS polymorphisms and progression of pharyngeal cancer. Because pharyngeal cancer was the predominant group among the cases and was associated with the combined variant genotypes of FAS, we further evaluated the association between the combined genotypes and progression of pharyngeal cancer. In this analysis, we found a statistically significant association between the combined genotypes and the extent of primary pharyngeal tumor (T) as well as regional lymph node metastasis at diagnosis (N; data not shown). However, when we incorporated the T and N data into three groups (i.e., T_1-2N_2-3, T_3-4N₀, and others), we found that the combined genotypes without −1377 (GG + AG)/−670 AA were associated with risk of T_3-4N₀ pharyngeal cancer compared with the combined genotypes with −1377 (GG + AG)/−670 AA (adjusted OR, 1.77; 95% CI, 1.07-2.94; Table 5). There was no such association observed between these combined genotypes and T_3-4N₀ oral cavity cancer and laryngeal cancer (data not shown). Lastly, we examined the distributions of the haplotypes and combined diplotype of both genes, but we did not find any significant difference (data not shown).

In the present study, we investigated the association between four known polymorphisms in the promoters of the FAS and FASLG genes, and risk of developing SCCHN in a hospital-based case-control study. We found that both the −1377 AA and −670 (GG + AG) genotypes were associated with an increased risk of SCCHN and a joint effect on risk, but not for the FASLG variant genotypes. The joint effects of the two FAS polymorphisms on risk of SCCHN were more pronounced among older female subjects, ever drinkers, and patients with pharyngeal cancer with larger tumors without regional lymph node metastases. To the best of our knowledge, no reported study has investigated the association between the FAS and FASLG polymorphisms and risk of SCCHN. Given the role of the FAS/FASLG pathway in carcinogenesis, it is biologically plausible that the FAS and FASLG polymorphisms may modulate risk of cancer.

There are several lines of evidence that support our findings. Cell death by the apoptotic pathway is essential for maintaining the normal function of cells, but it can be initiated or inhibited by a variety of stimuli (32). It has been shown that alterations of FAS and FASLG expression decrease the apoptotic capacity of cells and that many tumor cells might evade or suppress the immune system (33, 34). The loss of FAS or gain of FASLG expression are common features of most human malignancies and are associated with the progression of cancers, including SCCHN (9–12). Experimental data also suggest that this FAS/FASLG pathway may participate in immunosuppression, a phenomenon that has been observed in head and neck cancers as well (12). Therefore, the variants of the FAS and FASLG genes, if functional, could be expected to have an effect on cell death, and thus, carcinogenesis.

Several association studies have reported that the FAS and FASLG polymorphisms are associated with risk of diseases including cancer and systemic lupus erythematosus in different ethnic groups (14, 16, 18–20, 35, 36), although one study reported the lack of an association between the FAS −670 A>G polymorphism and skin cancer (22). Our data from this much larger study further support the notion that the FAS −1377 G>A and −670 A>G polymorphisms (located in the Sp1 and signal transducers and activators of transcription factor 1 binding sites, respectively) are potentially implicated in cancer risk.

In the present study, we observed a significantly increased risk of SCCHN among ever drinkers, suggesting that a gene-environment interaction may be involved in the development of SCCHN. It is reported that ethanol-mediated alteration of caspase-3 activation may play a role in cell apoptosis (37, 38), which may explain the possible role of the FAS polymorphisms in alcohol-induced SCCHN. However, this finding may be by chance, owing to the small number of observations in the stratified analysis. More interestingly, the finding that the risk associated with the combined genotypes without FAS −1377 (GG + AG)/−670 AA was significantly higher for pharyngeal cancer with larger tumors without regional lymph node metastasis, suggesting that the combined FAS genotypes may be associated with progression of pharyngeal cancer, or alternatively, that pharyngeal carcinoma may have a different etiology in terms of genetic susceptibility. It is possible that these results may be due to selection bias which is common in hospital-based case-control studies, and it is difficult to compare with genotyping data from other studies because few studies on FASLG polymorphisms have been published. However, the 42.3% of FASLG −844 CC and 44.7% of FASLG −844 CT genotypes of our 1,234 Caucasian controls were similar to previously published data in one study on American Caucasians (16). Because there were other genetic variants that were not assayed in this study, this hypothesis needs to be tested in future studies with a larger number of patients with pharyngeal cancer and data from dense gene maps, such as the HapMap database (39).

In conclusion, FAS (but not FASLG) polymorphisms have a main effect on risk of developing SCCHN. The combined genotypes of the FAS polymorphisms were associated with a significantly increased risk of SCCHN, which was more pronounced among older women, drinkers, and patients with pharyngeal cancer. We also found that the combined FAS genotypes were associated with a statistically significantly increased risk of pharyngeal cancer with larger tumors, but no regional lymph metastasis. These findings suggest that the FAS polymorphisms may jointly contribute to risk and progression of SCCHN, particularly for pharyngeal cancer. However, because the number of observations in the subgroup analyses were limited, the findings could be due to chance. In addition, because the frequency of the genotypes associated with risk of SCCHN was very low, the attributable risk in the general population might be low as well. Additional studies which include a larger number of patients with pharyngeal cancer, more detailed data on environmental exposure, inclusion of more single nucleotide polymorphisms in one gene or genes in the same biological pathway, and survival data are required to verify these findings. However, it is likely that the low participation rate relative to all SCCHN patients seen at M.D. Anderson may have introduced potential selection bias, particularly when the genotype may be associated with disease progression. Therefore, future population-based studies are needed to verify the findings.

We thank Margaret Lung, Peggy Schuber, and Leanel Fairly for their assistance in recruiting the subjects; Zhensheng Liu for his technical support; Qiuling Shi, Xiangjun Gu, and Shenying Fang for their help on data analysis; Jianzhong He and Kejin Xu for their laboratory assistance; Betty J. Larson and Joanne Sider for manuscript preparation; and Kathryn Hale for scientific editing.