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A polymorphism in FAS gene promoter associated with increased risk of nasopharyngeal carcinoma and correlated with anti-nuclear autoantibodies induction
Cancer Letters 233 (2006) 21–27 www.elsevier.com/locate/canlet
A polymorphism in FAS gene promoter associated with increased risk of nasopharyngeal carcinoma and correlated with anti-nuclear autoantibodies induction* Besma Bel Hadj Jrada,b, Wijden Mahfoutha, Noureddine Bouaouinaa,c, Sallouha Gabbouja, Slim Ben Ahmedd, Mondher Ltaı¨efe, Majida Jalbouta, Lotfi Chouchanea,* a
Laboratoire d’Immuno-Oncologie Mole´culaire, Faculte´ de Me´decine de Monastir, Universite´ du Centre, 5019 Monastir, Tunisia b Institut Supe´rieur de Biotechnologie de Monastir, Monastir, Tunisia c Department of Cance´rologie Radiothe´rapie, CHU Farhat Hached, Sousse, Tunisia d Department of Carcinologie Me´dicale, CHU Farhat Hached, Sousse, Tunisia e Department of Me´decine Communautaire, Faculte´ de Me´decine de Monastir, Monastir, Tunisia Received 1 January 2005; received in revised form 20 February 2005; accepted 25 February 2005
Abstract Loss of FAS (CD95) expression is a common feature of malignant transformation, which has been related to loss of epithelial cell differentiation and loss of sensitivity to apoptosis. We investigated the potential association between FAS promoter polymorphism and the genetic susceptibility to the Epstein–Barr virus (EBV)-related nasopharyngeal carcinoma. The in vivo functional significance of the FAS polymorphism was investigated by assessing the correlation between FAS genotypes and the presence of autoantibodies to cytoskeleton and nuclear antigens frequently detected in nasopharyngeal carcinoma. We determined the FAS polymorphism distributions by RFLP-PCR in 170 patients with nasopharyngeal carcinoma and in 224 sex and age-matched controls. We used ELISA and the immunofluorescence analysis to characterize the presence of IgG autoantibodies to the cytoskeleton and nuclear proteins in patients’ sera. A significantly increased risk of nasopharyngeal carcinoma was associated with heterozygote FAS-A/G (ORZ2.00, PZ0.001) and homozygote FAS-G/G (ORZ3.19, PZ0.0001) variants. The increased frequency of FAS-G/G genotype is correlated with the presence of anti-nuclear autoantibodies in patients with nasopharyngeal carcinoma (PZ0.0298). Our results demonstrated that FAS promoter polymorphism was significantly associated with the nasopharyngeal carcinoma in Tunisians. The anti-nuclear autoantibodies induction was also found to be related to FAS polymorphism. The FAS
* This work was supported by le Ministe`re de la Recherche Scientifique et de Technologie, by le Ministe`re de l’Enseignement Supe´rieur, by le Ministe`re de la Sante´ Publique de la Re´publique Tunisienne. * Corresponding author. Tel.: C216 73 462 200; fax: C216 73 460 737. E-mail address: [email protected] (L. Chouchane).
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.02.037
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promoter polymorphism associated not only with the increased risk of nasopharyngeal carcinoma in Tunisians but also with immune response deregulation observed in this cancer. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: FAS; Polymorphism; Nasopharyngeal carcinoma; Apoptosis; Autoantibodies
1. Introduction The nasopharyngeal carcinoma classification distinguishes two major types, squamous cell carcinoma and non-keratinizing carcinoma. The later includes the undifferentiated carcinoma of the nasopharyngeal type (UCNT). The association between EBV infection and nasopharyngeal carcinomas was initially suggested based on serological studies [1] and has been subsequently substantiated by the detection of viral genomes and gene products in the epithelial tumor cells [2,3]. The nasopharyngeal carcinoma (NPC) shows a distinct geographical distribution with welldefined high-risk ethnic groups. While it is rare in western countries, its incidence is high in certain regions such as Southeast Asia. In North Africa, the NPC has an intermediate incidence [4]. The NPC from North Africa and Asia share several common clinical and biological characteristics, but they differ with regard to their age distribution. In Asia, NPC mainly affects patients in the 4th or 5th decade of their life, whereas in North Africa an additional peak of incidence is found confined to young population [5]. In a recent study [6], we demonstrated that the autoantibodies to the cytoskeleton and nuclear proteins are associated with the nasopharyngeal carcinoma in Tunisians. Given the importance of apoptosis in immune response regulation and in antitumor immunity, aberrant apoptosis could conceivably associate with tumorigenesis and immune system deregulation. FAS (CD95, APO-1) is a cell surface receptor involved in apoptotic signal transmission in many cell types, including cells of the immune system [7]. The death signal cascade is initiated upon cross-linking of FAS by its natural ligand (FASL) [8]. Whereas FAS expression and susceptibility to FASL-mediated apoptosis is a common feature of most non-malignant tissues in humans, constitutive expression of FASL is restricted to activated T and NK cells and a few immuno-privileged sites [9]. FAS-mediated
apoptosis, which is progressively lost during tumorigenesis can be inhibited in several ways. There is now broad evidence showing that malignant cells take advantage of aberrant loss of FAS [10–12] and gain of FASL [13–15] expression compared to their non-malignant counterparts. Aberrant FAS and FASL expression reflects loss of differentiation in tumor cells resulting in loss of sensitivity to apoptosis [16,17]. Mutation of the FAS gene is another mechanism contributing to resistance of malignant cells to FASL mediated apoptosis. Studies have revealed the occurrence of mutations of the FAS gene in human germline resulting in autoimmune lymphoproliferative syndrome [18–20]. Somatic FAS mutations impairing the transduction of the apoptotic signal were described in lymphoid tumors [21–23]. More recently, somatic FAS gene mutations have also been found in solid tumors but the functional relevance of these findings remains to be established [24–27]. FAS-mediated apoptosis can also be inhibited by a soluble form of the FAS molecule generated by alternative splicing and lacking the transmembrane domain, which anchors the receptor molecule within the cell membrane of the target cell. Hence, soluble FAS no longer transduces the death signal after binding to FASL and competes with transmembrane FAS for FASL binding [28,29]. Competitive FASL antagonism by soluble FAS efficiently prevents lymphocyte killing both in vitro [9] and in vivo [30,31]. Several studies have shown increased levels of sFAS in several tumor types which correlated in certain cases with poor prognosis [32–34]. A genetic polymorphism (A to G substitution) in the promoter region of the FAS gene located at the nucleotide position of K670 from the transcriptional start site has been reported [35]. This polymorphism alters the binding site of the nuclear transcription element GAS. In vitro studies have shown that Stat1 binding activity was higher in K670A allele of FAS promoter than in K670G allele and that
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the transcriptional activity of A allele tended to be higher than that of G allele [36]. Hence, it was speculated that this polymorphism might result in the alteration of FAS gene expression leading to the decrease of FAS-mediated apoptosis. In line with this hypothesis, we investigated the potential association of the K670 (A/G) FAS polymorphism with the increased risk of nasopharyngeal carcinoma and that of autoantibodies induction in this cancer.
2. Materials and methods 2.1. Patients and controls The allele and genotype frequencies at the position K670 (A/G) of the FAS gene were determined in a group of 170 unrelated patients with undifferentiated nasopharyngeal carcinoma (UCNT), and in 224 unrelated control subjects. Controls and patients were age and sex matched and were selected from the same population living in the middle coast of Tunisia. Patients with UCNT were recruited from the Department of Radiation Oncology and Medical Oncology of Sousse Hospital, between 1991 and 2003. The patients with UCNT (118 males and 52 females) had a mean age of 41.5G16 years. The clinical stages ranged from II to IV (TNM classification, 1987). The diagnosis of cancer was confirmed by histopathology analyses. The histology was undifferentiated carcinoma (type III, WHO classification) in all cases [37]. Control subjects (147 males and 77 females) having a mean age of 40G12.5 years were healthy blood donors having no evidence of any personal or family history of cancer (or other serious illness). Written informed consent was obtained from all subjects. 2.2. DNA extraction Genomic DNA was extracted from peripheral blood leukocytes by using the salting out procedure [38]. Briefly, 5 ml of blood was mixed with Triton lysis buffer (0.32 M sucrose, 1% Triton X100, 5 mM MgCl2, H2O, 10 mM Tris–HCl, pH 7.5). Leucocytes were spun down
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and washed with H2O. The pellet was incubated with proteinase K at 56 8C and subsequently salted out at 4 8C using a saturated NaCl solution. Precipitated proteins were removed by centrifugation. The DNA in the supernatant fluid was dissolved in 400 ml H2O. 2.3. Polymorphism analysis of the K670 (A/G) FAS gene Based on the method described by Huang et al. [35], a polymorphism chain reaction followed by digestion with endonuclease MvaI was used to detect the A to G transition polymorphism at position K670 of FAS gene. Two sequence specific oligonucleotide primers were used for the polymerase chain reaction (PCR): the 3 0 primer (5 0 -GGCTGTCCATGTTGTGGCTGC-3 0 ) was used in combination with the 5 0 primer (5 0 CTACCTAAGAGCTATCTACCGTTC-3 0 ). PCR was performed by using 60 ng genomic DNA as template, 0.01 mM dNTPs, 1.5 mM MgCl2, 1! Taq polymerase buffer, 0.25 mM of each primer and 0.5 U of Taq DNA polymerase (Amersham, Paris, France) in a total reaction of 20 ml. Reaction conditions used with the thermal cycler (Biometra, Go¨ttingem, Germany) were as follows: 94 8C for 6 min followed by 28 cycles at 94 8C for 30 s, 62 8C for 30 s and 72 8C for 1 min. A final extension step was carried out at 72 8C for 10 min. The 331 bp PCR products were digested with MvaI and analyzed by agarose-gel electrophoresis. The presence of the A nucleotide at the position K670 of the FAS gene was indicated by the cleavage of the amplified product into two fragments of 233 and 98 bp. An additional MvaI site is generated by G substitution yielding to the cleavage of the amplified product into three fragments of 189, 98 and 44 bp. 2.4. Statistical analysis The allele frequencies of FAS were tested for the Hardy–Weinberg equilibrium for both patient and control groups using the chi-square test. The same test was used to evaluate for significant association between disease (nasopharyngeal carcinoma against controls) and FAS genotypes. Relative risk of nasopharyngeal carcinoma associated with a particular genotype was estimated by the odds ratio (OR). The chi-square test (or Fisher’s exact test when n!5) was used to determine whether significant
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washes with PBS, antibodies were revealed by antihuman Igs antibodies (IgG, IgA, and IgM) labeled with fluorescein.
differences (P-value) in the autoantibodies induction to cytoskeleton and nuclear antigens were observed between carriers of different genotypes. The non-parametric Mann–Whitney U-test was used to determine whether significant differences (P-value) in the soluble FAS (sFAS) levels were seen between carriers (patients and controls combined) of different genotypes. Statistical analyses were performed using the Statistical Package of Social Sciences for Windows (SPSS version 10.0). Differences between groups were tested for significance by the two-tailed test. A P-value less than 0.05 was considered significant.
3. Results 3.1. Polymorphism in the FAS gene as risk factor for nasopharyngeal carcinoma Table 1 shows genotype frequencies for FAS gene in patients with nasopharyngeal carcinoma and in the control group. These distributions were in Hardy– Weinberg equilibrium for both patient and control groups. The allelic frequency of the K670 (G) FAS was 0.568 in patients with nasopharyngeal carcinoma and 0.433 in control subjects (ORZ1.72, PZ0.0001). The frequency of FAS-A/G heterozygotes was 0.535 in the patient group and 0.509 in the control population resulting in a significantly OR associated with this genotype (ORZ2.00, PZ0.001). The frequency of the FAS-G/G homozygotes was 0.30 in the patient group and only 0.178 in controls (ORZ3.19, PZ0.0001). These data suggest an increased risk of developing nasopharyngeal carcinoma is associated with inheritance of the (K670) FAS-G allele in a dose-dependant manner. Since the highest incidence of the EBV-associated nasopharyngeal carcinomas in young subjects was found in North Africa, we stratified the patients according to age (younger or older than 25 years). No significant differences in FAS genotype distributions were found between the subgroups (data not shown).
2.5. ELISA for autoantibodies Sera autoreactivity to the cytoskeleton and nuclear proteins was determined on serum samples obtained prior to cancer therapy. Sera from 92 patients with primary UCNT were available before treatment. Each serum collected was tested individually. As negative control for the reaction, 82 sera from healthy individuals were pooled and used in each test. The autoantibodies detection was carried out as previously described [6]. 2.6. Immunofluorescence analysis Identification of anti-nuclear antibodies (ANA) by indirect immunofluorescence was carried out using cryostat sections prepared from liver tissue of young rats. These liver sections were deposed on glass and incubated overnight at K80 8C. Briefly, serum was diluted at 1/100 with PBS (pH 7.4) and deposited on liver sections. After 30 min of incubation and three
Table 1 The (K670) FAS genotype distribution in control subjects and in patients with nasopharyngeal carcinoma Genotype (K670) FAS
Patients (nZ170)
Controls (nZ224)
n
f
n
f
FAS-A/A FAS-A/G FAS-G/G
28 91 51
0.165 0.535 0.300
70 114 40
0.313 0.509 0.178
ORa
1 2.00 3.19
Confidence interval
P-value
[1.19 3.33] [1.76 5.77]
0.001 0.0001b
The chi-square test was used to determine whether significant differences (P-value) were observed when patient group was compared with control group. f, frequencies. a Risk estimates were evaluated by unconditional logistic regression analysis with adjustment for age and sex. b The association remains significant under the recessive model (genotype FAS-G/G compared to genotype FAS-A/GCFAS-A/A) with ORZ1.97 and PZ0.001.
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Table 2 Association between (K670) FAS polymorphism and autoantibodiesa induction in patients with nasopharyngeal carcinomab Genotype
MG, n (%) (C) (nZ12)
FAS-A/A FAS-A/G FAS-G/G P-value
4 (33.3) 5 (41.7) 3 (25)
MS, n (%) (K) (nZ80)
9 (11.2) 46 (57.6) 25 (31.2) 0.122
(C) (nZ11) 2 (18.2) 6 (54.5) 3 (27.3)
ACT, n (%) (K) (nZ81)
11 (13.6) 45 (55.4) 25 (31) 0.91
(C) (nZ11) 1 (9.1) 5 (45.5) 5 (45.4)
TUB, n (%) (K) (nZ81)
12 (14.8) 46 (57) 23 (28.4) 0.502
(C) (nZ16) 3 (18.7) 6 (37.5) 7 (43.8)
ANA, n (%) (K) (nZ76)
10 (13.2) 45 (59.2) 21 (27.6) 0.28
(C) (nZ22) 4 (18.2) 7 (31.8) 11 (50)
(K) (nZ70) 9 (12.8) 44 (62.9) 17 (24.3) 0.0298
The chi-square (or Fisher) test was used to determine whether significant differences (P-value) in the autoantibodies induction to cytoskeleton and nuclear antigens were observed between carriers of different genotypes. MS, myosin; ACT, actin; TUB, tubulin; MG, myoglobulin; ANA, anti-nuclear antibodies. a IgG autoantibodies, except for anti-nuclear antibodies where reactivity was deducted from different classes of Ig (IgG, IgM, and IgA). b Sera from patients were taken before cancer treatment.
3.2. Sera autoreactivity and Fas genetic polymorphism in nasopharyngeal carcinoma In a previous study [6], we showed a significant increase in the serum autoreactivity to tested cytoskeleton and nuclear proteins in the patients with nasopharyngeal carcinoma. The most frequent autoreactivity was found with nuclear antigens (NA) in patient’s sera compared to that of control subjects (22% in patients vs 1.2% in controls; PZ0.00003). Table 2 shows the sera autoreactivity of patients with nasopharyngeal carcinoma according to the FAS polymorphism. The frequency of FAS-G/G genotype is higher in patients producing anti-nuclear antibodies than in those without (0.50 vs 0.243, PZ0.0298), resulting in a significant association between the presence of anti-nuclear antibodies (ANA) and the FAS-G/G homozygote genotype. Conversely, the frequency of FAS-A/G is lower in patients having anti-nuclear antibodies (0.318 vs 0.629).
4. Discussion Although environmental factors, including exposure to EBV, are clearly important determinants of nasopharyngeal carcinoma susceptibility, a significant hereditary contribution to the etiology of this cancer has been suggested by several studies [4,39]. Genes encoding receptors involved in apoptosis could be attractive susceptibility candidate genes for this malignancy. Hence, in this study, we assessed the potential association of the variation in the promoter
of FAS gene with nasopharyngeal carcinoma in the Tunisian population. The present case/controlled study showed a substantially increased risk of nasopharyngeal carcinoma associated with inheritance of the (K670) FAS-G allele in a dose-dependant manner. Individuals homozygous for (K670) FAS-G have more than 3-fold risk to develop nasopharyngeal carcinoma (ORZ3.19, PZ0.0001), with heterozygotes having an intermediate risk (ORZ2.00, PZ0.001) compared with individuals homozygous for (K670) FAS-A. It has been shown that the FAS-G allele associated with reduced FAS transcriptional activity compared to the FAS-A allele [36]. Thus, it is expected that the FAS-G allele might result in the alteration of FAS gene expression leading to the decrease of FAS-mediated apoptosis of tumor cells. However, Lai et al. [40] reported recently that Japanese subjects carrying the FAS-A/A might have an increased risk of cervical cancer. To explain their finding, they speculate that more efficient FAS expression associated with FAS-A allele could lead to the apoptosis of immune cells. Although the (K670) FAS polymorphism has been found with potentially different transcriptional efficiency, no direct evidence of its involvement in FAS function or in any immune deregulation aspects has been shown. Therefore, we assessed the potential in vivo significance of FAS polymorphism by investigating the correlation between FAS genotypes and the presence of autoantibodies to cytoskeleton and nuclear antigens frequently detected in nasopharyngeal carcinoma. In a previous study [6], we showed that the frequency of sera containing autoantibodies to
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cytoskeleton and nuclear antigens was significantly increased in patients with nasopharyngeal carcinoma compared to that of healthy control subjects. In the present study, we showed that the (K670) FAS-G/G genotype is significantly higher in patients with nasopharyngeal carcinoma producing anti-nuclear antibodies than in those without. The frequency of FAS-G/G was higher in patients having autoantibodies to tubulin and actin than those without, but the difference in the genotype frequency did not reach statistical significance. Among possible mechanisms, which could explain the frequent presence of autoantibodies in patients carrying the FAS-G/G genotype, is the alteration of FAS expression on the surface of autoreactive B cells with FAS-G/G genotype, leading to their resistance to apoptosis and to their survival. These autoreactive cells will produce autoantibodies upon reactivation by EBV and/or exposure to nuclear and intracellular antigens generated by their shedding from viable cells or their release from dying tumor cells. Our findings are in agreement with those of several reports which suggest a causal effect of FAS alteration in the induction of autoimmune diseases [19,41–43]. Moreover, this FAS polymorphism was associated with SLE and with the presence of anti-RNP autoantibodies [44]. In conclusion, this study suggests that the FAS-G/G is associated with the susceptibility to nasopharyngeal carcinoma. Functional significance of FAS polymorphism was proved by its correlation with the antinuclear autoantibodies induction. Replication of this finding in a large population of NPC will be of use in determining whether the relation between the FAS polymorphism and anti-nuclear autoantibodies induction can be generalized.
Acknowledgements We gratefully acknowledge the technical assistance of O. Boughzala. We thank the staff of the Departments of Radiation Oncology, and Medical Oncology of CHU F. Hached, Sousse, and Dr A. Romdhane for providing samples and clinical information.
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A polymorphism in FAS gene promoter associated with increased risk of nasopharyngeal carcinoma and correlated with anti-nuclear autoantibodies induction* Besma Bel Hadj Jrada,b, Wijden Mahfoutha, Noureddine Bouaouinaa,c, Sallouha Gabbouja, Slim Ben Ahmedd, Mondher Ltaı¨efe, Majida Jalbouta, Lotfi Chouchanea,* a
Laboratoire d’Immuno-Oncologie Mole´culaire, Faculte´ de Me´decine de Monastir, Universite´ du Centre, 5019 Monastir, Tunisia b Institut Supe´rieur de Biotechnologie de Monastir, Monastir, Tunisia c Department of Cance´rologie Radiothe´rapie, CHU Farhat Hached, Sousse, Tunisia d Department of Carcinologie Me´dicale, CHU Farhat Hached, Sousse, Tunisia e Department of Me´decine Communautaire, Faculte´ de Me´decine de Monastir, Monastir, Tunisia Received 1 January 2005; received in revised form 20 February 2005; accepted 25 February 2005
Abstract Loss of FAS (CD95) expression is a common feature of malignant transformation, which has been related to loss of epithelial cell differentiation and loss of sensitivity to apoptosis. We investigated the potential association between FAS promoter polymorphism and the genetic susceptibility to the Epstein–Barr virus (EBV)-related nasopharyngeal carcinoma. The in vivo functional significance of the FAS polymorphism was investigated by assessing the correlation between FAS genotypes and the presence of autoantibodies to cytoskeleton and nuclear antigens frequently detected in nasopharyngeal carcinoma. We determined the FAS polymorphism distributions by RFLP-PCR in 170 patients with nasopharyngeal carcinoma and in 224 sex and age-matched controls. We used ELISA and the immunofluorescence analysis to characterize the presence of IgG autoantibodies to the cytoskeleton and nuclear proteins in patients’ sera. A significantly increased risk of nasopharyngeal carcinoma was associated with heterozygote FAS-A/G (ORZ2.00, PZ0.001) and homozygote FAS-G/G (ORZ3.19, PZ0.0001) variants. The increased frequency of FAS-G/G genotype is correlated with the presence of anti-nuclear autoantibodies in patients with nasopharyngeal carcinoma (PZ0.0298). Our results demonstrated that FAS promoter polymorphism was significantly associated with the nasopharyngeal carcinoma in Tunisians. The anti-nuclear autoantibodies induction was also found to be related to FAS polymorphism. The FAS
* This work was supported by le Ministe`re de la Recherche Scientifique et de Technologie, by le Ministe`re de l’Enseignement Supe´rieur, by le Ministe`re de la Sante´ Publique de la Re´publique Tunisienne. * Corresponding author. Tel.: C216 73 462 200; fax: C216 73 460 737. E-mail address: [email protected] (L. Chouchane).
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.02.037
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promoter polymorphism associated not only with the increased risk of nasopharyngeal carcinoma in Tunisians but also with immune response deregulation observed in this cancer. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: FAS; Polymorphism; Nasopharyngeal carcinoma; Apoptosis; Autoantibodies
1. Introduction The nasopharyngeal carcinoma classification distinguishes two major types, squamous cell carcinoma and non-keratinizing carcinoma. The later includes the undifferentiated carcinoma of the nasopharyngeal type (UCNT). The association between EBV infection and nasopharyngeal carcinomas was initially suggested based on serological studies [1] and has been subsequently substantiated by the detection of viral genomes and gene products in the epithelial tumor cells [2,3]. The nasopharyngeal carcinoma (NPC) shows a distinct geographical distribution with welldefined high-risk ethnic groups. While it is rare in western countries, its incidence is high in certain regions such as Southeast Asia. In North Africa, the NPC has an intermediate incidence [4]. The NPC from North Africa and Asia share several common clinical and biological characteristics, but they differ with regard to their age distribution. In Asia, NPC mainly affects patients in the 4th or 5th decade of their life, whereas in North Africa an additional peak of incidence is found confined to young population [5]. In a recent study [6], we demonstrated that the autoantibodies to the cytoskeleton and nuclear proteins are associated with the nasopharyngeal carcinoma in Tunisians. Given the importance of apoptosis in immune response regulation and in antitumor immunity, aberrant apoptosis could conceivably associate with tumorigenesis and immune system deregulation. FAS (CD95, APO-1) is a cell surface receptor involved in apoptotic signal transmission in many cell types, including cells of the immune system [7]. The death signal cascade is initiated upon cross-linking of FAS by its natural ligand (FASL) [8]. Whereas FAS expression and susceptibility to FASL-mediated apoptosis is a common feature of most non-malignant tissues in humans, constitutive expression of FASL is restricted to activated T and NK cells and a few immuno-privileged sites [9]. FAS-mediated
apoptosis, which is progressively lost during tumorigenesis can be inhibited in several ways. There is now broad evidence showing that malignant cells take advantage of aberrant loss of FAS [10–12] and gain of FASL [13–15] expression compared to their non-malignant counterparts. Aberrant FAS and FASL expression reflects loss of differentiation in tumor cells resulting in loss of sensitivity to apoptosis [16,17]. Mutation of the FAS gene is another mechanism contributing to resistance of malignant cells to FASL mediated apoptosis. Studies have revealed the occurrence of mutations of the FAS gene in human germline resulting in autoimmune lymphoproliferative syndrome [18–20]. Somatic FAS mutations impairing the transduction of the apoptotic signal were described in lymphoid tumors [21–23]. More recently, somatic FAS gene mutations have also been found in solid tumors but the functional relevance of these findings remains to be established [24–27]. FAS-mediated apoptosis can also be inhibited by a soluble form of the FAS molecule generated by alternative splicing and lacking the transmembrane domain, which anchors the receptor molecule within the cell membrane of the target cell. Hence, soluble FAS no longer transduces the death signal after binding to FASL and competes with transmembrane FAS for FASL binding [28,29]. Competitive FASL antagonism by soluble FAS efficiently prevents lymphocyte killing both in vitro [9] and in vivo [30,31]. Several studies have shown increased levels of sFAS in several tumor types which correlated in certain cases with poor prognosis [32–34]. A genetic polymorphism (A to G substitution) in the promoter region of the FAS gene located at the nucleotide position of K670 from the transcriptional start site has been reported [35]. This polymorphism alters the binding site of the nuclear transcription element GAS. In vitro studies have shown that Stat1 binding activity was higher in K670A allele of FAS promoter than in K670G allele and that
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the transcriptional activity of A allele tended to be higher than that of G allele [36]. Hence, it was speculated that this polymorphism might result in the alteration of FAS gene expression leading to the decrease of FAS-mediated apoptosis. In line with this hypothesis, we investigated the potential association of the K670 (A/G) FAS polymorphism with the increased risk of nasopharyngeal carcinoma and that of autoantibodies induction in this cancer.
2. Materials and methods 2.1. Patients and controls The allele and genotype frequencies at the position K670 (A/G) of the FAS gene were determined in a group of 170 unrelated patients with undifferentiated nasopharyngeal carcinoma (UCNT), and in 224 unrelated control subjects. Controls and patients were age and sex matched and were selected from the same population living in the middle coast of Tunisia. Patients with UCNT were recruited from the Department of Radiation Oncology and Medical Oncology of Sousse Hospital, between 1991 and 2003. The patients with UCNT (118 males and 52 females) had a mean age of 41.5G16 years. The clinical stages ranged from II to IV (TNM classification, 1987). The diagnosis of cancer was confirmed by histopathology analyses. The histology was undifferentiated carcinoma (type III, WHO classification) in all cases [37]. Control subjects (147 males and 77 females) having a mean age of 40G12.5 years were healthy blood donors having no evidence of any personal or family history of cancer (or other serious illness). Written informed consent was obtained from all subjects. 2.2. DNA extraction Genomic DNA was extracted from peripheral blood leukocytes by using the salting out procedure [38]. Briefly, 5 ml of blood was mixed with Triton lysis buffer (0.32 M sucrose, 1% Triton X100, 5 mM MgCl2, H2O, 10 mM Tris–HCl, pH 7.5). Leucocytes were spun down
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and washed with H2O. The pellet was incubated with proteinase K at 56 8C and subsequently salted out at 4 8C using a saturated NaCl solution. Precipitated proteins were removed by centrifugation. The DNA in the supernatant fluid was dissolved in 400 ml H2O. 2.3. Polymorphism analysis of the K670 (A/G) FAS gene Based on the method described by Huang et al. [35], a polymorphism chain reaction followed by digestion with endonuclease MvaI was used to detect the A to G transition polymorphism at position K670 of FAS gene. Two sequence specific oligonucleotide primers were used for the polymerase chain reaction (PCR): the 3 0 primer (5 0 -GGCTGTCCATGTTGTGGCTGC-3 0 ) was used in combination with the 5 0 primer (5 0 CTACCTAAGAGCTATCTACCGTTC-3 0 ). PCR was performed by using 60 ng genomic DNA as template, 0.01 mM dNTPs, 1.5 mM MgCl2, 1! Taq polymerase buffer, 0.25 mM of each primer and 0.5 U of Taq DNA polymerase (Amersham, Paris, France) in a total reaction of 20 ml. Reaction conditions used with the thermal cycler (Biometra, Go¨ttingem, Germany) were as follows: 94 8C for 6 min followed by 28 cycles at 94 8C for 30 s, 62 8C for 30 s and 72 8C for 1 min. A final extension step was carried out at 72 8C for 10 min. The 331 bp PCR products were digested with MvaI and analyzed by agarose-gel electrophoresis. The presence of the A nucleotide at the position K670 of the FAS gene was indicated by the cleavage of the amplified product into two fragments of 233 and 98 bp. An additional MvaI site is generated by G substitution yielding to the cleavage of the amplified product into three fragments of 189, 98 and 44 bp. 2.4. Statistical analysis The allele frequencies of FAS were tested for the Hardy–Weinberg equilibrium for both patient and control groups using the chi-square test. The same test was used to evaluate for significant association between disease (nasopharyngeal carcinoma against controls) and FAS genotypes. Relative risk of nasopharyngeal carcinoma associated with a particular genotype was estimated by the odds ratio (OR). The chi-square test (or Fisher’s exact test when n!5) was used to determine whether significant
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washes with PBS, antibodies were revealed by antihuman Igs antibodies (IgG, IgA, and IgM) labeled with fluorescein.
differences (P-value) in the autoantibodies induction to cytoskeleton and nuclear antigens were observed between carriers of different genotypes. The non-parametric Mann–Whitney U-test was used to determine whether significant differences (P-value) in the soluble FAS (sFAS) levels were seen between carriers (patients and controls combined) of different genotypes. Statistical analyses were performed using the Statistical Package of Social Sciences for Windows (SPSS version 10.0). Differences between groups were tested for significance by the two-tailed test. A P-value less than 0.05 was considered significant.
3. Results 3.1. Polymorphism in the FAS gene as risk factor for nasopharyngeal carcinoma Table 1 shows genotype frequencies for FAS gene in patients with nasopharyngeal carcinoma and in the control group. These distributions were in Hardy– Weinberg equilibrium for both patient and control groups. The allelic frequency of the K670 (G) FAS was 0.568 in patients with nasopharyngeal carcinoma and 0.433 in control subjects (ORZ1.72, PZ0.0001). The frequency of FAS-A/G heterozygotes was 0.535 in the patient group and 0.509 in the control population resulting in a significantly OR associated with this genotype (ORZ2.00, PZ0.001). The frequency of the FAS-G/G homozygotes was 0.30 in the patient group and only 0.178 in controls (ORZ3.19, PZ0.0001). These data suggest an increased risk of developing nasopharyngeal carcinoma is associated with inheritance of the (K670) FAS-G allele in a dose-dependant manner. Since the highest incidence of the EBV-associated nasopharyngeal carcinomas in young subjects was found in North Africa, we stratified the patients according to age (younger or older than 25 years). No significant differences in FAS genotype distributions were found between the subgroups (data not shown).
2.5. ELISA for autoantibodies Sera autoreactivity to the cytoskeleton and nuclear proteins was determined on serum samples obtained prior to cancer therapy. Sera from 92 patients with primary UCNT were available before treatment. Each serum collected was tested individually. As negative control for the reaction, 82 sera from healthy individuals were pooled and used in each test. The autoantibodies detection was carried out as previously described [6]. 2.6. Immunofluorescence analysis Identification of anti-nuclear antibodies (ANA) by indirect immunofluorescence was carried out using cryostat sections prepared from liver tissue of young rats. These liver sections were deposed on glass and incubated overnight at K80 8C. Briefly, serum was diluted at 1/100 with PBS (pH 7.4) and deposited on liver sections. After 30 min of incubation and three
Table 1 The (K670) FAS genotype distribution in control subjects and in patients with nasopharyngeal carcinoma Genotype (K670) FAS
Patients (nZ170)
Controls (nZ224)
n
f
n
f
FAS-A/A FAS-A/G FAS-G/G
28 91 51
0.165 0.535 0.300
70 114 40
0.313 0.509 0.178
ORa
1 2.00 3.19
Confidence interval
P-value
[1.19 3.33] [1.76 5.77]
0.001 0.0001b
The chi-square test was used to determine whether significant differences (P-value) were observed when patient group was compared with control group. f, frequencies. a Risk estimates were evaluated by unconditional logistic regression analysis with adjustment for age and sex. b The association remains significant under the recessive model (genotype FAS-G/G compared to genotype FAS-A/GCFAS-A/A) with ORZ1.97 and PZ0.001.
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Table 2 Association between (K670) FAS polymorphism and autoantibodiesa induction in patients with nasopharyngeal carcinomab Genotype
MG, n (%) (C) (nZ12)
FAS-A/A FAS-A/G FAS-G/G P-value
4 (33.3) 5 (41.7) 3 (25)
MS, n (%) (K) (nZ80)
9 (11.2) 46 (57.6) 25 (31.2) 0.122
(C) (nZ11) 2 (18.2) 6 (54.5) 3 (27.3)
ACT, n (%) (K) (nZ81)
11 (13.6) 45 (55.4) 25 (31) 0.91
(C) (nZ11) 1 (9.1) 5 (45.5) 5 (45.4)
TUB, n (%) (K) (nZ81)
12 (14.8) 46 (57) 23 (28.4) 0.502
(C) (nZ16) 3 (18.7) 6 (37.5) 7 (43.8)
ANA, n (%) (K) (nZ76)
10 (13.2) 45 (59.2) 21 (27.6) 0.28
(C) (nZ22) 4 (18.2) 7 (31.8) 11 (50)
(K) (nZ70) 9 (12.8) 44 (62.9) 17 (24.3) 0.0298
The chi-square (or Fisher) test was used to determine whether significant differences (P-value) in the autoantibodies induction to cytoskeleton and nuclear antigens were observed between carriers of different genotypes. MS, myosin; ACT, actin; TUB, tubulin; MG, myoglobulin; ANA, anti-nuclear antibodies. a IgG autoantibodies, except for anti-nuclear antibodies where reactivity was deducted from different classes of Ig (IgG, IgM, and IgA). b Sera from patients were taken before cancer treatment.
3.2. Sera autoreactivity and Fas genetic polymorphism in nasopharyngeal carcinoma In a previous study [6], we showed a significant increase in the serum autoreactivity to tested cytoskeleton and nuclear proteins in the patients with nasopharyngeal carcinoma. The most frequent autoreactivity was found with nuclear antigens (NA) in patient’s sera compared to that of control subjects (22% in patients vs 1.2% in controls; PZ0.00003). Table 2 shows the sera autoreactivity of patients with nasopharyngeal carcinoma according to the FAS polymorphism. The frequency of FAS-G/G genotype is higher in patients producing anti-nuclear antibodies than in those without (0.50 vs 0.243, PZ0.0298), resulting in a significant association between the presence of anti-nuclear antibodies (ANA) and the FAS-G/G homozygote genotype. Conversely, the frequency of FAS-A/G is lower in patients having anti-nuclear antibodies (0.318 vs 0.629).
4. Discussion Although environmental factors, including exposure to EBV, are clearly important determinants of nasopharyngeal carcinoma susceptibility, a significant hereditary contribution to the etiology of this cancer has been suggested by several studies [4,39]. Genes encoding receptors involved in apoptosis could be attractive susceptibility candidate genes for this malignancy. Hence, in this study, we assessed the potential association of the variation in the promoter
of FAS gene with nasopharyngeal carcinoma in the Tunisian population. The present case/controlled study showed a substantially increased risk of nasopharyngeal carcinoma associated with inheritance of the (K670) FAS-G allele in a dose-dependant manner. Individuals homozygous for (K670) FAS-G have more than 3-fold risk to develop nasopharyngeal carcinoma (ORZ3.19, PZ0.0001), with heterozygotes having an intermediate risk (ORZ2.00, PZ0.001) compared with individuals homozygous for (K670) FAS-A. It has been shown that the FAS-G allele associated with reduced FAS transcriptional activity compared to the FAS-A allele [36]. Thus, it is expected that the FAS-G allele might result in the alteration of FAS gene expression leading to the decrease of FAS-mediated apoptosis of tumor cells. However, Lai et al. [40] reported recently that Japanese subjects carrying the FAS-A/A might have an increased risk of cervical cancer. To explain their finding, they speculate that more efficient FAS expression associated with FAS-A allele could lead to the apoptosis of immune cells. Although the (K670) FAS polymorphism has been found with potentially different transcriptional efficiency, no direct evidence of its involvement in FAS function or in any immune deregulation aspects has been shown. Therefore, we assessed the potential in vivo significance of FAS polymorphism by investigating the correlation between FAS genotypes and the presence of autoantibodies to cytoskeleton and nuclear antigens frequently detected in nasopharyngeal carcinoma. In a previous study [6], we showed that the frequency of sera containing autoantibodies to
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cytoskeleton and nuclear antigens was significantly increased in patients with nasopharyngeal carcinoma compared to that of healthy control subjects. In the present study, we showed that the (K670) FAS-G/G genotype is significantly higher in patients with nasopharyngeal carcinoma producing anti-nuclear antibodies than in those without. The frequency of FAS-G/G was higher in patients having autoantibodies to tubulin and actin than those without, but the difference in the genotype frequency did not reach statistical significance. Among possible mechanisms, which could explain the frequent presence of autoantibodies in patients carrying the FAS-G/G genotype, is the alteration of FAS expression on the surface of autoreactive B cells with FAS-G/G genotype, leading to their resistance to apoptosis and to their survival. These autoreactive cells will produce autoantibodies upon reactivation by EBV and/or exposure to nuclear and intracellular antigens generated by their shedding from viable cells or their release from dying tumor cells. Our findings are in agreement with those of several reports which suggest a causal effect of FAS alteration in the induction of autoimmune diseases [19,41–43]. Moreover, this FAS polymorphism was associated with SLE and with the presence of anti-RNP autoantibodies [44]. In conclusion, this study suggests that the FAS-G/G is associated with the susceptibility to nasopharyngeal carcinoma. Functional significance of FAS polymorphism was proved by its correlation with the antinuclear autoantibodies induction. Replication of this finding in a large population of NPC will be of use in determining whether the relation between the FAS polymorphism and anti-nuclear autoantibodies induction can be generalized.
Acknowledgements We gratefully acknowledge the technical assistance of O. Boughzala. We thank the staff of the Departments of Radiation Oncology, and Medical Oncology of CHU F. Hached, Sousse, and Dr A. Romdhane for providing samples and clinical information.
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