Fas-1377A/G and fasL-844 T/C gene polymorphisms and epithelial ovarian cancer

Abstract.

Background: The Fas receptor is known to be

widely expressed in various tissues and FasL is highly expressed

on cells of the immune system and also on cells of

immune-privileged areas such as the eyes and brain. Ovarian cells are

known to exhibit marked FasL immunoreactivity throughout

follicular development; there may also be a relationship

between Fas and FasL polymorphisms and the immune

privileges of the epithelial ovarian cells. Patients and Methods:

The study included 47 epithelial ovarian carcinoma patients

and 41 healthy subjects. Polymerase chain reaction (PCR) and

restriction endonucleases were used to determine the

polymorphic Fas and FasL genes. Results: The FasL CC

genotype was found to increase the risk of ovarian carcinoma

and a protective effect of the GGCT genotype was observed.

Conclusion: Because of the expressional aspects of the FasL

-844T→C polymorphism, individuals carrying the FasL-844C

allele would be expected to have higher FasL expression on

tumour cells compared with those carrying the FasL-844T

allele. People with such a genotype show a tendency to develop

various tumours.

One of the two convergent pathways of apoptosis is the

extrinsic type occuring in response to activation of specific

cell membrane receptors termed “death receptors” (1, 2).

Fas is one such death receptor which usually works with its

ligand FasL. It is thought that tumour cells have the

capacity to escape from immune surveillance. Some obtain

immune privileges which prevent them from being

recognised by the immune system while others fight actively

with immune system cells (3-5). It is thought that the

Fas/FasL pathway plays an important role in the

development of those immune privileges (3, 6-9).

Fas (ApoI=CD95) is a type I membrane protein of 48

kDa consisting of 335 amino acids and belongs to one of the

subfamilies of death receptors which is part of the

TNF-receptor superfamily (10, 11). FasL is a type II

transmembrane protein that has been shown to be

expressed by NK cells and activated T-cells and by some

immune-privileged cells such as the eye and brain (8). It is a

~37 kDa member of the TNF-family and can be cleaved by

metalloproteinases into a 26 kDa soluble form (3). Both

forms have the ability to trimerize and then bind to their

receptor, Fas (3), but the membranal form is more efficient

than the soluble form (3). Some in vivo

models

demonstrated that tumors expressing cell surface

membranal FasL are rejected in association with a

neutrophilic inflamatory response (3). This means that the

tumour cells with the ability to express FasL have the

advantage of not being damaged by infiltrating lymphocytes,

which confers an immune privilege on the tumour (12).

Ovarian cancer is one of the most common tumors for

women and it is known that >90% of all ovarian cancers

are of epithelial origin (9). The ovaries are covered by the

ovarian surface epithelium (OSE); this single layer is an

important site of ovarian oncogenesis (13). Fas is known

to be expressed by human ovarian cells and several studies

have indicated a role for the Fas-FasL system in various

gynecological problems, (2, 5, 13-16). Thecal ovarian cells

are known to exhibit marked FasL immunoreactivity

throughout follicular development. The exact physiological

significance of this observation is not clear yet, but there

is a strong possibility that this death factor provides

immune privilige for the follicle as it does in the testes and

eyes (13, 16, 17).

The aim of this study was to determine the relationship

between Fas and FasL polymorphisms and the development

of ovarian cancer.

The most frequent polymorphisms of Fas and FasL that

each cause some expressional sequela of the gene products

991

Correspondence to: Prof. Dr. Turgay Isbir, Institute for Experimental Medical Research, Department of Molecular Medicine, Istanbul University, Capa, Istanbul, Turkey. Tel/Fax: +90 212 6351959, e-mail: tisbir@superonline.com

Key Words: Fas/FasL pathway, immune privilege, polymorphism, ovarian cancer.

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Fas-1377A/G and FasL-844 T/C Gene Polymorphisms

and Epithelial Ovarian Cancer

UZAY GORMUS

, ARZU ERGEN

, HULYA YILMAZ

,

BURAK DALAN

, SINAN BERKMAN

and TURGAY ISBIR

1

_{Istanbul University, Institute for Experimental Medical Research, Department of Molecular Medicine and}

_{Faculty of Medicine, Department of Obstetrics and Gynecology, Istanbul University, Capa, Istanbul, Turkey}

were investigated. It is known that the T

→C transition at

position -844 in the promoter region of FasL is located in

the binding motif for some important transcription factors.

It is thought that considerably higher basal expression of

FasL is associated with the FasL -844C allele compared with

the FasL-844T allele (5). Also G

→A transition at point

Fas-1377 decreases Fas expression (5).

Patients and Methods

Study groups. The study included 47 epithelial ovarian carcinoma patients and 41 healthy subjects. The healthy control subjects were chosen from women attending the Istanbul University Hospital, Obstetrics and Gynecology Clinics for non-specific minor complaints or regular health examinations. Exclusion criteria included a previous history of any kind of carcinoma in the subjects themselves or their first degree relatives. At recruitment, written informed consent was obtained from each subject. The mean age ± standard deviations was 48.62±11.50 years in the control group and 39.66±6.78 years in the patient group and 18.8% of the control group and 30.9% of the patient group were smokers.

Polymorphism analysis. The previously described method of Sun et al. was used (18). The primers used for polymerase chain reaction (PCR) for the -1377 G/A polymorphism were Fas forward, 5’-TGT GTG CAC AAG GCT GGC GC-3’ and Fas reverse, 5’-TGC ATC TGT CAC TGC ACT TAC CAC CA-3’; and for the FasL-844 T/C polymorphism the primer sequences were FasL forward, 5’-CAG CTA CTC GGA GGC CAA G-3’ and FasL reverse, 5’-GCT CTG AGG GGA GAG ACC AT-3’ from IDT (Integrated DNA Technologies Inc, Iowa, USA).

In the Fas procedure, in order to introduce a restriction endonuclease site, the 3’ end of the primer FasIF was changed from CAC to CGC to create a BstU1 site. These fragments were amplified using a 25 ÌL reaction mixture containing approximately 100 ng of template DNA, 1 ÌL of each primer, all four deoxyribonucleoside 5’ triphosphates (each at 0.2 mM), 2 mM MgCl₂ and 1 U of Taq polymerase in 1x reaction buffer (MBI Fermentas, Vilnius, Lithuania).

For FasL amplification, a similar reaction mixture was used, except that 2.5 mM MgCl₂was included.

For both Fas and FasL gene amplifications, the reactions were carried out with an initial melting step of 2 min at 94ÆC; followed by 35 cycles of 45 sec at 94ÆC, 45 sec at 62ÆC, 60 sec at 72ÆC; and a final elongation step of 7 min at 72ÆC.

PCR and restriction products of Fas were electrophoresed in 3% (w/v) and of FasL in 2% (w/v) agarose gels and stained with ethidium bromide.

The restriction endonuclease BstU1 was used to determine the Fas-1377 G/A polymorphism. The products of this reaction were separated on agarose gels containing ethidium bromide. BstU1 digestion generated the following fragments for the -1377G allele, fragments of 104 bp and 18 bp; Fas-1377A allele, a single fragment of 122 bp. For determinations of the FasL-844 T/C polymorphisms, the BsrDI restriction endonuclease was used which generated for the FasL-844 T allele, a single uncut fragment of 401 bp, and for the -844 C allele, two fragments of 233 and 168 bp.

Statistical analysis. The Student's t-test was used to determine whether or not significant differences in sex and ages existed between patient and control groups. Pearson's Chi-square analyses were used to examine differences in genotype distribution between cancer patients and control subjects.

Results

There was no significant difference in the distribution of the

Fas A and G alleles between cancer patients and the control

group (¯

: 0, 367; p>0.05; df:1) and also no difference of

distribution of the FasL T and C alleles between the study

groups (¯

: 2, 94; p>0.05; df:1) (Table I).

When the Fas and FasL genes were evaluated

independently of each other, the CC genotype individuals

had a statistically significant tendency to develop epithelial

ovarian cancer (¯

: 4, 15; p<0.05; df:1) (Table I). No other

statistically significant differences were found for any other

genotype.

Because Fas and FasL form a receptor–ligand system

working together in apoptotic cell death, the possibility of

an interaction between the single nucleotide polymorphism

(SNP) in Fas and FasL that was associated with the risk of

ovarian cancer was examined. No significant differences

were detected between any of the genotypic combinations

of the two genes except the GGCT combination. The

GGCT genotype had a protective effect against epithelial

ovarian cancer (¯

: 6, 604; p<0.025; df: 1) (Table II).

Discussion

Several studies of various carcinomas evaluated the

relationship between the risk of developing malignancy and

genetic polymorphisms. One focus of such investigation has

been the genes with a role in apoptotic pathways of which

the Fas/FasL system is one of the most important. Two

mechanisms of action of the Fas/FasL system in cancer have

been proposed (18, 19): Firstly, since FasL on

T-lymphocytes can promote apoptosis in Fas-expressing

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Table I. Genotype distribution of Fas and FasL.

Genotype Control subjects Cancer patients No. (%) No. (%) Fas position -1377 GG 29 (70.7) 28 (59.6) GA 12 (29.3) 19 (40.4) AA 0 0 FasL position -844 TT 14 (34.1) 13 (27.7) TC 21 (51.2) 18 (38.3) CC 6 (14.6) 16 (34)

cancer cells, this system could play an important role in

cell-mediated cytotoxic reactions against malignant cells and

secondly malignant cells could escape immune surveillance

by down-regulation of FasL and by killing the lymphocytes

which the express FasL.

Sun et al. have observed that subjects carrying the FasL

-844CC genotype had a three-fold increased risk of developing

cervical cancer compared with those carrying the TT genotype

(5), which is in agreement with our results where the FasL CC

genotype was found to increase the risk of ovarian carcinoma.

This could be explained by the considerably higher basal

expression of FasL associated with the FasL-844C allele

compared with the FasL-844T allele (5), which is consistent

with the increase of Fas and related protein expressions in

ovarian carcinoma suggested by some other studies (9, 20-22).

Further studies have indicated that in general CC genotype

women have a higher tendency to develop gynecological

carcinomas. Sun et al. also found that the heterozygous CT

genotype presented a higher risk of cervical cancer (5). No

such relationship between the CT genotype and ovarian

carcinoma was found in the present study.

Van Haaften-Day et al. have found increased expression

of Fas in borderline ovarian carcinoma compared to normal,

benign and malignant cases, but they did not find any

relationship between Fas and FasL expressions in malignant

ovarian tumours (9). The present study was confined to

malignant ovarian carcinoma patients and only a protective

effect of the GGCT genotype was observed.

In conclusion, because of the expressional aspects of

the FasL-844T→C polymorphism, it would be expected

that individuals carrying the FasL-844C allele would also

have higher FasL expression on tumour cells compared

with those carrying the FasL-844T allele. People with such

a genotype for this cell death pathway gene show a

tendency to develop various tumours possibly due to their

immune status.

References

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2 Dufournet C, Uzan C, Fauvet R, Cortez A, Siffroi JP and Darai E: Expression of apoptosis-related proteins in peritoneal, ovarian and colorectal endometriosis. J Reprod Immunol 70: 151-162, 2006.

3 Abrahams VM, Straszewski SL, Kamsteeg M, Hanczaruk B, Schwartz PE, Rutherford TJ and Mor G: Epithelial ovarian cancer cells secrete functional Fas ligand. Cancer Res 63: 5573-5581, 2003.

4 Liu K, Caldwell SA and Abrams SI: Cooperative disengagement of Fas and intercellular adhesion molecule-1 function in neoplastic cells confers enhanced colonization efficiency. Cancer Res 65(3): 1045-1054, 2005.

5 Sun T, Zhou Y, Li H, Han X, Shi Y, Wang L et al: FasL-844C polymorphism is associated with increased activation-induced T cell death and risk of cervical cancer. JEM 202(7): 967-974, 2005. 6 Myong NH: Tissue microarray analysis of Fas and FasL expressions in human non-small cell lung carcinomas; with reference to the p53 and bcl-2 overexpressions. J Korean Med Sci 20: 770-776, 2005.

7 Zhang X, Miao X, Sun T, Tan W, Qu S, Xiong P, Zhou Y and Lin D: Functional polymorphisms in cell death pathway genes Fas and FasL contribute to risk of lung cancer. J Med Genet 42: 479-484, 2005.

8 Niederkorn JY: See no evil, hear no evil, do no evil: the lessons of immune privilege. Nat Immunol 7(4): 354-359, 2006.

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13 Quirk SM, Cowan RG and Huber SH: Fas antigen-mediated apoptosis of ovarian surface epithelial cells. Endocrinology 138(11): 4558-4566, 1997.

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Gormus et al: Gene Polymorphisms and Ovarian Cancer

993 Table II. The genotype combinations of Fas and FasL.

TT CT CC Total

Case Control Case Control Case Control Case Control

AG 3 6 11 5 5 1 19 12

GG 10 8 7 16 11 5 28 29

16 Kim JM, Yoon YD and Tsang BK: Involvement of the Fas/Fas Ligand system in p53-mediated granulosa cell apoptosis during follicular development and atresia. Endocrinology 140(5): 2307-2317, 1999.

17 Quirk SM, Cowan RG, Joshi SG and Henrikson KP: Fas antigen-mediated apoptosis in human granulosa/luteal cells. Biology of Reproduction 52: 279-287, 1995.

18 Sun T, Miao X, Zhang X, Tan W, Xiong P and Lin D: Polymorphisms of death pathway genes Fas and FasL in esophageal squamous cell carcinoma. J Nat Cancer Inst 96(13): 1030-1036, 2004.

19 Liu K, Caldwell SA and Abrams SI: Immune selection and emergence of aggresive tumor variants as negative consequences of Fas-mediated cytotoxicity and altered IFN-Á-regulated gene expression. Cancer Res 65(10): 4376-4388, 2005.

20 Gopalan B, Litvak A, Sharma S, Mhashilkar AM, Chada S and Ramesh R: Activation of the Fas-FasL signaling pathway by MDA-7/IL-24 kills human ovarian cancer cells. Cancer Res 65(8): 3017-3024, 2005.

21 Akhmedkhanov A, Lundin E, Guller S, Lukanova A, Micheli A, Ma Y et al: Circulating soluble Fas levels and risk of ovarian cancer. BMC Cancer 3: 1-7, 2003.

22 Konno R, Takano T, Sato S and Yajima A: Serum soluble Fas level as a prognostic factor in patients with gynecological malignancies. Clin Cancer Res 6: 3576-3580, 2000.

Received November 27, 2006

Revised January 26, 2007

Accepted January 29, 2007

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