Tumor Biol. DOI 10.1007/s13277-014-2896-7

RESEARCH ARTICLE

Promoter polymorphism of FASL confers protection against female-specific cancers and those of FAS impact the cancers divergently Sateesh Reddy Nallapalle & Sarika Daripally & V. T. S Vidudala Prasad

Received: 17 July 2014 / Accepted: 26 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract We investigated risk association of FAS (−1377 G>A and −670 A>G) and FASL (−844 T>C) promoter polymorphisms with breast, ovarian, cervical, and endometrial cancers and report that the FASL −844 CC genotype was protective against breast, ovarian, cervical, and endometrial cancers (P≤0.01). On the other hand, FAS −1377 GA and AA variants increased risk of breast cancer. However, the GA variant of FAS −1377 was also found to be a risk factor for cervical cancer. In contrast, FAS −670 AG variant significantly lowered risk of breast cancer. Further, we also observed that risk association of co-occurrence of FAS and/or FASL variants with the cancers varied as compared to the presence of individual polymorphisms. Although risk and protective haplotypes of FAS SNPs were observed across the cancer phenotypes, the association of the haplotypes was significant for breast cancer alone with a 3-fold enhanced risk. The protective effect of the FASL CC genotype seen in this study suggests that similar biomolecular mechanisms involving FASL might play a role in female-specific cancers.

Keywords FAS . FASL . Breast cancer . Ovarian cancer . Cervical cancer . Endometrial cancer . Gynecological cancers

S. R. Nallapalle : S. Daripally : V. T. S. V. Prasad (*) Research and Development, Basavatarakam Indo-American Cancer Hospital and Research Institute (BIACH&RI), Road No. 10, Banjara Hills, Hyderabad 500034, India e-mail: [email protected] V. T. S. V. Prasad e-mail: [email protected] S. Daripally Acharya Nagarjuna University, Andhra Pradesh, India

Introduction Cancers of female breast, cervix, ovary, and endometrium together account for 41.5 and 28.6 % of overall cancer prevalence and mortality in females, respectively [1]. Among the female-specific malignancies, breast cancer is the most prevalent followed by ovarian, cervical, and endometrial tumors [1]. All types of cancers, irrespective of causative factors, origin, and age of onset, are characterized by uncontrolled cell proliferation and inability of cancerous cells to undergo apoptosis required for controlling tumorous growth. Defective apoptotic machinery has been shown to be a major contributing factor for cancer, including those of female-specific cancers [2, 3]. In females, it is known that primary cancer of one organ predisposes females to other primary cancers, albeit at a later age [4–6]. Unraveling precise mechanisms that initiate cancer, a multifactorial disease, and gaining insights into how one cancer might lead to another may help in devising novel strategies for early intervention, diagnosis, and effective treatment. FAS/FASL system plays a significant role in maintaining homeostasis of cells essential for controlling unwanted growth through apoptosis. FAS, FS7-associated cell surface antigen (also known as CD95 or APO-1) and FASL, FAS ligand (also known as CD95L) are members of tumor necrosis factor superfamily. They are particularly expressed in immune system cells apart from other cells. FAS, a cell surface type 1 transmembrane glycoprotein, is expressed widely, whereas its cognate ligand, FASL, a type II transmembrane protein expression, is restricted to few types of cells [7]. However, various cancerous tissues, such as cervical, ovarian, endometrial, breast, lung, gastrointestinal, head and neck, renal prostate, and skin, have been shown to express FASL [8–15]. FAS receptor transduces cell death signal through its cytosolic domain when FASL binds to its extracellular domain, triggering apoptosis in many types of cells. Genes encoding FAS and

Tumor Biol.

FASL located at chromosomes 10q24.1 and 1q23, respectively, exhibit polymorphisms in various ethnic groups. Promoter region single nucleotide polymorphisms (SNPs) of FAS (−1377G>A; rs2234767 and −670A>G; rs1800682) and FASL (−844 T>C; rs763110) are reported to alter expression of FAS and FASL genes resulting in perturbed apoptosis [16, 17]. Presence of these gene variants were found to be associated with various cancers, including the cancers that are specific to females, such as breast, ovarian, cervical, and endometrial cancers [18–22]. These female-specific tissues are sensitive to female hormones such as estrogen/estradiol which regulate expression of FAS/FASL genes [23–26]. However, reports on the association of these FAS and FASL SNPs with breast, cervical, and ovarian cancers are scattered and contradictory [21, 22, 27–35] and to our knowledge, there are no reports on the association of FAS −1377 G>A and FASL −844 T>C polymorphisms in endometrial cancers. Occurrence of one of the female-specific cancers, for example, breast cancer, may predispose patients to ovarian or endometrial cancers [36, 37]. Breast, ovarian, cervical, and endometrial tissues and the malignancies of these tissues are sensitive to estrogen/estradiol [38–43], age at menarche, child bearing, and menopausal age [44]. Further, hormonal therapy and anticancer drugs also have been shown to predispose these patients to cancer [45–48]. Keeping the abovementioned commonalities, we wanted to examine the impact of FAS (−1377 G>A and −670 A>G) and FASL (−844 T>C) polymorphisms on breast, ovarian, cervical, and endometrial cancers. To better understand the association between SNPs and disease phenotypes, study subjects were drawn from less heterogeneous population with similar genetic makeup and environment.

Materials and methods Study design and subjects The hospital-based study was designed to investigate whether FAS (−1377 G>A and −670 A>G) and FASL (−844 T>C) promoter SNPs have any association with female-specific cancers. The study participants hailing from Andhra Pradesh, India, were divided into a total of groups: (i) control and (ii) breast, (iii) ovarian, (iv) cervical, and (v) endometrial cancer patients. Participants were enrolled during the years 2009–2013. A total of 1000 female subjects, 212 controls and 788 cancer (breast cancer, 245; ovarian cancer, 216; cervical cancer, 192; and endometrial cancer, 135 patients) subjects, residing in Andhra Pradesh were enrolled for the study. Age of the subjects ranged between 15 and 85 years. Control subjects were unrelated to each other, apparently healthy and with no known family history of female-specific cancers. Both cancer and control subjects, to our knowledge, were neither habitual

tobacco nor alcohol users. The study was approved by the institute’s ethics committee. Cancer subjects were cancer patients visiting our institute. All the cancer patients were confirmed subjects of cancer. Cancers were diagnosed and confirmed by biopsy and histopathological evaluation of the excised cancer tissue. TNM staging system was used for breast cancer determining disease progression, whereas as for ovarian, cervical, and endometrial cancers. FIGO staging system was used. DNA isolation and identification of polymorphisms Peripheral blood samples were drawn from consenting subjects, and the genomic DNA was extracted from the blood samples by salting out protocol [49] with few modifications. The DNA was stored at −20 °C, until use. The gene polymorphisms of FAS (−1377 G>A and −670 A>G) and FASL (−844 T>C) were identified by PCR-RFLP. The desired gene fragments were amplified, using specific primers by PCR (PCR Kit, Fermentas, USA), under optimal conditions. Primers used for FAS −1377 G>A were (fp. 5′-tgtgtgcacaaggctggcgc-3′ and rp. 5′-tgcatctgtcactgcacttaccacca-3′), −670 A>G (fp. 5′atagctggggctatgcgatt-3′ and rp. 5′-catttgactgggctgtccat -3′), and for FASL −844 T>C were (fp. 5′-caatgaaaatgaacacattg3′ and rp. 5′-cccactttagaaattagatc-3′). The PCR reaction mixture (50 μl) contained the following: for FAS −1377 G>A, 6.1 μl of 10× buffer, 10 nmol of dNTPs each, 115 nmol of MgCl2, 1 unit of Taq, and about 150–200 ng of genomic DNA; for FAS −670 A>G and FASL −844 T>C SNPs, the concentration of the PCR mixture was same as above, but for concentrations of buffer and MgCl2; for FAS −670 A>G, 5 μl 10× buffer and 100 nmol of MgCl2; and for FASL −844 T>C, 5 μl buffer and 125 nmol of MgCl2, were added in the PCR reaction mixture. The PCR mixture volume was made up to 50 μl with ultrapure water. The PCR reaction conditions were as follows: FAS −1377 G>A was carried out at an initial denaturation temperature of 94 °C for 5 min, followed by 37 cycles of 35 s at 94 °C, 35 s at 59 °C, and 35 s at 72 °C. The expected size of the amplicon was 122 bp. For FAS −670 A>G, PCR was carried out at an initial denaturation temperature of 95 °C for 5 min, followed by 35 cycles of 45 s at 95 °C, 35 s at 54.6 °C, and 40 s at 72 °C. The expected size of amplicon was 193 bp. For FASL −844 T>C, PCR was carried out at an initial denaturation temperature of 95 °C for 5 min, followed by 37 cycles for 40 s at 94 °C, 40 s at 44.0 °C, and 40 s at 72 °C. The expected size of amplicon was 85 bp. After the set cycles, a final elongation step of 7 min at 72 °C was added to all three PCRs. PCR products were verified by 2 % agarose gel electrophoresis (AGE). PCR products, encompassing FAS −1377 G>A, FAS −670 A>G, and FASL −844 T>C variants, were digested with BstUI, ScrFI, and DraIII, respectively, as per the manufacturer’s instructions. The genotypes were identified by 3 % AGE, and the bands

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were detected under UV light. The products obtained were of desired size, and the SNPs were validated by sequencing few random amplicons.

computing ORs and P values. The haplotype G-A which comprises the wild-type alleles of both SNPs was used as a reference haplotype.

Statistical analysis Allele and genotype frequencies of the SNPs were compared between cancer subjects and controls. Association of the polymorphisms with disease risk or protection was interpreted by calculating odds ratio (OR) and 95 % confidence interval (CI) using chi-squared test and P value by Fisher’s exact test (two-sided) using GraphPad Instat (version 5.0). The effect of the SNPs toward disease susceptibility was assessed by ORs with 1 as a reference value; OR less than 1.0 was considered to be protective, and a value more than 1.0 was considered as risk. The difference in the SNP frequency between cancer subjects and controls was considered statistically significant at P≤0.05. Hardy-Weinberg Equilibrium (HWE) conformation of the three polymorphisms was tested in control and cancer subjects using an online tool (http://www.oege.org/ software/hardy-weinberg.html). The combined ORs were computed for various genotype combinations of the FAS and FASL SNPs with wild-type genotypes as a reference to study the additive effect of the SNPs on the disease susceptibility. LD and haplotype analysis Two-marker analysis was performed for the FAS SNPs to compute D′ and r2 values, linkage disequilibrium (LD) plots, and haplotypes from the genotype data using Haploview (version 4.2). The haplotype frequencies were compared in different patient groups with control subjects. The association of haplotypes with disease susceptibility was analyzed by Table 1

Results Demographic data and clinicopathological information of the subjects are summarized in Tables 1, 2, 3, and 4. As shown in Table 1, the average age of breast, ovarian, cervical, and endometrial cancer subjects, respectively, was 50.3, 46.8, 47, and 57.7 years, whereas the average age of control females was 40 years. However, BMI of the subjects did not differ between cancer subjects and controls. As shown in Table 2, ductal carcinoma of breast, adenocarcinoma of ovaries and endometrium and squamous cell carcinoma of cervical cancer were found to be the major subtypes of the subjects. The breast cancer subjects (for whom the data was available) were stratified based on the status of the hormone receptors and progression of the cancers (Tables 3 and 4). Our data showed that most breast cancer subjects were at stage II/III, and ovarian cancer subjects were at stage III as opposed to the cervical cancer subjects (stage I/II) who were at early stages of the disease. Genotype frequencies of FAS −1377 G>A, FAS −670 A>G, and FASL −844 T>C in controls and cancer subjects were within the HWE (P>0.05), except for −1377 G>A polymorphism in ovarian cancer (P≤0.05). Data presented in Tables 5 and 6 showed the presence of the three SNPs of FAS and FASL in both controls and in cancer subjects, albeit at varying frequencies. In controls, frequencies of FAS −1377 G>A wild-type, heterozygous, and homozygous genotypes were 56.7, 37.4, and 5.9 %, respectively, whereas frequencies

Demographic data of the subjects

Age (years) (available/total) Average±SE ≤47 years >47 years Available/Total Average weight (kg)±SE Average Height (m)±SE Average BMI±SE Geographical distribution (available/total) State of Andhra Pradesh Other statesa

Breast cancer

Ovarian cancer

Cervical cancer

Endometrial cancer

Controls

241/245 50.3±0.7 96 (40.0) 145 (60.0) 241/245 61±1.07 1.52±0.005 26.2±0.4 241/245 239 2

215/216 46.8±0.9 104 (48.4) 111 (51.6) 215/216 56.4±1.1 1.51±0.008 24.5±0.5 215/216 214 1

191/192 47±0.8 104 (54.4) 87 (45.6) 191/192 56.1±1.1 1.54±0.006 23.7±0.5 191/192 190 1

135/135 57.7±0.9 22 (16.3) 113 (83.7) 135/135 67.7±2.2 1.52±0.008 29.5±0.9 135/135 134 1

189/212 40±1.3 138 (73) 51 (27) 28/212 64.8±2.7 1.62±0.02 24.6±0.9 212/212 212 0

Available: number of subjects from whom data could be collected. Total: total number of subjects of the study SE standard error a

Other states: Odisha, Karnataka, Maharashtra, SE standard error

Tumor Biol. Table 2

Histological subtypes of the female cancers

Breast cancer Available/total Ductal carcinoma (all subtypes) Lobular carcinoma Squamous cell carcinoma Malignant melanoma of breast Ovarian cancer Available/total Adenocarcinoma Serous papillary carcinoma Mucinous carcinoma Serous carcinoma Germ cell tumor Granulosa cell tumors Infiltrating carcinoma Clear cell carcinoma Dysgerminoma Round cell tumor Cervical cancer Available/total Squamous cell carcinoma Adenocarcinoma Small cell carcinoma Endometrial cancer Available/total Adenocarcinoma Adenosquamous carcinoma Squamous cell carcinoma Mullerian tumor

161/243 155 (96.3) 3 (1.9) 1 (0.6) 2 (1.2) 92/216 69 (75.0) 6 (6.5) 4 (4.3) 4 (4.1) 2 (2.2) 2 (2.2) 2 (2.2) 1 (1.1) 1 (1.1) 1 (1.1) 130/192 111 (85.4) 18 (13.8) 1 (0.8) 109/135 100 (91.7) 4 (3.7) 4 (3.7) 1 (0.9)

Available: number of subjects from whom data could be collected. Total: total number of subjects of the study

of FAS −670 A>G wild-type, heterozygous, and homozygous genotypes were 33.5, 46.9, and 19.6 %, respectively. On the other hand, frequencies of FASL −844 T>C genotypes wildtype, heterozygous, and homozygous were 24.3, 44.2, and 35.5 %, respectively. Prevalence of FAS −1377 AA variant was significantly more in breast (P≤0.003) but not in ovarian, cervical, and endometrial cancers. However, FAS −1377 GA variant was significantly more prevalent both in breast and cervical cancers (P≤0.005). We found that the breast cancer risk was significantly increased with both GA and AA variants of FAS −1377 G>A, whereas the risk of cervical cancer was elevated by the heterozygous variant, GA. On the other hand, FAS −670 AG variant was found to be less frequent (P≤ 0.004) in breast cancer and the carriers of the variant were at lowered risk for breast cancer (OR=0.51, 95 % CI=0.33– 0.80). Contrary to the distribution of FAS SNPs, the CC genotype of FASL −844 T>C polymorphism was found to be significantly low in all the four female-specific cancers and was found to be protective against all the four cancers studied (Table 6). As shown in Table 7, risk of breast cancer associated with GA and AA variants of FAS −1377 G>A polymorphism

showed a significant increase in the ER- and PR-negative subjects, whereas the CC variant of FASL −844 T>C polymorphism decreased the risk of breast cancer risk in triplenegative subjects. Combined effect of the FAS SNPs We also found that the presence of combined genotypes of FAS SNPs had a variable effect on the cancer phenotypes (Table 8). The highest risk was conferred by genotype combination: AA of −1377 G>A + AA of −670 A>G. A 10-fold risk was observed in breast cancer patients with this genotype while it had no effect on other cancers. A significant but contrasting effect was noted with one genotype combination (GA + AA) with increased risk for breast cancer (OR=3.7, 95 % CI=1.80–7.65) and reduced risk for endometrial cancer (OR=0.2, 95 % CI=0.04–0.93). The other genotypes which exhibited association with increased risk for cervical (OR= 2.6, 95 % CI=1.21–5.48) and endometrial (OR=3.25, 95 % CI=1.12–9.44) cancers were GA + GG and AA + GG, respectively. The only cancer phenotype with no additive effect of the SNPs was ovarian cancer. LD and haplotype analysis of the FAS SNPs The D′ and r2 values varied across the case and control groups. While the SNPs were independent of each other (r2 =0.0) with no LD between them in breast cancer subjects (D′=0.0, 95 % Table 3

ER, PR, and Her-2 status of the breast cancer patients

Estrogen receptor status (available/total) Positive Negative Progesterone receptor status (available/total) Positive Negative ER/PR status (available/total) Positive/positive Positive/negative Negative/positive Negative/negative Her-2 status (available/total) Positive Negative ER/PR/Her-2 status (available/total) Triple positive Positive/positive/negative Positive/negative/positive Positive/negative/negative Negative/negative/positive Triple negative

88/245 38 (43.2) 50 (56.8) 88/245 32 (36.4) 56 (63.6) 88/245 32 6 0 50 21/245 4 (19) 17 (81) 21/245 2 6 1 1 1 10

Available: number of subjects from whom data could be collected. Total: total number of subjects of the study

Tumor Biol. Table 4

Progression of the female cancers

Stage (available/total) I II III IV Metastasis (no./available) Recurrence (no./available) Relapse (no./available) Secondary cancer (no./available) Second primary (no./available)

Breast cancer

Ovarian cancer

Cervical cancer

Endometrial cancer

51/245 0 24 (47.1) 26 (51) 1 (2) 19/245 9/245 0 0 0

58/216 5 (8.6) 0 51 (87.9) 2 (3.4) 17/216 22/216 10/216 0 1/216

75/192 43 (57.3) 26 (34.7) 5 (6.7) 1 (1.3) 5/192 10/192 1/192 1/192 0

7/135 7 (100) 0 0 0 1/135 1/135 1/135 0 0

Available: number of subjects from whom data could be collected. Total: total number of subjects of the study

CI=0.01–0.22), there was mild to strong LD among other cancers; the values of D′ in cervical, ovarian, and endometrial cancers were 0.47, 0.53, and 0.83 respectively (Fig. 1a–e). In controls, there was moderate LD between the SNPs (D′=0.44, 95 % CI=0.272–0.59). The various haplotypes observed for the FAS SNPs (−1377 G>A and −670 A>G) were G-A, G-G, A-G, and A-A (Table 9). With G-A as the reference haplotype, the remaining haplotypes were found to confer risk or protection toward ovarian or endometrial cancer while conferring only risk for breast and cervical cancers. However, a significant association of the haplotypes was noted only for breast cancer with at least a 3-fold enhanced risk for the disease. Interestingly, we also noted that the frequency of the G-A haplotype was much lower in breast cancer patients (22.5 %) as compared to controls (49.3 %) unlike in other cancers. The frequency of G-A haplotype in ovarian, endometrial, and cervical cancers were 47.9, 47.2, and 41.3 %, respectively.

groups, while most of the endometrial cancer patients belong to the age group 51–65 years (81/135). However, average age of cancers across the cancer phenotypes was similar to the median age. The average ages of patients were 50.3 (24– 85 years), 46.8 (15–84), 47 (25–75), and 57.7 years (34–82) for breast, ovarian, cervical, and endometrial cancer subjects, respectively. When the data was stratified based on the average menopausal age for Indians, reference age points for premenopausal and postmenopausal subjects were ≤47 and >47 years, respectively [50, 51]. Number of cervical cancer subjects was more in premenopausal females compared to postmenopausal females (54.4 vs 45.6 %). In contrast, majority of the breast, ovarian, and endometrial subjects were postmenopausal females. This trend was more predominant in endometrial cancer wherein 83.7 % of the subjects were postmenopausal females (Table 1).

Two locus analysis of FAS and FASL SNPs Co-occurrence of FAS −1377 GA/AA and FAS −670 AG/GG and/or FASL −844 TC/CC variants with varying frequencies were noted in both control and cancer subjects (Table 10). Analysis of two locus polymorphisms revealed that combination of genotypes at FAS −670 A>G and FASL −844 T>C altered the risk of breast cancer alone. The presence of AG + TC/CC or GG + CC genotypes of the SNPs were less prevalent in breast cancer subjects as compared to the reference genotypes AA + TT (OR=1.0) and were protective against the breast cancer (P≤0.01). Distribution of female-specific cancers across different age groups As shown in Fig. 2, majority of breast (100/241), ovarian (96/ 215), and cervical (101/191) cancer patients were between age group 36–50 years as compared to younger or older age

Discussion To the best of our knowledge, this is the first study to assess risk association of FAS and FASL promoter SNPs with female-specific cancers within the same cohort and report that the SNPs alter the risk of the cancers, divergently. Our investigations on the risk association of the FAS (−1377 G>A and −670 A>G) and FASL (−844 T>C) promoter polymorphisms with female-specific cancers indicated that women with FASL −844 CC genotype are less susceptible to the breast, ovarian, cervical, and endometrial cancers. We also found that FAS −670 AG genotype lowers risk of breast cancer, while the FAS −1377 G>A polymorphism is a risk factor for breast and cervical cancers. Our findings of increased breast cancer risk with GA and AA genotypes of FAS −1377 G>A polymorphism and protective effect of AG genotype of the FAS −670 A>G against breast cancer are in partial agreement with earlier reports [33, 52]. However, in Austrian [53] and Chinese

*P≤0.05, statistically significant

N number of samples

Endometrial cancer (N=127)

Cervical cancer (N=173)

Ovarian cancer (N=195)

FAS −670 A>G Controls (N=194) Breast cancer (N=216)

Endometrial cancer (N=129)

Cervical cancer (N=179)

Ovarian cancer (N=192)

FAS −1377 G>A Controls (N=203) Breast cancer (N=222)

OR (95 % CI) P≤

OR (95 % CI) P≤

OR (95 % CI) P≤

OR (95 % CI) P≤

OR (95 % CI) P≤

OR (95 % CI) P≤

OR (95 % CI) P≤

OR (95 % CI) P≤

Parameter

38 (29.9) 1

48 (27.7) 1

54 (27.7) 1

AA, N (%) 65 (33.5) 100 (46.3) 1

66 (51.2) 1

74 (41.3) 1

113 (58.9) 1

GG, N (%) 115 (56.7) 87 (39.2) 1

GA, N (%) 76 (37.4) 107 (48.2) 1.86 (1.24–2.8) 0.003* 60 (31.2) 0.80 (0.52–1.23) 0.3 90 (50.3) 1.84 (1.20–2.80) 0.005* 47 (36.4) 1.08 (0.67–1.73) 0.81 AG, N (%) 91 (46.9) 72 (33.3) 0.51 (0.33–0.8) 0.004* 104 (53.3) 1.37 (0.87–2.17) 0.2 78 (45.1) 1.16 (0.71–1.87) 0.63 53 (41.7) 1.0 (0.58–1.68) 1.0

Frequency of genotypes

Association of FAS polymorphisms with breast, ovarian, cervical, and endometrial cancers

Polymorphism/sample size (N)

Table 5

AA, N (%) 12 (5.9) 28 (12.6) 3.08 (1.48–6.41) 0.003* 19 (9.9) 1.61 (0.74–3.47) 0.25 15 (8.4) 1.94 (0.86–4.4) 0.14 16 (12.4) 2.32 (1.04–5.21) 0.06 GG, N (%) 38 (19.6) 44 (20.4) 0.75 (0.44–1.28) 0.34 37 (19.0) 1.17 (0.65–2.09) 0.66 47 (27.2) 1.67 (0.95–2.96) 0.086 36 (28.4) 1.62 (0.88–2.97) 0.13

GA + AA, N (%) 88 (43.3) 135 (60.8) 2.02 (1.37–2.98) 0.0003* 79 (41.1) 0.91 (0.61–1.36) 0.68 105 (58.7) 1.85 (1.23–2.78) 0.003* 63 (48.8) 1.24 (0.80–1.94) 0.36 AG + GG, N (%) 129 (66.5) 116 (53.7) 0.58 (0.39–0.87) 0.008* 141 (72.3) 1.31 (0.85–2.02) 0.22 125 (72.3) 1.31 (0.83–2.05) 0.25 89 (70.1) 1.18 (0.72–1.91) 0.54 129 (50.8) 1

174 (50.3) 1

212 (54.4) 1

A allele, N (%) 221 (57.0) 272 (63.0) 1

179 (69.4) 1

238 (66.5) 1

286 (74.5) 1

G allele, N (%) 306 (75.4) 281 (63.3) 1

Frequency of alleles A allele, N (%) 100 (24.6) 163 (36.7) 1.77 (1.31–2.38) 0.0001* 98 (25.5) 1.04 (0.75–1.44) 0.80 120 (33.5) 1.54 (1.12–2.11) 0.008* 79 (30.6) 1.35 (0.95–1.91) 0.10 G allele, N (%) 167 (43.0) 160 (37.0) 0.77 (0.58–1.03) 0.08 178 (45.6) 1.11 (0.84–1.47) 0.47 172 (49.7) 1.30 (0.97–1.75) 0.07 125 (49.2) 1.28 (0.93–1.76) 0.14

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Tumor Biol. Table 6

Association of FASL −844 T>C polymorphism with breast, ovarian, cervical, and endometrial cancers

Subjects/sample size

Parameter

Frequency of genotypes TT, N (%) TC, N (%)

Controls (N=206) Breast cancer (N=228)

50 (24.3) 82 (36.0) OR (95 % CI) 1 P≤ Ovarian cancer (N=203) 70 (34.5) OR (95 % CI) 1 P≤ Cervical cancer (N=179) 61 (34.1) OR (95 % CI) 1 P≤ Endometrial cancer (N=127) 49 (38.6) OR (95 % CI) 1 P≤

91 (44.2) 102 (44.7) 0.68 (0.43–1.07) 0.11 87 (42.9) 0.68 (0.42–1.08) 0.125 82 (45.8) 0.73 (0.45–1.19) 0.23 57 (44.9) 0.64 (0.38–1.1) 0.09

Frequency of alleles CC, N (%)

TC + CC, N (%) T allele, N (%) C allele, N (%)

65 (35.5) 44 (19.3) 0.41 (0.24–0.69) 0.001* 46 (22.6) 0.51 (0.3–0.86) 0.012* 36 (20.1) 0.45 (0.26–0.79) 0.006* 21 (16.5) 0.33 (0.18–0.62) 0.0005*

156 (75.7) 146 (64.0) 0.57 (0.37–0.86) 0.009* 133 (65.5) 0.60 (0.39–0.93) 0.02* 118 (65.9) 0.62 (0.39–0.96) 0.04* 78 (61.4) 0.51 (0.31–0.82) 0.006*

191 (46.4) 266 (58.3) 1 227 (55.9) 1 204 (57.0) 1 155 (61.0) 1

221 (53.6) 190 (41.7) 0.61 (0.47–0.80) 0.0005* 179 (44.1) 0.68 (0.51–0.89) 0.006* 154 (43.0) 0.65 (0.49–0.86) 0.003* 99 (39.0) 0.55 (0.40–0.75) 0.0002*

N number of samples *P≤0.05, statistically significant

women [52], FAS −1377 G>A polymorphism but not that of FAS −670 A>G was shown to be risk factor for breast cancer. On the contrary, FAS −670 A>G polymorphism but not that of FAS −1377 G>A was found to increase breast cancer risk in Iranian women [32]. Adding to the inconsistent findings, a New York-based study found no correlation between breast cancer risk and the FAS SNPs [28]. Underlying the importance of ethnicities of the study participants, two metaanalysis studies linked the FAS −1377 G>A polymorphism with increased risk for breast cancer in Chinese but not in Caucasian women [54, 55]. Wang et al. [56], contrary to our observation, failed to find association between the FAS −670 A>G polymorphism and breast cancer risk. The variations among the reports may be due to the differences in the ethnic groups used in the studies, risk behavior of the subjects and environmental factors. Our data also indicated differential distribution of the FAS and FASL SNPs among the breast cancer subjects stratified based on the status of the hormone receptors. Our finding of lack of association between ovarian cancer risk and the FAS SNPs is in agreement with the reports that suggested risk of ovarian cancer in Turkish [22] and Japanese [21] women is independent of the FAS polymorphisms. However, concomitant presence of GG (FAS −1377 G>A) and TC (FASL −844 T>C) genotypes was reported to be protective against ovarian cancer in Turkish population [22]. Our observation of null association of the FAS −670 A>G polymorphism with cervical cancer is in agreement with reports on Taiwanese, Koreans, Chinese, and Black populations [27, 29–31], but differed from studies that linked increased cervical cancer risk with FAS −670 A>G polymorphism in North Indian women [34], and in Brazilian females who were

under 48 years of age [35], and a study that reported lack of association between the FAS SNP and with cervical cancer [27]. Our finding of increased risk of cervical cancer with FAS −1377 G>A differed from meta-analysis studies which found lack of association of the polymorphism with cervical cancer [57, 58]. Our study, which is the first one to assess the impact of both FAS (−1377 G>A and −670 A>G) and FASL polymorphisms on endometrial cancer risk, found no link between the FAS polymorphisms and the cancer and is in agreement with a Japanese study which reported lack of correlation between FAS −670 A>G polymorphism and endometrial cancer risk in Japanese women [21]. However, in Taiwanese women, A-A haplotype of the FAS −1377 G>A and −670 A>G polymorphisms was shown to enhance cervical cancer risk [30]. In line with the report, we found this haplotype increased risk for breast cancer by 3.86-fold. The most significant finding of our study was protective effect conferred by the FASL −844 CC genotype against breast, ovarian, cervical, and endometrial cancers. This finding, although not in line with some reports, was in agreement with other studies. Our data on the protective effect of FASL CC genotype differed from reported association of the FASL −844 CC genotype with an increased risk of cancers in diverse ethnic groups [33, 53]. The FASL genotype was associated with elevated risk of breast cancer in Chinese [33, 53], ovarian cancer in Turkish and Chinese [22, 53], and cervical cancer in Chinese [27] females. However, null association between the FASL −844 T>C polymorphism and breast cancer in Caucasians [28], cervical cancer in South Koreans [29], and in South African blacks [31] has been reported. Two metaanalysis studies also failed to find association between the FASL −844 T>C polymorphism with cervical cancer [59, 60].

25

1

0

3.19 (0.31– 33.18); 0.34 0

–

1.0 (0.16– 6.16); 1.0

4.53 (0.18– 112.7); 0.4

4.54 (0.89– 9.58 (1.23– 23.09); 0.07 74.34); 0.06

2 5.23 (1.08– 25.24); 0.05*

8

3.1 (0.12– 0.43 (0.02– 79.77); 1.0 10.82); 1.0

0.5 (0.02– 12.52); 1.0

6

0

0

0.6 (0.23– 1.55); 0.37

0.24 (0.01– 0.57 (0.02– 5.95); 0.42 14.28); 1.0 0 0

3.06 (1.24– 7.57); 0.02*

0

0.49 (0.19– 1.3); 0.21

1.02 (0.55– 1.89); 1.0 0

9

TC

20

G allele

0.31 (0.09– 1.04); 0.08 0

4

CC

FASL −844 T>C

0.1 (0.005– 2.14); .0.12 3

0

0.5 (0.25– 1.0); 0.07

1

0

0.82 (1.33– 5.1); 1.0

3

0.38 (0.03– 4.36); 0.58 0

0.26 (0.01– 6.45); 0.44 1

0

0.71 (0.17– 0.85 (0.15– 0.76 (0.21– 2.96); 0.72 4.89); 1.0 2.8); 0.74

0.88 (0.35– 0.11 (0.01– 2.21); 0.82 0.97); 0.03*

0.61 (0.16– 2.41); 0.51

0.17 (0.007– 0.26 (0.01– 0.18 (0.007– 0.26 (0.01– 4.2); 0.34 5.55); 0.51 4.6); 0.36 6.45); 0.44 6 8 1 4

0.17 (0.007– 0.26 (0.01– 0.18 (0.007– 0.260.01– 4.2); 0.34 5.55); 0.51 4.6); 0.36 6.45); 0.44 0 0 0 0

0

0.95 (0.29– 3.03); 1.0

5

0.14 (0.008– 0.54 (0.03– 2.75); 0.14 8.98); 1.0

0

0.67 (0.41– 0.59 (0.27– 0.46 (0.19– 1.12); 0.14 1.28); 0.22 1.41); 0.12

0.17 (0.007– 0.26 (0.01– 0.18 (0.007– 0.26 (0.01– 4.2); 0.34 5.55); 0.51 4.6); 0.36 6.45); 0.44 20 27 16 9

0.65 (0.27– 1.57); 0.36 0

13

AG + GG

0.24 (0.02– 1.14 (0.18– 0.5 (0.1– 2.34); 0.31 7.14); 1.0 2.57); 0.41 0 0 0

0.14 (0.007– 0.34 (0.34– 3.03); 0.18 7.28); 0.53 1 2

0

0.46 (0.22– 1.0); 0.06

0.61 (0.03– 0.24 (0.01– 0.57 (0.02– 12.82); 1.0 5.95); 0.42 14.28); 1.0 10 4 2

3.9 (0.16– 3.06 (0.19– 97.34); 0.44 49.41); 0.43 0 0

1

1.53 (0.45– 5.19); 0.5

1.31 (0.26– 6.63); 1.0 1

4

1.02 (0.10– 9.92); 1.0

1

2.38 (1.45– 3.9); 0.0009*

3

0

1

1

3.08 (0.12– 1.31 (0.08– 79.76); 1.0 21.2); 1.0

3.92 (1.82– 8.45); 0.0003*

2

*P≤0.05, statistically significant

Triple negative OR (95 % CI); P≤

Negative/ negative/ positive OR (95 % CI); P≤

Positive/ negative/ positive OR (95 % CI); P≤

Positive/ positive/ negative OR (95 % CI); P≤

7

0.43 (0.15– 1.2 (0.42– 1.24); 0.12 3.41); 0.79 0 0

6

GG

3.9 (0.16– 3.06 (0.19– 0.24 (0.009– 0.57 (0.02– 97.34); 0.44 49.4); 0.43 5.95); 0.42 14.28); 1.0 30 35 13 7

4.79 (1.4– 16.35); 0.02*

5

4.53 (0.18– – 112.73); 0.4

1

3.78 (1.72– 8.32); 0.0007* ER/PR/Her-2 status 1 Triple positive OR (95 % 1.5 (0.09– CI); P≤ 24.58); 1.0

Negative/ negative OR (95 % CI); P≤

Positive/ negative OR (95 % CI); P≤

3.59 (0.84– 2.45 (0.99– 1.97 (1.04– 15.39); 0.1 6.04); 0.05* 3.71); 0.05* 0 1 1

2.3 (0.089– 5.8); 0.1

18

3

12

15

A allele

AG

GG + AA

GA

AA

FAS −670 A>G

FAS −1377 G>A

Association of FAS and FASL SNPs with breast cancer, based on the hormonal status

ER/PR status Positive/ positive OR (95 % CI); P≤

Table 7

17

C allele

0

34

5

0.29 (0.029– 2.79); 0.34

1

0

0

9 0.32 (0.89– 0.71 (0.29– 1.15); 0.13 1.74); 0.5

5

0.12 (0.004– 0.17 (0.008– 2.68); 0.25 3.63); 0.22

0

0.12 (0.004– 0.17 (0.008– 2.68); 0.25 3.63); 0.22

0

0.64 (0.11– 0.62 (0.19– 3.61); 0.64 2.0); 0.56

4

0.32 (0.02– 5.2); 0.43

1

0.53 (0.26– 0.64 (0.39– 1.09); 0.12 1.04); 0.86

25

0.12 (0.004– 0.17 (0.01– 2.68); 0.25 3.63); 0.22

0

0.42 (0.17– 0.51 (0.27– 1.01); 0.08 0.9); 0.04*

13

TC + CC

Tumor Biol.

Tumor Biol. Table 8

Combined effect of FAS polymorphisms toward breast, ovarian, cervical, and endometrial cancers

FAS −1377 G>A and FAS −670 A>G

CF (%), N=191

BC (%), N=209

OC (%), N=186

CC (%), N=162

EC (%), N=122

GG + AA GA + AG OR (95 P≤ GA + GG OR (95 P≤ AA + AG OR (95 P≤ AA + GG OR (95 P≤ GG + AG OR (95 P≤ GG + GG OR (95 P≤ GA + AA OR (95 P≤ AA + AA OR (95 P≤

48 (25.1) 39 (20.4)

38 (18.2) 38 (18.2) 1.23 (0.66–2.28) 0.53 22 (10.5) 1.54 (0.73–3.3) 0.34 7 (3.3) 2.2 (0.6–8.1) 0.34 5 (2.4) 1.0 (0.30–3.72) 1.0 25 (12.0) 0.69 (0.36–1.31) 0.33 14 (6.7) 1.36 (0.57–3.24) 0.51 44 (21.0) 3.7 (1.80–7.65) 0.0003* 16 (7.7) 10.10 (2.19–46.7) 0.0005*

44 (23.7) 38 (20.4) 1.06 (0.58–1.94) 0.88 16 (8.6) 0.97 (0.44–2.1) 1.0 9 (4.8) 2.5 (0.70–8.5) 0.24 8 (4.3) 1.5 (0.47–4.5) 0.58 52 (28.0) 1.23 (0.70–2.18) 0.56 11 (5.9) 0.92 (0.37–2.27) 1.0 6 (3.2) 0.44 (0.16–1.22) 0.14 2 (1.1) 1.09 (0.15–8.08) 1.0

28 (17.3) 40 (24.7) 1.76 (0.93–3.34) 0.11 27 (16.6) 2.6 (1.21–5.48) 0.015* 5 (3.1) 2.14 (0.53–8.65) 0.30 7 (4.3) 2.0 (0.61–6.55) 0.36 29 (17.9) 1.08 (0.56–2.09) 0.87 10 (6.2) 1.32 (0.51–3.4) 0.63 16 (9.9) 1.83 (0.79–4.26) 0.2 0 (0.0) 0.34 (0.02–7.35) 0.53

32 (26.2) 23 (18.9) 0.88 (0.45–1.75) 0.73 19 (15.6) 1.58 (0.72–3.47) 0.32 1 (0.8) 0.38 (0.04–3.5) 0.64 13 (10.7) 3.25 (1.12–9.44) 0.04* 27 (22.1) 0.88 (0.46–1.69) 0.74 4 (3.3) 0.46 (0.14–1.54) 0.27 2 (1.6) 0.2 (0.04–0.93) 0.03* 1 (0.8) 0.75 (0.06–8.6) 1.0

% CI) 18 (9.4) % CI) 4 (2.1) % CI) 6 (3.1) % CI) 46 (24.1) % CI) 13 (6.8) % CI) 15 (7.9) % CI) 2 (1.1) % CI)

N number of common samples, CF control female, BC breast cancer, OC ovarian cancer, CC cervical cancer, EC endometrial cancer *P≤0.05, statistically significant

On the other hand, in line with our findings, the FASL −844 CC genotype was found to confer protection against childhood acute lymphoblastic leukemia [61], and esophageal [62] and non-small-cell lung [63] cancers. Zhao et al. [62] found that FASL −844 CC genotype was protective against esophageal cancer in females irrespective of their age, but the protective effect was limited to males who are below 60 years of age [62]. FASL −844 CC was reportedly associated with lower prevalence of second primary cancer of head and neck squamous cell carcinoma [64] and with good prognosis for breast cancer in Tunisian patients with diagnosis age ≤50 years [65]. A recent report also indicated that FASL TC genotype lowers risk of breast cancer in Chinese women [66]. Negative expression of FASL was correlated with oral-cancer-related deaths and shortened disease-free survival (DFS) [67]. The “G” and “T” alleles of FAS −1377 G>A and FASL −844 T>C, respectively, were found to be risk factors for bladder cancer in Turkish population [68]. These findings taken together with our data indicate a protective role for FASL T>C polymorphism against cancer. We have also noted that the combined presence of FAS SNPs modulate cancer risk independent of the presence of individual SNPs. Susceptibility toward endometrial cancer

which was unaltered with the individual polymorphisms was found to be elevated with the combined presence of AA (FAS −1377 G>A) and GG (FAS −670 A>G) and decreased with the concomitant presence of GA (FAS −1377 G>A) and AA (FAS −670 A>G) genotypes. Concomitant presence of GA (FAS −1377 G>A) and AA (FAS −670 A>G), and AA (FAS −1377 G>A) and AA (FAS −670 A>G) genotypes caused a profound increase in breast cancer risk. Cervical cancer risk was also found to be elevated with the combined presence of GA (FAS −1377 G>A) and GG (FAS −670 A>G) genotypes. Our data also revealed that the effect of gene-gene interaction may also impact cancer risk. We observed that co-occurrence of FAS −670 AG polymorphic variant with the FASL −844 CC genotype was more protective against breast cancer with an OR=0.31 as compared to the presence of FAS −670 AG (OR= 0.51) or FASL −840 CC genotype (OR=0.41). All cancers, notwithstanding their heterogeneity, are characterized by diminished apoptotic ability of tumor cells and acquired ability to evade and escape from the immune cells. Aberrations in FAS/FASL genes, especially the promoter region SNPs, have been linked to resistance to apoptosis and enhanced evasion of immune surveillance and escape by

Tumor Biol. Fig. 1 a–e Linkage disequilibrium plots generated for the FAS SNPs using Haploview from the genotype data as input file across different study groups. Values indicated in the squares are D′ values, the color of the square varies according to the measure of LD. Low or no LD is indicated as white, mild LD as shades of pink, and strong LD is indicated as dark red

counter attacking infiltrating immune cells observed in cancer [10]. Paradoxically, the FAS/FASL system has also been implicated in cell proliferation and promoting tumor growth Table 9

in epithelial cancer, melanoma, myeloma and thyroid cancer, and primary chronic lymphocytic leukemia B cells [69–72]. Although FAS is the first death receptor identified and

Haplotype frequencies of FAS SNPs across controls and female cancer subjects

Haplotypes

G-A G-G OR (95 % CI) P value A-G OR (95 % CI) P value A-A OR (95 % CI) P value

Frequency distribution of haplotypes across different cancers CF (%), N=191

BC (%), N=209

OC (%), N=186

CC (%), N=162

EC (%), N=122

94 (49.3) 49 (25.6)

47 (22.5) 82 (39.2) 3.34 (2.0–5.50) 0.0001* 51 (24.4) 3.09 (1.76–5.41) 0.0001* 29 (13.8) 3.86 (1.89–7.90) 0.0002*

89 (47.9) 48 (25.8) 1.03 (0.63–1.69) 0.90 37 (19.6) 1.18 (0.68–2.05) 0.57 12 (6.7) 0.84 (0.37–1.90) 0.83

67 (41.3) 42 (25.7) 1.20 (0.71–2.01) 0.51 39 (24.3) 1.65 (0.94–2.90) 0.08 14 (8.7) 1.30 (0.59–2.89) 0.54

57 (47.2) 28 (22.5) 0.94 (0.53–1.66) 0.69 34 (27.9) 1.69 (0.95–3.03) 0.07 3 (2.4) 0.32 (0.09–1.18) 0.11

33 (17.1)

15 (8.0)

Frequencies are given in percentage with respect to total number of subjects in each study group N number of common samples, CF control female, BC breast cancer, OC ovarian cancer, CC cervical cancer, EC endometrial cancer *P≤0.05, statistically significant

12 (6.1) 15 (7.6) 0.85 (0.31–2.32); 0.8 12 (6.1) 1.87 (0.58–6.06); 0.38 11 (5.6) 1.37 (0.43–4.32); 0.77 5 (2.5) 1.25 (0.29–5.35); 1.0 1 (0.5) 1.25 (0.07–22.15); 1.0 1 (0.5)

CF (%), N=188 20 (10.6) 44 (23.4)

15 (8.1) 22 (11.9)

FAS −670 A>G & FASL −844 T>C AA + TT AG + TC OR (95 % CI); P≤ AG + CC OR (95 % CI); P≤ GG + TC OR (95 % CI); P≤ GG + CC OR (95 % CI); P≤ Any one wild type OR (95 % CI); P≤ FAS −1377 G>A & FAS −844 T>C GG + TT GA + TC OR (95 % CI); P≤ GA + CC OR (95 % CI); P≤ AA + TC OR (95 % CI); P≤ AA + CC OR (95 % CI); P≤ Any 1 wild OR (95 % CI); P value Co-occurrence of FAS −1377 G>A, FAS −670 A>G and FASL −844 T>C polymorphisms GG + AA+ TT GA + AG+ TC OR (95 % CI); P≤ GA + AG+ CC OR (95 % CI); P≤ GA + GG+ TC OR (95 % CI); P≤ GA + GG+ CC OR (95 % CI); P≤ AA + AG+ TC OR (95 % CI); P≤ AA + AG+ CC 2 (1.1)

1 (0.5)

5 (2.7)

10 (5.4)

8 (4.3)

CF (%), N=185

102 (51.8)

3 (1.5)

5 (2.6)

17 (8.6)

CF (%), N=197 28 (14.2) 42 (21.3)

69 (36.7)

15 (8.0)

16 (8.5)

24 (12.8)

BC (%), N=203 37 (18.2) 31 (15.3) 0.38 (0.19–0.78); 0.009* 14 (6.9) 0.31 (0.13–0.74); 0.01* 22 (10.8) 0.74 (0.32–1.72); 0.52 5 (2.5) 0.18 (0.06–0.57); 0.003* 94 (46.3) 0.74 (0.39–1.38); 0.35 BC (%), N=209 30 (14.4) 35 (16.7) 0.78 (0.39–1.54); 0.49 30 (14.3) 1.65 (0.75–3.62); 0.24 11 (5.3) 2.05 (0.63–6.66); 0.27 4 (1.9) 1.24 (0.25–6.06); 1.0 99 (47.4) 0.90 (0.50–1.62); 0.77 BC (%), N=198

Female cancer phenotype

Two locus analysis of FAS and FASL polymorphisms for female-specific cancers

Polymorphisms

Table 10

10 (5.6) 17 (9.5) 1.16 (0.42–3.22); 0.80 8 (4.5) 1.5 (0.42–5.32); 0.75 6 (3.4) 0.9 (0.25–3.27); 1.0 4 (2.2) 1.2 (0.26–5.59); 1.0 3 (1.7) 4.5 (0.41–49.66); 0.30 3 (1.7)

OC (%), N=184 13 (7.1) 47 (25.5) 1.64 (0.73–3.70); 0.31 23 (12.5) 1.47 (0.60–3.64); 0.49 10 (5.4) 0.96 (0.33–2.76); 1.0 7 (3.8) 0.72 (0.23–2.24); 0.77 84 (45.7) 1.87 (0.87–4.03); 0.12 OC (%), N=182 31 (17.0) 27 (14.8) 0.58 (0.29–1.17); 0.15 13 (7.1) 0.69 (0.28–1.67); 0.50 4 (2.2) 0.72 (0.17–2.96); 0.73 5 (2.8) 1.5 (0.33–6.88); 0.71 102 (56.1) 0.90 (0.50–1.6); 0.77 OC (%), N=178 6 (3.8) 15 (9.6) 1.7 (0.54–5.39); 0.41 9 (5.7) 2.8 (0.73–10.78); 0.18 13 (8.3) 3.25 (0.92–11.41); 0.07 3 (1.9) 1.5 (0.27–8.35); 0.67 2 (1.3) 5.0 (0.38–66.05); 0.25 3 (1.9)

CC (%), N=163 13 (8.0) 34 (20.9) 1.19 (0.52–2.72); 0.83 15 (9.2) 0.96 (0.37–2.49); 1.0 21 (12.9) 2.02 (0.78–5.24); 0.16 4 (2.4) 0.41 (0.11–1.51); 0.23 76 (46.6) 1.69 (0.78–3.66); 0.25 CC (%), N=171 21 (12.3) 35 (20.5) 1.11 (0.54–2.29); 0.85 19 (11.1) 1.49 (0.63–3.54); 0.39 7 (4.1) 1.87 (0.52–6.71); 0.36 3 (1.7) 1.33 (0.24–7.28); 1.0 86 (50.3) 1.12 (0.60–2.12); 0.75 CC (%), N=157

12 (10.3) 14 (12.1) 0.79 (0.29–2.19); 0.8 1 (0.9) 0.15 (0.02–1.43); 0.11 7 (6.0) 0.87 (0.26–2.99); 1.0 5 (4.3) 1.25 (0.29–5.35); 1.0 1 (0.9) 1.25 (0.07–22.15); 1.0 0 (0.0)

EC (%), N=121 14 (11.6) 25 (20.6) 0.81 (0.35–1.88); 0.67 6 (5.0) 0.36 (0.11–1.10); 0.10 14 (11.6) 1.25 (0.46–3.37); 0.80 7 (5.8) 0.67 (0.21–2.06); 0.56 55 (45.4) 1.14 (0.53–2.46); 0.84 EC (%), N=121 26 (21.5) 22 (18.2) 0.56 (0.26–1.18); 0.14 6 (5.0) 0.38 (0.13–1.11); 0.08 8 (6.6) 1.72 (0.50–5.94); 0.54 1 (0.8) 0.36 (0.03–3.67); 0.61 58 (47.9) 0.61 (0.33–1.14); 0.15 EC (%), N=116

Tumor Biol.

Combination of wild-type genotypes at two of the three SNPs versus mutant genotype at the third SNP

Combination of mutant genotypes at two of the three SNPs versus wild-type genotype at the third SNP b

a

N number of samples genotyped for all the three SNPs, CF control female, BC breast cancer, OC ovarian cancer, CC cervical cancer, EC endometrial cancer

*P≤0.05, statistically significant

70 (37.8)

48 (26.0)

1 (0.6)

3 (1.6)

OR (95 % CI); P≤ AA + GG+ TC OR (95 % CI); P≤ AA + GG+ CC OR (95 % CI); P≤ Any two wild types vs mutanta OR (95 % CI); P≤ Any one wild type vs two mutantsb OR (95 % CI); P≤

Polymorphisms

Table 10 (continued)

Female cancer phenotype

0.62 (0.05–7.75); 1.0 2 (1.0) 0.83; (0.11–5.82); 1.0 0 (0.0) 0.41 (0.01–11.06); 1.0 64 (32.3) 1.67 (0.71–3.89); 0.28 75 (37.9) 1.34 (0.59–3.06); 0.53

2.25 (0.32–15.98); 0.63 1 (0.6) 0.5 (0.04–5.52); 1.0 2 (1.1) 3.0 (0.24–37.70); 0.56 54 (30.3) 1.69 (0.69–4.11); 0.27 70 (39.3) 1.5 (0.63–3.57); 0.39

3.75 (0.49–28.40); 0.30 4 (2.5) 3.33 (0.57–19.6); 0.21 0 (0.0) 0.79 (0.03–22.20); 1.0 40 (25.5) 2.08 (0.74–5.87); 0.22 62 (39.5) 2.21 (0.81–6.06); 0.16

0.24 (0.01–5.66); 0.50 6 (5.1) 2.5 (0.51–12.14); 0.44 1 (0.9) 1.25 (0.07–22.15); 1.0 34 (29.3) 0.88 (0.37–2.13); 0.82 35 (30.2) 0.62 (0.26–1.48); 0.37

Tumor Biol.

characterized and is linked to cancer [73, 74], its apoptotic and non-apoptotic functions relevant to cancer remain enigmatic. The FAS −1377 G>A and FAS −670 A>G germline SNPs in the promoter region of the gene block the binding of Sp1 transcription factor binding site present in the silencer region and STAT1 transcriptional factor binding site present at the enhancer region of the gene, respectively, leading to downregulation of the FAS gene expression [75, 76]. Although the FAS −1377 G>A was shown to decrease the FAS expression, the FAS −670 A>G polymorphism was reported to have no major impact on the transcriptional activity of the FAS gene [77]. On the other hand, the FASL −844 T>C SNP at the binding site for CAAT/enhancer binding protein, particularly the CC variant was shown to upregulate FASL gene compared to the TT variant [78]. The increased risk of breast and cervical cancers with FAS −1377 G>A polymorphism found in the present study and by others may be explained by the downregulation expression of the aberrant FAS gene, leading to reduced ability of cancer cells to apoptose, and upregulation of mutated FASL gene expression resulting in elevated ligand levels on T cells. This overexpression of the FASL gene results in enhanced activation induced T cell death leading to diminished immune surveillance [79] and heightened immune evasion of cancer cells [52]. Contrary to this view, Das et al. reported elevated levels of FAS messenger RNA (mRNA) but not that of FASL [80], in various gynecological cancers than in corresponding control tissue. Takagi et al. also failed to find upregulation of FASL gene expression in endometrial and ovarian carcinomas [81]. These reports taken, together with our data on protective effect of FASL, indicate that the notion of FASL-mediated evasion of immune escape and attack on the T cells may not be entirely applicable to female-specific cancers. Das et al. have also noted higher levels of FASL but not that of FAS expression in non-cancerous endometrial tissue, as compared to ovaries or cervix [80]. Protective effect of the FASL −844 CC genotype observed in the present study appears to be more complex and may not be solely reasoned by the FASL −844 CC genotype-driven increased expression of the ligand. Soluble FAS (sFAS) levels were found to be more in breast cancer [82] and uterine cancer [83]. In uterine cancer study, soluble FASL (sFASL) was also found to be increased and levels of sFAS and FASL were correlated with stage of the malignancy (FIGO) [83]. In ovarian cancer, the FAS levels were reported to help differentiate between benign ovarian cysts and cancer [84]. Similarly, the protective effect of FAS −670 AG genotype against breast cancer, seen in the study, may not be explained by the altered FAS gene expression, as the transcription activity of FAS gene was reported to be unaltered with the FAS −670 AG polymorphic variant [85]. A recent study also reported that the FAS promoter polymorphisms did not alter the FAS mRNA levels

Tumor Biol. Fig. 2 Prevalence of breast, ovarian, cervical, and endometrial cancers in different age groups

in breast cancer tissue as compared to the non-cancerous tissue [66]. However, Xu et al. [66] reported increased levels of sFAS in the serum of the patients carrying FAS polymorphic gene variants and suggested that sFAS may play a role in the FAS-associated functions related to cancer. Despite the inconsistencies in the reports, associations of the FAS and FASL polymorphisms noted in the present study taken together with other studies indicate relevance of the FAS and FASL SNPs to the cancers. The discrepancies in the reports may be due to the different ethnicities of the study subjects, lifestyle, and environmental factors. Factors affecting cancers are diverse and might not be linked to a single factor, and may vary between males and females. The femalespecific cancer risk factors, such as age at menarche, child birth and menopause, and hormonal status, play a role in the female-specific cancers, like breast, ovarian, cervical, and endometrial malignancies. These associations of the promoter region polymorphisms of FAS/FASL with the cancers may not be directly correlated with the levels of gene expression as the expression is regulated by other SNPs and epigenetic factors, like methylation and acetylation [86, 87]. Additionally, functional outcome of the FAS/FASL system may not be assessed by protein levels of the FAS/FASL, as the posttranslational modifications such as palmitoylation, sumolation, or phosphorylation of the proteins [88, 89] may also affect apoptotic ability of the FAS/FASL system. Further, sFASL resulting from proteolytic cleavage of FASL by metalloproteases has been shown to have reduced apoptotic ability and may have roles independent of FAS [88]. We hypothesize that the role(s) of FAS/FASL system in females, at least in part, may be controlled by the levels of mFASL and/or sFASL versus FAS, which in turn may be regulated by the status of estrogen/estradiol. The variations reported on the impact of FAS and FASL promoter region SNPs on the cancers may be accounted by different genetic backgrounds and aberrations of the receptor/ ligand coding genes. In addition, gene-gene and geneenvironment interactions which possibly differ in different

ethnic populations may also contribute to the inconsistent results. Hence, studies on the association of FAS/FASL polymorphisms with female cancers in cohorts with similar ethnic background and geographical location may provide better insights into the genotype-phenotype correlation as opposed to the data collected from different ethnic groups. The major strength of the study is the less heterogeneity of the study participants and simultaneously assessing the risk association of the FAS and FASL genotypes on the female-specific cancers. Our study is hospital-based, and limitations of the study include unavailability of age at menarche, menopausal age, and clinicopathological information from some subjects. In summary, our hospital-based study is unique as we investigated the impact of three promoter region SNPs of FAS and FASL on female-specific cancers in subjects with similar genetic background and environment, and demonstrated that the homozygous CC variant of FASL −844 T>C polymorphism confers protection against breast, ovarian, cervical, and endometrial cancers. In addition, we reported that FAS −1377 G>A polymorphism is a risk factor for breast and cervical cancers, while FAS −670 A>G SNP confers protection against breast cancer. Further, we also observed that the risk of cancers varies with the combined presence of the SNPs as compared to presence of the individual SNPs. Protective effect of the FASL polymorphism observed in the present study warrants for larger studies to ascertain anticancer potential of FASL. Acknowledgments The research was partly funded by a grant from Indian Council of Medical Research (ICMR), New Delhi (to VVTSP; grant no. 5/8/10-3(Oto)/CFP/11-NCD-1), and by Basavatarakam IndoAmerican Cancer Hospital and Research Institute (BIACH&RI), Hyderabad, India. Mr. Sateesh works for the ICMR grant. Mrs. Sarika is thankful to Council for Scientific and Industrial Research, Government of India, for the award of Junior Research Fellow. We also like to thank Acharya Nagarjuna University, Nagarjuna Nagar, AP, India, for registering Mrs. Sarika for her doctoral degree. We would like to thank Dr. SarithaKatta,volunteer researcher at the R&D, BIACH&RI, for her help in preparing the manuscript. We also acknowledge the technical help provided by the research assistants of the R&D.

Tumor Biol.

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