Functional implication of TRAIL 2716 C/T promoter ...

Breast Cancer Res Treat DOI 10.1007/s10549-010-0900-5

PRECLINICAL STUDY

Functional implication of TRAIL 2716 C/T promoter polymorphism on its in vitro and in vivo expression and the susceptibility to sporadic breast tumor Ranjana Pal • Sailesh Gochhait • Shilpi Chattopadhyay • Pawan Gupta Neeraj Prakash • Gaurav Agarwal • Arun Chaturvedi • Nuzhat Husain Syed Akhtar Husain • Rameshwar N. K. Bamezai

• •

Received: 8 November 2009 / Accepted: 15 April 2010 Ó Springer Science+Business Media, LLC. 2010

Abstract Recently, TRAIL function has been elucidated beyond its known classical role of mediating cellular homeostasis and immune surveillance against transformed cells. Here, we show how CC genotype of -716 TRAIL promoter SNP rendered risk for sporadic breast cancer as compared to the CT and TT genotypes (Precessive model = 0.018, OR = 1.4, 95% CI = 1.1–1.9; Pallele model = 0.010, OR = 1.3, 95% CI = 1.1–1.7). The in silico prediction of the introduction of core Sp1/Sp3-binding motif suggested the functional significance of the SNP variation. This functional implication was validated by luciferase assay in HeLa (P = 0.026), MCF-7 (P = 0.022), HepG2 (P = 0.024), and HT1080 (P = 0.030) cells and also by real-time expression studies on tumor tissues (P = 0.01), revealing the transcriptionally repressed status of -716 T when compared to -716 C allele. The SNP–SNP interactions reflected an Electronic supplementary material The online version of this article (doi:10.1007/s10549-010-0900-5) contains supplementary material, which is available to authorized users. R. Pal  S. Gochhait  S. Chattopadhyay  R. N. K. Bamezai (&) National Centre of Applied Human Genetics, School of Life Sciences, Jawaharlal Nehru University, Aruna Asafali Road, New Delhi 110067, India e-mail: [email protected] P. Gupta Dharamshila Cancer Hospital and Research Centre, Dharamshila Marg, Vasundhara Enclave, Delhi 110096, India N. Prakash Rajiv Gandhi Cancer Institute and Research Center, Sector V, Rohini, New Delhi 110085, India

enhanced protective effect of CT and TT genotypes with the protective genetic backgrounds of TP53-BRCA2 (P = 0.002, OR = 0.2, 95% CI = 0.1–0.6), IFNG (P = 0.0000002, OR = 0.3, 95% CI = 0.2–0.4), and common variant Casp8 (P = 0.0003, OR = 0.5, 95% CI = 0.3– 0.7). Interestingly, a comparison with clinical parameters showed overrepresented CT and TT genotypes in progressing (P = 0.041) and ER/PR negative tumors (P = 0.024/ 0.006). This was explained by increased apoptotic index, calculated as a ratio of selected pro-apoptotic and antiapoptotic gene expression profiles, in CC genotyped tumors, favoring either intrinsic (P = 0.008,0.018) or extrinsic (P = 0.025,0.217) pathway depending upon the ER/PR status. Our study reveals for the first time that a promoter SNP of TRAIL functionally modulates the gene and consequently its role in breast cancer pathogenesis, cautioning to consider the -716 TRAIL SNP status in patients undergoing TRAIL therapy. Keywords

TRAIL  Polymorphism  Apoptosis

A. Chaturvedi  N. Husain Chhatrapati Shahuji Maharaj Medical University, Chowk, Lucknow, Uttar Pradesh 226003, India S. A. Husain Human Genetics Laboratory, Department of Biosciences, Jamia Milia Islamia, Maulana Mohammad Ali Zohar Road, New Delhi 110025, India Present Address: R. N. K. Bamezai SMVDU, Katra, Jammu and Kashmir 180003, India

G. Agarwal Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226 014, India

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Introduction Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an important component of the apoptotic machinery which has evolved in the higher eukaryotes to maintain cellular homeostasis failing which facilitates oncogenic proliferation. TRAIL has attained the centre stage in antitumor drug discovery because of its efficacy in killing tumor cells without lethal toxicity in pre-clinical models apart from the inherent property to activate both the extrinsic and intrinsic apoptotic pathways [1–3]. The allogenic graft versus tumor and graft versus host experiments have shown conclusively that the activated T cells mediate apoptosis in tumor via TRAIL [4]. Further, the specificity for the tumor killing over normal cells has been shown by the effect of transplanted hematopoietic cells retrovirally transduced to overexpress TRAIL on mammary carcinoma [5]. The TRAIL-mediated host immune surveillance of tumors is further reinforced by interferon-gamma (IFNG) produced by T cells and/or by TRAIL production by a variety of immune cells induced by IFNG [6]. Paradoxically, TRAIL has also been shown to induce death of activated leukocytes [7–10]; thus, playing a vital role in escape of certain cancer cells from immune surveillance, a possible contributing factor to the metastasis of cancers [10], supported by the recent idea of tumor counter attack [11, 12]. However, what determines and influences the direction of action remains to be ascertained. TRAIL activates various intracellular signaling pathways consisting of NFjB, PI3K, and MAPK family proteins in specific cell types or in cells with defective death receptor signaling besides triggering apoptotic signal through caspase activation [13]. The TRAIL-induced NFjB can stimulate apoptosis or cell survival by suitably modulating the expression of DR4/5, IAP1, IAP2, and survivin depending on whether RELA or REL constitute the NFjB transcription factor [13–15]. TRAIL has been shown to inhibit the cyclic AMP responsible element mediated gene expression [16] and also induce CHEK2, a component of the DNA damage response (DDR), as a positive feedback loop involving the mitochondria-dependent activation of caspases, independent of p53 [17]. p53 also transactivates TRAIL and its receptors DR4/5 thus regulating apoptosis [18–20]. A plethora of cytokines including TGFB1, TNFA, IFNG, IL10, and IL6 have been reported to modulate the TRAIL expression directly or indirectly [6, 21–24]. TRAIL is emerging as a promising target for therapeutic intervention but is yet to be correlated with tumor etiology. Interacting partners in the pathway have been suggested to have a role in cancer pathogenesis which could attribute to functional redundancy. SNPs present along the TRAIL gene located in the 3q26 region have been extensively

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studied in multiple sclerosis and fatty liver disease [25–30]. However, similar studies in cancer including that of breast are lacking. In the present study, we, for the first time, show how -716 C/T (rs12488654) SNP in the promoter region of TRAIL is associated with breast cancer with functional implications both in vitro and in vivo studies. The study further dissects the role of the functional promoter SNP of TRAIL and its interactions with functional SNPs in other cancer-related genes and also the status of pro- and anti-apoptotic gene expression in breast tumor tissues. Finally, a model is proposed to show how SNP genotypes in -716 TRAIL promoter could differentially influence tumor onset and progression.

Materials and methods Cases and controls A total of 749 peripheral blood samples from north India were studied for the germline status of polymorphisms in the promoter of TRAIL gene along with DNA damage response (DDR)—apoptotic—cytokine pathway genes such as TP53, BRCA2, Casp8, and IFNG. This included 337 patients with sporadic breast tumor and 412 unrelated, healthy female controls with no history of breast tumor. Care was taken that both cases and controls were ethnically and geographically matched. Controls were chosen with no significant difference in distribution of age when compared with cases (P = 0.622), with average age of patient being 45.82 years and that of controls 41.29 years. Of the 337 breast cancer cases, a representative set of 79 tumor tissues with the same germline and somatic genotype background for TRAIL -716 nucleotide position were further studied for the status of expression of 11 candidate genes, TRAIL, DR4, DR5, DcR1, DcR2, cytochrome c, Bcl2, Casp8, Casp8L, FlipL, and FlipS, belonging to the apoptotic pathway. Prior approval was obtained from the Jawaharlal Nehru University ethical committee and the informed consent of the concerned subjects for sample collection and study. The normal and tumor tissue samples collected from the patients were frozen immediately and stored at -80°C until use. DNA was extracted from the peripheral blood leucocytes and from tissues according to the standard phenol–chloroform method. Polymorphism study PCR was performed with the following primer sequences with respective annealing temperatures mentioned alongside: -692A/G, -716C/T, -760T/C, -802C/T polymorphisms in TRAIL, forward 50 -GGCCAGCTTATGACATC TGA-30 and reverse 50 -TCTCCTTCTCAGATCTCCAT

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CC-30 (60°C); -26G/A polymorphism in BRCA2, forward 50 -CTCAGTCACATAATAAGGAAT-30 and reverse 50 -A CACTGTGACGTACTGGGTTTT-30 (55°C); TP53 exon 4 codon 72 Arg/Pro polymorphism, forward 50 -TGCTCTT TTCACCCATCTAC-30 and reverse 50 -ATACGGCCAGG CATTGAAGT-30 (62°C); CA-repeat polymorphism in IFNG Intron 1, forward 50 -CAGACATTCACAATTGATT TTATTC-30 and reverse 50 -CTGTGCCTTCCTGTAGGG TA-30 (60°C); and -652 6N del polymorphism in Casp8, forward 50 -CTGCATGCCAGGAGCTAAGT-30 and reverse 50 -GCCATAGTAATTCTTGCTCTGC-30 (64°C). TRAIL, BRCA2, and TP53 polymorphisms were detected by sequencing of the PCR products [31] and SSLP carried out [32] to detect IFNG CA-repeat length polymorphism. Casp8 -652 6N deletion polymorphism was genotyped by performing denaturing high performance liquid chromatography (DHPLC) [33].

polymerase (Finnzymes, Keilaranta, Espoo, Finland). TRAIL -913 amplified product was cloned in TA cloning vector (Fermentas Canada Inc., Ontario, Canada). TRAIL -913 TA clone was digested with KpnI and SmaI (New England Biolabs, Ipswich, MA, USA) for cloning into pGL3 basic vector (Promega Corporation, Madison, WI, USA). Sequencing (Applied Biosystems, Foster City, CA, USA) was performed to confirm positive clones with no change in sequence. Site directed mutagenesis was performed in order to generate T at position -716, using the following primers: sense 50 -GTGTCAACTACTTCCTACT TGTCCAGCCTAACACAC-30 and anti-sense 50 -GTGTGT TAGGCTGGACAAGTAGGAAGTAGTTGACAC-30 with phusion polymerase enzyme followed by DpnI treatment. The sequence of the mutagenized insert was also confirmed by sequencing. Cell culture

Cloning of TRAIL promoter Promoter region clone of TRAIL (Fig. 1) with C at -716 position (HUGO nomenclature) was amplified from already genotyped healthy control DNA, using the following primers: TRAIL -913 (869 bp amplicon): forward 50 -TGGGCCAGCTTATGACATCT-30 and reverse 50 -TC ACTGAAGCCCTTCCTTCT-30 with Phusion DNA

Human breast adenocarcinoma, MCF-7, cervical carcinoma, HeLa cells, liver carcinoma, HepG2, and fibrosarcoma, HT1080, were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum and maintained at 37°C with 5% CO2 (cell culture reagents were obtained from Life Technologies, Inc., now part of Invitrogen Corporation).

Fig. 1 Schematic representation of TRAIL gene depicting the regions analyzed and the reporter gene assay with chimeric TRAIL promoter with C or T at -716 position

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Transient transfection and luciferase assays Plasmid DNA was isolated using the plasmid maxi kit (Qiagen Inc., Valencia, CA, USA) for transient transfection. All nucleofections were carried out using amaxa biosystems nucleofector kit for MCF-7 cells, HeLa cells, HepG2 cells, and HT1080 cells according to the manufacturer’s instructions (AMAXA GmbH, Cologne, Germany). MCF-7, HeLa, HepG2, and HT1080 cells were nucleofected at a density of 1 9 105 cells per well in sixwell plates and grown overnight in DMEM with 10% bovine calf serum, prior to luciferase assay. A total of 1 lg of TRAIL promoter construct and 0.1 lg of pRL-TK Renilla luciferase vector (Promega Corporation, Madison, WI, USA) with 100 ll of nucleofection solution were used for each transfection. The pRL-TK Renilla luciferase activity was used as a control for transfection efficiency. Each transfection experiment was performed in duplicate and repeated a minimum of three times. Firefly luciferase and Renilla luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA). Approximately 16 h after nucleofection, cells were washed twice with 19 phosphatebuffered saline and harvested with 600 ll of passive lysis buffer (Promega Corporation, Madison, WI, USA). Cell lysates were cleared by centrifugation, and 5 ll was added to 100 ll of firefly luciferase substrate, and light units were measured in a luminometer (TD-20/20, DLReady; Turner Designs, Inc., Sunnyvale, CA, USA; and Promega Corporation, Madison, WI, USA). Renilla luciferase activities were measured in the same tube after addition of 100 ll of Stop and Glo reagent.

ABI Prism 7900 Sequence Detection System (Applied Biosystems). Threshold cycle (Ct) numbers were established by using SDS 1.1 RQ software (Applied Biosystems). All the reactions were carried out in duplicates. GeNorm software was used to identify two most stable internal control genes, raw Ct value of which was used to calculate the normalization factor. It calculates the gene expression stability measure (M) for a reference gene and allows stepwise exclusion of the gene with highest M value, resulting in ranking of the tested genes according to their expression stability [34]. For geNorm input, we require sample quantity (Q) relative to average expression (avg Ct) which can be obtained by the formulae Q ¼ Efficiencyðavg Ct sample Ct Þ : The geNorm input values for endogenous control were used to find the most stable internal control gene followed by the calculation of normalization factor for each tissue sample. GeNorm input values for target genes were then divided by the normalization factor to obtain the normalized target gene expression value. The normalization factor was used to compute normalized expression for the target genes. Categorization of the results was done on the basis of pro- versus anti- apoptotic genes involved in extrinsic [(Casp8 ? DR4 ? DR5)/(Casp8L ? DcR1 ? DcR2 ? FlipL ? FlipS)] and intrinsic [Cytochrome c/Bcl2] pathways. Since ER/PR negative breast cancer cell lines are predominantly sensitive to TRAIL, an index was developed [35] where tumor expression for these candidate genes were studied against the genotype background of TRAIL -716 SNP and the ER/PR status of the tumor.

Statistical analysis Relative gene expression of tumor samples Total RNA was extracted from 79 tumor samples by using TRIzol (Sigma, Saint Louis, MO) according to manufacturer’s instructions. RNA quality from each sample was determined by the A260/A280 absorbance ratio and by electrophoresis on 1.2% agarose formaldehyde gel. Four micrograms of total RNA was reverse transcribed into single strand cDNA, using High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems). Commercially available Taqman Gene expression Assay system for quantitating transcript levels of TRAIL, DR4, DR5, DcR1, DcR2, cytochrome c, Bcl2, Casp8, Casp8L, FlipL, and FlipS (Supplementary Table 1) was used for studying the mRNA expression in 79 representative samples (Applied Biosystems, Foster City, CA). GAPDH, B-Actin, PUM1, and MRPL19 (Applied biosystems) were used as endogenous controls. The primer-probe sets were selected to avoid amplification of contaminating genomic DNA. Quantitative real-time PCR was carried out using an

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Overall genotype frequencies of breast cancer patients and control subjects were compared using the 3 9 2 v2 test. Once a significant overall difference between patients and control subjects was detected (P \ 0.05), the individual genotypes were analyzed using an unconditional logistic regression model with correction for age using the SPSS statistical package, version 13 (SPSS Inc., Chicago, IL, USA). Haploview 4.1 was used to calculate linkage disequilibrium between -760, -716, and -692 SNPs located on same chromosome of the TRAIL locus. In order to compare the relative luciferase activity, the t test was used. For evaluating the real-time PCR generated data, the average raw Ct values were used for computing normalized target gene expression level using geNorm software. Mann–Whitney U test and Kruskal–Wallis H test were used for comparison between two groups and among multiple groups, respectively (http://udel.edu/*mcdonald/ statkruskalwallis.html). P value was considered significant at and below 0.05.

Breast Cancer Res Treat Table 1 Genotype and allele frequencies of -760, -716, and -692 TRAIL polymorphisms in sporadic breast cancer patients and normal controls Genotypes

Patients N = 337

Controls N = 412

Pa

Pb

Pc

OR (95% CI)d

1.0 (referent)

TRAIL promoter, -760T [ C TT

318 (94.4%)

392 (95.1%)

–

TC

19 (5.6%)

20 (4.9%)

0.627

1.2 (0.6–2.2)

CC

0 (0.0%)

0 (0.0%)

–

–

T

97.20%

97.60%

0.636

0.9 (0.5–1.6)

TRAIL promoter, -716C [ T CC 183 (54.3%)

0.594, 0.614

0.741

188 (45.6%)

–

1.0 (referent)

CT

131 (38.9%)

181 (43.9%)

0.053

0.7 (0.5–1.0)

TT

23 (6.8%)

43 (10.4%)

C

73.70%

67.60%

0.946, 0.954

0.035

CC vs. CT ? TT

0.033

0.6 (0.3–0.9)

0.010

1.3 (1.1–1.7)

0.018

1.4 (1.1–1.9)

TRAIL promoter, -692A [ G AA

219 (67.5%)

278 (67.5%)

-

1.0 (referent)

AG

105 (31.2%)

123 (29.9%)

0.605

1.1 (0.8–1.5)

GG

13 (3.9%)

11 (2.7%)

A

80.60%

82.40%

0.925, 0.550

0.579

0.320

1.5 (0.7–3.5)

0.361

0.9 (0.7–1.2)

CI confidence interval, OR odds ratio The referent genotypes were considered on the basis of highest proportion of a homozygous genotype at any particular locus in the control group a

P value for Hardy–Weinberg equilibrium testing

b

P value for 3 9 2 v2 test of comparison of overall genotype frequencies between breast cancer patients and controls

c,d

P values and corresponding age-adjusted ORs with 95% CIs for comparison of genotype frequencies between breast cancer patients and controls by logistic regression (not adjusted for age in allele frequency comparisons)

Results Polymorphism in TRAIL and other DDR-apoptoticcytokine pathway genes in association with breast cancer The study of polymorphisms in the promoter region of the TRAIL gene indicated a significant difference in the genotype distribution between patients and controls only at nucleotide position -716 (P = 0.035) (Table 1). The predominant CC genotype, when compared against combined CT and TT genotypes, provided risk (Precessive model = 0.018, odds ratio [OR] = 1.4, 95% confidence interval [CI] = 1.1–1.9). As expected, a significant overrepresentation of ‘‘C’’ as major allele in cases as compared to controls (Pallele model = 0.010, OR = 1.3, 95% CI = 1.1– 1.7) indicated it to be the risk allele. The CT and TT germline genotype backgrounds were also observed in a substantial percentage of sporadic breast cancer patients (45.7%), though the analysis showed these to be protective in nature (P = 0.018) (Table 1). The haploview data showed -716 to be in strong linkage disequilibrium with flanking SNPs -760 and -692 in patients and controls (Supplementary Fig. 1). Furthermore, the

-760T/-716T/-692A haplotype was found to be significantly over represented in controls as compared to patients (P = 0.0034) (Supplementary Table 2). Stratification of genotypic data to assess the combined influence of TRAIL, TP53, and BRCA2 polymorphisms on breast cancer risk revealed that in the presence of protector genotypes of TP53 codon 72, BRCA2 -26, and TRAIL -716 CT and TT genotypes, a synergistic effect of fivefold protection was observed (P = 0.002, OR = 0.2, 95% CI = 0.1–0.6). Further, the risk associated with TRAIL -716 CC genotype with respect to breast tumor was reduced in the presence of TP53 codon 72 and BRCA2 -26 protector genotype (P = 0.005, OR = 0.2, 95% CI = 0.1–0.6; although not significant if Bonferroni correction was applied) (Supplementary Table 3). Similar analysis to assess the combined influence of TRAIL and IFNG polymorphisms on breast cancer risk revealed that in the presence of protector genotype of IFNG Intron 1 long CArepeat, TRAIL -716 protector genotype provided more than threefold protection (P = 0.0000002, OR = 0.3, 95% CI = 0.2–0.4) (Supplementary Table 4). Studies carried out on TRAIL -716 and Casp8 -652 interaction showed the common variant of Casp8 to have a greater protective effect when present in combination with TRAIL protector

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genotype (P = 0.0003, OR = 0.5, 95% CI = 0.3–0.7) as compared to heterozygous and del homozygous combination of Casp8 (P = 0.028, OR = 0.6, 95% CI = 0.4–0.9). Even the CC risk genotype of TRAIL in the presence of heterozygous and del homozygous combination of Casp8 provided protection from breast tumor (P = 0.008, OR = 0.6, 95% CI = 0.4–0.9) (Supplementary Table 4). Functional relevance of -716C/T TRAIL promoter polymorphism In vitro analysis In order to understand the functional significance of TRAIL -716 C/T promoter polymorphism on the expression of TRAIL gene, HeLa, MCF-7, HepG2, and HT1080 cell lines were transiently transfected with TRAIL promoter construct (pGL3Basic) with -716 C/T polymorphic site. Figure 1 shows TRAIL -913 construct containing the C allele with a significantly higher expression than the T allele in HeLa (P = 0.026), MCF-7 (P = 0.022), HepG2 (P = 0.024), and HT1080 (P = 0.030) cell lines. In vivo analysis In order to characterize the functional implication of TRAIL -716 polymorphism in in vivo condition, a representative set of 79 sporadic breast tumor samples were selected possessing the same genetic background at germline and somatic level for the SNP position under study. These were then subjected to real-time expression analysis for TRAIL transcript. The comparison of TRAIL transcript levels in tumors depicted that a significant difference was observed for TRAIL expression in tumors with different -716 genotype backgrounds (P = 0.010), where the normalized expression values were used for statistical analysis. The significant difference increased (P = 0.003) when the protector (CT ? TT) genotypes were combined and compared against the CC risk genotype, depicting a decreasing trend with the change in major genotype of CC to CT ? TT (Fig. 2). Association of the functional promoter polymorphism with clinicopathological parameters in breast tumors followed by real-time expression analysis of apoptotic pathway genes The comparison of tumor samples with various clinicopathological parameters on the basis of TRAIL -716 genotype background (Fig. 3) showed no significant variation in distribution with increasing nodal status and tumor stage. However, an increasing trend of CT and TT percentage and a decreasing trend of CC percentage with advancing T size were observed (P = 0.041) (Fig. 3).

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Fig. 2 Scatter plot of normalized TRAIL gene expression with respect to different TRAIL -716 genotypic background in breast tumors. Normalization factor, using PUM1 and MRPL19, was used for calculating the normalized expression of the TRAIL gene. P values shown are for: overall distribution, CC vs. CT ? TT

Also, CT and TT germline genotype backgrounds were predominant in ER negative (P = 0.024) and PR negative (P = 0.006) breast tumors. In order to understand the tumor heterogeneity, if any, between TRAIL -716 CC and CT ? TT genotype bearing tumors with respect to pro- and anti- apoptotic gene, the representative set of 79 tumor samples with uniform germline and somatic genetic background for the -716 SNP were studied for real-time expression of 11 genes (TRAIL, DR4, DR5, DcR1, DcR2, cytochrome c, Bcl2, Casp8, Casp8L, FlipL, and FlipS) in the apoptotic pathway. The results of expression for these genes were categorized in pro- apoptotic versus anti- apoptotic signals belonging to intrinsic and extrinsic pathways. The results showed that TRAIL -716 genotype influenced apoptosis that occurred through extrinsic pathway; CC with increased apoptosis and CT ? TT with decreased apoptosis (P = 0.009) (Fig. 4, Supplementary Fig. 2). Similar comparison with further stratification according to the ER/PR status, where details were available in 50 out of 79 tumors, revealed higher apoptotic index for CC genotype bearing ER/PR positive tumors favoring the extrinsic pathway (P = 0.025/0.217) while the same genotype favored the intrinsic pathway in ER/PR negative tumors (P = 0.008/0.018) (Fig. 5).

Discussion Despite being a key regulator of cell death and a major candidate for anti-cancer therapeutic intervention, the

Breast Cancer Res Treat Fig. 3 Association of -716 TRAIL promoter polymorphism with clinicopathological parameters of breast tumors

Fig. 4 Categorization of expression pattern of genes involved in extrinsic [(Casp8 ? DR4 ? DR5)/(Casp8L ? DcR1 ? DcR2 ? FlipL ? FlipS)] and intrinsic [Cytochrome c/Bcl2] pro-/anti-

apoptotic pathways, stratified with respect to TRAIL -716 genotype. P values shown are for: overall distribution, CC vs. CT ? TT

TRAIL gene till date has not been critically analyzed for association with any cancer pathogenesis. The recent reports of a possibility of tumor counter-attack on immunesurveillance mechanism makes such an attempt pertinent. Here, we show how the TRAIL -716 promoter SNP, which functionally regulates its expression, modulates sporadic breast cancer onset and progression under differential genetic background. The case–control association study showed that the major genotype CC at -716 TRAIL promoter followed a recessive model for providing risk to breast cancer. The functional characterization of the -716 polymorphism in TRAIL promoter by reporter gene assay revealed that the C allele resulted in a higher expression than the T allele in in vitro assays in four different cancer cell lines indicating that the polymorphism has a role in the regulation of TRAIL expression. Bioinformatics analysis revealed the creation of a stimulatory protein 1 (Sp1) transcription factor binding site by the change from C to T at -716 position. It has been reported that the transcription factor

Sp3 exhibits a similar DNA binding affinity for Sp1 consensus sequence [36–43] and represses the Sp1-mediated trans-activation of promoters with two or more Sp1 sites [36, 37, 39–41, 44–50]. TRAIL has two Sp1 consensus sequences in the basal promoter [22, 29]. We suggest that TRAIL promoter with TT genotype at -716 nucleotide position and with additional Sp1 consensus sequence shows lower expression due to the repression caused by binding of Sp3. Whereas, the higher expression in CC genotype background at the same position has a lower probability of Sp3 driven repression as compared to CT and TT genotypes. The expression variation observed in both in vitro and in vivo assays, with respect to different SNP genotype backgrounds, would suggest the important role of Sp1:Sp3 ratio in TRAIL gene regulation. SNP–SNP interaction analysis depicted how the lowpenetrating SNP influenced other relevant gene SNPs, supporting the polygenic model of differential risk to the sporadic form of breast cancer. The concurrent presence of the protector genotypes: TRAIL -716 CT ? TT, TP53

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Breast Cancer Res Treat Fig. 5 Real time analysis of TRAIL expression when categorized with respect to estrogen receptor (ER) and progesterone receptor (PR) status in tumors with specific TRAIL -716 genotype background. P values shown are for: CC—receptor pos vs. neg; CT ? TT—receptor pos vs. neg; Receptor pos—CC vs. CT ? TT; Receptor neg—CC vs. CT ? TT

codon 72 Pro/Pro, and BRCA2 -26 GA, provided a fivefold protection from sporadic breast cancer. We propose that this may be due to a preferential shift toward cell cycle arrest followed by DNA damage repair over the apoptotic induction driven by TRAIL, under protector genotype background condition of TP53 codon 72 and BRCA2 50 UTR [31]. Similarly, TRAIL -716 CT ? TT genotype when present in combination with protector genotype of IFNG CA-repeat showed an additive effect, providing 3.3fold protection from breast cancer. This result apparently indicated that ‘‘high-producer-longer-IFNG-CA-repeat’’ mediated enhanced tumor immune-surveillance [51], probably facilitated by decreased TRAIL arbitrated activation induced cell death (AICD) of lymphocytes observed in case of -716 CT ? TT genotype and decreasing the likelihood of tumor pathology. Previous studies also have suggested that Casp8 -652 6N del polymorphism reduces the expression of caspase 8 by destroying a binding element for stimulatory protein 1 (Sp1) [52]. We suggest that Casp8 high producer common variant genotype when present in combination with TRAIL low producer heterozygous and rare variant protector genotype, it enhances the protective effect by decreasing AICD of T lymphocytes, i.e., despite high expressing Casp8 genotype, reduced TRAIL expression activates less of caspase 8 and decreases apoptosis of incoming T lymphocytes. Conversely, Casp8 heterozygous and del homozygous genotypes also provide protection on interaction with TRAIL risk genotype by decreasing AICD of T lymphocytes, i.e., in spite of high production of TRAIL, we believe, low caspase 8

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production reduces the intensity of death receptor apoptotic pathway, thereby reducing apoptosis of T lymphocytes and acting as protector in cancer pathology. On the basis of our findings and existing literature, we propose a dual role for TRAIL -716 promoter polymorphism (Fig. 6). We propose that during the process of tumor initiation, the cells express TRAIL but are death receptor negative or weakly positive. This enables the transforming cells to evade recognition by immune cells; however, the incoming activated leukocytes are killed with endogenously produced TRAIL [10, 12, 53, 54]. Under these circumstances, high TRAIL expressing individuals with -716 CC genotype are at a greater risk of developing tumor which can be inferred from our observation of the predominance of the major genotype, CC, in the initial stages of breast cancer, ER/PR positive cases and small tumor size. However, once the tumor is established, its progression is likely to be compromised because of increased apoptosis by activated leucocytes producing TRAIL and acting via death receptor which the tumor acquires with time. This is supported by the observation of CC genotype bearing tumors showing increased apoptotic indices via the extrinsic apoptotic pathway as compared to CT ? TT genotype bearing tumors. This difference, however, was not apparent for a similar comparison in the intrinsic pathway. Also, CC genotype bearing tumors showed preference for extrinsic apoptotic pathway in ER/ PR positive tumors and for intrinsic apoptotic pathway in ER/PR negative tumors. On the other hand, low TRAIL expressing individuals with -716 CT and TT genotypes

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Fig. 6 Hypothesis showing the role of TRAIL -716 polymorphism in breast cancer susceptibility Assumption: (1) premalignant cells first express mTRAIL on cell surface followed by DR4/5 expression, (2)

tumor cells produce membrane TRAIL and soluble TRAIL, (3) death receptor pathway is intact in tumor cells and immune cells

are likely to be at a lower risk to develop tumor initially since they will not be able to kill the incoming lymphocytes efficiently. However, once tumor is established by other initiating factors, low TRAIL expressed by the immune cells would provide an escape route and allow the tumor cells to proliferate and help in tumor progression. This is indicated by the observation of CT and TT genotype backgrounds conferring risk for tumor progression as its prevalence increased in tumors of late stages, ER/PR negative status and large size. It is likely we surmise that such tumors might have been initiated due to the malfunction of other known/ unknown genes/factors. The proposed hypothesis on the basis of the observations made in the present study attempts to explain (i) why a majority of the human beings are protected to get cancer, reflected by the neutralizing effect of protective functional variants in cancer related genes such as the TRAIL -716 SNP and (ii) the dual role of certain cytokine genes as seen in the onset and progression of cancer, also observed for the TRAIL -716 SNP. Finally, although the use of TRAIL as a target for mono/ combined therapy is in its nascent stage (in the form of rTRAIL and agonistic anti-TRAILR mABs), the complete clinical success is evaded by limitations such as tumor TRAIL sensitivity, induction of apoptosis in normal cells (especially hepatocytes and keratinocytes) and immune

suppression. Our study strongly reflects the role of -716 TRAIL promoter in functionally modulating TRAIL as well as other related candidates in the antitumor network, finally shaping breast cancer pathogenesis. The lessons drawn and the proposed hypothesis need to be considered seriously while delineating the patients and causes of limitations of TRAIL therapy administration to address the question, ‘‘for whom the TRAIL as a target would work efficiently.’’ Acknowledgments The authors would like to thank the patient and control subjects for their participation. RP is grateful to the Council of Scientific and Industrial Research, New Delhi, India, for the predoctoral fellowship. RB was supported by a research grant from the University Grants Commission to the National Centre of Applied Human Genetics and the University with Potential of Excellence projects.

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