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Caspase (CASP) 3, 7, and 8 are important caspases in the apoptosis pathway and play an important role in the development and progression of cancer. We examined the association between genetic variants in the CASP 3, 7, and 8 genes and risk of endometrial cancer among Chinese women. Genotypes for 1,028 women with endometrial cancer and 1,003 healthy controls were determined with the Affymetrix MegAllele Targeted Genotyping System and Molecular Inversion Probe (MIP) method. Of 35 selected SNPs, four in the CASP7 gene were in high linkage disequilibrium (rs11593766, rs3124740, rs11196445, and rs11196418) and associated with the risk of endometrial cancer. The AA genotype of rs11196418 (OR=0.36;95%CI:0.14–0.94) and the G allele of rs11593766 were associated with reduced risk (OR=0.75;95%CI=0.59–0.96 for carriers of one G allele; OR=0.70;95%CI=0.24–2.03 for carriers of two G alleles). The AA genotype of rs11196445 (OR=1.74;95%CI= 0.99–3.05), the CC genotype of rs3124740 (OR=1.36;95%CI= 1.06–1.75), and the GG genotype of rs10787498 in the CASP7 gene (OR=1.90;95%CI=1.16–3.11) were associated with increased risk compared with homozygotes of the major alleles. The gene-disease association appeared to be more pronounced among pre-menopausal women, although tests for multiplicative interaction between genes and menopausal status failed to reach statistical significance. The GG genotype of rs2705901 in the CASP3 gene was significantly associated with increased cancer risk compared with the CC genotype (OR=2.25;95%CI=1.03–4.95). No association was observed between polymorphisms of the CASP8 gene and risk of endometrial cancer. These findings suggest that genetic variants in CASP3 and CASP7 may play a role in endometrial cancer susceptibility.
Endometrial cancer is the second most common gynecologic cancer in the world . Early age at menarche, late age at menopause, nulliparity, obesity, use of hormone-replacement therapy, and use of tamoxifen are well-established risk factors for endometrial cancer. Recently, it has been increasingly recognized that genetic polymorphisms may play an important role in the development of endometrial cancer. The most studied are the steroid metabolism pathway genes [2–4], cell-cycle control pathway genes [5–7], and DNA repair pathway genes [8, 9].
Apoptosis is a selective process for deleting cells in various biological systems and plays an essential role in the development and maintenance of tissue homeostasis in multicellular organisms . During apoptosis in humans, initiator caspases (CASPs) integrate molecular signals into proteolytic activity , and subsequently activate the downstream effector CASPs, thus transmitting and amplifying the apoptotic signal . Inappropriate regulation of apoptosis is believed to be the cause of many human diseases, including cancer . CASP3 and 7 have been identified as key executors of apoptosis in mammalian cells, and play a central role in the execution phase of apoptosis [14, 15]. CASP8 is a cysteine protease, which cleaves downstream substrates, such as effector CASPs, to initiate the apoptotic cascade and transmit apoptotic signals downstream of death receptors . The expression levels of CASPs in tumors are found to be distinct from levels in normal tissue in a series of cancers. For example, expression of CASP3 was observed to be down-regulated in pediatric neuroblastoma , breast cancer , and gastric carcinoma , while CASP7 was found to be down-regulated in colonic carcinoma , breast cancer , and gastric cancer . Decreased expression of CASP8 was also reported in neuroblastoma  and pediatric tumors .
Genetic polymorphisms in the CASP genes may affect cancer risk through altering expression levels and functions of these genes. Several polymorphisms have been linked to susceptibility to a growing list of cancers in recent years. Yang et al  found that a six-nucleotide deletion polymorphism in the CASP8 gene (-652 6N ins-->del) decreased the risk of pancreatic cancer. Hosgood et al  studied the associations between polymorphisms in CASP3, 8, 9, and 10 and susceptibility to multiple myeloma, and found that the TT genotype of the CASP3 rs1049216 SNP was associated with a five-fold decrease in risk, and the AG and AA genotypes of the CASP9 rs1052576 SNP were associated with decreased cancer risk. Other malignancies evaluated include cancers of lung , colon and rectum , stomach , breast , and ovary . Little is known about associations with endometrial cancer.
In this study, we analyzed a set of functional and tagging SNPs in the CASP3, 7, and 8 genes in association with endometrial cancer.
Details of the study methods have been described elsewhere [32, 33]. Briefly, 1,449 women newly diagnosed with endometrial cancer aged 30 to 69 years were identified between 1997 and 2003 through the population-based Shanghai Cancer Registry; 1,199 cases (82.7%) participated in the study. Controls were randomly selected from the general population of urban Shanghai using the Shanghai Resident Registry according to the age distribution of endometrial cancer cases in 1996. Women with a history of any cancer or hysterectomy were not eligible. Of the 1,629 eligible women contacted, 1,212 (74.4%) participated in the study. Study protocols were approved by the Institutional Review Boards of all institutes involved in the study, and all participants provided written, informed consent prior to participating in the study.
Study participants were interviewed in person by trained retired medical professionals using a structured questionnaire. Detailed information on demographic factors, menstrual and reproductive history, hormone use, prior disease history, physical activity, tobacco and alcohol use, diet, weight history, and family history of cancer was collected for all participants. Body weight, height, and circumferences of the waist and hips were measured according to a standardized protocol at the time of interview. Menopause was defined as the cessation of the menstrual period for at least 12 months before the reference date (diagnosis date for the cases and interview date for the controls), excluding lapses caused by pregnancy, breastfeeding, or estrogen hormone use. Body mass index (BMI, weight in kilograms/height in meters2) and waist-to-hip circumference ratio (WHR) were calculated using measured anthropometrics.
Of the study participants who completed an in-person interview, 850 cases and 853 controls donated a blood sample and 280 cases and 274 controls provided a buccal cell sample. 187 cases and 186 controls provided samples using a mouthwash method; and 93 cases and 88 controls provided samples using a buccal swab method. Due to the very low DNA yield of the buccal swab method, we did not include buccal swab DNA samples in the genotyping. In addition, DNA samples from 19 controls who donated a blood sample were used up in other studies. Thus, DNA samples from 1,037 cases (86.5%, 850 blood and 187 buccal cell) and 1,020 controls (84.2%, 834 blood and 186 buccal cell) were included in the genotyping study. Genotyping data for the CASP genes were obtained from 1,028 cases and 1,003 controls, a success rate of 99.1% and 99.6%, respectively.
Both known non-synonymous SNPs and tagging SNPs were selected for the study. Tagging SNPs were selected by searching Han Chinese data from the HapMap project1 using the Tagger program  and non-synonymous SNPs were identified by review of literature and existing databases. The following criteria were used to identify tagging SNPs: 1) SNPs were located in the genes or within the 5 kb region flanking the genes, 2) had a minor allele frequency (MAF) ≥ 0.05, and 3) the other unselected SNPs could be captured by one of the tagging SNPs with a LD of r2 ≥ 0.90. SNP selection was completed in December of 2005. As a result, a total of 34 htSNPs and 1 non-synonymous SNP were identified.
The SNPs were genotyped using the Affymetrix MegAllele Targeted Genotyping System with the Molecular Inversion Probe (MIP) method  as part of a large-scale genotyping effort that included 1,737 SNPs. Genotyping was conducted at the Vanderbilt Microarray Shared Resource following the manufacturer’s protocol. Briefly, 2.01 μg of genomic DNA was annealed to the assay panel overnight at 58°C. After annealing, the samples were split into 4 equal aliquots. Each aliquot was gap filled with 4 different aliquots receiving a different dNTP. The dNTP was ligated to produce a padlocked probe and then digested with exonucleases. The padlocked probe was then cleaved at a specific cleavage site and inverted. The inverted probe was the substrate for two rounds of PCR. After passing quality control (QC) tests, samples were hybridized to the arrays. Arrays were then washed, stained, and detected via the scanner and analyzed by using the Affymetrix protocol.
As a QC procedure, we included 39 blinded duplicate samples and 12 HapMap DNA samples in the genotyping. The average consistency rate for these samples was 99.6%; the lowest consistency rate was 97.4%. The genotyping of SNPs was highly successful, with call rates of 99.5–100% (median: 99.95%). Finally, the laboratory staff remained blind to the case-control status and identity of all samples.
We used SAS software (version 9.1, SAS Institute, Inc., Cary, NC) for the statistical analyses. Chi-squared statistics and the t-test were used to evaluate case-control differences in the distribution of risk factors and genotypes and to determine whether allele frequencies in healthy controls deviated from Hardy-Weinberg equilibrium. Linkage disequilibrium (LD) between the polymorphisms in the CASP7 and CASP8 genes was assessed by HaploView version 4.0 . Haplotypes were reconstructed using HAPSTAT software , which uses an expectation maximization algorithm to calculate maximum likelihood estimates of haplotype frequencies, while taking into account phase ambiguity . Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (95%CIs) with adjustment for age. We used HAPSTAT software to evaluate associations between haplotypes and endometrial cancer risk and interactions between haplotype and environmental exposures on endometrial cancer risk . We made additional adjustments for non-genetic risk factors, including having a first-degree relative with cancer, age at menarche, age at menopause, years of menstruation, number of live births, use of oral contraceptives, body mass index (BMI), waist-to-hip ratio (WHR), and physical activity status, which did not alter the gene-disease associations (data not shown). Thus, only the age-adjusted results are reported.
Our study population consisted of 1,028 endometrial cancer cases and 1,003 healthy controls. Table 1 presents a comparison of characteristics of cases and controls. Endometrial cancer cases and controls were comparable with respect to age, with a mean age of 54.3 (±8.5) years for cases and 54.4 (±8.5) years for controls. More cases (35.7%) had a family history of any cancer than did controls (29.6%), P<0.01. Compared to controls, cases were more likely to be diagnosed with diabetes, have earlier age at menarche, later age at menopause, longer duration of menstruation, fewer pregnancies, and higher BMI and WHR, but were less likely to regularly drink alcohol, use oral contraceptives, or regularly participate in physical activity.
The associations of endometrial cancer with SNPs in the CASP3, 7, and 8 genes are presented in Table 2. The genotype distributions of all 35 polymorphisms under study were in Hardy–Weinberg equilibrium among controls. In the CASP 3 gene, the GG genotype of rs2705901 was significantly associated with increased cancer risk compared with the CC genotype (OR=2.25, 95%CI=1.03–4.95). Among the 18 SNPs examined in the CASP7 gene, five variants were significantly or marginally significantly associated with endometrial cancer risk. Specifically, the CC genotype of rs3124740 (OR=1.36, 95%CI=1.06–1.75, ptrend = 0.03), the GG genotype of rs10787498 (OR=1.90, 95%CI=1.16–3.11), and the AA genotype of rs1196445 (OR=1.74, 95%CI=0.99–3.05, ptrend = 0.06) were related to an increased risk of endometrial cancer compared with their respective common homozygous genotype. The AA genotype of rs11196418 (OR=0.36, 95%CI: 0.14–0.94) and the G allele (GG or GT) of the rs11593766 SNP (OR=0.75, 95%CI=0.59–0.96 for the GT genotype and OR=0.70, 95%CI: 0.24–2.03 for the GG genotype (ptrend = 0.02)) were associated with reduced risk of endometrial cancer. None of the 13 SNPs under study in CASP8 were associated with the risk of endometrial cancer.
We further evaluated the association of the rs11196418, rs11593766, rs3124740, rs11196445 and rs10787498 polymorphisms in the CASP7 gene with endometrial cancer risk stratified by menopausal status (Table 3). The gene-disease associations appeared to be more pronounced among pre-menopausal women. However, no significant interaction between SNPs and menopausal status was observed (p >0.05) (Table 3), nor were any significant interactions observed for rs2705901 in the CASP3 gene with menopausal status (pinteraction =0.72, data not shown in table). We further evaluated the interactive effect of other risk factors such as smoking, alcohol consumption, age at menarche, and years of menstruation on the rs11196418, rs11593766, rs3124740, rs11196445, and rs10787498 polymorphisms, and no significant interaction was observed.
The genetic variants under study in CASP7 fall into two major haplotype blocks as shown in Figure 1. rs11196418, rs3124740, rs11593766, and rs11196445 were located in the same haplotype block and were in close LD (r2 ≥ 0.95).
Using Hapstat software, we constructed 6 haplotypes in block 1 and 7 haplotypes in block 2 of the CASP7 gene. Table 4 lists the frequencies of the haplotypes among cases and controls. Haplotypes with frequencies below 3.0% among both cases and controls were excluded from the analysis. We found that a decreased risk of endometrial cancer was associated with Hap6 in block 1, consisting of SNPs rs12415607, rs11196418, rs7922608, rs11593766, rs11196438, rs11196444, rs3124740, rs11196445, rs3127075, rs17090904, and rs12359418 (dominant: OR=0.78, 95%CI=0.61–0.1.00; additive: OR=0.80, 95%CI=0.63–1.02 relative to Hap1) and Hap6 in block 2, consisting of SNPs rs6585241, rs10787498, rs12247479, rs1127687, rs17090919, and rs10885497 (dominant: OR=0.77, 95%CI=0.60–0.98; additive: OR=0.79, 95%CI=0.62–1.00 relative to Hap1). Three protective alleles and one risk allele were contained in the significant Hap6 in block 1, and one protective allele was included in Hap6 in block 2.
The associations between CASP7 haplotypes and the risk of endometrial cancer were further examined by stratifying participants according to menopausal status (Table 5). Associations of both Hap6 in block 1 and Hap6 in block 2 with endometrial cancer were more pronounced in pre-menopausal women. However, tests for multiplicative interaction were not significant. In addition, we found that Hap2 in block 2 was associated with increased risk among post-menopausal women under dominant and additive models, and a significant multiplicative interaction between the Hap2 in block2 and menopausal status was observed (pinteraction = 0.0006).
This work is the first attempt to evaluate the relationship between polymorphisms in the CASP3, 7, and 8 genes and susceptibility to endometrial cancer. We found that one SNP (rs2705901) in CASP3 and five SNPs (rs11196418, rs11593766, rs3124740, rs11196445, and rs10787498) in CASP7 were associated with the risk of endometrial cancer. No significant associations were observed for variants of CASP8.
Soung et al  previously investigated the entire coding region and all splice sites of the CASP7 gene in human solid cancer tissues and normal tissues for carcinomas of the stomach, colon, head/neck, esophagus, urinary bladder, and lung, and expressed the tumor-derived CASP7 mutants in 293 T cells. Their data suggested that inactivating mutations of the CASP7 gene leads to the loss of its apoptotic function and contributes to the pathogenesis of some human solid cancers. However, no epidemiologic studies have evaluated the association of CASP7 polymorphisms with cancer risk. In our study, we found that five SNPs in the CASP7 gene, one variant in the promoter (rs11196418), one non-synonymous mutation (rs11593766), two intron mutations (rs3124740, and rs11196445) and one variant in the 3′ UTR (rs10787498), were associated with the risk of endometrial cancer. Of these SNPs, four are in close proximity and in high LD. SNP rs11593766, which was related to decreased risk of endometrial cancer, is located in exon 2 and causes a Glu to Asp change at the N-terminal end of the protein. However, the functional significance of this change is unknown. The two polymorphisms located in intron 2 are in very close contig positions. rs11196418 and rs10787498 are located in the promoter and 3′ UTR of the CASP7 gene, and thus may be involved in the regulation of gene expression. Studies are needed to verify our findings and to investigate the functionality of these SNPs and other SNPs in the region.
Recent studies have shown the utility of haplotype analysis in studying gene-disease associations . In our study, Hap6 in block 1 of the CASP7 gene, which included three single, low-risk alleles (rs11593766 (G), rs3124740 (G) and rs11196445 (G)) and one single, high-risk allele (rs11196418 (G)), was strongly associated with decreased risk of endometrial cancer compared with Hap1 in block1, which included one more high risk allele (rs11593766(T)), confirming the findings of our single SNP analyses. We found that Hap6 in block 2 of the CASP7 gene, which contained one low risk allele (rs10787498 (T)), was associated with decreased risk of cancer, particularly among pre-menopausal women. However, the only difference in alleles between Hap6 and Hap1 in block 2 was at rs1127687, which was not a variant significantly linked to endometrial cancer risk. Also, Hap4 in block 2, which contained the same allele as Hap6, was not related to cancer risk. We observed a similar situation for the increased risk associated with Hap2 compared to Hap1 in block 2. These results suggest that polymorphisms may exert independent or interactive effects on the development of endometrial cancer.
It is believed that early age at menarche, late age at menopause, and long duration of menstruation over the course of a lifetime increases the risk of endometrial cancer due to prolonged exposure to estrogens . Estrogens have also been reported to affect the activity of caspases and the apoptosis of cells. For example, Thiantanawat et al  found that withdrawal of estrogen from MCF-7Ca cells results in higher CASP7 activity. Zhang et al  also reported that 17-beta-estradiol may prevent neuronal apoptosis and that 17-beta-estradiol-treated neuronal extracts directly inhibit the recombinant activity of CASP3, 6, 7, and 8. Therefore, it is plausible that estrogen exposure may interact with CASP7 SNPs in the etiology of endometrial cancer. Our results suggest that gene-disease associations are more pronounced among pre-menopausal women, although no significant interaction was observed. However, our study was not adequately powered to detect moderate interactions. Interestingly, the association of endometrial cancer risk with Hap2 in block 2 of CASP7 was observed to be significantly modified by menopausal status, suggesting complex gene-gene and gene-environment interactions.
One previous study has reported that the C allele of CASP3 Ex8+567T>C (rs1049216) was associated with a decreased risk of non-Hodgkin lymphoma (OR = 0.4, 95% CI = 0.3–0.7) in a US population . Variant alleles at the −928A>G, 77G>A, and 17532A>C positions in the CASP3 gene, as well as the haplotypes constructed with these polymorphisms, were linked to decreased risk for lung cancer . In our study, we found that a variant at CASP3, rs2705901, a SNP located in the boundary region of the CASP3 gene, was significantly associated with endometrial cancer risk.
Studies on associations between the CASP8 gene and cancer risk have generated conflicting results. Sun et al  reported that the -652 6N insertion/deletion variant in CASP8 was associated with several kinds of tumors, including lung, esophageal, gastric, colorectal, cervical, and breast cancers in a Chinese population. A UK study  observed a 1.37-fold increased risk of glioma (95% CI = 1.10–1.70; P = 0.004) in carriers of the CASP8 D302H variant allele. However, neither of these variants was associated with the risk of colorectal cancer in another UK study . A recent multi-ethnic study failed to find an association between the CASP8 -652 6N ins/del polymorphism and cancers of the breast, colorectum, or prostate . The D302H polymorphism is not present in Asian populations, and in the current study, we did not find a significant association of endometrial cancer with any tagging or known non-synonymous SNPs in the CASP8 gene, including rs6747918, the SNP in close LD with rs3834129 (the -652 6N ins/del variant).
Our study has several strengths. First, we used a combination of functional and tagging SNP approaches to capture polymorphisms, which is the most comprehensive evaluation of genetic markers in the genes included in the study. Second, this study has a large sample size from a population with a relatively homogeneous ethnic background (>98% Han Chinese). Finally, the relatively high participation rate (82.8% for cases and 74.4% for controls), high DNA sample donation rate (86.5% for cases and 84.2% for controls), and low frequency of hysterectomy (5.1%) mitigates concern about selection bias. Nevertheless, chance findings cannot be excluded. Further studies are needed to replicate our findings and evaluate the mechanisms underlying the associations of CASP genes with endometrial cancer risk.
In summary, of the 35 non-synonymous and tagging SNPs in the CASP3, 7, and 8 genes that were investigated in this study, five variants (rs11593766, rs3124740, rs11196445, rs1196418 and rs10787498) in CASP7, and one (rs2705901) in CASP3, were associated with risk of endometrial cancer. These results warrant replication in other study populations.
We would like to thank Dr. Fan Jin for her contributions to implementing the study in Shanghai, Ms. Regina Courtney, Ms. Qing Wang, Dr. Shawn Levy, and the Vanderbilt Microarray Shared Resource for their contributions to the genotyping, and Ms. Bethanie Hull for her assistance in the preparation of this manuscript. The Vanderbilt Microarray Shared Resource is supported by the Vanderbilt-Ingram Cancer Center (P30 CA68485), the Vanderbilt Diabetes Research and Training Center (P60 DK20593), the Vanderbilt Digestive Disease Center (P30 DK58404), and the Vanderbilt Vision Center (P30 EY08126). This study would not have been possible without the support of the study participants and research staff of the Shanghai Endometrial Cancer Study. This work was supported by USPHS grant R01 CA92585 from the National Cancer Institute.
Sources of Support: This work was supported by USPHS grant R01CA92585 from the National Cancer Institute.