Genetic Polymorphisms in the Apoptosis-Associated Gene CASP3 and the Risk of Lung Cancer in Chinese Population

Caspase-3 (CASP3) plays a central role in executing cell apoptosis and thus in carcinogenesis. We previously investigated the relationship between functional polymorphisms in CAPS3 829 A>C and 20541 C>T and risk of esophageal squamous cell carcinoma. However little is known about the role of CASP3 variants in susceptibility to lung cancer. To figure out the contribution of CASP3 polymorphisms to lung cancer risk, genotypes of 1000 lung cancer patients and 1000 controls were conducted by RFLP-PCR (restriction fragment length polymorphism PCR). The transcriptional activity of CASP3 829 A>C was examined by dual luciferase reporter assay. Logistic regression was applied to calculate Odds ratios (OR) and 95% confidence intervals (95%CI). Compared with CASP3 829 AA genotype, AC and CC genotype had significantly increased risk of lung cancer with OR (95% CI) of 1.33 (1.09–1.63) and 1.55 (1.19–2.01), respectively. To further explore the possible impact of 829 A>C SNP on CASP3 transcriptional activity, we detected the dual luciferase activity of PGL3-promoter vectors containing 829A or 829C alleles in lung cancer cell lines and found that report gene expressions driven by 829A containing CASP3 promoter were 1.64-fold, 1.94-fold greater than those driven by CASP3 829C containing counterparts in A549 and NCI-H1975 cells (P<0.001). When stratified by sex, the significantly increased risk associated with CASP3 829 AC or CC genotype was obviousl in males with OR (95% CI) of 1.42 (1.11–1.81) and 1.51 (1.11–2.05), but not in females. When stratified by age, we found that CASP3 829 AC or CC genotype contributed to the risk of lung cancer in youngers with OR (95% CI) of 2.73 (1.71–4.34) and 4.02 (2.20–7.32), but not in elder group. We also found that 829AC or 829CC genotype increased adenocarcinoma risk compared with the AA genotype with OR (95%CI) of 1.33 (1.04–1.70) and 1.51(1.09–2.07). CASP3 polymorphism and smoking interaction was demonstrated related with higher risk of lung cancer. We achieved that the CASP3 829AC or 829CC genotypes was associated with increased risk of lung cancer in both non-smoker and smoker group, with OR (95%CI) of 1.48 (1.08–2.02) and OR (95%CI) of 1.64 (1.09–2.48) among non-smokers and OR (95%CI) of 2.68 (1.89–3.81) and OR (95%CI) of 3.23 (2.21–4.92) among smokers, respectively. Among carriers with 20541CT genotype, the ORs (95%CI) of risk with lung cancer for smoking <16, 16–28, or > 28 pack-years were 1.16(0.65–2.07), 1.66(0.98–2.82) and 5.01(3.31–7.58) compared with the 20541CC carriers. And among carriers with 20541CT genotype, the ORs (95%CI) were 0.86(0.33–2.20), 2.12(0.83–5.41) and 5.71(2.68–12.16). These results highlight apoptosis-related CASP3 as an important gene in human carcinogenesis and further support the CASP3 polymorphisms confer to the lung cancer susceptibility.


Introduction
Lung cancer is a malignant lung tumor and leads to massive death worldwide. Many factors including tobacco smoking, living habit, environmental and eating factors are vital causes of lung cancer [1]. We all have known that smoking is a major factor for lung cancer, but only some of smokers suffer form lung cancer through lifetime. It is concluded that gene differences of each individual partly determine the susceptibility to lung cancer [2]. Thus, we develop a further study to discover the molecular gene markers which can give rise to the high risk developing lung cancer.
Along the apoptosis process, some kinds of death proteases are activated and cell changes biochemically and morphologically [3,4]. Therefore apoptosis may cause the somatic mutations and now thought to contribute to a number of human diseases, ranging from neurodegenerative disorders to malignancy [5,6]. The dislocation of apoptosis contributes to tumor development and progression [7]. Caspases (CASPs) is a kind of cysteine-dependent aspartatespecific proteases, and in charge of the initiation and execution of apoptosis. Based on their functions, CASPs can be devided into initiator CASPs and effector CASPs based on their proapoptotic functions. CASP8, CASP9, and CASP10 belong to initiator CASPs, and they transmit apoptotic signals; CASP3, CASP6, and CASP7 belong to activate effector CASPs, and they perform the final cell death process [3]. Caspase-3 (CASP3) plays an essential role during apoptotic cell death by proteolytic cleaving a variety of key proteins required for cellular functioning and survival [8]. PARP-1 (poly ADP-ribose polymerase 1) is one main substrate of CASP3. When apoptosis begins, CASP3 cleaves PARP-1 into two fragments to inactivate the enzymatic activity of PARP-1. It increases the activity of one kind of endonucleases which can induce cell apoptosis though DNA cleaving [8].
Takata et al. indicated that caspase-3 was expressed in both the nucleus and the cytoplasm of lung cancer cells [9]. CASP3 mutations were detected many types of tumor, including colon carcinomas, non-small cell lung cancers, non-Hodgkin lymphomas, stomach carcinomas, hepatocellular carcinomas, and multiple myelomas [10]. Xie et al. sequenced 261 DNA samples from healthy individuals of Han Chinese population to search for genetic variants within the regulatory region, exons 2-7 and their flanking sequences of CASP3. They identified three single nucleotide polymorphisms (SNPs), 829 A>C, 17532 A>C, and 20541 C>T, which located in 5'-regulatory region, intron 4, and 3'-regulatory region of CASP3, respectively. They also found that 17532 A>C and 20541 C>T were in complete linkage disequilibrium [11]. Based on these, we final investigated CASP3 829 A>C and 20541 C>T polymorphisms in this lung cancer case-control study.

Study subjects
Our case-control study collected 1000 lung cancer patients and 1000 healthy controls. Keep all participators genetically unrelated ethnic Han Chinese. All the cases were newly diagnosed, histopathologically confirmed, and previously untreated (by radiotherapy or chemotherapy) primary lung cancer. The patients were recruited between January 2008 and December 2012 at Tangshan Gongren Hospital. There were no age, sex, stage, or histology restrictions; however, patients with previous cancer or metastasized cancer from other organs were excluded. The controls were randomly selected from a pool of cancer-free subjects recruited from a nutritional survey conducted in the same region during the same period as the cases were collected. The selection criteria include no prior history of cancer, and controls were matched to the cases by age (±5 years) and sex. At recruitment, informed consent was obtained from each subject who was then interviewed for detailed information on demographic characteristics and lifetime history of tobacco use. The study was approved by the institutional review board of North China University of Science and Technology. All participants provide their written informed consent to participate in this study.

Genotype Analysis
Genomic DNA of all controls and patients was extracted from peripheral blood lymphocytes. RFLP-PCR (restriction fragment length polymorphism PCR) analysis was applied for genotyping the CASP3 SNPs. Briefly, to produce CASP3 region containing the 829 A>C (rs4647602) site, the PCR primer pairs was 5'-TAG TTG CAG GGT TTA AAC TCC AAT GC-3' and 5'-CTA ACT CCT CAC GGC CTG GGA T-3' . The primer pairs used to amplify CASP3 20541 C>T (rs1049216) was 5'-GTG AAA AAG TTA AAC ATT GAA TTA A-3' and 5'-TTC TTC CAC ATC ATC ATT TCT A-3' . The two primer pairs were coincident in our previous epidemiological study of esophageal squamous cell carcinoma [12]. In brief, PCR was performed using a 25-μl reaction mixture containing 100 ng DNA, 0.1 μmol⁄/L each primer, 0.2 mmol/L deoxynucleoside triphosphate, and 1.0 U Taq DNA polymerase (TaKaRa). The PCR profile consisted of an initial melting step of 95°C for 4 min, followed by 35 cycles 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a final extension step of 72°C for 7 min. The amplified PCR products for 829 A>C (137bp) and 20541 C>T (103bp) were digest with BglI and AseI (New England Biolabs, Beverly, MA, USA) and separated on 3% agarose gel (Figs 1A and 2A). The 829C allele had one BglI restriction site that resulted in two bands (112 bp and 25 bp) and the 20541C allele had one AseI restriction site that resulted in two bands of 82 bp and 21 bp. Genotyping was carried out without knowledge of the case-control status of the subjects. The CASP3 829 A>C and 20541 C>T genotypes revealed by PCR-RFLP analysis were further confirmed by direct DNA sequencing (Figs 1B and 2B). Ten percent of the samples were randomly selected for repeated assays, and the results were 100% consistent.

Construction of promoter-reporter plasmids
To verify whether the 829 A>C SNP influences the transcriptional activity of CASP3, The primers used for amplifying CASP3 promoter were 5'-ata cGCTAGCTACCCAGT GACCAG CAAGTG-3'and 5'-gataAAGCTTGGTGG CAAAACAAACACTCC-3', which contain Nhe I and Hind III (NEB, MA, USA) cloning sites (underlined sequences), respectively [13]. The resulting PCR product were subsequently digested with Nhe I and Hind III and cloned into the pGL3-basic vector (Promega, Madison, USA) containing the firefly luciferase gene as a reporter. To produce the luciferase construct containing the 829A allele, a pair of primers 5'-GGT TTAAAC TCCAATTCATTT TCGGCC C-3' and 5'-GAA TTG GAGTTTAAACCC TGCAACTATCTC-3' was used to make the single site mutagenesis (Invitrogen, Carlsbad, CA, USA). The constructs were all confirmed by DNA sequencing.

Cell culture, transfection and luciferase assay
Human lung cancer cells (A549, NCI-H1975) used for the luciferase reporter analysis were provided from Cobioer Biosciences (Cobioer, Nanjing, China). A549 and NCI-H1975 lung cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (GIBCO, NY, USA) and 1% of penicillin and streptomycin in a humidified environment at 37°C with 5% CO 2 . Cells (2×10 5 ) were plated in a 24-well plate and grown to 80-90% confluence. Cells were co-transfected with 1ug firefly luciferase reporter plasmid and 1ng pRL-SV40 (Promega, Madison, USA) using Lipofectamine 2000 reagent (Invitrogen, CA, USA). Luciferase activity was determined according the manufacturer's protocol using a dual-luciferase reporter assay system (Promega, Madison, USA). For each plasmid construct, three independent transfection experiments were carried out, and each luciferase assay was performed in triplicate. The empty pGL3 Basic vector cotransfected with pRL-SV40 plasmid served as a control. Fold increase was calculated by defining the activity of empty pGL3 Basic vector as 1. Differences were determined by t test, and P .01 was considered significant.

Statistical analysis
Student's t-test was used to compare the means of age andχ 2 -test was used to compare the frequency distributions among cases and controls. Odds ratios (OR) and 95% confidence intervals (CI) were computed to evaluate the susceptibility of lung cancer using multivariate logistic regression analysis adjusted by age, sex, and smoking. All statistical tests were two-sided, and a P -value of <0.05 was considered significant using Statistical Analysis System software (Version 16.0; IBM, New York, USA).

Subject characteristics
The characteristics of the two study groups were summarized in Table 1. No statistically significant differences in terms of age and gender distributions were found between the cases and controls. However, more smokers were present among lung-cancer patients than that among cancer-free controls, and the ratios respectively are 61.4% and 48.4% (P < 0.001). Moreover, there were 67.6% smokers over 28 pack-years in the case group and just 41.1% over 28 packyears smokers in control group, showing that smoking causes the majority of lung cancers in the participants. Based on the pathological types of cases, 43.0% belonged to squamous-cell carcinoma, 50.4% belonged to adenocarcinoma, and 6.6% belonged to other types, including undifferentiated cancer, bronchi alveolar carcinoma, and mixed-cell carcinoma.
Association of CASP3 genotypes with lung cancer risk Interaction of CASP3 Genotypes and gender, age and classification of lung cancer The factors including age, gender and smoking status were selected to identify whether CASP3 829A>C and 20541 C>T polymorphisms had some relations with lung cancer. We operated a multivariate regression model to obtain the association between CASP3 829A>C and 20541

Interaction of CASP3 Genotypes and smoking
Because Tobacco smoking is an accepted aetiological predisposition for lung cancer, we investigated the interaction between the CAPS3 polymorphisms and smoking, which was shown in Tables 5 and 6. We achieved that the CASP3 829AC or CC genotype compared with the AA genotype was associated with increased risk of lung cancer in non-smoker group (OR =

Effects of CASP3 829 A>C SNP on the transcriptional activity
To explored the possible impact of 829 A>C SNP on CASP3 transcriptional activity, we constructed promoter vectors containing 829A or 829C alleles and dual luciferase assay was carried out in lung cancer cell lines, A549 and NCI-H1975. Report gene expressions driven by 829A containing CASP3 promoter were 1.64-fold, 1.94-fold greater than those driven by CASP 829C containing counterparts in A549 and NCI-H1975 cells (P<0.001) (Fig 3). These results suggest that the 829 A>C polymorphism influences CASP3 promoter activity.

Discussion
In the previous study, we reported that FAS/FASL, as another apoptosis associated protein, was associated with the risk of esophageal cancer [14]. In the current study, we investigated the potential association of CASP3 polymorphisms (829 A>C and 20541 C>T) and with the risk of lung cancer in Chinese population. We observed that individuals who carried CASP3 829C allele were at significantly increased risk for lung cancer. Moreover, the risk was more evident in the subgroups of male subjects, younger subjects and smokers. These results are consistent with our previous study for esophageal squamous cell carcinoma [14]. This finding further provided evidences that the CASP3 played a significant role in human carcinogenesis. By means of eliminating DNA-damaged cells, apoptosis can keep hosts cells away from cancer development [15]. Caspases are important mediators of apoptosis. CASP3, as an executionphase caspase, is known to play a crucial role during apoptosis. Some research demonstrated the mutation of CASP3 existed in human tumor tissues and cell lines as expected [9,10,16]. Other studies demonstrated that CASP3 was essential to the regulation of B-cell homeostasis by the way of DNA fragmentation, chromatin margination, nuclear collapse and cleavage of many key participators involved in apoptosis [17][18][19][20][21]. Importantly, one of the essential features of tumor cells is the ability of cells to avoid apoptosis, and this capability can help them destroy the anticancer defense mechanisms [22]. Mandruzzato and Wang have reported that a large number of caspases mutations in human tumor cells, which caused reduced apoptotic activities [23,24]. It suggested that the mutation of CASP3 was particularly prone to occur in human cancer tissues, or CASP3 mutation genotype resulted in carcinogenesis. Moreover, in many tumors, it was probably that an analogous loss or down regulation of CASP3 expression may happen [8,25,26]. It is possible that this CASP3 mutation may have resulted that the target tissue operated the apoptosis disadvantageously and thus raised the potential risk of carcinogenesis. This presence indicates that the CASP3 gene mutates in human cancer on occasion. Luciferase activity profiles were assayed following transfection of the constructs into A549 and NCI-H1975. pGL3-829A and pGL3-829C denote caspase-3 promoter constructs containing the 829A or 829C allele, respectively. All of the constructs were cotransfected with pRL-SV40 to standardize the transfection efficiency. Values were means±SD from more than 3 separate experiments that were each performed in triplicate. **P < .001 compared with each of the construct counterparts. One study determined nine potentially functional polymorphisms in the Caspase on survival of early-stage NSCLC patients. Their conclusion was that the CASP7 rs2227310 and CASP9 rs4645981 polymorphisms may affect survival in early-stage NSCLC [27]. Some association studies have suggested possible links between CASP3 polymorphism and the susceptibility to several of cancers, including endometrial cancer, prostrate cancer and head and neck cancer [28][29][30]. One case-control study was demonstrated that CASP3 rs4647601 TT genotype was related with an increased dangerous impact of squamous cell carcinoma of the Head and Neck [29]. A meta-analysis showed that the homozygote (CC) of rs2705897 (A/C) in the CASP3 gene had a positive association with cancer susceptibility [31]. In addition, Jang et al. demonstrated that individuals carried at least one variant allele of the CASP3 -928A>G, 77G>A, and 17532A>C polymorphisms contributed to the genetic susceptibility to lung cancer [32]. Similarity, our present study showed that functional CASP3 829 A>C polymorphism, another significant SNP of CASP3, increased the susceptibility of lung cancer. Our previous real-time PCR analyses indicated that individuals carried CASP3 829AA genotype had obviously higher RNA levels than that carried 829 AC and 829 CC genotypes [12]. This is consistent with our reporter gene results in lung cancer cells. We found that CASP3 829A allele containing promoter had higher reporter gene transcriptional activity than 829C allele containing CASP3 promoter. Evasion of apoptosis is a common feature of malignancy. The acquired ability to resist apoptotic stimuli is one of the primary characteristics of a malignant cell. This result implied that the 829 A>C polymorphism caused the decline of CASP3 transcriptional activity and further contribute to the increased risk of developing lung cancer.
Our findings of a significantly elevated risk, most evident in male and younger subjects with a tendency of increased risk with more variant alleles, suggested that for genetic susceptibility the CASP3 SNPs might be typical markers for lung cancer, because characteristics of genetic susceptibility include an early age of lung cancer onset. It is also possible that gender was important for lung cancer susceptibility. Quite a lot studies showed that gender is an independent or interactional factor for lung cancer susceptibility [33]. Interestingly, another finding of our study CASP3 829C allele was associated with higher risk of lung cancers, suggesting that these polymorphisms might be ecumenical risk causation for most of frequent tumor. These results are accordance with our previous conclusion of esophageal squamous cell carcinoma. All these findings indicated that apoptosis-interrelated CASP3 is a cancer susceptibility gene and plays an important role in human carcinogenesis.
Because tobacco smoking is a proverbial etiological factor for many kinds of cancer including lung cancer, it has been shown to mediate an alteration of the intracellular balance between pro-and anti-apoptogenic factors [34]. An analysis of CASP9 promoter polymorphisms contributing to genetic susceptibility to lung cancer suggested that CASP9 polymorphisms and their haplotypes interacted with tobacco smoking [35]. One of our published study observed that tobacco smoking worsened the trend of the susceptibility of lung cancer by genetic variant [36]. Therefore we investigated gene-environment interaction between the CASP3 polymorphisms and smoking. An important point of our study was CASP3 polymorphisms had an interaction with cigarette. The CASP3 829CC genotype modified the susceptibility of lung cancer in habitual smokers but outside non-smokers, especially in heavy smokers with OR (95% CI) of 7.29 (4.23-12.59), suggesting a gene-environment interaction. We also found the increased risk of lung cancer among both all the smoker group and heavy smoker subgroup, who took with the CASP3 20541 CT and TT genotype, however the similar significant association with incremental risk of lung cancer was not proved in nonsmoker, different gender and age group.
In summary, we identified that two SNPs of CASP3 gene increased the susceptibility of lung cancer with a large sample size in this case-control study. Tabaco smoking modulated and was interacted the association with CASP3 polymorphisms to lung cancer. The present results are in line with our prior conclusions in the esophageal squamous cell carcinoma research; further indicating that apoptosis-interrelated CASP3 is a cancer susceptibility gene and plays a significant role in human carcinogenesis.