Effects of NFKB1 and NFKBIA Gene Polymorphisms on Susceptibility to Environmental Factors and the Clinicopathologic Development of Oral Cancer

Background Oral cancer, which is the fourth most common cancer in Taiwanese men, is associated with environmental carcinogens. The possibility that genetic predisposition in nuclear factor-kappa B (NF-κB)-signaling pathways activation is linked to the development of oral squamous cell carcinoma (OSCC) requires investigation. The current study examines associations between polymorphisms within promoter regions of NFKB1 encoding NF-κB1 and NFKBIA encoding IkappaBalpha (IκBα) with both the susceptibility to develop OSCC and the clinicopathological characteristics of the tumors. Methodology/Principal Findings Genetic polymorphisms of NFKB1 and NFKBIA were analyzed by a real-time polymerase chain reaction (real-time PCR) for 462 patients with oral cancer and 520 non-cancer controls. We found that NFKB1 −94 ATGG1/ATGG2, −94 ATGG2/ATGG2, and the combination of −94 ATGG1/ATGG2 and ATGG2/ATGG2 genotypes NFKBIA −826 T (CT+TT) and −881 G (AG+GG) allelic carriages, were more prevalent in OSCC patients than in non-cancer participants. Moreover, we found that NFKB1 or NFKBIA gene polymorphisms seem to be related to susceptibility to develop oral cancer linked to betel nut and tobacco consumption. Finally, patients with oral cancer who had at least one −519 T allele of the NFKBIA gene were at higher risk for developing distant metastasis (P<.05), compared with those patients CC homozygotes. Conclusions Our results suggest that NFKB1 −94 ATTG2, NFKBIA −826 T, and −881 G alleles are associated with oral carcinogenesis. The combination of NFKB1 or NFKBIA gene polymorphisms and environmental carcinogens appears related to an increased risk of oral cancer. More importantly, the genetic polymorphism of NFKBIA −519 might be a predictive factor for the distal metastasis of OSCC in Taiwanese.


Introduction
Oral squamous cell carcinoma (OSCC), a common malignant cancer of the head and neck region, is the fourth most common cancer among men and the sixth leading cause of cancer deaths in Taiwan [1]. OSCC development is a multistep process requiring the accumulation of multiple genetic alterations, influenced by a patient's genetic predisposition and by environmental factors. The environmental factors include alcohol and tobacco consumption, betel (areca) quid chewing, chronic inflammation, and viral infection [1][2][3][4][5][6]. Gene expression is affected by a single-nucleotide polymorphism (SNP) located within the promoter or other regulatory regions of the gene and are thought to be associated with the development of certain diseases [7]. To elucidate the complex process of carcinogenesis and improve the scientific basis for preventive interventions, the identification of major genes influencing a patient's susceptibility to OSCC should be prioritized.
Activation of nuclear factor-kappa B (NF-kB), a hallmark of the inflammatory response, is detected frequently in tumors and may play an important role in linking inflammation to tumor development and progression [8,9]. NF-kB was originally identified as a nuclear factor specific to B cells bound to the B site of the k-light chain gene enhancer. NF-kB is a heterodimer in the Rel family, which contains 5 members: RelA, RelB, c-Rel, p50/105 (NF-kB1), and p52/p100; the dimeric form of NF-kB p50/RelA is the most common form. NF-kB plays a central role in coordinating the expression of a wide variety of genes that control innate and adaptive immune responses, and also plays a critical role in cancer development and progression [8]. In normal cells, NF-kB is inactivated in the cytoplasm by binding to its inhibitor IkB. When IkB proteins are phosphorylated and degraded, NF-kB is released and further translocated to the nucleus, where gene transcription is initiated [10].
The IkB family includes IkappaB alpha (IkBa), IkBb, IkBc, IkBd, IkBe, IkB-f, IkB-R, Bcl-3, p100, and p105, which are all constitutively expressed except for IkBa, which is inducibly expressed. IkBa is a classic form of the IkB family that can be found in the cytoplasm and nuclei [11]. Previous studies demonstrated that NF-kB suppression in cancer inhibits cell proliferation, causes cell-cycle arrest, and results in apoptosis, suggesting that NF-kB plays an important role in cell proliferation and survival [12]. Furthermore, NF-kB is known to prevent apoptosis by inducing antiapoptotic proteins, and suppresses the apoptotic potential of chemotherapeutic agents, leading to chemoresistance [13]. Aberrant expression of NF-kB proteins has been well documented in several types of cancer, including OSCC [14], with the level increasing gradually from premalignant lesions to invasive cancer [15]. Moreover, NF-kB1, which comprises p50 homodimers, transcriptionally regulates the antiapoptotic protein Bcl-2, which is overexpressed in a high proportion of oral cancer cases [16]. These various findings suggest that NF-kB signaling plays an important role in oral carcinogenesis.
Prior research has reported that polymorphic variations in promoter regions of the NF-kB1 gene NFKB1 and the IkBa gene NFKBIA and in the 39-untranslated region (39-UTR) of NFKBIA were associated with a risk for Hodgkin's lymphoma, multiple myeloma, breast cancer, prostate cancer, gastric cancer, colorectal cancer, and melanoma [17][18][19][20][21][22][23]. In Taiwan Lin et al. investigated the 294 ins/del ATTG polymorphism in the NFKB1 promoter among 3 population groups, namely, OSCC patients, male areca chewers with oral submucous fibrosis, and controls (non-areca chewers). They found no significant difference in NFKB1 genotypes among the 3 groups [24]. However, after stratification by age, OSCC was shown to have a higher frequency of the insertion allelotype in patients older than 50 years. No significant difference was found for the NFKB1 allelotype or genotype in patients with OSCC who exhibited different statuses of lymph node metastasis or clinical stage. Apart from the Lin et al. study, no reports have focused on the association between NFKB1 and NFKBIA polymorphisms and OSCC development.
The current study investigated relationships between SNPs in the promoter regions of the NFKB1 and NFKBIA genes and the risk of oral cancer. In addition, we evaluated the influences of these SNPs combined with betel nut and tobacco consumption, which lead to the susceptibility of oral cancer. We also investigated the relationship among genetic influences, environmental exposure, and the clinicopathological characteristics of oral cancer. To our knowledge, this is the first study to demonstrate a significant association between NFKB1 and NFKBIA polymorphisms and oral carcinogenesis.

Subjects and specimen collection
We recruited 462 patients (444 men and 18 women, with a mean age of 54.4611.4 years) at Chung Shan Medical University Hospital in Taichung and Changhua Christian Hospital and Show Chwan Memorial Hospital in Changhua, Taiwan. Patients were enrolled as a case group between 2007 and 2011. For the control group, we randomly chose 520 non-cancer people (426 men and 94 women, with a mean age of 52.4614.7 years) who visited the same hospitals and resided in the same geographic area. Before commencement of the study, approval was obtained from the Institutional Review Board of Show Chwan Memorial Hospital, and each person provided written informed consent to participate in the study.
For both cases and controls, we used a questionnaire to obtain information on patient exposure to betel quid chewing, tobacco use, and alcohol consumption. Medical information for the cases was obtained from their medical records, and included TMN clinical staging, primary tumor size, lymph node involvement, and histologic grade. Oral cancer patients were clinically staged at the time of diagnosis according to the TNM staging system of the American Joint Committee on Cancer (AJCC) Staging Manual (7 th ed.) [25]. Tumor differentiation was examined by a pathologist according to the AJCC classification. Whole blood specimens collected from controls and OSCC patients were placed in tubes containing ethylenediaminetetraacetic acid (EDTA), and were immediately centrifuged and stored at 280uC.

Genomic DNA Extraction
Genomic DNA was extracted using QIAamp DNA blood mini kits (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. We dissolved DNA in a TE buffer (10 mM Tris, 1 mM EDTA; pH 7.8), which was subsequently quantified by measuring the OD260. The final preparation was stored at 220uC and used to create templates for the polymerase chain reaction (PCR).

Real-time PCR
The allelic discrimination of the NFKB1 294 ATTG, NFKBIA 2519, NFKBIA 2826, and NFKBIA 2881 gene polymorphisms were assessed with the ABI StepOne TM Real-Time PCR System (Applied Biosystems) and analyzed with SDS v3.0 software (Applied Biosystems) using the TaqMan assay. The final volume for each reaction was 5 mL, containing 2.5 mL TaqMan Genotyping Master Mix, 0.125 mL TaqMan probes mix, and 10 ng genomic DNA. The real-time PCR included an initial denaturation step at 95uC for 10 min, followed by 40 cycles at of 95uC for 15 s and then at 60uC for 1 min.

Statistical analysis
Differences between the 2 groups were considered significant if p-values were less than .05. Hardy-Weinberg equilibrium (HWE) was assessed using a goodness-of-fit X 2 -test for biallelic markers. The Mann-Whitney U-test and Fisher's exact test were used to compare differences in the distributions of patient demographic characteristics between the non-cancer (control) and oral cancer groups. The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) of the association between genotype frequencies and risk in addition to clinicopathological characteristics were estimated using multiple logistic regression models, after controlling for other covariates. We analyzed all data using the Statistical Analytic System (SAS Institute, Cary, NC, USA) software (v. 9.1, 2005) for Windows.

Results
The statistical analysis of demographic characteristics is shown in Table 1. We found significantly different distributions of age (p = 0.023), sex (p,0.001), betel quid chewing (p,0.001), alcohol consumption (p,0.001), and tobacco use (p,0.001) between control participants and OSCC patients. To reduce the possible interference of environmental factors, the adjusted ORs (AORs) with 95% CIs were estimated by multiple logistic regression models after controlling for other covariates in each comparison.
We also compared the results from participants with the polymorphic gene to those from participants showing the WT gene. We found significantly higher risks for OSCC in participants with the NFKBIA 2826 C/T, 2826 C/T+T/T, 2881 A/G, and 2881 A/G+GG polymorphic genotypes. These risks were, respectively, 1.6-fold (95% CI = 1.0-2.6), 1.7-fold (95% CI = 1.1-2.7), 1.6-fold (95% CI = 1.0-2.6), and 1.7-fold (95% CI = 1.2-2.7), compared with the WT. However, people with the NFKBIA 2519 polymorphic gene were not at a significantly higher risk than those with the WT gene.
Among betel nut consumers in the cohort, tobacco smoking elevated oral cancer risk significantly in participants polymorphic for NFKB1 in the 294 locus or NFKBIA in three loci (2519, 2826, and 2881), compared with people with the WT gene who did not smoke tobacco (Table 4). Moreover, people who were either polymorphic for NFKBIA in 3 loci (2519, 2826, and 2881) or who smoked were at 4.67-to 5.91-fold risk (p,0.05) of developing oral cancer, compared with people with the WT gene who did not smoke (Table 4). These results suggest that NFKB1 and NFKBIA gene polymorphisms exert a strong influence on oral cancer susceptibility in men who chew betel nut and/or smoke tobacco.
To explore the effects of polymorphic genotypes of NFKB1 and NFKBIA on the clinical status of OSCC, we classified OSCC patients into 2 subgroups. In the first subgroup, patients had homozygous WT alleles, and in the second subgroup, they had at least one polymorphic allele. For the genotypic frequencies of the SNPs, only NFKBIA 2519 showed a significant association with clinical pathological variables in OSCC patients. Compared with the WT genotype (C/C), patients with at least one polymorphic T allele of NFKBIA 2519 showed a higher risk (4.88-fold; 95% CI = 1.2-20.4) for distant metastasis (Table 5).

Discussion
The NF-kB pathway plays an important role in tumor development and aggressiveness by enhancing tumor angiogenesis, antiapoptosis, and proliferation, and by repressing the immune response [12,26]. NF-kB is rarely found to be constitutively active in normal cells, but it is constitutively active in most tumor cell lines [27][28][29]. Prior research has demonstrated that NF-kB is constitutively activated in OSCC [15]. Furthermore, the 294 insertion/deletion ATTG polymorphism was shown to have a regulatory influence on NFKB1 gene expression; the promoter sequence containing the ATTG2 allele displayed a 26 higher activity than comparable sequences containing the ATTG1 allele [30]. The allele plays an important role in susceptibility to prostate cancer, cervical squamous cell carcinoma, gastric cancer, and hepatocellular carcinoma [22,23,31,32]. Thus, increased risk for oral cancer associated with the NFKB1 ATGG1/ATGG2 variant might have resulted from its positive regulation of NF-kB expression ( Table 2).
Alcohol consumption, tobacco smoking, and betel quid chewing are the main known etiologic factors of oral cancer. In this study, we observed that higher ratios of people in the OSCC group had consumed alcohol and tobacco and chewed betel quid (61.7%, 84.0%, and 76.4%, respectively), compared with the controls (40.3%, 40.4%, and 17.5%, respectively) ( Table 1). This finding indicated that alcohol and tobacco consumption and betel quid chewing are highly associated with increased risks for oral cancer. Long-term tobacco smoking and betel nut chewing have been shown to contribute to carcinogenesis [3][4][5]33]. Betel nut constituents can increase protein levels of c-fos and c-jun protooncogenes, and tobacco consumption can significantly increase nuclear hypoxia-inducible factor (HIF)-1a expression in oral cancer [34,35].
People carrying the murine double minute 2 (MDM2) SNP 309 GG genotype have oral mucosa that is more susceptible to environ-  Odds ratios (ORs) and their 95% confidence intervals (CIs) were estimated by logistic regression models. The adjusted odds ratios (AORs) with 95% CIs were estimated by multiple logistic regression models after controlling for age, sex, betel quid chewing, alcohol consumption, and tobacco use. doi:10.1371/journal.pone.0035078.t002 Table 3. Adjusted odds ratio (AOR) and 95% CI for oral cancer associated with NFKB and NFKBIA genotypic frequencies and betel nut chewing among 598 smokers. ORs with 95% CIs were estimated by logistic regression models. AORs with 95% CIs were estimated by multiple logistic regression models after controlling for age and sex. a People with wild genotype but not chewers of betel nut. b People with either one or more mutated genotypes, or chewers of betel nut (but not both).
c People with at least one mutated genotype and also chewers of betel nut. doi:10.1371/journal.pone.0035078.t003 mental carcinogens, including tobacco, alcohol, and betel nuts [36]. Therefore, exposure to these factors can result in earlier onset of tumor formation [36]. Accordingly, we evaluated the combined effect of environmental carcinogens and NFKB1 gene polymorphisms on the risk of oral cancer (Tables 2, 3). We found that the NFKB1 294 insertion/deletion ATTG polymorphism Odds ratios (ORs) with their 95% confidence intervals were estimated by logistic regression models. Adjusted odds ratios (AORs) with their 95% confidence intervals were estimated by multiple logistic regression models after controlling for age and sex. a People with wild genotype but not cigarette smokers. b People with either one or more mutated genotypes, or smokers (but not both).
c People with at least one mutated genotype and also cigarette smokers. doi:10.1371/journal.pone.0035078.t004 combined with betel quid chewing and smoking further increased risk of oral carcinogenesis. This phenomenon might be caused by alterations in the binding affinities between betel nut and tobacco constituents and the promoter of the polymorphic NFKB1 gene. Consequently, expression or activity of NF-kB was further changed. Several earlier reports have indicated that tobacco and betel nut constituents can induce NF-kB activation in oral keratinocytes [37,38]. Moreover, evidence has shown that alkaline saliva generated by chewing betel quid may play a role in cigarette-related nicotine-induced DNA damage, and reactive oxygen species may be involved in generating this DNA damage [39]. These findings provide a possible molecular explanation for the synergistic effect of betel quid chewing and smoking in oral cancer development. However, details underlying the mechanism must be verified by other well-designed experiments. An imbalance between NF-kB and IkB is a critical step in tumor development and response to treatment [40]. Polymorphisms of the 3 loci (2519, 2826, and 2881) of the NFKBIA promoter were investigated in this study. The 2881 position was selected based on potential functional effects because of its location within the transcription factor, retinoic acid-related orphan receptor a (RORa), and binding sites [41]. NFKBIA 2881G might alter the binding ability of RORa, which plays a potential role in cancer development [41]. Moreover, 2881 and 2826 in the NFKBIA promoter showed strong local linkage disequilibrium (LD) in 2 Asian populations (Chinese and Japanese). The current study findings showed that Taiwanese men with at least one polymorphic G allele of NFKBIA 2881 or the T allele of NFKBIA 2826 are at a high risk for oral carcinogenesis ( Table 2). The synergistic effect of environmental factors (betel quid and smoking) and NFKBIA 2881 and 2826 polymorphisms on the risk of oral cancer was also demonstrated adequately (Tables 3 and 4).
Although to date no direct evidence has been presented proving that 2881G or 2826T at the promoter of NFKBIA decreases promoter activity, indirect evidence indicates this possibility. For instance, NF-kB expression was twice as high in patients with sarcoidosis, compared with that of control participants. In contrast, the 2881G and 2826T allelic carriages were more prevalent in sarcoidosis patients than in controls [42], indicating that the 2881G and 2826T allelic carriages might be associated with increased activity of NF-kB. Because IkBa is an absolute requirement for normal termination of the NF-kB response [11], NFKBIA 2881G and 2826T allelic carriages were thought to retard efficient IkBa expression.
In conclusion, our results suggest that NFKB1 gene polymorphisms might be correlated with oral cancer susceptibility, and the combined effect of NFKB1 or NFKBIA gene polymorphisms with environmental carcinogens increases risk of oral cancer development significantly. Patients with oral cancer carrying at least one T allele of NFKBIA 2519 are at higher risk of developing distal metastasis, compared with patients carrying C/C homozygotes.

Author Contributions
Conceived and designed the experiments: CWL MHC. Performed the experiments: YSH CWS. Analyzed the data: CHL CHH LHW. Contributed reagents/materials/analysis tools: MHC SFY. Wrote the paper: MHC.