Insertion/Deletion Polymorphism in the Promoter Region of NFKB1 Gene Increases Susceptibility for Superficial Bladder Cancer in Chinese

Abstract

Recently, a functional insertion/deletion polymorphism (−94 insertion/deletion ATTG) in the promoter of NFKB1 gene, which encodes the p50 subunit of nuclear factor-κB protein complex, was identified. The aim of this study was to explore the association between this polymorphism and bladder cancer in a Chinese population. The NFKB1 polymorphism was assessed in 207 patients with superficial transitional cell carcinomas in bladder and in 228 age-, sex-, and smoking-matched healthy volunteers. The polymerase chain reaction assay was used to determine the NFKB1 genotypes. Genomic DNA used for the assay was extracted from peripheral blood lymphocytes. This study found that the frequency of ATTG₂ allele in bladder cancer patients was significantly higher than that in control subjects (65.2% vs. 56.1%, p = 0.006, odds ratio = 1.465), suggesting that the functional NFKB1 promoter polymorphism is associated with increased risk for superficial transitional cell carcinoma of the bladder.

Introduction

I n the United States, bladder cancer is the fifth most common cancer in men and ninth in women, and in 2008, there were 68,810 cases of newly diagnosed bladder cancer and 14,100 related deaths; 74.5% of the cases occurred in men (Jemal et al., 2008). In China, bladder cancer is the 10th most common cancer, accounting for 17,365 deaths in 2005, and mortality has steadily increased between 1991 and 2005 (Yang et al., 2004). Although evidence has suggested that it is the result of interaction of multiple factors, including ethnic, environmental, genetic, and dietary factors, the etiology of bladder cancer is unclear. Tobacco smoking, industrially related aromatic amines and amides, and some anticancer drugs are suggested to be associated with risk of bladder cancer (Cohen et al., 2000). Many people are exposed to these risk factors, but only a portion of exposed individuals develop bladder cancer, suggesting a variation in individual susceptibility to bladder carcinogenesis.

Abnormal regulation of apoptosis, a biological process that plays an important role in maintaining homeostasis, is likely to promote carcinogenesis (Thompson, 1995; Zornig et al., 2001). Defects in apoptosis genes have been identified as causative or contributing pathogenetic factors. Accumulating evidence has pointed out that carcinogenesis is associated not only with prolonged cell survival but also with delayed apoptosis in precancerous cells and, therefore, abnormal apoptosis may allow for uncontrolled cell growth (Thompson, 1995).

Nuclear factor-κB (NF-κB) is a major transcription regulator of apoptosis, cell growth control, and immune response genes, and there are five members of the NF-κB family in mammals: p50/p105, p65/RelA, c-Rel, RelB, and p52/p100. Although many dimeric forms of NF-κB have been detected, the major form of NF-κB is a heterodimer of the p50 and p65/RelA subunits, encoded by the NFKB1 and RelA genes, respectively (Chen et al., 1999). Various stimuli such as bacterial and viral products and cytokines are able to activate NF-κB, mainly through phosphorylation and subsequent degradation of the specific inhibitors, that is, the IκB proteins that retain NF-κB in the cytoplasm. After activation, NF-κB enters the nucleus where it modulates transcription of many genes (Barnes and Karin, 1997). As a transcription factor, NF-κB has been suggested by several groups to protect cells against apoptosis; further, it may play an important role in carcinogenesis (Neiman et al., 1991; Beg et al., 1995). In bladder cancer, NF-κB activation is also able to protect cancer cells from apoptotic cell death as well as upregulate the production of several cytokines that may induce cancer-associated paraneoplastic syndromes (Sumitomo et al., 1999).

A common insertion/deletion (−94 insertion/deletion ATTG) in the NFKB1 gene was identified, which seems to be the first potential functional NFKB1 genetic variation, the presence of a 4 base pair (bp) deletion resulted in the loss of binding to nuclear proteins, leading to reduced promoter activity (Karban et al., 2004). The acknowledged oncogenic potential of NF-κB has led to recent investigations on the relationship between this polymorphism and carcinogenic processes in renal cell carcinoma, colorectal carcinoma, and chronic lymphocytic leukemia, but no correlation was found with the clinical outcome of these tumors (Riemann et al., 2006). Epidemiological studies have investigated the association between NFKB1 polymorphism and risk of bladder cancer in Caucasians but the result did not suggest the relation between NFKB1 polymorphism and risk of bladder cancer (Riemann et al., 2007). We analyzed this polymorphism in our ongoing hospital-based case–control study of bladder cancer in a southwestern Chinese population.

Materials and Methods

Study population

This study was approved by the hospital ethics committee, and all subjects gave written informed consent to participate. The case–control study contained 207 patients with superficial transitional cell carcinoma of the bladder and 228 control subjects. All subjects were Han population living in Sichuan province of southwest China. Bladder cancer patients were recruited from Affiliated Hospital of North Sichuan Medical College between October 2006 and May 2009. Data on sociodemographic characteristics, recent and previous tobacco use, other lifestyle habits, and family history of cancer were collected.

All the patients had newly diagnosed cancer, previously untreated (with intravesical instillation therapies or chemotherapy or radiotherapy), and the diagnoses were histologically confirmed. The clinical stages are T_a or T₁ in all patients with transitional cell carcinoma of bladder. The age- and sex-matched control subjects, in whom there was no previous diagnosis of any cancer type or any other chronic disease (chronic obstructive lung disease, diabetes mellitus, blood diseases, autoimmune disorders, etc.), were selected from a routine health survey in the same hospital.

Determination of genotypes

Genomic DNA was extracted from 1 mL ethylenediaminetetraacetic acid–anticoagulated peripheral blood samples by a standard phenol–chloroform extraction method. The polymerase chain reaction (PCR)–polyacrylamide gel electrophoresis method was used to genotype the −94 insertion/deletion ATTG polymorphisms of NFKB1. DNA fragments containing the polymorphism were amplified, and the primer sequences were as follows: forward, 5′-tggaccgcatgactctatca-3′ and reverse, 5′-ggctctggcttcctagcag-3′. PCR was performed in a total volume of 50 μL, including 5 μL 10 × PCR buffer, 1.5 mM MgCl₂, 0.25 mM dNTPs, 0.5 μM each primer, 100 ng of genomic DNA, and 1.5 U of Taq DNA polymerase. The PCR conditions were 94°C for 5 min, followed by 36 cycles of 45 s at 94°C, 45 s at 62°C, and 45 s at 72°C, with a final elongation at 72°C for 10 min. Four microliters of PCR products was separated by a 6% polyacrylamide gel and staining with 1.5 g/L argent nitrate. Allele (ATTG)₁ yields a 154 bp band and allele (ATTG)₂ yields a 158 bp band. About 10% of the samples were randomly selected and the assays repeated; the results were 100% concordant.

Statistical analyses

All data analyses were carried out using SPSS 13.0 statistical software. Allelic and genotypic frequencies of NFKB1 gene −94 insertion/deletion ATTG polymorphism were obtained by directed counting, and Hardy–Weinberg equilibrium were evaluated by chi-square test. Odds ratio (OR) and respective 95% confidence intervals were reported to evaluate the effects of any difference between allelic and genotypic distribution. Probability values of 0.05 or less were regarded as statistically significant in patients with bladder cancer compared with control subjects.

Results

The characteristics of the cases and controls are presented in Table 1. The cases and controls appeared to be well matched on age, sex, and smoking status: mean age was 62.3 ± 8.0 years for cases and 61.5 ± 8.2 years for controls (p = 0.333); 81.2% and 18.8% of the cases and 79.4% and 20.6% of the controls were men and women, respectively (p = 0.661); mean pack-years was 40.4 ± 9.07 for cases and 39.0 ± 9.7 for controls (p = 0.309); and 79.2% and 20.8% of the cases and 75.4% and 24.6% of the controls were ever smoking and never smoking, respectively (p = 0.203).

Table 1.

The Distribution of Selected Characteristics by Case–Control Status

Variable	Cases (207)	Controls (228)	p
Sex, n (%)
Male	168 (81.2)	181 (79.4)	NS
Female	39 (18.8)	47 (20.6)
Mean (SD) age, years	62.3 (8.0)	61.5 (8.2)	NS
Smoking status, n (%)
Never	43 (20.8)	56 (24.6)	NS
Ever	164 (79.2)	172 (75.4)
Mean (SD) pack-years	40.4 (9.07)	39.0 (9.7)	NS

No significant differences between the cases and controls in the age, sex, and smoking status (p > 0.05).

NS, not significant; SD, standard deviation.

Three genotypes of the NFKB1 −94 insertion/deletion ATTG polymorphism (ATTG₁/ATTG₁, ATTG₁/ATTG₂, and ATTG₂/ATTG₂) were successfully identified. Genotype distributions had no deviation from Hardy–Weinberg equilibrium both in bladder cancer patients and control subjects. Differences in allelic and genotypic distributions of NFKB1 −94 insertion/deletion ATTG polymorphism were tested between bladder cancer patients and control subjects, and observed differences are presented in Table 2.

Table 2.

The Allelic and Genotypic Distributions of NFKB1 Polymorphism Among Bladder Cancer Patients and Controls

Variables		Patients (n = 207)	Controls (n = 228)	p	OR	95% CI
NFKB1 −94 genotype (%)	ATTG₁/ATTG₁	26 (12.6)	46 (20.2)	Ref.
	ATTG₁/ATTG₂	92 (44.4)	108 (47.4)	NS	1.507	0.865–2.627
	ATTG₂/ATTG₂	89 (43.0)	74 (32.5)	0.009	2.128	1.202–3.767
NFKB1 −94 allele (%)	ATTG₁	144 (34.8)	200 (43.9)	0.006	1.465	1.114–1.927
	ATTG₂	270 (65.2)	256 (56.1)

Bold-faced values indicate significant difference at the 5% level.

Ref., reference; OR, odds ratio; CI, confidence interval.

The frequency of ATTG₂ allele in bladder cancer patients was significantly higher than that in control subjects (65.2% and 56.1%, in bladder cancer patients and control subjects, respectively), and the p-value and OR were 0.006 and 1.465, respectively. No significant difference was observed between bladder cancer patients and control subjects in ATTG₁/ATTG₁ versus ATTG₁/ATTG₂ comparison. The frequency for ATTG₂/ATTG₂ genotype was significantly overrepresented in bladder cancer patients (p = 0.009, OR = 2.128, 95% confidence interval = 1.202–3.767 for ATTG₂/ATTG₂ vs. ATTG₁/ATTG₁). The NFKB1 −94 insertion/deletion ATTG polymorphism was associated with bladder cancer, and ATTG₂ homozygote subjects were at significant risks for bladder cancer.

Discussion

It is increasingly clear that genetic factors play a critical role in determining risk of bladder cancer, and many polymorphisms associated with bladder cancer in various genes have been reported (Chen et al., 2000, 2002; Mittal et al., 2005; Srivastava et al., 2005). It is possible that individual variations in the genetic makeup of humans (single-nucleotide polymorphism) may have an effect on the relationship between host and cancer cells and thus, at least in part, contribute to this extraordinary variation. Recent studies in humans have revealed that polymorphisms of the NFKB1 gene are associated with oral squamous cell carcinoma (Lin et al., 2006), nasopharyngeal carcinoma (Zhou et al., 2009), colorectal carcinoma (Lewander et al., 2007), and multiple myeloma (Berenson et al., 2001). In bladder cancer, Riemann et al. (2007) reported that NFKB1 polymorphism is not associated with an increased susceptibility to transitional cell carcinomas of the bladder in Caucasians, but it is a possible new prognostic marker for recurrence in superficial bladder cancer.

In this case–control study, we have identified a significant association between the −94 insertion/deletion ATTG polymorphism in NFKB1 promoter and the risk of superficial transitional cell carcinoma of the bladder. Individuals with ATTG₂ allelotype have 1.465 times greater risks for bladder cancer, compared with those carrying ATTG₁ allelotype (p = 0.006). Individuals homozygous for ATTG₂ have the highest risk compared with individuals homozygous for ATTG₁ (OR = 2.128, p = 0.009). The ATTG₂ polymorphism in NFKB1 promoter is fairly frequent, and it may be valuable for bladder cancer risk prediction in general population. Currently, one published study (Lin et al., 2006) assessed the relationship between the −94 insertion/deletion ATTG polymorphism in NFKB1 promoter and the risks of oral squamous cell carcinoma occurring on older male areca chewers, with the OR in oral squamous cell carcinoma carrying ATTG₂ allelotype being 1.78 relative to control subjects in individuals more than 50 years old.

Cancer cells are distinguished from normal cells by several hallmarks, including evasion of apoptosis, self-sufficiency in growth signaling, insensitivity to antigrowth signals, sustained angiogenesis, limitless replicative potential, propensity toward tissue invasion, and metastasis (Hanahan and Weinberg, 2000). NF-κB regulates the transcription of many genes for apoptosis, cell adhesion, differentiation, proliferation, angiogenesis, and immune response (Beinke and Ley, 2004). Abnormalities in NF-κB regulation are involved in multiple human pathologies including inflammatory diseases, immune deficiencies, diabetes, and atherosclerosis as well as tumors. NF-κB is inhibited by binding to NF-κB inhibitor (IκB), and release of activated NF-κB follows proteasome-mediated degradation of IκB resulting from phosphorylation of the inhibitor and finally conjugation with ubiquitin (Berenson et al., 2001; Beinke and Ley, 2004). Inhibition of NF-κB abrogated tumor cell survival and carcinogenesis, and in a recent study, inhibition of NF-κB activity in melanocytes results in reduction of melanoma tumor growth in vivo (Sunwoo et al., 2001; Loercher et al., 2004; Yang et al., 2007). In vitro, Baeuerle and Henkel (1994) have concluded that NF-κB has a role in the control of cell proliferation based on the following observations. First, NF-κB is involved in the transcriptional activation of several genes encoding growth factors and their receptors. Second, the viral counterpart of c-rel, v-rel, is a potent oncogene in avian cells. Third, in some cell types, NF-κB is activated by mitogenic stimuli. Finally, some members of the NF-κB/IκB family are encoded in chromosomal regions frequently altered in neoplasia. Therefore, NF-κB may act as a key factor for cytokine production in cells and may also be associated with tumor cell growth. At the same time, some kinds of bladder cancer cells can acquire the ability to activate NF-κB, which directly protects cancer cells from apoptotic cell death as well as upregulates the production of several cytokines that may induce cancer-associated paraneoplastic syndromes (Sumitomo et al., 1999). In this study, we found that ATTG₂ allelic frequency in bladder cancer patients is higher than that in control subjects, which is different from the result of study in Caucasians (Riemann et al., 2007). This difference may be associated with the different ethnic group and diverse genetic background. Considering that the promoter sequence containing ATTG₂ allele had activity two times higher than comparable sequences containing ATTG₁ allele, a detrimental role of NF-κB-activation in bladder cancer initiation and development has been suggested.

We identified the association between the −94 insertion/deletion ATTG polymorphism in NFKB1 promoter and the risk of superficial bladder cancer in Chinese. Thus, we report that the ATTG₂ allele is associated with the increased risk of superficial transitional cell carcinoma of the bladder.

Footnotes

Acknowledgments

This work was supported by the Applied Basic Research Programs of Science and Technology Commission Foundation of Sichuan Province (No. 2008JY0088-2) and the Applied Basic Research Programs of Education Commission Foundation of Sichuan Province (No. 2006B032).

Disclosure Statement

No competing financial interests exist.

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