The role of XRCC6/Ku70 in nasopharyngeal carcinoma

The role of XRCC6/Ku70 in nasopharyngeal carcinoma

Chung-Yu Huang

, Chia-Wen Tsai

, Chin-Mu Hsu

, Liang-Chun Shih

, Wen-Shin Chang

, Hao-Ai Shui

, Da-Tian Bau

1

Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, R.O.C.

2

Taichung Armed Forces General Hospital, Taichung, Taiwan, R.O.C.

3

Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, R.O.C.

4

Terry Fox Cancer Research Laboratory, China Medical University Hospital, Taichung, Taiwan, R.O.C.

5

Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan, R.O.C.

* These Authors contributed equally to this study

Correspondence to: Da-Tian Bau, Department of Medical Research, Terry Fox

Cancer Research Lab, China Medical University Hospital, 2 Yuh-Der Road, Taichung, 404 Taiwan, Tel: +886 422052121 Ext 7534

e-mail: [email protected]

Running title: XRCC6/Ku70 in Nasopharyngeal Carcinoma

Keywords

Genotype; Immunohistochemistry; Nasopharyngeal carcinoma; Non-

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homologous end-joining; Polymorphism; Real-time quantitative reverse transcription; XRCC6/Ku70.

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Abstract

XRCC6/Ku70, an upstream player in the DNA double strand break repair

system, was examined its association with the risk of nasopharyngeal carcinoma (NPC). In the case–control study, 176 NPC patients and 352 cancer-free controls were genotyped and the associations of XRCC6 promoter T-991C (rs5751129); G-57C (rs2267437); G-31A (rs132770), and intron 3 (rs132774) polymorphisms with NPC risk were evaluated.

NPC tissue samples of variant genotypes were also estimated their XRCC6 mRNA and protein expression by real-time quantitative reverse transcription and Western blotting, respectively. As for XRCC6 promoter T-991C, the TC and CC genotypes had a significantly increased risk of NPC compared with wild-type TT genotype [adjusted odds ratio (aOR) = 2.04 and 3.41, 95% confidence interval (CI) = 1.23-3.28 and 1.30-9.17, P = 0.0072 and 0.0165, respectively]. The mRNA and protein expression levels with NPC tissues revealed that a statistically significantly lower XRCC6 mRNA and protein expressions in the NPC samples with TC/CC genotypes compared with those with TT genotype (P = 0.0210 and 0.0164, respectively). The findings suggested that XRCC6 may play an important role in the NPC carcinogenesis and could serve as a chemotherapeutic target for personalized medicine and therapy.

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Introduction

Nasopharyngeal carcinoma (NPC), the cancer originating in the nasopharynx, occurs relatively few in the West (with the age-standardized

incidence rate (ASR) < 1/100,000), but keeps its leading rank among tumors in Southern China (ASR = 30-50/100,000), Southeast Asia (ASR = 9-12/100,000) and Taiwan (ASR = 8.2-8.4/100,000).

The geographical pattern of incidence may suggest an interaction of complicated environmental and genetic factors. Although the etiology of NPC remains to be elucidated, those people with infection of Epstein-Barr virus (EBV),

environmental risk factor exposure,

risky dietary habits

and risky genotypes such as single nucleotide polymorphisms (SNPs) may have higher susceptibility to NPC.

The human genome integrity was taken care by the human DNA repair system, in which the accumulated mutations or defects are thought to be essential for carcinogenesis.

Therefore, it is reasonable to hypothesis that the loss or lower of function of DNA repair capacity via genomic variation might have a significant influence on NPC carcinogenesis. In humans, genetic variations on the predominant non-homologous end-joining (NHEJ), together with those on the alternative homologous recombination DNA double strand break (DSB) repair system, has been postulated as an

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important contributor to the etiology of cancer.

In recent years, several proteins involved in the NHEJ pathway have been identified, including ligase IV, XRCC4, XRCC6 (Ku70), XRCC5 (Ku80), DNA-PKcs, Artemis and XLF.

Also, Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells.

As for NHEJ, some genetic polymorphisms were reported to influence DNA repair capacity and confer predisposition to several types of cancer, including skin,

breast,

bladder,

lung

and oral cancers.

However, there is no information regarding NPC and NHEJ gene polymorphisms. Recently, some epidemiological studies have investigated the association between the XRCC6 polymorphism and the risk for various types of cancer, including

gastric cancer,

oral cancer,

breast cancer,

lung cancer

and renal cell carcinoma.

We hypothesize that differential XRCC6 genotypes together with its RNA and protein expression may also contribute to NPC susceptibility.

To test this hypothesis, our present study was designed to investigate the association of XRCC6 genotypes with risk of NPC in our case-control study in a central Taiwan population. In addition, we also investigated the association of the XRCC6 mRNA and protein expression patterns with NPC risk by real-time polymerase chain reaction (PCR) and Western

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blotting respectively, to assess the potential functional effect of XRCC6 genotype in NPC risk. To the best of our knowledge, this is the first study to evaluate the association between the XRCC6 genotypes and NPC susceptibility and to explore the potential function of XRCC6 in NPC at the same time.

Methods

Study population

One hundred and seventy six patients diagnosed with NPC were recruited at the outpatient clinics of general surgery between 2003-2009 at the China Medical University Hospital, Taichung, Taiwan, Republic of China. All the patients voluntarily participated, completed a self-administered questionnaire and provided peripheral blood samples. The questionnaire included questions on history and frequency of alcohol consumption, betel quid chewing and smoking habits and “ever” was defined as more than twice a week for years. Self-reported alcohol consumption, betel quid chewing and smoking habits were evaluated and classified as categorical variables. Double amounts of non-NPC or other types of cancer, healthy people as controls were selected by matching for age and gender after initial random sampling from the Health Examination Cohort of the hospital. The study was approved by the Institutional Review Board of the

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China Medical University Hospital and written-informed consent was obtained from all the participants.

Genotyping protocol

The total genomic DNA of each subject was extracted from the leucocytes of peripheral blood using a QIAamp Blood Mini Kit (Qiagen, Taipei, Taiwan) and stored as previously published.

The primers used for XRCC6 promoter T-991C were: forward 5’-

AACTCATGGACCCACGGTTGTGA-3’, and reverse 5’-

CAACTTAAATACAGGAATGTCTTG-3’; for promoter G-57C were:

forward 5’- AAACTTCAGACCACTCTCTTCT-3’, and reverse 5’- AAGCCGCTGCCGGGTGCCCGA-3’; for promoter G-31A were: forward

5’-TACAGTCCTGACGTAGAAG-3’, and reverse 5’-

AAGCGACCAACTTGGACAGA-3’; for intron 3 were forward 5’-

GTATACTTACTGCATTCTGG-3’, and reverse 5’-

CATAAGTGCTCAGTACCTAT-3’. The following cycling conditions were performed: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min.

Restriction fragment length polymorphism (RFLP) conditions

As for the XRCC6 promoter T-991C, the resultant 301 bp PCR product was

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mixed with 2 U Dpn II. The restriction site was located at -991 with a T/C polymorphism, and the C form PCR products could be further digested while the T form could not. Two fragments 101 bp and 200 bp were present if the product was digestible C form. The reaction was incubated for 2 h at 37°C. Then, 10 l of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either (a) C/C homozygote (digested), (b) T/T homozygote (undigested), or (c) C/T heterozygote. As for the XRCC6 promoter G-57C, the resultant 298 bp PCR products were mixed with 2 U Hae II. The restriction site was located at -57 with a G/C polymorphism, and the G form PCR products could be further digested while the C form could not. Two fractions 103 and 195 bp were present if the product was digestible G form. The reaction was incubated for 2 h at 37°C. Then, 10 l of product was loaded into a 3%

agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either (a) G/G homozygote (digested), (b) C/C homozygote (undigested), or (c) C/G heterozygote. As for the XRCC6 promoter G-31A, the resultant 226 bp PCR products were mixed

with 2 U Mnl I. The restriction site was located at -31 with a G/A polymorphism, and the A form PCR products could be further digested while the G form could not. Two fractions 80 and 146 bp were present if the product was digestible A form. The reaction was incubated for 2 h at

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37°C. Then, 10 l of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either (a) A/A homozygote (digested), (b) G/G homozygote (undigested), or (c) A/G heterozygote. As for the XRCC6 intron 3, the resultant 160 bp PCR products were mixed with 2 U Msc I. The restriction site was located at intron 3 with a TGG/CCA polymorphism, and the CCA form PCR products could be further digested while the TGG form could not. Two fractions 46 and 114 bp were present if the product was digestible CCA form. The reaction was incubated for 2 h at 37°C. Then, 10 l of product was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. The polymorphism was categorized as either (a) CCA/CCA homozygote (digested), (b) TGG/TGG homozygote (undigested), or (c) CCA/TGG heterozygote.

mRNA XRCC6 expression pattern

To evaluate the correlation between the XRCC6 mRNA expression and XRCC6 polymorphism, twenty surgically removed NPC tissue samples

adjacent to tumors with different genotypes were subjected to extraction of the total RNA using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The total RNA was measured by real-time quantitative RT-PCR using FTC-3000 real-time quantitative PCR

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instrument series (Funglyn Biotech Inc., Canada). GAPDH was used as an internal quantitative control. The primers used for amplification of XRCC6 mRNA were: forward 5’-CGATAATGAAGGTTCTGGAAG-3’ and reverse 5’-CTGGAAGTGCTTGGTGAG-3’, while for GAPDH the primers were: forward 5’-GAAATCCCATCACCATCTTCCAGG-3’ and reverse 5’-GAGCCCCAGCCTTCTCCATG-3’. Fold changes were normalized by the levels of GAPDH expression, and each assay was done in at least triplicate.

Western blotting analysis

The NPC specimens were homogenized in RIPA lysis buffer (Upstate Inc., Lake Placid, NY, USA), the homogenates were centrifuged at 10000g for 30 min at 4°C, and the supernatants were used for western blotting.

Samples were denatured by heating at 95°C for 10 min, then separated on a 10

SDS-PAGE gel, and transferred to a nitrocellulose membrane (BioRad Laboratories, Hercules, CA). The membrane was blocked with 5

non-fat milk and incubated overnight at 4°C with mouse monoclonal anti-human XRCC6 antibody (1:1000; Transduction Lab Inc., Franklin Lakes, NJ), then with the corresponding horseradish peroxidase-conjugated goat anti- mouse IgG secondary antibody (Chemicon, Temecula, CA) for 1 h at room temperature. After reaction with ECL solution (Amersham, Arlington

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Heights, IL, USA), a bound antibody was visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK). Finally, the blots were incubated at 56°C for 18 min in stripping buffer (0.0626 M Tris-HCl, pH 6.7, 2

SDS, 0.1M mercaptoethanol) and re-probed with a monoclonal mouse anti--actin antibody (Sigma, St. Louis, MO, USA) as the loading control. The optical density of each specific band was measured using a computer-assisted imaging analysis system (Gene Tools Match software; Syngene).

Statistical analyses

To ensure that the controls used were representative of the general population and to exclude the possibility of genotyping error, the deviation of the genotype frequencies of XRCC6 single nucleotide polymorphisms in the control subjects from those expected under the Hardy-Weinberg equilibrium was assessed using the goodness-of-fit test. Pearson’s Chi- square test or Fisher’s exact test (when the expected number in any cell was less than five) was used to compare the distribution of the XRCC6 genotypes between cases and controls. The associations between the XRCC6 polymorphisms and NPC risk were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible

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confounders. P < 0.05 was considered statistically significant, and all statistical tests were two-sided.

Results

Basic comparisons between the case and control groups

The selected characteristics of the control and case subjects are summarized in Table 1. As shown in the Table, all the characteristics of patients and controls were matched and none of the frequency distribution between the two groups was statistically different from each other (P >

0.05). In this population, the personal habits include smoking, alcohol consumption, and betel quid chewing seemed not to be direct risk factors for NPC.

Association of XRCC6 genotypes and NPC risk

The genotypic distributions of the XRCC6 polymorphisms in the cases and controls are presented in Table 2. Among the polymorphic sites, the most significant findings were the results about the XRCC6 promoter T-991C genotyping. The ORs after adjusting those confounding factors (age, gender, smoking, alcohol drinking and betel quid chewing status) for the people carrying TC and CC genotypes were 2.04 (95% CI = 1.23-3.28) and 3.41 (95% CI = 1.30-9.17) respectively, compared to those carrying TT

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wild-type genotype. The P for trend was significant (P = 0.0014). In the dominant (TC plus CC versus TT) and recessive (CC versus TT plus TC) models, the association between XRCC6 promoter T-991C polymorphism and the risk for NPC was also statistically significant (adjusted OR = 2.23 and 2.31, 95% CI = 1.44-3.36 and 1.17-7.84, respectively). As for the XRCC6 promoter G-57C, promoter G-31A, and intron 3 polymorphisms,

there was no difference between NPC and control groups in the distribution in the genotype frequency at these polymorphic sites (Table 2). To sum up, the genotyping results indicated that individuals carrying TC or CC genotype at XRCC6 promoter T-991C may at higher risk for NPC.

XRCC6 T-991C genotype-phenotype correlation about the expression levels of the XRCC6 mRNA and proteins

We have also collected 20 surgically removed NPC tissue samples adjacent to tumors for phenotype study. These samples were obtained from the NPC patients before any therapy with the identified genotypes of the XRCC6 T- 991C polymorphisms with the blood, and their frequencies of the TT, TC, and CC genotypes of the XRCC6 T-991C were 14, 5, and 1, respectively.

The transcriptional expressions of mRNA levels were examined by real- time quantitative RT-PCR of these patients with variant XRCC6 T-991C genotype (Fig. 1). The levels of XRCC6 mRNA for TC, and CC genotypes

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of the XRCC6 T-991C were 0.85-, and 0.81-fold, compared with TT genotypes, respectively. The one sample with CC genotype were added to the five samples of TC genotype for effective statistical analysis, and a significantly lower levels of XRCC6 mRNA expression were identified in samples from patients with TC/CC genotypes than from those with the TT genotype (P = 0.0210) (Fig. 1).

We have also examined the XRCC6 protein expression patterns in the tumor sites from NPC patients with XRCC6 T-991C TT, TC and CC genotypes (Fig. 2A). The Western blotting results showed that the XRCC6 was much higher expressed in the tissues of XRCC6 T-991C TT genotype than those with TC or CC genotypes (Fig. 2B). Following the central dogma of molecular biology, the results at DNA, RNA and protein levels all showed that XRCC6 T-991C genotype together with the downstream RNA and protein consequences played an important role in NPC etiology.

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Discussion

In the current study, the association of XRCC6 polymorphism and NPC risk was firstly investigated in Taiwan. As for the DNA level, we found that individuals carrying the TC and CC genotypes were of higher risk of NPC compared with those carrying TT genotype at XRCC6 T-991C (Table 2).

Then we have also investigated the effects of XRCC6 T-991C genotype on its mRNA expression level, finding that NPC tissues from patients with TC or CC genotypes were of lower mRNA expression than those with TT genotype (P = 0.0210) (Fig. 1). Last, we have investigated the protein expression patterns of XRCC6 in NPC tumor tissues. The results from Western blotting (Fig. 2A) showed that tissues from patients with TC or CC genotypes were also of lower level of protein expression than those with TT genotype (P = 0.0164) (Fig. 2B). To the best of our knowledge, this is the first study for the role of XRCC6 in NPC with multi-approach positive findings.

The XRCC6 can team up with XRCC5 as a heterodimer, or independent of XRCC5.

The XRCC6-knockout mice, but not the XRCC5-knockout ones, have less mature T-lymphocytes, higher incidence of thymic lymphomas, and higher rate of fibroblast transformation. However, the detail mechanisms causing the differences remain unclear.

As for the protein level, it was reported that proteomic defects in XRCC6 may cause not only

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lower DSB repair capacity, but also growth retardation, ionizing radiation hypersensitivity, and severe combination immune deficiency due to severely impaired variable division joining (V[D]J) recombination capacity.

Based on the above findings, it was proposed that small genomic variations in XRCC6 such as polymorphisms might escape the cell cycle checking mechanisms, and also lead to suboptimal DNA repair capacity which would accumulate DNA variations step by step triggering NPC tumorigenesis.

In literature, XRCC6 T-991C polymorphism was found to be associated with the risk for gastric,

oral

and breast cancer.

In this paper, we first found that the genotypes of XRCC6 were associated with the susceptibility of NPC. The XRCC6 T-991C variation mapped to the promoter region of XRCC6 does not direct result in amino acid coding alteration, it is plausible

to influence the expression level or stability of the XRCC6 protein, similar to the case in XRCC4.

Therefore, we designed functional experiment to investigate whether the T-991C SNP could influence the mRNA and protein levels of XRCC6. In the results of real-time quantitative RT-PCR, we found that the tissues of C allele indeed had a lower expression level of the XRCC6 mRNA than those of T (Fig. 1). Similarly, the results from the protein level also supported this hypothesis (Fig. 2). The T allele may increase the expression levels of the XRCC6 mRNA, which may lead to

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increased expression of XRCC6 protein and elevated DSB repair capacity, performing cancer preventive effects on normal tissues.

In 2005, it was first described in NPC that a higher proportion of XRCC6 (+) tumor cells was associated with poor response to radiation therapy and with higher risk of locoregional recurrence.

Their work was the first one to investigate the expression of XRCC6 in paraffin-embedded NPC tumor samples, but they did not investigate the alterations at mRNA and DNA levels of XRCC6 in NPC as we did in this study. In esophageal cancer cell lines, XRCC6 expression level was correlated with radiosensitivity,

where as uterine cervical cancer patients with low levels of XRCC6 expression in their biopsy samples were radiosensitive and with a netter radiotherapy outcome. Thus, the expression of XRCC6 may serve as a good candidate for a predictive and prognostic marker for NPC, as it is for other types of cancer.

Our present study indicate that the functional XRCC6 T-991C polymorphism is associated with Taiwan NPC risk, and this novel functional XRCC6 polymorphism may lead to differential XRCC6 mRNA and protein expression levels. In the future, XRCC6 may be a good chemotherapeutic target for NPC early prediction and pharmacogenomic

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therapy.

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Acknowledgements

We thank Prof. Wood for his critical review of the manuscript and his precious scientific advice and English editing. The assistance from Ping- Fang Wang in sample collection, and from Hong-Xue Ji in genotyping were highly appreciated by the authors. This study was supported by research grants from the Terry Fox Cancer Research Foundation, and Taichung Armed Forces General Hospital (grant number 101-25).

Competing interests

The authors declare that they have no competing interest.

Funding

None

Ethical approval

Not required.

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Captions to illustrations

Fig. 1. Analysis of XRCC6 mRNA expression levels among NPC patients.

(A) Quantitative RT-PCR for XRCC6 from NPC tissue samples of three genotypes was performed and GAPDH was used as an internal quantitative control. Fold changes were normalized by the levels of GAPDH expression, and each assay was performed in at least triplicate. (B) The groups of TC and CC in (A) were pulled together and compared with TT group.

Fig. 2. The expression levels of XRCC6 in NPC tissues from patients of

different XRCC6 genotypes. (A) Western blotting analysis of XRCC6 expression in tumor tissues from cases with TT, TC and CC XRCC6 genotypes. (B) Quantification of the Western blotting data from Figure 2A.

-actin was used as the loading control. Data are averaged from at least three repeats from the tissues of each group with 15 μg total sample protein for each lane.

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