Gene 537 (2014) 238–244
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A nonsense mutation in the Xeroderma pigmentosum complementation group F (XPF) gene is associated with gastric carcinogenesis Zhong-Hua Wei 1, Wen-Huan Guo 1, Jun Wu, Wen-Hao Suo, Guo-Hui Fu ⁎ Pathology Center, Shanghai First People's Hospital / Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China
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Article history: Accepted 25 December 2013 Available online 8 January 2014 Keywords: XPF SNP Mutation Gastric cancer Ubiquitination
a b s t r a c t XPF/ERCC1 endonuclease is required for DNA lesion repair. To assess effects of a C2169A nonsense mutation in XPF at position 2169 in gastric cancer tissues and cell lines, genomic DNA was extracted from blood samples of 488 cancer patients and 64 gastric tumors. The mutation was mapped using a TaqMan MGB probe. In addition, gastric cancer cell lines were transfected with mutated XPF to explore XPF/ERCC1 interaction, XPF degradation, and DNA repair by a comet assay. The C2169A mutation was not detected in 488 samples of blood genomic DNA, yet was found in 32 of 64 gastric cancer tissue samples (50.0%), resulting in a 194C-terminal amino acid loss in XPF protein and lower expression. Laser micro-dissection confirmed that this point mutation was not present in surrounding normal tissues from the same patients. The truncated form of XPF (tXPF) impaired interaction with ERCC1, was rapidly degraded via ubiquitination, and resulted in reduced DNA repair. In gastric cancers, the mutation was monoallelic, indicating that XPF is a haplo-insufficient DNA repair gene. As the C2169A mutation is closely associated with gastric carcinogenesis in the Chinese population, our findings shine light on it as a therapeutic target for early diagnosis and treatment of gastric cancer. © 2013 Published by Elsevier B.V.
1. Introduction Exposure to environmental DNA damaging agents impairs DNA integrity, leading to carcinogenesis (Ciccia et al., 2008; Hoeijmakers, 2001; Klarer and McGregor, 2011; Reddy and Vasquez, 2005; Woods, 1998). Fortunately, intrinsic defense systems have evolved to overcome these damages through a number of DNA repair pathways, including nucleotide excision repair (NER), base excision repair (BER), and mismatch repair (MMR) (Diderich et al., 2011; Dip et al., 2004; Fagbemi et al., 2011; Gregg et al., 2011). The BER system affects a wide range of nucleotide modifications that subtly alter DNA structure (Fortini et al., 2003; Krokan et al., 2000). The MMR functions to correct replication mistakes like single base–base mismatches and small insertion/deletion loops during erroneous base incorporation of DNA polymerases in replication or recombination (Jiricny, 2006). The NER pathway is crucial to maintaining cellular DNA integrity. The gene mutation undermining the NER pathway is either inherited from the germline or arises somatically, leading to genomic instability and elevated cancer risk (Beckman and Abbreviations: XPF, Xeroderma pigmentosum complementation group F; SNP, single nucleotide polymorphism; ERCC1, excision repair cross-complimentary group 1; NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; MTHFR, methylenetetrahydrofolate reductase. ⁎ Corresponding author at: Pathology Center, Shanghai First People's Hospital, Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine, No. 280, South Chong-Qing Road, Shanghai 200025, PR China. Tel.: +86 21 63846590x776601. E-mail address: [email protected] (G.-H. Fu). 1 These authors contributed equally to this work. 0378-1119/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.gene.2013.12.061
Loeb, 2005; Diderich et al., 2011; Hyka-Nouspikel et al., 2012; Lagerwerf et al., 2011; Yeh et al., 2012). Many genes are involved in gastric carcinogenesis and give rise to various tumor types through DNA mutations, polymorphisms, and/or gene–environment interactions (Knudson, 2001; Lopez-Lazaro, 2010). Inherited or somatic mutations in E-cadherin genes are present in 30– 50% of gastric cancer cells (Fitzgerald et al., 2010). Additionally, polymorphisms, the interleukin 1β, interferon-γ chain 1, and methylenetetrahydrofolate reductase (MTHFR) genes, are believed to be oncogenic (Figueiredo et al., 2002; Thye et al., 2003; Zintzaras, 2006). Environmental factors, such as Helicobacter pylori infections, also influence these polymorphisms (Figueiredo et al., 2002; Thye et al., 2003). The Xeroderma pigmentosum complementation group F protein (XPF, also known as ERCC4) is 916 amino acids long and includes an excision repair cross-complimentary group 1 (ERCC1) binding domain (Choi et al., 2005; Masuyama et al., 2005; Tripsianes et al., 2005). XPF plays a key role in NER (de Laat et al., 1999; Volker et al., 2001), directly interacting with ERCC1 to remove inter-strand cross-links in damaged DNA (Fagbemi et al., 2011; Gregg et al., 2011). Impaired function of these two proteins and defective interplay between them are considered potential causes of carcinogenesis (Kirschner and Melton, 2010). XPF-deficient patients are at increased risk for skin cancer, accelerated aging, or cerebrooculo-facio-skeletal syndrome (Gregg et al., 2011; Suzumura and Arisaka, 2010). Recently, studies disclosed a correlation between XPF mutations and primary tumors (Wang et al., 2010). XPF mutations, however, remain elusive in gastric cancer development (Bevan and Houlston, 1999; Chun and Ford, 2012). We assessed the
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effects of the rs2020959CNA SNP at position 2169 of XPF on gastric carcinoma epidemiology and carcinogenic machinery.
Table 1 Analysis of genotype distribution between different tumors and peripheral blood. Test group
2. Materials and methods 2.1. Ethics statement
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Blood Gastric cancer
Genotype
p-value
CC (%)
CA (%)
AA (%)
Total
488 (100.00) 32 (50.00)
0 (0.00) 32 (50.00)
0 (0.00) 0 (0.00)
488 64
b0.001
p-values were obtained from Fisher's exact test.
This study was performed according to the guidelines approved by the Ethics Committees of Shanghai Jiao Tong University. Blood and tissue samples were obtained with informed consent from patients, and all patient data was analyzed anonymously.
2.2. Sample collection A total of 682 patients of Han nationality were investigated in this study. Blood samples from 488 Chinese cancer patients hospitalized at the Jiang-Xi provincial Tumor Hospital between February 1 and 15, 2012 were included. Formalin-fixed paraffin-embedded gastric cancer specimens were obtained from the Department of Pathology of Shanghai Jiao Tong University Affiliated Sixth People's Hospital between January 2010 and December 2012. The 64 cases of gastric cancer specimens for genotyping included 27 cases of tubular adenocarcinoma, 18 cases of papillary adenocarcinoma, 9 cases of mucinous adenocarcinoma, and 10 cases of poorly differentiated carcinoma among which 25 cases were in early stage and 39 cases were in advanced stage. And the 130 cases of gastric cancer specimens for immunohistochemistry consisted of 51 cases of tubular adenocarcinoma, 37 cases of papillary adenocarcinoma, 15 cases of mucinous adenocarcinoma, and 27 cases of poorly differentiated carcinoma among which 47 cases were in early stage and 83 cases were in advanced stage.
2.5. Construction of expression vector PCR products of full length XPF and ERCC1 genes were separated by agarose gel electrophoresis after DNA amplification and ligated to pCDNA3.1 + (Takara Bio, Otsu, Japan) to generate expression vectors pCDNA-XPF and pCDNA-ERCC1. Additionally, XPF PCR products were ligated to pEGFP-C1 to generate pEGFP-XPF. The pEGFP-tXPF vector was generated by site-directed mutagenesis on the pEGFP-XPF backbone using the Fast Mutagenesis System kit (Beijing TransGene Biotech Co., Beijing, China). All constructs were amplified in Escherichia coli TOP10 cells (Tiangen Biotech Co., Ltd., Beijing, China), purified using the Plasmid Maxiprept kit (Axygen, Corning, Tewkesbury, MA, USA), sequenced, and stored at −20 °C for transient transfection. 2.6. Immunohistochemistry Histological sections (4 μm) were dewaxed with xylene and rehydrated in ethanol. Deparaffinized slides were incubated at 98 °C in 10 mmol/l citrate buffer at pH 6.0 for 30 min. Endogenous peroxidase was blocked with 0.05% (v/v) hydrogen peroxide (H2O2) in PBS.
2.3. Selection and genotyping of the XPF gene We conducted a bioinformatic analysis of 954 previously reported SNPs in the general population as listed in the NCBI SNP databank (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?showRare=on& chooseRs=all&go=Go&locusId=2072). The PFS database was utilized as a complementary source to verify the results obtained from the NCBI database (http://pfs.nus.edu.sg/(S(svpmljuq4h5ped1mbc1qhu5y))/ FuncDetail_V1.aspx?Func=NCBI_Gene_LinkOut&FuncAddInfo=2072). The rs2020959C N A (NM_005236.2: c. 2169C N A) SNP, considered a nonsense mutation, was selected for further study. Blood DNA was isolated with the TIANamp Blood DNA Maxi Kit (DP333, Tiangen Biotech Co., LTD., Beijing, China). DNA was extracted from formalin-fixed paraffin embedded tissue samples using the TIAN quick FFPE DNA Kit (DP330, Tiangen Biotech Co., LTD., Beijing, China). Both gastric cancer and surrounding normal tissues were obtained by laser capture microdissection (LCM) followed by DNA extraction using the Arcturus® PicoPure® DNA Extraction Kit (KIT0103, Applied Biosystems, Foster City, CA, USA) according to manufacturer's instructions. The rs2020959 SNP was genotyped via qPCR with TaqMan MGB probes and primers (C_11189219_10) for XPF/rs2020959. 17.4% of tissue samples were reassayed to verify results and reproducibility, and 100% concordance was confirmed.
2.4. Cell culture Human gastric cancer AGS, SGC7901, and MKN45 cell lines, human embryonic kidney 293 T (HEK293T) cells, and Chinese Hamster Ovary (CHO) K1 cells were purchased from Shanghai cell bank (Chinese academy of Sciences). Cells were cultured and grown in DMEM medium at 37 °C in 5% carbon dioxide and supplemented with penicillin/ streptomycin and 10% fetal bovine serum (FBS).
Fig. 1. Western blot analysis of XPF expression levels in three human gastric cancer cell lines and in HEK293T cells. (A) Representative XPF western blot image in four cell lines. XPF and tXPF are as indicated in the top panel. tXPF was not detected in HEK293T cells, but in gastric cancer MKN45, SGC 7901, and AGS cell lines. (B) Quantification of XPF and tXPF protein after normalized to β-actin by using Image J software. Data presented as mean ± standard deviation (SD).
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Slides were incubated with rabbit anti-XPF polyclonal antibody (Santa Cruz, CA, USA) overnight at 4 °C. Immunostaining was performed with the UltraSenstive™ S-P kit (MaiXin, Fuzhou, China). Staining optimization, evaluation, and analysis were performed by two scientists (Zhong-Hua Wei and Wen-Huan Guo). A four-point scale method measured the percentage of positively stained cells as described previously (b5% = 0; ≥5% to b20% = + 1; ≥ 20% to 50% = +2; ≥50% = +3). Cases with 0 and + 1 staining scores were in the low expression group, and +2 and +3 staining scores were in the high group.
2.7. Transient transfection and immunoprecipitation HEK293T cells were seeded in 6-well plates at a 1 × 105 cells per well density and grown for 16 h prior to transfection. Plasmid transfections were performed with X-TREME GENE HP DNA Transfection Reagent (Roche Molecular Systems, CA, USA). Cells were either cotransfected with HA-ub expression vectors or treated with proteasome inhibitor MG132 (5 μM) for 12 h, 24 h and 48 h. After transfection, cells were lysed in buffer B (pH 8.0, 50 mM Tris–HCl, 150 mM NaCl, 1% Triton-X 100, 100 μg/ml PMSF). Clarified lysates (2 mg) were incubated with either 20 μl protein A/G PLUS Agarose (Santa Cruz, CA, USA) or with anti-ERCC1 antiserum (Santa Cruz, CA, USA) for 2 h at 4 °C. After extensive washing with Buffer B, immunoprecipitated proteins were separated by 8% SDS-PAGE. The following antibodies were used for immunoblotting: anti-GFP (Santa Cruz, CA, USA), anti-ERCC1 (Santa Cruz,
CA, USA), anti-HA (Santa Cruz, CA, USA) and anti-XPF (GeneTex, CA, USA). 2.8. Western blot MKN45, SGC7901, AGS, and HEK293T cell lines were harvested and pelleted by centrifugation. Proteins were extracted on ice for 20 min in lysis buffer (pH8.0, 50 mM Tris–HCl, 150 mM NaCl, 1% Triton-X 100, 100 μg/ml PMSF). Protein concentrations were determined by the Quick Start™ Bradford Protein Assay Kit 2 (Bio-Rad). Equivalent amounts of total protein were separated by 8% SDS-PAGE and transferred onto nitrocellulose membranes (Protran™ Whatman Inc., USA). After blocking with 5% non-fat milk, membranes were incubated overnight at 4 °C with monoclonal primary antibodies against XPF (GeneTex, CA, USA) or β-actin (Sigma, MO, USA). After washes in PBS, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma, MO, USA) and visualized using an enhanced chemiluminescence system (ProteinSimple, CA, USA). Quantification of each band was performed using Image J (National Institutes of Health, MD, USA). XPF levels were normalized to β-actin. 2.9. Comet assay The comet assay was performed according to previous literature (Arora et al., 2010; Usanova et al., 2010). Briefly, cells were incubated
Fig. 2. Immunohistochemistry analysis of XPF in gastric cancer and surrounding normal tissues. (A), (B), and (C). High expression of XPF in gastric mucosa in surrounding normal tissues and low expression in different stage of cancer tissues. Red arrow, well differentiated cancer tissue; green arrow, moderately differentiated cancer tissue; yellow arrow, poorly differentiated cancer tissue. Scale bar = 50 μm. (D) Statistical diagram depicting XPF expression levels in 130 cases of gastric cancer and surrounding normal tissues. p-values obtained from chi-square test.
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with cisplatin (15 μg/ml) for 2 h and further treated with 100 μM H2O2 for 15 min to induce DNA strand breaks. After harvest, 25 μl of the cell suspension (2 × 105 cells/ml) was mixed with 75 μl of low melting point (LMP) agarose at 37 °C. The mixture was transferred onto a microscope slide pre-coated with 1% normal melting point (NMP) agarose, covered, and placed at 4 °C until agarose solidified. The coverslip was removed and the slide was placed in chilled lysis solution at 4 °C for 6 h. Slides were then transferred into an electrophoresis chamber containing ice-cold alkaline solution for 20 min. The single cell gel electrophoresis assay (comet) lasted 20 min (25 V, 300 mA). After, the slide was neutralized three times for 10 min each, stained with DAPI (2 μg/ml), and visualized under a fluorescence microscope (200×, Leica DM2000). Fifty cells per sample were analyzed using the CASP Image Analysis software (CASPLab, Wroclaw, Poland). The value of the tail moment was used to determine the decrease in tail moment (DTM) according to the fomula: DTM % = [1 − (TMpht − TMcu)/(TMcht − TMcu)] × 100%, where the TMpht is the mean tail moment of both cisplatin and H2O2 treated samples, TMcu stands for the mean tail moment of the untreated control sample, and the TMcht is the mean tail moment of H2O2 treated control sample. The DTM reflected the repair function by the XPF protein. 2.10. Statistical analysis XPF expression in gastric cancer and surrounding normal tissues was analyzed by chi-square tests. Distributions of the allele and genotype
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frequencies were calculated by Fisher's exact test. Statistical significance was evaluated by one-way analysis of variance (ANOVA) with Student– Newman–Keuls (SNK) test for post hoc analysis. All analyses were performed in SPSS v16.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. The C2169A mutation in XPF in the Chinese population To investigate the C N A nonsense mutation in the Chinese population and to understand the role of tXPF in carcinogenesis, blood genomic DNA was extracted from 488 Chinese cancer patients to identify mutations by the TaqMan SNP allelic discrimination assay. Surprisingly, we did not detect the C N A in any of the 488 Chinese cancer patients' blood samples (Table 1), suggesting that this SNP is uncommon in Chinese patients. The heterozygous C N A mutation position 2169 of the XPF gene was detected in 32/64 of gastric cancer tissues that included 25 early stage and 39 advanced stage samples. Specifically, 14 samples (56%) were from early stage and 18 samples (46.2%) were from advanced stage cancers, for an overall detection rate was 50%. We thus highlight this somatic mutation in pathogenesis of gastric cancer. Five gastric cancer tissues were compared to their respective normal counterparts obtained by laser capture microdissection (LCM). Wildtype copies of XPF were identified in all five tissue samples, while the C2169A mutation was detected in 2 gastric cancer specimens. Interestingly, the same mutation was found in three gastric cancer cell lines tested: AGS, MKN45, and SGC7901. The heterozygous C N A
Fig. 3. Interaction between XPF and ERCC1 and the effect of the rs2020959 SNP on the XPF/ERCC1 complex. (A) Schematic representation of the XPF and ERCC1 interaction domain. The normal nucleotide and corresponding amino acid sequences distributed around the mutation site of XPF are presented. (B) Schematic representation of the nonsense mutation at position 2169 in XPF (upper panel). The mutation introduces a premature stop codon, resulting in tXPF expression (lower left panel). pEGFP-XPF or pEGFP-tXPF expression constructs were transfected into gastric cancer AGS cells for 24 h, and XPF or tXPF expression were detected by western blot, using anti-GFP antibody. Arrows indicate XPF (upper band) and tXPF (lower band) (lower right panel).
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mutation in XPF introduces a premature stop codon, so these cell lines express both a tXPF protein and wild-type variant. XPF expression was reduced in these cells compared to HEK293T cells that express only wild-type XPF (Fig. 1). The C N A mutation is not an SNP, but a cancer-related nonsense mutation common in tumors of Chinese gastric cancer patients. We next investigated XPF protein expression in 130 gastric cancer specimens compared to surrounding normal tissues by immunohistochemistry. High XPF expression was observed in 113 normal tissues (86.9%) and in only 24 cancer tissues (18.5%) (P b 0.001), suggesting the instability of native XPF in gastric cancer tissues (Fig. 2).
3.2. Prediction and characterization of tXPF expression in AGS gastric cancer cells According to the dbSNP database, a total of 954 SNPs in the XPF gene have been documented in humans (http://www.ncbi.nlm.nih. gov/projects/SNP/snp_ref.cgi?showRare=on&chooseRs=all&go= Go&locusId=2072). We report that rs2020959C N A SNP is a nonsense mutation resulting in tXPF expression that lacks 194C-terminal amino
Fig. 4. Determination of tXPF/ERCC1 interaction and the tXPF protein stability. (A) XPF or tXPF interact with ERCC1 in vitro. GFP tagged XPF or tXPF expression construct and ERCC1 vector were co-transfected into AGS cells for 24 h. Cells were lysed and equal amounts of protein were immunoprecipitated with anti-ERCC1 antibody and analyzed by Western blotting. Normal mouse IgG served as a negative control. The XPF/ERCC1 interaction was used as a positive control (lane 2). XPF and tXPF were visualized using specific anti-GFP antibody. WCL, whole cell lysis. (B) and (C) Comparison of XPF and tXPF protein stabilities. XPF or tXPF expression vectors were transfected into AGS cells with ERCC1, and expression levels were detected by anti-GFP antibody. (B) Protein quantification was performed using the Image J software. Data presented as mean ± SD of three replicate experiments.
acids (Fig. 3A). We confirmed this by cloning wild-type XPF and the mutant gene into a pEGFP-C1 vector to generate two constructs: pEGFP-XPF and pEGFP-tXPF. These plasmids were independently transfected into AGS cells for 24 h. Immunoblotting confirmed that C2169A results in the truncated tXPF (Fig. 3B). 3.3. Impaired interaction between tXPF and ERCC1 and degradation via the ubiquitin pathway Human XPF interacts with ERCC1 through its 265 amino acid residues (AA559 to AA823) (Rahn et al., 2010). To characterize tXPF's affinity to ERCC1, either XPF or tXPF was co-expressed with ERCC1 in AGS cells for 24 h. Interplay between ERCC1 and XPF or tXPF was detected by immunoprecipitation and determined by western blot. Immunostaining results demonstrated that binding affinity of tXPF to ERCC1 was reduced compared to XPF (Fig. 4A). To evaluate tXPF's stability, either XPF or tXPF constructs were cotransfected with ERCC1 in AGS cells for 1, 2, and 3 days, respectively. Western blotting analysis showed the XPF protein remained stable for 3 days. Contrastingly, tXPF levels significantly decreased 3 days posttransfection (Figs. 4B and C). We speculated that the XPF protein level depended on integrity, and deletion of the 194C-terminal amino acids compromised this. To test this, pCDNA3.1-HAtXPF was transfected into AGS cells, and MG132, a proteasome inhibitor, was added the second day. Cells were harvested 3 days after transfection for western
Fig. 5. XPF and tXPF are degraded through the ubiquitin pathway. (A) AGS cells were transfected with pCDNA-HAtXPF plasmids, and treated with MG132 (5 μmol). The tXPFHA expression was detected by western blot using anti-HA antibody. (B) AGS cells were transfected with plasmids as indicated, and then treated with MG132 (5 μmol) or had no treatment for 12 h. Whole cell lysis (WCL) was subject to western blot assay, and XPF and tXPF were detected by anti-GFP antibody. (C) Immunoprecipitation was performed using anti-GFP antibody with WCL protein. tXPF and XPF were detected using anti-HA antibody.
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blot analysis. tXPF remarkably accumulated after MG132 treatment (Fig. 5A), implicating ubiquitination in tXPF degradation. Further, XPF or tXPF was co-expressed with HA ubiquitin in AGS cells and protein levels were determined by anti-GFP or anti-HA antibodies (Figs. 5B and C). Immunoprecipitation with anti-GFP antibody revealed polyubiquitin chains found conjugated to XPF and tXPF after MG132 treatment (Fig. 5C). Notably, accumulation of polyubiquitinated tXPF was more significant than XPF, indicating that tXPF is most likely more vulnerable to proteasomal degradation. 3.4. The C2169A XPF mutation significantly impairs DNA repair capacity XPF is essential for DNA repair. We assessed whether its truncation affects DNA repair capacity by the comet assay (Figs. 6A and B). The pCDNA-ERCC1 construct was co-transfected into CHO-K1 cells (absent of the endogenous XPF (Wood and Burki, 1982)) with either pEGFP-XPF or pEGFP-tXPF. After 24 h, cells were treated with cisplatin for 2 h and further treated with H2O2 for 15 min to induce intra- and inter-strand DNA crosslinks. DNA crosslink repair was evaluated at 7 h and 24 h, respectively, after the cisplatin treatment. In pEGFP-XPF/pCDNA-ERCC1 co-transfected cells, DNA crosslink level was reduced by nearly 50% at 24 h. In contrast, no reduction was detected in the DNA crosslink level after the pEGFP-tXPF/pCDNA-ERCC1 transfection (Fig. 6C). Collectively, this data demonstrates impaired DNA repair from tXPF expression. 4. Discussion Gastric cancer is the third most common cancer in China (Hu et al., 2007). Although its etiology remains unclear, genetic aberrations play an important role in gastric carcinogenesis (Abnet et al., 2010). We revealed that a nonsense mutation in XPF at position 2169 tremendously attenuates DNA repair and stimulates degradation. Intriguingly, the mutation was not found in peripheral blood samples or in normal tissues of cancer patients. This mutation was specific to gastric cancer tissues, implicating it as a putative biomarker. Five major pathways orchestrate DNA repair in mammalian cells involving over 130 genes. The NER pathway is most important (Friedberg, 2001; Wood et al., 2001) and mediated by nine major participant proteins including ERCC1. ERCC1 heterodimerizes with XPF, so compromising ERCC1/XPF dimerization would undermine NER (Kirschner and Melton, 2010). We found that deleting the C-terminal portion of XPF impaired the XPF/ERCC1 dimerization and attenuated the repair of cisplatin-induced DNA damages. Additionally, the truncated protein was more susceptible to ubiquitinmediated degradation. According to epidemiological databases, an “A” and not a “C” at position 2169 in XPF was identified in 4.1% and 1.2% of the Mexican and Japanese populations respectively, but not in Chinese or European populations (NCBI, 2013). In accordance with these databases, we did not detect the SNP in 488 blood samples obtained from Chinese patients. Conversely, this mutation was observed in 50% of gastric cancer tissues in Chinese patients, suggesting that this nonsense mutation is closely correlated with gastric carcinogenesis. It is well accepted that cancers are complex diseases and that one sporadic genetic variant is usually not sufficient to increase risk. It is more likely that disrupted DNA repair machinery first leads to an accumulation of mutated that synergize into malignance (Knudson, 2001; Lopez-Lazaro, 2010). Although the exact etiology of gastric carcinoma remains obscure, evidences from other cancer research suggests relevancy between abnormality of DNA repair nuclease ERCC1/XPF and gastric carcinogenesis. Matoka et al. utilized tissue recombination models to investigate potential roles of XPF/ERCC1 and NER in prostate cancer (Matoka et al., 2012), observing that XPF/ERCC1 contributed to protection against prostate carcinogenesis, while pathway disruption predisposes prostate epithelial cells for transformation (Matoka et al., 2012). Moreover, Facista et al.
Fig. 6. The C N A nonsense mutation affected DNA crosslink repair capacity. CHO-K1 cells were co-transfected with pCDNA-ERCC1 and pEGFP-XPF or pEGFP-tXPF constructs, followed by cisplatin treatment for 2 h and then 100 μM H2O2 for 15 min. (A) and (B) show fluorescence images of alkaline comet assays with indicated protein. Slides were stained with DAPI (2 μg/ml) (blue) and visualized under a fluorescence microscope (200×). (C) DNA cross-link levels were measured 7 h and 24 h after treatment and reduced tail moments were calculated by the formula described in Materials and methods. Values were presented as mean ± SD of triplicate samples.
revealed that decreased expression of ERCC1 and XPF happens early in colon cancer progression (Facista et al., 2012). A previous study surrounding the Eastern Chinese population uncovered 3 polymorphisms in the ERCC1 gene that associated with increased gastric cancer risk. The consequence of XPF polymorphisms was poorly defined, as no correlation was found between the rs2276466C N G, rs6498486A N C, and gastric cancer (He et al., 2012). Meta-analysis results offered clues on the interrelation of SNPs in XPF and increased cancer risk (Vineis et al., 2009). In our study, the rs2020959C N A mutation in XPF introduces a premature stop codon in cancer tissues, implicating XPF as an oncogene in early steps of transformation. Since the rs2020959 SNP perturbs a critical aspect of XPF function, we refer to it as a mutation rather than an SNP. This mutation is predominately monoallelic, implicating it as a haplo-insufficient DNA repair gene. To best of our knowledge, the present study is the first to reveal that C2169A in XPF is associated with an increased risk of gastric cancer and may be a key determinant for malignant transformation of normal gastric
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cells. Intervention to restore the DNA repair system to normal when tumors express tXPF may merit further exploration. Conflict of interest There is no conflict of interest. Acknowledgments This work was supported in part by the National Natural Science Foundation of China (NO30570697; NO30770960) and by the Shanghai Municipal Science and Technology Commission Research Project (NO11JC1406700). We would like to thank Xi-Dai Long, Chun-Yin Luo, Shu-Guang Liu, and Yang-Mei Shen for collecting blood and tissue samples. References Abnet, C.C., et al., 2010. A shared susceptibility locus in PLCE1 at 10q23 for gastric adenocarcinoma and esophageal squamous cell carcinoma. Nat. Genet. 42 (9), 764–767. Arora, S., et al., 2010. Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells. DNA Repair (Amst) 9 (7), 745–753. Beckman, R.A., Loeb, L.A., 2005. Genetic instability in cancer: theory and experiment. Semin. Cancer Biol. 15 (6), 423–435. Bevan, S., Houlston, R.S., 1999. Genetic predisposition to gastric cancer. QJM 92 (1), 5–10. Choi, Y.J., et al., 2005. Biophysical characterization of the interaction domains and mapping of the contact residues in the XPF–ERCC1 complex. J. Biol. Chem. 280 (31), 28644–28652. Chun, N., Ford, J.M., 2012. Genetic testing by cancer site: stomach. Cancer J. 18 (4), 355–363. Ciccia, A., McDonald, N., West, S.C., 2008. Structural and functional relationships of the XPF/MUS81 family of proteins. Annu. Rev. Biochem. 77, 259–287. de Laat, W.L., Jaspers, N.G., Hoeijmakers, J.H., 1999. Molecular mechanism of nucleotide excision repair. Genes Dev. 13 (7), 768–785. Diderich, K., Alanazi, M., Hoeijmakers, J.H., 2011. Premature aging and cancer in nucleotide excision repair-disorders. DNA Repair (Amst) 10 (7), 772–780. Dip, R., Camenisch, U., Naegeli, H., 2004. Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair. DNA Repair (Amst) 3 (11), 1409–1423. Facista, A., et al., 2012. Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer. Genome Integr. 3 (1), 3. Fagbemi, A.F., Orelli, B., Scharer, O.D., 2011. Regulation of endonuclease activity in human nucleotide excision repair. DNA Repair (Amst) 10 (7), 722–729. Figueiredo, C., et al., 2002. Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma. J. Natl. Cancer Inst. 94 (22), 1680–1687. Fitzgerald, R.C., et al., 2010. Hereditary diffuse gastric cancer: updated consensus guidelines for clinical management and directions for future research. J. Med. Genet. 47 (7), 436–444. Fortini, P., et al., 2003. The base excision repair: mechanisms and its relevance for cancer susceptibility. Biochimie 85 (11), 1053–1071. Friedberg, E.C., 2001. How nucleotide excision repair protects against cancer. Nat. Rev. Cancer 1 (1), 22–33. Gregg, S.Q., Robinson, A.R., Niedernhofer, L.J., 2011. Physiological consequences of defects in ERCC1-XPF DNA repair endonuclease. DNA Repair (Amst) 10 (7), 781–791.
He, J., et al., 2012. Polymorphisms in ERCC1 and XPF genes and risk of gastric cancer in an Eastern Chinese population. PLoS One 7 (11), e49308. Hoeijmakers, J.H., 2001. Genome maintenance mechanisms for preventing cancer. Nature 411 (6835), 366–374. Hu, Z., Ajani, J.A., Wei, Q., 2007. Molecular epidemiology of gastric cancer: current status and future prospects. Gastrointest. Cancer Res. 1 (1), 12–19. Hyka-Nouspikel, N., et al., 2012. Deficient DNA damage response and cell cycle checkpoints lead to accumulation of point mutations in human embryonic stem cells. Stem Cells 30 (9), 1901–1910. Jiricny, J., 2006. The multifaceted mismatch-repair system. Nat. Rev. Mol. Cell Biol. 7 (5), 335–346. Kirschner, K., Melton, D.W., 2010. Multiple roles of the ERCC1-XPF endonuclease in DNA repair and resistance to anticancer drugs. Anticancer Res. 30 (9), 3223–3232. Klarer, A.C., McGregor, W., 2011. Replication of damaged genomes. Crit. Rev. Eukaryot. Gene Expr. 21 (4), 323–336. Knudson, A.G., 2001. Two genetic hits (more or less) to cancer. Nat. Rev. Cancer 1 (2), 157–162. Krokan, H.E., et al., 2000. Base excision repair of DNA in mammalian cells. FEBS Lett. 476 (1–2), 73–77. Lagerwerf, S., et al., 2011. DNA damage response and transcription. DNA Repair (Amst) 10 (7), 743–750. Lopez-Lazaro, M., 2010. A new view of carcinogenesis and an alternative approach to cancer therapy. Mol. Med. 16 (3–4), 144–153. Masuyama, M., et al., 2005. Resonance assignments for the DNA binding domain of ERCC1/XPF heterodimer. J. Biomol. NMR 32 (2), 175. Matoka, D.J., et al., 2012. Deficiency of DNA repair nuclease ERCC1-XPF promotes prostate cancer progression in a tissue recombination model. Prostate 72 (11), 1214–1222. NCBI, 2013. Reference SNP (refSNP) Cluter Reports: rs2020959. 01-29 Available from: http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=2020959. Rahn, J.J., Adair, G.M., Nairn, R.S., 2010. Multiple roles of ERCC1-XPF in mammalian interstrand crosslink repair. Environ. Mol. Mutagen. 51 (6), 567–581. Reddy, M.C., Vasquez, K.M., 2005. Repair of genome destabilizing lesions. Radiat. Res. 164 (4 Pt 1), 345–356. Suzumura, H., Arisaka, O., 2010. Cerebro-oculo-facio-skeletal syndrome. Adv. Exp. Med. Biol. 685, 210–214. Thye, T., et al., 2003. Genomewide linkage analysis identifies polymorphism in the human interferon-gamma receptor affecting Helicobacter pylori infection. Am. J. Hum. Genet. 72 (2), 448–453. Tripsianes, K., et al., 2005. The structure of the human ERCC1/XPF interaction domains reveals a complementary role for the two proteins in nucleotide excision repair. Structure 13 (12), 1849–1858. Usanova, S., et al., 2010. Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression. Mol. Cancer 9, 248. Vineis, P., et al., 2009. A field synopsis on low-penetrance variants in DNA repair genes and cancer susceptibility. J. Natl. Cancer Inst. 101 (1), 24–36. Volker, M., et al., 2001. Sequential assembly of the nucleotide excision repair factors in vivo. Mol. Cell 8 (1), 213–224. Wang, M., et al., 2010. A novel XPF-357A N C polymorphism predicts risk and recurrence of bladder cancer. Oncogene 29 (13), 1920–1928. Wood, R.D., Burki, H.J., 1982. Repair capability and the cellular age response for killing and mutation induction after UV. Mutat. Res. 95 (2–3), 505–514. Wood, R.D., Mitchell, M., Sgouros, J., Lindahl, T., 2001. Human DNA repair genes. Science 291 (5507), 1284–1289. Woods, C.G., 1998. DNA repair disorders. Arch. Dis. Child. 78 (2), 178–184. Yeh, J.I., et al., 2012. Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair. Proc. Natl. Acad. Sci. U. S. A. 109 (41), E2737–E2746. Zintzaras, E., 2006. Association of methylenetetrahydrofolate reductase (MTHFR) polymorphisms with genetic susceptibility to gastric cancer: a meta-analysis. J. Hum. Genet. 51 (7), 618–624.
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Gene journal homepage: www.elsevier.com/locate/gene
A nonsense mutation in the Xeroderma pigmentosum complementation group F (XPF) gene is associated with gastric carcinogenesis Zhong-Hua Wei 1, Wen-Huan Guo 1, Jun Wu, Wen-Hao Suo, Guo-Hui Fu ⁎ Pathology Center, Shanghai First People's Hospital / Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, PR China
a r t i c l e
i n f o
Article history: Accepted 25 December 2013 Available online 8 January 2014 Keywords: XPF SNP Mutation Gastric cancer Ubiquitination
a b s t r a c t XPF/ERCC1 endonuclease is required for DNA lesion repair. To assess effects of a C2169A nonsense mutation in XPF at position 2169 in gastric cancer tissues and cell lines, genomic DNA was extracted from blood samples of 488 cancer patients and 64 gastric tumors. The mutation was mapped using a TaqMan MGB probe. In addition, gastric cancer cell lines were transfected with mutated XPF to explore XPF/ERCC1 interaction, XPF degradation, and DNA repair by a comet assay. The C2169A mutation was not detected in 488 samples of blood genomic DNA, yet was found in 32 of 64 gastric cancer tissue samples (50.0%), resulting in a 194C-terminal amino acid loss in XPF protein and lower expression. Laser micro-dissection confirmed that this point mutation was not present in surrounding normal tissues from the same patients. The truncated form of XPF (tXPF) impaired interaction with ERCC1, was rapidly degraded via ubiquitination, and resulted in reduced DNA repair. In gastric cancers, the mutation was monoallelic, indicating that XPF is a haplo-insufficient DNA repair gene. As the C2169A mutation is closely associated with gastric carcinogenesis in the Chinese population, our findings shine light on it as a therapeutic target for early diagnosis and treatment of gastric cancer. © 2013 Published by Elsevier B.V.
1. Introduction Exposure to environmental DNA damaging agents impairs DNA integrity, leading to carcinogenesis (Ciccia et al., 2008; Hoeijmakers, 2001; Klarer and McGregor, 2011; Reddy and Vasquez, 2005; Woods, 1998). Fortunately, intrinsic defense systems have evolved to overcome these damages through a number of DNA repair pathways, including nucleotide excision repair (NER), base excision repair (BER), and mismatch repair (MMR) (Diderich et al., 2011; Dip et al., 2004; Fagbemi et al., 2011; Gregg et al., 2011). The BER system affects a wide range of nucleotide modifications that subtly alter DNA structure (Fortini et al., 2003; Krokan et al., 2000). The MMR functions to correct replication mistakes like single base–base mismatches and small insertion/deletion loops during erroneous base incorporation of DNA polymerases in replication or recombination (Jiricny, 2006). The NER pathway is crucial to maintaining cellular DNA integrity. The gene mutation undermining the NER pathway is either inherited from the germline or arises somatically, leading to genomic instability and elevated cancer risk (Beckman and Abbreviations: XPF, Xeroderma pigmentosum complementation group F; SNP, single nucleotide polymorphism; ERCC1, excision repair cross-complimentary group 1; NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; MTHFR, methylenetetrahydrofolate reductase. ⁎ Corresponding author at: Pathology Center, Shanghai First People's Hospital, Faculty of Basic Medicine, Shanghai Jiao Tong University School of Medicine, No. 280, South Chong-Qing Road, Shanghai 200025, PR China. Tel.: +86 21 63846590x776601. E-mail address: [email protected] (G.-H. Fu). 1 These authors contributed equally to this work. 0378-1119/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.gene.2013.12.061
Loeb, 2005; Diderich et al., 2011; Hyka-Nouspikel et al., 2012; Lagerwerf et al., 2011; Yeh et al., 2012). Many genes are involved in gastric carcinogenesis and give rise to various tumor types through DNA mutations, polymorphisms, and/or gene–environment interactions (Knudson, 2001; Lopez-Lazaro, 2010). Inherited or somatic mutations in E-cadherin genes are present in 30– 50% of gastric cancer cells (Fitzgerald et al., 2010). Additionally, polymorphisms, the interleukin 1β, interferon-γ chain 1, and methylenetetrahydrofolate reductase (MTHFR) genes, are believed to be oncogenic (Figueiredo et al., 2002; Thye et al., 2003; Zintzaras, 2006). Environmental factors, such as Helicobacter pylori infections, also influence these polymorphisms (Figueiredo et al., 2002; Thye et al., 2003). The Xeroderma pigmentosum complementation group F protein (XPF, also known as ERCC4) is 916 amino acids long and includes an excision repair cross-complimentary group 1 (ERCC1) binding domain (Choi et al., 2005; Masuyama et al., 2005; Tripsianes et al., 2005). XPF plays a key role in NER (de Laat et al., 1999; Volker et al., 2001), directly interacting with ERCC1 to remove inter-strand cross-links in damaged DNA (Fagbemi et al., 2011; Gregg et al., 2011). Impaired function of these two proteins and defective interplay between them are considered potential causes of carcinogenesis (Kirschner and Melton, 2010). XPF-deficient patients are at increased risk for skin cancer, accelerated aging, or cerebrooculo-facio-skeletal syndrome (Gregg et al., 2011; Suzumura and Arisaka, 2010). Recently, studies disclosed a correlation between XPF mutations and primary tumors (Wang et al., 2010). XPF mutations, however, remain elusive in gastric cancer development (Bevan and Houlston, 1999; Chun and Ford, 2012). We assessed the
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effects of the rs2020959CNA SNP at position 2169 of XPF on gastric carcinoma epidemiology and carcinogenic machinery.
Table 1 Analysis of genotype distribution between different tumors and peripheral blood. Test group
2. Materials and methods 2.1. Ethics statement
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Blood Gastric cancer
Genotype
p-value
CC (%)
CA (%)
AA (%)
Total
488 (100.00) 32 (50.00)
0 (0.00) 32 (50.00)
0 (0.00) 0 (0.00)
488 64
b0.001
p-values were obtained from Fisher's exact test.
This study was performed according to the guidelines approved by the Ethics Committees of Shanghai Jiao Tong University. Blood and tissue samples were obtained with informed consent from patients, and all patient data was analyzed anonymously.
2.2. Sample collection A total of 682 patients of Han nationality were investigated in this study. Blood samples from 488 Chinese cancer patients hospitalized at the Jiang-Xi provincial Tumor Hospital between February 1 and 15, 2012 were included. Formalin-fixed paraffin-embedded gastric cancer specimens were obtained from the Department of Pathology of Shanghai Jiao Tong University Affiliated Sixth People's Hospital between January 2010 and December 2012. The 64 cases of gastric cancer specimens for genotyping included 27 cases of tubular adenocarcinoma, 18 cases of papillary adenocarcinoma, 9 cases of mucinous adenocarcinoma, and 10 cases of poorly differentiated carcinoma among which 25 cases were in early stage and 39 cases were in advanced stage. And the 130 cases of gastric cancer specimens for immunohistochemistry consisted of 51 cases of tubular adenocarcinoma, 37 cases of papillary adenocarcinoma, 15 cases of mucinous adenocarcinoma, and 27 cases of poorly differentiated carcinoma among which 47 cases were in early stage and 83 cases were in advanced stage.
2.5. Construction of expression vector PCR products of full length XPF and ERCC1 genes were separated by agarose gel electrophoresis after DNA amplification and ligated to pCDNA3.1 + (Takara Bio, Otsu, Japan) to generate expression vectors pCDNA-XPF and pCDNA-ERCC1. Additionally, XPF PCR products were ligated to pEGFP-C1 to generate pEGFP-XPF. The pEGFP-tXPF vector was generated by site-directed mutagenesis on the pEGFP-XPF backbone using the Fast Mutagenesis System kit (Beijing TransGene Biotech Co., Beijing, China). All constructs were amplified in Escherichia coli TOP10 cells (Tiangen Biotech Co., Ltd., Beijing, China), purified using the Plasmid Maxiprept kit (Axygen, Corning, Tewkesbury, MA, USA), sequenced, and stored at −20 °C for transient transfection. 2.6. Immunohistochemistry Histological sections (4 μm) were dewaxed with xylene and rehydrated in ethanol. Deparaffinized slides were incubated at 98 °C in 10 mmol/l citrate buffer at pH 6.0 for 30 min. Endogenous peroxidase was blocked with 0.05% (v/v) hydrogen peroxide (H2O2) in PBS.
2.3. Selection and genotyping of the XPF gene We conducted a bioinformatic analysis of 954 previously reported SNPs in the general population as listed in the NCBI SNP databank (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?showRare=on& chooseRs=all&go=Go&locusId=2072). The PFS database was utilized as a complementary source to verify the results obtained from the NCBI database (http://pfs.nus.edu.sg/(S(svpmljuq4h5ped1mbc1qhu5y))/ FuncDetail_V1.aspx?Func=NCBI_Gene_LinkOut&FuncAddInfo=2072). The rs2020959C N A (NM_005236.2: c. 2169C N A) SNP, considered a nonsense mutation, was selected for further study. Blood DNA was isolated with the TIANamp Blood DNA Maxi Kit (DP333, Tiangen Biotech Co., LTD., Beijing, China). DNA was extracted from formalin-fixed paraffin embedded tissue samples using the TIAN quick FFPE DNA Kit (DP330, Tiangen Biotech Co., LTD., Beijing, China). Both gastric cancer and surrounding normal tissues were obtained by laser capture microdissection (LCM) followed by DNA extraction using the Arcturus® PicoPure® DNA Extraction Kit (KIT0103, Applied Biosystems, Foster City, CA, USA) according to manufacturer's instructions. The rs2020959 SNP was genotyped via qPCR with TaqMan MGB probes and primers (C_11189219_10) for XPF/rs2020959. 17.4% of tissue samples were reassayed to verify results and reproducibility, and 100% concordance was confirmed.
2.4. Cell culture Human gastric cancer AGS, SGC7901, and MKN45 cell lines, human embryonic kidney 293 T (HEK293T) cells, and Chinese Hamster Ovary (CHO) K1 cells were purchased from Shanghai cell bank (Chinese academy of Sciences). Cells were cultured and grown in DMEM medium at 37 °C in 5% carbon dioxide and supplemented with penicillin/ streptomycin and 10% fetal bovine serum (FBS).
Fig. 1. Western blot analysis of XPF expression levels in three human gastric cancer cell lines and in HEK293T cells. (A) Representative XPF western blot image in four cell lines. XPF and tXPF are as indicated in the top panel. tXPF was not detected in HEK293T cells, but in gastric cancer MKN45, SGC 7901, and AGS cell lines. (B) Quantification of XPF and tXPF protein after normalized to β-actin by using Image J software. Data presented as mean ± standard deviation (SD).
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Slides were incubated with rabbit anti-XPF polyclonal antibody (Santa Cruz, CA, USA) overnight at 4 °C. Immunostaining was performed with the UltraSenstive™ S-P kit (MaiXin, Fuzhou, China). Staining optimization, evaluation, and analysis were performed by two scientists (Zhong-Hua Wei and Wen-Huan Guo). A four-point scale method measured the percentage of positively stained cells as described previously (b5% = 0; ≥5% to b20% = + 1; ≥ 20% to 50% = +2; ≥50% = +3). Cases with 0 and + 1 staining scores were in the low expression group, and +2 and +3 staining scores were in the high group.
2.7. Transient transfection and immunoprecipitation HEK293T cells were seeded in 6-well plates at a 1 × 105 cells per well density and grown for 16 h prior to transfection. Plasmid transfections were performed with X-TREME GENE HP DNA Transfection Reagent (Roche Molecular Systems, CA, USA). Cells were either cotransfected with HA-ub expression vectors or treated with proteasome inhibitor MG132 (5 μM) for 12 h, 24 h and 48 h. After transfection, cells were lysed in buffer B (pH 8.0, 50 mM Tris–HCl, 150 mM NaCl, 1% Triton-X 100, 100 μg/ml PMSF). Clarified lysates (2 mg) were incubated with either 20 μl protein A/G PLUS Agarose (Santa Cruz, CA, USA) or with anti-ERCC1 antiserum (Santa Cruz, CA, USA) for 2 h at 4 °C. After extensive washing with Buffer B, immunoprecipitated proteins were separated by 8% SDS-PAGE. The following antibodies were used for immunoblotting: anti-GFP (Santa Cruz, CA, USA), anti-ERCC1 (Santa Cruz,
CA, USA), anti-HA (Santa Cruz, CA, USA) and anti-XPF (GeneTex, CA, USA). 2.8. Western blot MKN45, SGC7901, AGS, and HEK293T cell lines were harvested and pelleted by centrifugation. Proteins were extracted on ice for 20 min in lysis buffer (pH8.0, 50 mM Tris–HCl, 150 mM NaCl, 1% Triton-X 100, 100 μg/ml PMSF). Protein concentrations were determined by the Quick Start™ Bradford Protein Assay Kit 2 (Bio-Rad). Equivalent amounts of total protein were separated by 8% SDS-PAGE and transferred onto nitrocellulose membranes (Protran™ Whatman Inc., USA). After blocking with 5% non-fat milk, membranes were incubated overnight at 4 °C with monoclonal primary antibodies against XPF (GeneTex, CA, USA) or β-actin (Sigma, MO, USA). After washes in PBS, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma, MO, USA) and visualized using an enhanced chemiluminescence system (ProteinSimple, CA, USA). Quantification of each band was performed using Image J (National Institutes of Health, MD, USA). XPF levels were normalized to β-actin. 2.9. Comet assay The comet assay was performed according to previous literature (Arora et al., 2010; Usanova et al., 2010). Briefly, cells were incubated
Fig. 2. Immunohistochemistry analysis of XPF in gastric cancer and surrounding normal tissues. (A), (B), and (C). High expression of XPF in gastric mucosa in surrounding normal tissues and low expression in different stage of cancer tissues. Red arrow, well differentiated cancer tissue; green arrow, moderately differentiated cancer tissue; yellow arrow, poorly differentiated cancer tissue. Scale bar = 50 μm. (D) Statistical diagram depicting XPF expression levels in 130 cases of gastric cancer and surrounding normal tissues. p-values obtained from chi-square test.
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with cisplatin (15 μg/ml) for 2 h and further treated with 100 μM H2O2 for 15 min to induce DNA strand breaks. After harvest, 25 μl of the cell suspension (2 × 105 cells/ml) was mixed with 75 μl of low melting point (LMP) agarose at 37 °C. The mixture was transferred onto a microscope slide pre-coated with 1% normal melting point (NMP) agarose, covered, and placed at 4 °C until agarose solidified. The coverslip was removed and the slide was placed in chilled lysis solution at 4 °C for 6 h. Slides were then transferred into an electrophoresis chamber containing ice-cold alkaline solution for 20 min. The single cell gel electrophoresis assay (comet) lasted 20 min (25 V, 300 mA). After, the slide was neutralized three times for 10 min each, stained with DAPI (2 μg/ml), and visualized under a fluorescence microscope (200×, Leica DM2000). Fifty cells per sample were analyzed using the CASP Image Analysis software (CASPLab, Wroclaw, Poland). The value of the tail moment was used to determine the decrease in tail moment (DTM) according to the fomula: DTM % = [1 − (TMpht − TMcu)/(TMcht − TMcu)] × 100%, where the TMpht is the mean tail moment of both cisplatin and H2O2 treated samples, TMcu stands for the mean tail moment of the untreated control sample, and the TMcht is the mean tail moment of H2O2 treated control sample. The DTM reflected the repair function by the XPF protein. 2.10. Statistical analysis XPF expression in gastric cancer and surrounding normal tissues was analyzed by chi-square tests. Distributions of the allele and genotype
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frequencies were calculated by Fisher's exact test. Statistical significance was evaluated by one-way analysis of variance (ANOVA) with Student– Newman–Keuls (SNK) test for post hoc analysis. All analyses were performed in SPSS v16.0 (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. The C2169A mutation in XPF in the Chinese population To investigate the C N A nonsense mutation in the Chinese population and to understand the role of tXPF in carcinogenesis, blood genomic DNA was extracted from 488 Chinese cancer patients to identify mutations by the TaqMan SNP allelic discrimination assay. Surprisingly, we did not detect the C N A in any of the 488 Chinese cancer patients' blood samples (Table 1), suggesting that this SNP is uncommon in Chinese patients. The heterozygous C N A mutation position 2169 of the XPF gene was detected in 32/64 of gastric cancer tissues that included 25 early stage and 39 advanced stage samples. Specifically, 14 samples (56%) were from early stage and 18 samples (46.2%) were from advanced stage cancers, for an overall detection rate was 50%. We thus highlight this somatic mutation in pathogenesis of gastric cancer. Five gastric cancer tissues were compared to their respective normal counterparts obtained by laser capture microdissection (LCM). Wildtype copies of XPF were identified in all five tissue samples, while the C2169A mutation was detected in 2 gastric cancer specimens. Interestingly, the same mutation was found in three gastric cancer cell lines tested: AGS, MKN45, and SGC7901. The heterozygous C N A
Fig. 3. Interaction between XPF and ERCC1 and the effect of the rs2020959 SNP on the XPF/ERCC1 complex. (A) Schematic representation of the XPF and ERCC1 interaction domain. The normal nucleotide and corresponding amino acid sequences distributed around the mutation site of XPF are presented. (B) Schematic representation of the nonsense mutation at position 2169 in XPF (upper panel). The mutation introduces a premature stop codon, resulting in tXPF expression (lower left panel). pEGFP-XPF or pEGFP-tXPF expression constructs were transfected into gastric cancer AGS cells for 24 h, and XPF or tXPF expression were detected by western blot, using anti-GFP antibody. Arrows indicate XPF (upper band) and tXPF (lower band) (lower right panel).
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mutation in XPF introduces a premature stop codon, so these cell lines express both a tXPF protein and wild-type variant. XPF expression was reduced in these cells compared to HEK293T cells that express only wild-type XPF (Fig. 1). The C N A mutation is not an SNP, but a cancer-related nonsense mutation common in tumors of Chinese gastric cancer patients. We next investigated XPF protein expression in 130 gastric cancer specimens compared to surrounding normal tissues by immunohistochemistry. High XPF expression was observed in 113 normal tissues (86.9%) and in only 24 cancer tissues (18.5%) (P b 0.001), suggesting the instability of native XPF in gastric cancer tissues (Fig. 2).
3.2. Prediction and characterization of tXPF expression in AGS gastric cancer cells According to the dbSNP database, a total of 954 SNPs in the XPF gene have been documented in humans (http://www.ncbi.nlm.nih. gov/projects/SNP/snp_ref.cgi?showRare=on&chooseRs=all&go= Go&locusId=2072). We report that rs2020959C N A SNP is a nonsense mutation resulting in tXPF expression that lacks 194C-terminal amino
Fig. 4. Determination of tXPF/ERCC1 interaction and the tXPF protein stability. (A) XPF or tXPF interact with ERCC1 in vitro. GFP tagged XPF or tXPF expression construct and ERCC1 vector were co-transfected into AGS cells for 24 h. Cells were lysed and equal amounts of protein were immunoprecipitated with anti-ERCC1 antibody and analyzed by Western blotting. Normal mouse IgG served as a negative control. The XPF/ERCC1 interaction was used as a positive control (lane 2). XPF and tXPF were visualized using specific anti-GFP antibody. WCL, whole cell lysis. (B) and (C) Comparison of XPF and tXPF protein stabilities. XPF or tXPF expression vectors were transfected into AGS cells with ERCC1, and expression levels were detected by anti-GFP antibody. (B) Protein quantification was performed using the Image J software. Data presented as mean ± SD of three replicate experiments.
acids (Fig. 3A). We confirmed this by cloning wild-type XPF and the mutant gene into a pEGFP-C1 vector to generate two constructs: pEGFP-XPF and pEGFP-tXPF. These plasmids were independently transfected into AGS cells for 24 h. Immunoblotting confirmed that C2169A results in the truncated tXPF (Fig. 3B). 3.3. Impaired interaction between tXPF and ERCC1 and degradation via the ubiquitin pathway Human XPF interacts with ERCC1 through its 265 amino acid residues (AA559 to AA823) (Rahn et al., 2010). To characterize tXPF's affinity to ERCC1, either XPF or tXPF was co-expressed with ERCC1 in AGS cells for 24 h. Interplay between ERCC1 and XPF or tXPF was detected by immunoprecipitation and determined by western blot. Immunostaining results demonstrated that binding affinity of tXPF to ERCC1 was reduced compared to XPF (Fig. 4A). To evaluate tXPF's stability, either XPF or tXPF constructs were cotransfected with ERCC1 in AGS cells for 1, 2, and 3 days, respectively. Western blotting analysis showed the XPF protein remained stable for 3 days. Contrastingly, tXPF levels significantly decreased 3 days posttransfection (Figs. 4B and C). We speculated that the XPF protein level depended on integrity, and deletion of the 194C-terminal amino acids compromised this. To test this, pCDNA3.1-HAtXPF was transfected into AGS cells, and MG132, a proteasome inhibitor, was added the second day. Cells were harvested 3 days after transfection for western
Fig. 5. XPF and tXPF are degraded through the ubiquitin pathway. (A) AGS cells were transfected with pCDNA-HAtXPF plasmids, and treated with MG132 (5 μmol). The tXPFHA expression was detected by western blot using anti-HA antibody. (B) AGS cells were transfected with plasmids as indicated, and then treated with MG132 (5 μmol) or had no treatment for 12 h. Whole cell lysis (WCL) was subject to western blot assay, and XPF and tXPF were detected by anti-GFP antibody. (C) Immunoprecipitation was performed using anti-GFP antibody with WCL protein. tXPF and XPF were detected using anti-HA antibody.
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blot analysis. tXPF remarkably accumulated after MG132 treatment (Fig. 5A), implicating ubiquitination in tXPF degradation. Further, XPF or tXPF was co-expressed with HA ubiquitin in AGS cells and protein levels were determined by anti-GFP or anti-HA antibodies (Figs. 5B and C). Immunoprecipitation with anti-GFP antibody revealed polyubiquitin chains found conjugated to XPF and tXPF after MG132 treatment (Fig. 5C). Notably, accumulation of polyubiquitinated tXPF was more significant than XPF, indicating that tXPF is most likely more vulnerable to proteasomal degradation. 3.4. The C2169A XPF mutation significantly impairs DNA repair capacity XPF is essential for DNA repair. We assessed whether its truncation affects DNA repair capacity by the comet assay (Figs. 6A and B). The pCDNA-ERCC1 construct was co-transfected into CHO-K1 cells (absent of the endogenous XPF (Wood and Burki, 1982)) with either pEGFP-XPF or pEGFP-tXPF. After 24 h, cells were treated with cisplatin for 2 h and further treated with H2O2 for 15 min to induce intra- and inter-strand DNA crosslinks. DNA crosslink repair was evaluated at 7 h and 24 h, respectively, after the cisplatin treatment. In pEGFP-XPF/pCDNA-ERCC1 co-transfected cells, DNA crosslink level was reduced by nearly 50% at 24 h. In contrast, no reduction was detected in the DNA crosslink level after the pEGFP-tXPF/pCDNA-ERCC1 transfection (Fig. 6C). Collectively, this data demonstrates impaired DNA repair from tXPF expression. 4. Discussion Gastric cancer is the third most common cancer in China (Hu et al., 2007). Although its etiology remains unclear, genetic aberrations play an important role in gastric carcinogenesis (Abnet et al., 2010). We revealed that a nonsense mutation in XPF at position 2169 tremendously attenuates DNA repair and stimulates degradation. Intriguingly, the mutation was not found in peripheral blood samples or in normal tissues of cancer patients. This mutation was specific to gastric cancer tissues, implicating it as a putative biomarker. Five major pathways orchestrate DNA repair in mammalian cells involving over 130 genes. The NER pathway is most important (Friedberg, 2001; Wood et al., 2001) and mediated by nine major participant proteins including ERCC1. ERCC1 heterodimerizes with XPF, so compromising ERCC1/XPF dimerization would undermine NER (Kirschner and Melton, 2010). We found that deleting the C-terminal portion of XPF impaired the XPF/ERCC1 dimerization and attenuated the repair of cisplatin-induced DNA damages. Additionally, the truncated protein was more susceptible to ubiquitinmediated degradation. According to epidemiological databases, an “A” and not a “C” at position 2169 in XPF was identified in 4.1% and 1.2% of the Mexican and Japanese populations respectively, but not in Chinese or European populations (NCBI, 2013). In accordance with these databases, we did not detect the SNP in 488 blood samples obtained from Chinese patients. Conversely, this mutation was observed in 50% of gastric cancer tissues in Chinese patients, suggesting that this nonsense mutation is closely correlated with gastric carcinogenesis. It is well accepted that cancers are complex diseases and that one sporadic genetic variant is usually not sufficient to increase risk. It is more likely that disrupted DNA repair machinery first leads to an accumulation of mutated that synergize into malignance (Knudson, 2001; Lopez-Lazaro, 2010). Although the exact etiology of gastric carcinoma remains obscure, evidences from other cancer research suggests relevancy between abnormality of DNA repair nuclease ERCC1/XPF and gastric carcinogenesis. Matoka et al. utilized tissue recombination models to investigate potential roles of XPF/ERCC1 and NER in prostate cancer (Matoka et al., 2012), observing that XPF/ERCC1 contributed to protection against prostate carcinogenesis, while pathway disruption predisposes prostate epithelial cells for transformation (Matoka et al., 2012). Moreover, Facista et al.
Fig. 6. The C N A nonsense mutation affected DNA crosslink repair capacity. CHO-K1 cells were co-transfected with pCDNA-ERCC1 and pEGFP-XPF or pEGFP-tXPF constructs, followed by cisplatin treatment for 2 h and then 100 μM H2O2 for 15 min. (A) and (B) show fluorescence images of alkaline comet assays with indicated protein. Slides were stained with DAPI (2 μg/ml) (blue) and visualized under a fluorescence microscope (200×). (C) DNA cross-link levels were measured 7 h and 24 h after treatment and reduced tail moments were calculated by the formula described in Materials and methods. Values were presented as mean ± SD of triplicate samples.
revealed that decreased expression of ERCC1 and XPF happens early in colon cancer progression (Facista et al., 2012). A previous study surrounding the Eastern Chinese population uncovered 3 polymorphisms in the ERCC1 gene that associated with increased gastric cancer risk. The consequence of XPF polymorphisms was poorly defined, as no correlation was found between the rs2276466C N G, rs6498486A N C, and gastric cancer (He et al., 2012). Meta-analysis results offered clues on the interrelation of SNPs in XPF and increased cancer risk (Vineis et al., 2009). In our study, the rs2020959C N A mutation in XPF introduces a premature stop codon in cancer tissues, implicating XPF as an oncogene in early steps of transformation. Since the rs2020959 SNP perturbs a critical aspect of XPF function, we refer to it as a mutation rather than an SNP. This mutation is predominately monoallelic, implicating it as a haplo-insufficient DNA repair gene. To best of our knowledge, the present study is the first to reveal that C2169A in XPF is associated with an increased risk of gastric cancer and may be a key determinant for malignant transformation of normal gastric
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cells. Intervention to restore the DNA repair system to normal when tumors express tXPF may merit further exploration. Conflict of interest There is no conflict of interest. Acknowledgments This work was supported in part by the National Natural Science Foundation of China (NO30570697; NO30770960) and by the Shanghai Municipal Science and Technology Commission Research Project (NO11JC1406700). We would like to thank Xi-Dai Long, Chun-Yin Luo, Shu-Guang Liu, and Yang-Mei Shen for collecting blood and tissue samples. References Abnet, C.C., et al., 2010. A shared susceptibility locus in PLCE1 at 10q23 for gastric adenocarcinoma and esophageal squamous cell carcinoma. Nat. Genet. 42 (9), 764–767. Arora, S., et al., 2010. Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells. DNA Repair (Amst) 9 (7), 745–753. Beckman, R.A., Loeb, L.A., 2005. Genetic instability in cancer: theory and experiment. Semin. Cancer Biol. 15 (6), 423–435. Bevan, S., Houlston, R.S., 1999. Genetic predisposition to gastric cancer. QJM 92 (1), 5–10. Choi, Y.J., et al., 2005. Biophysical characterization of the interaction domains and mapping of the contact residues in the XPF–ERCC1 complex. J. Biol. Chem. 280 (31), 28644–28652. Chun, N., Ford, J.M., 2012. Genetic testing by cancer site: stomach. Cancer J. 18 (4), 355–363. Ciccia, A., McDonald, N., West, S.C., 2008. Structural and functional relationships of the XPF/MUS81 family of proteins. Annu. Rev. Biochem. 77, 259–287. de Laat, W.L., Jaspers, N.G., Hoeijmakers, J.H., 1999. Molecular mechanism of nucleotide excision repair. Genes Dev. 13 (7), 768–785. Diderich, K., Alanazi, M., Hoeijmakers, J.H., 2011. Premature aging and cancer in nucleotide excision repair-disorders. DNA Repair (Amst) 10 (7), 772–780. Dip, R., Camenisch, U., Naegeli, H., 2004. Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair. DNA Repair (Amst) 3 (11), 1409–1423. Facista, A., et al., 2012. Deficient expression of DNA repair enzymes in early progression to sporadic colon cancer. Genome Integr. 3 (1), 3. Fagbemi, A.F., Orelli, B., Scharer, O.D., 2011. Regulation of endonuclease activity in human nucleotide excision repair. DNA Repair (Amst) 10 (7), 722–729. Figueiredo, C., et al., 2002. Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma. J. Natl. Cancer Inst. 94 (22), 1680–1687. Fitzgerald, R.C., et al., 2010. Hereditary diffuse gastric cancer: updated consensus guidelines for clinical management and directions for future research. J. Med. Genet. 47 (7), 436–444. Fortini, P., et al., 2003. The base excision repair: mechanisms and its relevance for cancer susceptibility. Biochimie 85 (11), 1053–1071. Friedberg, E.C., 2001. How nucleotide excision repair protects against cancer. Nat. Rev. Cancer 1 (1), 22–33. Gregg, S.Q., Robinson, A.R., Niedernhofer, L.J., 2011. Physiological consequences of defects in ERCC1-XPF DNA repair endonuclease. DNA Repair (Amst) 10 (7), 781–791.
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