| Background and purposeOxaliplatin-based chemotherapy has been proved to be effective in treating advanced colorectal cancer. However, individual efficacy of oxaliplatin demonstrates significant variations. It is known that individual difference in drug sensitivity is mainly determined by genetic factors such as single nucleotide polymorphisms (SNPs) of genes which related to drug mechanism and drug metabolism. The nucleotide excision repair (NER) is one of the major pathways of DNA repair system and closely related to platinum-based drug resistance. Furthermore, it is the only known mechanism for the removal of bulky, helix-distorting DNA adducts produced by platinum agents in mammalian cells. Although oxaliplatin compounds has became the first line regimen in advanced colorectal cancer treatment, only limited studies reported the relationship between SNPs in NER pathway. And the clinical outcome of platinum-based chemotherapy in CRC and the conclusions of some studies were conflicting. Therefore, it is significant to find a predictor to evaluate the response to oxaliplatin chemotherapy. The excision repair cross complementation group 5 (ERCC5) gene, also known as Xeroderma pigmentosum group G (XPG), is one of the essential DNA repair enzymes of the NER pathway. ERCC5 gene expression has been proved to exist in various tumor cell lines or tissues and its expression level is correlated with the response to platinum-based chemotherapy.The promoter, due to containing numerous transcription factor binding sites, is the central to the regulation of gene transcription. There is a growing body of evidences indicating that genetic variations in this region might affect the transcription of target genes. It was early to make a conclusion but some study implied that SNPs in promoter region might play a functional role in ERCC5 transcription and/or function. We searched the SNPs information of ERCC5 gene in NCBI dbSNP and Ensembl database and found that there were numerous SNPs in gene region including the 5'-UTR which were coincide with the SNPs of this gene in Chinese Han population inquired in Hapmap database.On the basis of the aforementioned data, we hypothesized that some SNPs in ERCC5 promoter might closely relate to gene expression and then affect clinical outcome of oxaliplatin chemotherapy, and it is necessary to reveal the relationship between SNPs of ERCC5 gene and platinum-base chemotherapy.In the study,we investigated the association between the ERCC5 polymorphisms at promoter region in Chinese Han population and the clinical outcome in patients with advanced CRC treated with oxaliplatin-based chemotherapy, then further validated the biological functions of positive associated SNPs, and observed the expression of ERCC5 gene to reveal the relationship of SNP, gene expression and clinical outcome.Methods1. Bioinformatics methods were used to analyze SNPs of ERCC5 promoter region in Chinese Han population.2. The distribution of selected polymorphic sites in patients with colorectal cancer treated with oxaliplatin-based chemotherapy was detected using polymerase chain reaction-ligation detection reaction (PCR-LDR) method. Genotype distribution was analyzed usingχ2 test for Hardy-Weinberg equilibrium. Linkage disequilibrium (LD) was assessed via Linkage disequilibrium analyzer software (LDA). Haplotype frequency was estimated using the PHASE package. Theχ2 or Fisher's exact test was used to summarize the association between response to oxaliplatin and ERCC5 polymorphism. The Kaplan-Meier method was adopted to estimate survival curves, and the log-rank test was used to compare patients'Time to progression (TTP) between genotype or haplotype groups. Univariate and multivariate analyses using Cox regression were used to assess the importance of genotypes with adjustment for clinical features.3. A total of four different haplotype DNA which contain ERCC5 -763A>G and +25A>G sites were prepared by using extension PCR site-directed mutagenesis technology and were cloned into a luciferase expression plasmid (promoterless pGL3-Basic) respectively. Then the different plasmids were transfected into LOVO cells to observe promoter activities. EMSA were used for identifying nuclear protein binding ability between -763A and -763G allele, +25A and +25G allele.4. The mRNA and protein expression of ERCC5 in tumor tissues of colorectal cancer patients with different genotypes were detected by real-time PCR and Western blot respectively.Results1. A total of 6 polymorphic sites in ERCC5 promoter region, -1415C>T (rs2094258), -763A>G (rs2016073), -413C>T (rs943245), +25A>G (rs751402), +202C>T (rs2296147) and +372C>T (rs2296148) were selected for further study.2. Among the 6 polymorphic sites,five (-1415C>T, -763A>G, +25A>G, +202C>T and +372C>T) were identified in our study group. The genotype distribution for each polymorphism was found to be in Hardy-Weinberg equilibrium. No genetic variation was observed at site -413.3. In 105 patients, the objective response rate among patients with -763GG genotype was significantly higher than that with -763AA genotype (60.0% vs. 26.3%, P=0.021) while the objective response rate among patients with +25AA genotype was significantly higher than that with +25GG genotype (56.3% vs. 27.0%, P=0.042). Similarly, the disease control rate among patients with -763GG genotype was significantly higher than that with -763AA genotype (86.7% vs. 52.6%, P=0.028) while the disease control rate among patients with +25AG genotype was significantly higher than that with +25GG genotype (73.1% vs. 51.4%, P=0.035). No significant association was found between the -1415C>T, +202C>T or +372C>T polymorphism and chemotherapy response in our study.4. We conducted linkage disequilibrium analysis among these five SNPs and found that only two SNPs, -763A>G and +25A>G, were in tight linkage disequilibrium (r2=0.73,D'=0.87). Haplotype analysis was conducted in 105 patients and we found that -763A/+25G and -763G/+25A were two major haplotypes. Among them, patients with the -763A/+25G haplotype had a higher risk of non-response (SD+PD) to oxaliplatin chemotherapy compared to those carrying the -763G/+25A haplotype (62.5% vs. 29.7%, OR 2.000, 95% CI 1.110~3.602, P=0.021). Similarly, patients with the -763A/+25G haplotype had a higher risk of disease progression (PD) to oxaliplatin chemotherapy compared to those carrying the -763G/+25A haplotype (66.7% vs. 25.0%, OR 2.148, 95% CI 1.130~4.085, P=0.020).5. The combination of -763A>G and +25A>G polymorphisms were analyzed in 105 patients and we found that there were three major genotype combinations: -763AA+25GG, -763AG+25AG and -763GG+25AA. Among them, individuals with the -763GG+25AA demonstrated higher objective response rates to oxaliplatin chemotherapy compared to those with the -763AA+25GG (61.5% vs. 27.3%, P=0.044). While individuals with the -763GG+25AA or -763AG+25AG demonstrated higher disease control rates to oxaliplatin chemotherapy compared to those with the -763AA+25GG (84.6% vs. 51.5% and 73.3% vs. 51.5% respectively, P=0.049 and P=0.047 respectively).6. The total median TTP of 95 patients was 8 months. The median TTP among patients with the -763AA genotype (36/95 cases, 6 months) was significantly lower than that of other genotypes (48/95 case, 8 months for -763AG genotype and 11/95 cases, 10 months for -763GG genotype, P=0.009). Similarly, the median TTP among patients with the +25GG genotype (35/95 cases, 5 months) was significantly lower than that of other genotypes (48/95 case, 8 months for +25AG genotype and 12/95 cases, 6 months for +25AA genotype, P=0.029). No significant difference was found between genotypes of -1415C>T, +202C>T or +372C>T respectively in our study. In the multivariate model, a significantly higher risk of progression was associated with -763AA genotype and +25GG genotype, and patient with these risk genotypes demonstrated a significantly increasing risk of progression (HR 1.948, 95% CI 1.211~3.134, P=0.006).7. Four haplotype DNA which contain ERCC5 -763A>G and +25A>G sites were cloned into a promoterless pGL3-Basic vector respectively. Dual luciferase reporter assay showed that promoter activities was observed at -910~ +292bp fragment of ERCC5 gene, and the promoter activity was different in each haplotype. The relative luciferase activity of the -763A/+25G haplotype was significantly higher than other three haplotypes (P<0.05).8. EMSA showed that both -763A and -763G allele had nuclear protein binding ability, but the intensity of binding band for the -763A allele was higher than that for -763G allele. However, +25A allele did not show any nuclear protein binding ability while +25G allele did.9. The expression level of ERCC5 mRNA and protein were variant in tumor tissues with different genotype. The expression of ERCC5 mRNA was significantly higher in tumor tissues with -763AG+25GG, -763AA+25GG or -763AA+25AG genotype combination than that in -763GG+25AA genotype combination (P<0.001). However, the expression in tumor tissues with -763AG+25AG genotype combination were significantly lower than that in -763GG+25AA genotype combination (P<0.01). Similarly, the expression of ERCC5 protein was significantly higher in tumor tissues with -763AA+25GG, -763AG+25GG genotype combination than that in -763GG+25AA genotype combination (P<0.05). However, the expression in tumor tissues with -763AG+25AG genotype combination were significantly lower than that in -763GG+25AA genotype combination (P<0.05). Among the three major genotype combinations, the expression of ERCC5 mRNA and protein in -763AA+25GG was the highest, in -763GG+25AA the second, and in -763AG+25AG the lowest (P<0.05, respectively).Conclusion1. The -763A>G and +25A>G polymorphisms of ERCC5 gene are associated with clinical outcome of oxaliplatin-based chemotherapy in Chinese patients with advanced colorectal cancer.2. The haplotype -763A/+25G can significantly increase the promoter activity. The expression level of ERCC5 mRNA and protein is high in tumor tissues of patients with -763AA+25GG genotype combination.3. The nuclear protein binding ability between -763A and -763G allele,+25A and +25G allele is different. -763A>G and +25A>G polymorphisms may alter the binding affinity with nuclear protein.4. The -763A allele and +25G allele might increase the binding affinity with transcription factors and gene transcription activity,thus increase expression level of ERCC5 in related genotypes and lead to poor response to platinum-based chemotherapy.
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