A variant of the Cockayne syndrome B gene ERCC6 confers risk of lung cancer

Abstract

Cockayne syndrome B protein (ERCC6) plays an essential role in DNA repair. However, the Cockayne syndrome caused by the ERCC6 defect has not been linked to cancer predisposition; likely due to the fact that cells with severe disruption of the ERCC6 function are sensitive to lesion-induced apoptosis, thus reducing the chance of tumorigenesis. The biological function and cancer susceptibility of a common variant rs3793784:C>G (c.-6530C>G) in the ERCC6 was examined. We show that the c.-6530C allele has lower binding affinity of Sp1 by EMSA and displays a lower transcriptional activity in vitro and in vivo. We then examined the contribution of this polymorphism to the risk of lung cancer in a case-control study with 1,000 cases and 1,000 controls. The case-control analysis revealed a 1.76-fold (P= x 10(-9)) excess risk of developing lung cancer for the c.-6530CC carriers compared with noncarriers. The c.-6530CC interacts with smoking to intensify lung cancer risk, with the odds ratio (OR)=9 for developing lung cancer among heavy smokers. Our data constituted strong evidence that ERCC6 rs3793784:C>G alters its transcriptional activity and may confer personalized susceptibility to lung cancer.

Figure 1

Scheme of the 5′-flanking region of human ERCC6 and promoter activity of fragments flanking the rs3793784:C>G SNP in ERCC6. A:ERCC6 5′-untranslated region identified in the NCBI database. Boxes E1 and E2 represent exon 1 and exon 2. The SNP is located at c. −6530, which is 471 bp upstream the transcription start site located at c. −6061. The ERCC6 genomic reference sequence with GenBank accession no. AY204752.1 was used with the A of the translation initiation codon as nucleotide+1. Other numbers represent primer positions for cloning reporter constructs. B: Fragments F1–F4 were PCR-amplified to make reporter constructs, and their lengths and positions are shown in parentheses. C: Fragments F1–F4 with the C allele were cloned into the pGL3-Basic vector and transfected into H460 cells. Their activities were measured by dual luciferase assays. Results are expressed as fold increases in luciferase activity relative to the empty pGL3-Basic vector.

Figure 2

Comparison of promoter activities between constructs with different allele. Fragments F1–F3 with the C or G allele were cloned into the pGL3-Basic vector and transfected into H460 cells. The lengths and positions of F1–F3 were the same as those in Fig.1. Their activities were measured by dual luciferase assays and the results are expressed as fold increases in luciferase activity relative to the empty pGL3-Basic vector. *Constructs with the G allele had higher activities compared with those with the C allele (P<0.01).

Figure 3

UV irradiation impacts on allele-specific promoter activity. A: Comparison of promoter activities between C allele-containing construct and G allele-containing construct in A549 cells with or without UV irradiation. B: Comparison of promoter activities between C allele-containing construct and G allele-containing construct in 16HBE cells with or without UV irradiation. #After UV irradiation, constructs containing the −6530G allele induced higher luciferase expressions (P<0.01) in two cell lines.

Figure 4

Electrophoretic mobility shift assays using infrared dye IRD™-labeled probes and nuclear extracts from H460 cells. A: Nuclear extracts were incubated with infrared dye IRD™-labeled C allele probe or G allele probe. The DNA-protein complex (indicated by arrow A) showed density deference between the C and G allele. The competition assay was done by the addition of 25-, 100-, or 250-fold molar excess of unlabeled probes to the incubation mixtures. B: A faint supershift band was detected when 200 and 400 ng anti-Sp1 antibody was incubated with the nuclear extracts prior to the addition of the infrared dye IRD™-labeled G probe at room temperature. C: DNA-protein complex formed by incubation of IRD™-labeled C or G allele probe with nuclear extracts had the same pattern as that formed by incubation of IRD™-labeled Sp1 consensus probe with nuclear extracts. The formation of DNA-protein complex formed by the G or C probe was inhibited by 100-fold molar excess of unlabeled Sp1 probe.

Figure 5

Levels of ERCC6 mRNA expression in lung tissues as a function of ERCC6 genotype. Columns, mean; bars, ±SE normalized to β-actin. Expression level among the CC genotype was significantly lower than that among the CG or GG genotype; *P<0.05.