A novel functional DEC1 promoter polymorphism -249T>C reduces risk of squamous cell carcinoma of the head and neck

Abstract

Human DEC1 (deleted in esophageal cancer 1) gene is located on chromosome 9q, a region frequently deleted in various types of human cancers, including squamous cell carcinoma of the head and neck (SCCHN). However, only one epidemiological study has evaluated the association between DEC1 polymorphisms and cancer risk. In this hospital-based case-control study, four potentially functional single-nucleotide polymorphisms -1628 G>A (rs1591420), -606 T>C [rs4978620, in complete linkage disequilibrium with -249T>C (rs2012775) and -122 G>A(rs2012566)], c.179 C>T p.Ala60Val (rs2269700) and 3' untranslated region-rs3750505 as well as the TP53 tumor suppressor gene codon 72 (Arg72Pro, rs1042522) polymorphism were genotyped in 1111 non-Hispanic Whites SCCHN patients and 1130 age-and sex-matched cancer-free controls. After adjustment for age, sex and smoking and drinking status, the variant -606CC (i.e. -249CC) homozygotes had a significantly reduced SCCHN risk (adjusted odds ratio = 0.71, 95% confidence interval = 0.52-0.99) compared with the -606TT homozygotes. Stratification analyses showed that a reduced risk associated with the -606CC genotype was more pronounced in subgroups of non-smokers, non-drinkers, younger subjects (defined as ≤57 years), carriers of the TP53 Arg/Arg (rs1042522) genotype, patients with oropharyngeal cancer or late-stage SCCHN. Further in silico analysis revealed that the -249 T-to-C change led to a gain of a transcription factor-binding site. Additional functional analysis showed that the -249T-to-C change significantly enhanced transcriptional activity of the DEC1 promoter and the DNA-protein-binding activity. We conclude that the DEC1 promoter -249 T>C (rs2012775) polymorphism is functional, modulating susceptibility to SCCHN among non-Hispanic Whites.

Fig. 1.

Interactions among genetic and environmental factors by CART analysis. The DEC1 −606T>C SNP was classified as having common genotypes (CT + TT) and protective variant (CC); smoking and drinking status was defined as never or ever (former and current) users; Five terminal nodes are highlighted in gray. The numbers in each node may not add up to the total numbers due to the missing information from some subjects. The reference group was the least percentage of cases, and ORs and 95% CIs for each factor combination were obtained from logistic regression models adjusted by age and sex.

Fig. 2.

The DEC1 promoter reporter gene constructs and DEC1-luciferase assays. (A) Schematic presentation of a linealized reporter gene construct containing a 1202 bp fragment of the DEC1 promoter (−1178 to +24 relative to the transcriptional starting site) with both variants at the −606/−249/−122 sites was inserted into the pGL3-basic luciferase expression vector. (B) Luciferase assay for the two DEC1 promoter constructs containing either −249T and −249C alleles, respectively. Both the constructs were sequenced to confirm the orientation and integrity (C). Significant differences between two alleles were indicated by asterisk (*P < 0.01).

Fig. 3.

Nuclear factor binding to the −249 T>C polymorphic region of the DEC1 promoter. (A) The putative Nkx-2.5 TFBS at −249 T and C of the DEC1 promoter was predicted by TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess). (B) EMSA was performed using UM-SCC-22A nuclear extracts with biotin-labeled probes corresponding to both −249 T and C of DEC1 gene (T allele: lanes 1–4 or C allele: lanes 5–8), along with or without competition from unlabeled probes. (C) Biotin-labeled probe containing the T allele at −249 site and UM-SCC-22A nuclear extracts in the presence of Nkx-2.5 antibody (lane 3) or normal IgG (lane 4).