Allele-specific up-regulation of FGFR2 increases susceptibility to breast cancer

Abstract

The recent whole-genome scan for breast cancer has revealed the FGFR2 (fibroblast growth factor receptor 2) gene as a locus associated with a small, but highly significant, increase in the risk of developing breast cancer. Using fine-scale genetic mapping of the region, it has been possible to narrow the causative locus to a haplotype of eight strongly linked single nucleotide polymorphisms (SNPs) spanning a region of 7.5 kilobases (kb) in the second intron of the FGFR2 gene. Here we describe a functional analysis to define the causative SNP, and we propose a model for a disease mechanism. Using gene expression microarray data, we observed a trend of increased FGFR2 expression in the rare homozygotes. This trend was confirmed using real-time (RT) PCR, with the difference between the rare and the common homozygotes yielding a Wilcox p-value of 0.028. To elucidate which SNPs might be responsible for this difference, we examined protein-DNA interactions for the eight most strongly disease-associated SNPs in different breast cell lines. We identify two cis-regulatory SNPs that alter binding affinity for transcription factors Oct-1/Runx2 and C/EBPbeta, and we demonstrate that both sites are occupied in vivo. In transient transfection experiments, the two SNPs can synergize giving rise to increased FGFR2 expression. We propose a model in which the Oct-1/Runx2 and C/EBPbeta binding sites in the disease-associated allele are able to lead to an increase in FGFR2 gene expression, thereby increasing the propensity for tumour formation.

Figure 1. Diagram of the FGFR2 Gene

Genetic linkage is taken from HapMap, and the positions of the eight candidate SNPs (red lines) within intron 2 are indicated. The two SNPs for which data are presented in this study are shown in green. Red circle: original tagging SNP rs2981582.

Figure 2. Correlation of FGFR2 Expression in Breast Tumours with Genotype

Quantitative RT-PCR was carried out using a probe targeting the 3' UTR of FGFR2.

Figure 3. Protein–DNA Interactions at FGFR2–33 and FGFR2–13 In Vitro and In Vivo

EMSAs on (A) FGFR2–33 and (B) FGFR-13 minor (m) and common (c) alleles, using 5 μg (FGFR2–33) and 2 μg (FGFR2–13) of HCC1954 nuclear extracts. Competitor oligonucleotides (minor, common, and ER as negative control) and antisera are indicated above each lane. (C) Alignment of the sequence around FGFR2–33 with binding site of C/EBPβ in the IL-6 promoter [15] and of FGFR2–13 with the Oct/Runx site in the β-casein gene [18]. The SNP is shown in red and the allele binding the transcription factor is shown. (D) ChIP assays for FGFR2–13 and FGFR2–33. Enrichment for the minor (HCC70^–/–) and the common (T47D^+/+) genotype is given relative to a negative control (TRXR2, located on 22q11.2) after normalisation against rabbit IgG.

Figure 4. Transcriptional Activation by the Minor and Common Alleles of Oct-1/Runx2 and C/EBPβ Binding Sites of FGFR2

(A) Diagram of the concatemerised binding sites cloned into pGL3Enh. (B) Luciferase assays in HCC70 cells. Results are given as fold increase over pEnh activity. CMV-β-gal served as transfection control. The binding sites are indicated beneath each data point, with [Oct/Runx] and [C-EBPβ] being trimerized, while [C/O/R] contained only a single binding site for C/EBPβ, Oct-1, and Runx2. The asterisk denotes p-values <0.05 in a Student's t-test comparing the common versus the minor allele of each site.