Evidence that breast cancer risk at the 2q35 locus is mediated through IGFBP5 regulation

Abstract

GWAS have identified a breast cancer susceptibility locus on 2q35. Here we report the fine mapping of this locus using data from 101,943 subjects from 50 case-control studies. We genotype 276 SNPs using the 'iCOGS' genotyping array and impute genotypes for a further 1,284 using 1000 Genomes Project data. All but two, strongly correlated SNPs (rs4442975 G/T and rs6721996 G/A) are excluded as candidate causal variants at odds against >100:1. The best functional candidate, rs4442975, is associated with oestrogen receptor positive (ER+) disease with an odds ratio (OR) in Europeans of 0.85 (95% confidence interval=0.84-0.87; P=1.7 × 10(-43)) per t-allele. This SNP flanks a transcriptional enhancer that physically interacts with the promoter of IGFBP5 (encoding insulin-like growth factor-binding protein 5) and displays allele-specific gene expression, FOXA1 binding and chromatin looping. Evidence suggests that the g-allele confers increased breast cancer susceptibility through relative downregulation of IGFBP5, a gene with known roles in breast cell biology.

Figure 1. Genetic mapping and epigenetic landscape at the 2q35 locus.

Manhattan plot of the 2q35 breast cancer susceptibility locus. Genotyped (black dots) and imputed (red dots) SNPs are plotted based on their chromosomal position on the x axis and their overall P values (log₁₀ values, likelihood ratio test) from the European BCAC studies (46,451 cases and 42,599 controls) on the y axis. The shaded region represents an area bounded by SNPs correlated with rs4442975 at r²=0.8. Data from the UCSC Genome Browser, including epigenetic marks for methylation of histone H3 at lysine 4 (H3K4me1, H3K4me3) and acetylation of H3 at lysine 27 (H3K27ac) in seven cell types from ENCODE. The positions of all analysed iCOGS SNPs are marked. LD, using data from the BCAC population, is depicted beneath—white represents r²=0 and black r²=1.

Figure 2. Allele-specific binding of FOXA1 at the rs4442975 site.

(a) Epigenetic and transcriptional landscape of the 2q35 risk interval. Coloured histogram denotes histone modification ChIP-seq data from ENCODE. Data from the UCSC Genome Browser, including epigenetic marks for H3K4me1 in seven cell types from ENCODE, H3K4me2 from MCF7 cells, DNaseI hypersensitivity clusters in 125 cell types from ENCODE, and TF ChIP-seq data from MCF7 and T47D ER+ breast cancer cells, which are homozygous for the g-allele of rs4442975 and rs6721996 (ENCODE). The PRE contains SNP rs4442975. (b) Position weight matrix of FOXA1 from JASPAR, with homology to the risk (g) and cancer-protective (t) alleles of rs4442975 coloured below. (c) IGR histogram for SNP rs4442975 predicting the binding intensity of FOXA1 using a seven-nucleotide affinity model. The top row of coloured numbers shows the number of instances for each K-mer found genome wide within H3K4me2 elements in MCF7 cells. The bottom row shows the averaged binding intensities at the K-mers (50 bp window). Control profiles, shown in grey, are generated by scrambling the probed sequence. (d) Allele-specific FOXA1 ChIP-qPCR results assessed at the rs4442975 SNP in heterozygous BT474 breast cancer cells. Error bars denote s.d. P values were determined with a two-tailed t-test. **P<0.01.

Figure 3. Chromatin interactions at the 2q35 risk region with IGFBP5 in breast cell lines.

(a) 3C interaction profiles between the PRE (containing rs4442975) and the IGFBP5 promoter region (grey box). 3C libraries were generated with EcoRI, with the anchor point set at the PRE. A physical map of the region interrogated by 3C is shown above, with the grey bar representing the position of the IGFBP5 promoter (not to scale). Graphs represent three biological replicates assayed in duplicate. Error bars denote s.d. (b) 3C followed by sequencing for the rs4442975-containing region in heterozygous BT474 breast cancer cells shows allele-specific chromatin looping. Chromatograms represent one of the three independent 3C libraries generated and sequenced. (c) Luciferase reporter assays in breast cell lines demonstrating enhancer activity of the PRE at the 2q35 risk locus. The PRE was cloned upstream of an IGFBP5 promoter-driven luciferase reporter with and without SNP rs4442975. Cells were transiently transfected with each of these constructs and assayed for luciferase activity after 24 h. Graphs represent two independent experiments assayed in triplicate. Error bars denote s.d. P values were determined with a two-tailed t-test. ****P<0.0001.

Figure 4. IGFBP5 expression in breast cancer cell lines and normal breast tissue.

(a) Endogenous IGFBP5 expression measured by qPCR in untreated ER+ human breast cancer cell lines and (b) oestrogen-stimulated breast cancer cell lines. Graphs represent three independent experiments. Error bars denote s.e.m. P values were determined by a two-tailed t-test. ****P<0.0001. (c) Allele-specific IGFBP5 expression measured by allelic amplification of intronic marker variant pos271557291. Chromatograms represent one of the three independent experiments performed and sequenced.