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

Meyer KB, Maia AT, O'Reilly M, Teschendorff AE, Chin SF, Caldas C, Ponder BA - PLoS Biol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom. Kerstin.Meyer@cancer.org.uk

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.

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Protein–DNA Interactions at FGFR2–33 and FGFR2–13 In Vitro and In VivoEMSAs 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.
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pbio-0060108-g003: Protein–DNA Interactions at FGFR2–33 and FGFR2–13 In Vitro and In VivoEMSAs 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.

Mentions: This correlation suggests that the functional SNPs map to a regulatory region within the gene, possibly by altering one or more transcription factor binding sites. Interactions between proteins from nuclear extracts and DNA were examined for the eight most strongly disease-associated alleles (Figure 1). Two of these candidate functional SNPs showed distinct binding patterns in electrophoretic mobility shift assays (EMSA). The common allele of rs7895676 (FGFR2–33) formed strong protein–DNA complexes with nuclear extracts from the breast carcinoma cell lines HCC1954 (Figure 3A) and PMC42 and from HeLa cells (unpublished data), whereas no binding was detected on the minor allele. Competition studies and supershift experiments identify the bound protein as C/EBPβ (Figure 3A). We note that the FGFR2–33 sequence has considerable homology to the C/EBPβ binding site from the interleukin 6 (IL-6) promoter [15] (Figure 3C). The heterogeneity of the observed protein–DNA complexes is most likely due to the presence of multiple C/EBPβ isoforms. For rs2981578 (FGFR2–13), both alleles give rise to a strong protein–DNA complex in HCC1954 cell extracts. However, a second more slowly migrating complex was only seen on the rarer genotype (Figure 3B). Interestingly, both alleles are able to compete for both bands, suggesting that the formation of the upper complex depends on the presence of the lower complex. Inspection of the FGFR2 DNA indicated the presence of a perfect octamer binding site immediately adjacent to the SNP, while the SNP itself lay within a sequence with homology to Runx binding sites (Figure 3C). Competition studies and incubation with specific antisera shows that both alleles bind Oct-1, while only the minor allele binds Oct-1 and Runx2 in HCC1954 nuclear extracts (Figure 3B), as well as in PMC42 cells (Figure S1).


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

Meyer KB, Maia AT, O'Reilly M, Teschendorff AE, Chin SF, Caldas C, Ponder BA - PLoS Biol. (2008)

Protein–DNA Interactions at FGFR2–33 and FGFR2–13 In Vitro and In VivoEMSAs 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2365982&req=5

pbio-0060108-g003: Protein–DNA Interactions at FGFR2–33 and FGFR2–13 In Vitro and In VivoEMSAs 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.
Mentions: This correlation suggests that the functional SNPs map to a regulatory region within the gene, possibly by altering one or more transcription factor binding sites. Interactions between proteins from nuclear extracts and DNA were examined for the eight most strongly disease-associated alleles (Figure 1). Two of these candidate functional SNPs showed distinct binding patterns in electrophoretic mobility shift assays (EMSA). The common allele of rs7895676 (FGFR2–33) formed strong protein–DNA complexes with nuclear extracts from the breast carcinoma cell lines HCC1954 (Figure 3A) and PMC42 and from HeLa cells (unpublished data), whereas no binding was detected on the minor allele. Competition studies and supershift experiments identify the bound protein as C/EBPβ (Figure 3A). We note that the FGFR2–33 sequence has considerable homology to the C/EBPβ binding site from the interleukin 6 (IL-6) promoter [15] (Figure 3C). The heterogeneity of the observed protein–DNA complexes is most likely due to the presence of multiple C/EBPβ isoforms. For rs2981578 (FGFR2–13), both alleles give rise to a strong protein–DNA complex in HCC1954 cell extracts. However, a second more slowly migrating complex was only seen on the rarer genotype (Figure 3B). Interestingly, both alleles are able to compete for both bands, suggesting that the formation of the upper complex depends on the presence of the lower complex. Inspection of the FGFR2 DNA indicated the presence of a perfect octamer binding site immediately adjacent to the SNP, while the SNP itself lay within a sequence with homology to Runx binding sites (Figure 3C). Competition studies and incubation with specific antisera shows that both alleles bind Oct-1, while only the minor allele binds Oct-1 and Runx2 in HCC1954 nuclear extracts (Figure 3B), as well as in PMC42 cells (Figure S1).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Cambridge Research Institute, Li Ka Shing Centre, Cambridge, United Kingdom. Kerstin.Meyer@cancer.org.uk

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.

Show MeSH
Related in: MedlinePlus