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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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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.
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pbio-0060108-g004: 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.

Mentions: To test whether differential protein binding could alter the ability of the susceptibility alleles to activate transcription, we multimerised oligonucleotides overlapping both the Oct-1/Runx2 and the C/EBPβ binding sites, cloned these in both orientations upstream of the luciferase reporter gene in pGL3Enh (Figure 4A), and assayed them in three breast cancer cell lines (PMC42, HCC70, and T47D). Transfections were carried out in triplicate and repeated at least twice for each cell line. A representative transfection into HCC70 cells is shown in Figure 4B (see Figure S2 for PMC42 and T47D). In all three cell lines tested, the minor allele at the Oct-1/Runx2 site stimulated transcription 2- to 5-fold over the common allele, independent of orientation, with the average being just above a 3-fold increase (p < 0.01). In contrast, the minor and common alleles of the multimerised C/EBPβ binding site did not show a consistent pattern of activation relative to each other. It varied with the cell lines and the orientation in which constructs were tested. Nevertheless, relative to the parental vector, the common allele always showed transcriptional activation. Compared to the common allele, the minor allele was either not significantly different or gave rise to a smaller degree of activation. However, in the latter case, the rare allele still activated transcription significantly above the enhancer-only construct (p < 0.01). Presumably this reflects the fact that the minor allele of FGFR2–33 still binds C/EBPβ above background levels in vivo (Figure 3D). By comparing the two different sites, we found that for Oct-1/Runx2 the minor allele was more active, while for C/EBPβ, the common site yielded higher levels of transcription in the majority of experiments. Hence their effects were opposing. We therefore assayed a synthetic construct consisting of single sites for C/EBPβ, Oct-1, and Runx2. In this arrangement, the effect of Oct-1/Runx2 clearly predominates, with the minor allele expressed at higher levels, reflecting the situation at the endogenous locus.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2365982&req=5

pbio-0060108-g004: 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.
Mentions: To test whether differential protein binding could alter the ability of the susceptibility alleles to activate transcription, we multimerised oligonucleotides overlapping both the Oct-1/Runx2 and the C/EBPβ binding sites, cloned these in both orientations upstream of the luciferase reporter gene in pGL3Enh (Figure 4A), and assayed them in three breast cancer cell lines (PMC42, HCC70, and T47D). Transfections were carried out in triplicate and repeated at least twice for each cell line. A representative transfection into HCC70 cells is shown in Figure 4B (see Figure S2 for PMC42 and T47D). In all three cell lines tested, the minor allele at the Oct-1/Runx2 site stimulated transcription 2- to 5-fold over the common allele, independent of orientation, with the average being just above a 3-fold increase (p < 0.01). In contrast, the minor and common alleles of the multimerised C/EBPβ binding site did not show a consistent pattern of activation relative to each other. It varied with the cell lines and the orientation in which constructs were tested. Nevertheless, relative to the parental vector, the common allele always showed transcriptional activation. Compared to the common allele, the minor allele was either not significantly different or gave rise to a smaller degree of activation. However, in the latter case, the rare allele still activated transcription significantly above the enhancer-only construct (p < 0.01). Presumably this reflects the fact that the minor allele of FGFR2–33 still binds C/EBPβ above background levels in vivo (Figure 3D). By comparing the two different sites, we found that for Oct-1/Runx2 the minor allele was more active, while for C/EBPβ, the common site yielded higher levels of transcription in the majority of experiments. Hence their effects were opposing. We therefore assayed a synthetic construct consisting of single sites for C/EBPβ, Oct-1, and Runx2. In this arrangement, the effect of Oct-1/Runx2 clearly predominates, with the minor allele expressed at higher levels, reflecting the situation at the endogenous locus.

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