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. 2011 Jul 18;13(4):R72.
doi: 10.1186/bcr2917.

Allele-specific regulation of FGFR2 expression is cell type-dependent and may increase breast cancer risk through a paracrine stimulus involving FGF10

Petra E A Huijts  1 , Minka van Dongen, Moniek C M de Goeij, Adrian J van Moolenbroek, Freek Blanken, Maaike P G Vreeswijk, Esther M de Kruijf, Wilma E Mesker, Erik W van Zwet, Rob A E M Tollenaar, Vincent T H B M Smit, Christi J van Asperen, Peter Devilee
Affiliations

Allele-specific regulation of FGFR2 expression is cell type-dependent and may increase breast cancer risk through a paracrine stimulus involving FGF10

Petra E A Huijts et al. Breast Cancer Res. .

Abstract

Introduction: SNPs rs2981582 and rs2981578, located in a linkage disequilibrium block (LD block) within intron 2 of the fibroblast growth factor receptor 2 gene (FGFR2), are associated with a mildly increased breast cancer risk. Allele-specific regulation of FGFR2 mRNA expression has been reported previously, but the molecular basis for the association of these variants with breast cancer has remained elusive to date.

Methods: mRNA levels of FGFR2 and three fibroblast growth factor genes (FGFs) were measured in primary fibroblast and epithelial cell cultures from 98 breast cancer patients and correlated to their rs2981578 genotype. The phosphorylation levels of downstream FGFR2 targets, FGF receptor substrate 2α (FRS2α) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), were quantified in skin fibroblasts exposed to FGF2. Immunohistochemical markers for angiogenesis and lymphocytic infiltrate were semiquantitatively assessed in 25 breast tumors.

Results: The risk allele of rs2981578 was associated with increased FGFR2 mRNA levels in skin fibroblasts, but not in skin epithelial cell cultures. FGFR2 mRNA levels in skin fibroblasts and breast fibroblasts correlated strongly in the patients from whom both cultures were available. Tumor-derived fibroblasts expressed, on average, eight times more FGFR2 mRNA than the corresponding fibroblasts from normal breast tissue. Fibroblasts with higher FGFR2 mRNA expression showed more FRS2α and ERK1/2 phosphorylation after exposure to FGF2. In fibroblasts, higher FGFR2 expression correlated with higher FGF10 expression. In 25 breast tumors, no associations between breast tumor characteristics and fibroblast FGFR2 mRNA levels were found.

Conclusions: The influence of rs2981578 genotypes on FGFR2 mRNA expression levels is cell type-dependent. Expression differences correlated well with signaling levels of the FGFR2 pathway. Our results suggest that the increased breast cancer risk associated with SNP rs2981578 is due to increased FGFR2 signaling activity in stromal fibroblasts, possibly also involving paracrine FGF10 signaling.

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Figures

Figure 1
Figure 1
The relationship between FGFR2 mRNA expression and the rs2981578 genotype in skin fibroblast and skin epithelial cell cultures. (A) A significant correlation between the rs2981578 genotype and FGFR2 mRNA expression level as measured by quantitative real-time PCR was present in the 68 skin fibroblast cultures (P = 0.02; one-way ANOVA). The expression levels were log2-transformed and normalized to HNRPM and TBP expression. (B) FGFR2 mRNA expression and rs2981578 genotypes in 25 skin epithelial cell cultures (P = 0.73; one-way ANOVA). The expression levels were log2-transformed and normalized to HNRPM, SRPR and TBP expression. Each dot represents the expression level in one patient.
Figure 2
Figure 2
FGFR2 mRNA expression levels in fibroblasts cultured from different locations. (A) We found a significant correlation between FGFR2 mRNA expression in skin fibroblasts and normal breast fibroblasts from the same patient (n = 22). The FGFR2 expression levels were log2-transformed and normalized to HNRPM and TBP expression. (B) FGFR2 mRNA expression in tumor-derived fibroblasts is significantly higher than in fibroblasts cultured from normal breast tissue. The FGFR2 expression levels were log2-transformed and normalized to HNRPM and TBP expression. Each dot represents FGFR2 expression in one patient. The lines connect the fibroblast cultures from one patient. The horizontal bars shows the mean expression for all fibroblast cultures from that origin (skin -3.8 (±1.9 SD), normal breast -2.9 (±1.4 SD), tumor tissue 0.2 (±1.5 SD)). *P < 0.001.
Figure 3
Figure 3
Correlation of expression levels of FGFR2 and FGF10 mRNA in 68 skin fibroblast cultures. No correlation was found for FGFR2 and FGF2 mRNA or FGFR2 and FGF7 mRNA levels. The data were normalized to HNRPM and TBP and log2-transformed. Each dot represents the expression levels from one patient. (A) FGFR2 and FGF10 (P = 8 × 10-8; Pearson's correlation). (B) FGFR2 and FGF7 (P = 0.02; Pearson's correlation). (C) FGFR2 and FGF2 (P = 0.07; Pearson's correlation).
Figure 4
Figure 4
Response of skin fibroblast cultures with high and low FGFR2 mRNA levels to stimulation with ligand FGF2. Phosphorylation of downstream targets FRS2α and ERK1/2 were measured before and after 10 minutes of stimulation with FGF2. The average difference of the increase in phosphorylation is shown, which was calculated by dividing the increase in samples with high FGFR2 mRNA levels by the increase in samples with low FGFR2 mRNA levels. The 95% confidence intervals are also shown. Phosphorylation of FRS2α was measured in four fibroblast cultures: two with high and two with low FGFR2 mRNA levels. Phosphorylation of ERK1/2 was measured in 10 fibroblast cultures, five with high and five with low FGFR2 mRNA levels.
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