The online version of this article (doi:10.1186/bcr2917) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
The experiments were performed by PH, MD, MG and AM. The patient material was provided by RT and VS. The cells were cultured by FB and MV. The stroma percentage of 50 tumors was analyzed by EK and WM. The experiments were designed, analyzed and interpreted by PH, CA and PD. EZ helped with the statistical analyses and the interpretation of the data. The manuscript was drafted by PH and critically revised by MV, CA and PD. All authors read and approved the final manuscript.
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.
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.
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.
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.
Additional file 1: Figure S1. (A) Typical fibroblast and (B) typical epithelial cell cultures (original magnification, ×10). (TIFF 5 MB)13058_2010_2713_MOESM1_ESM.TIFF
Additional file 2: Supplementary methods. This file contains supplementary methods regarding the quantitative real-time PCR and Western blot analyses. (DOC 49 KB)13058_2010_2713_MOESM2_ESM.DOC
Additional file 3: Figure S2. Phosphorylation levels of downstream targets of FGFR2 after different periods of stimulation with FGF2 in one fibroblast sample (sample from experiment 1 with high FGFR2 mRNA levels). (A) Using one of the blots as an example, we show phosphorylated ERK1/2 examined after 0, 1, 5, 10 and 20 minutes. H: lanes with total protein from a fibroblast sample with high FGFR2 mRNA level; L: lanes from a fibroblast sample with low FGFR2 mRNA level; M: lane with the length marker; T: tubulin, E1/2 ERK1/2. (B) Phosphorylation levels of FRS2α, ERK1 and ERK2 are shown as fold increases compared to the phosphorylation levels at 0 minutes of stimulation. (TIFF 406 KB)13058_2010_2713_MOESM3_ESM.TIFF
Additional file 4: Figure S3. The relationship between FGF10 mRNA expression and the rs2981578 genotype in 68 skin fibroblast cultures (P = 0.06; one-way ANOVA). The expression levels were log2-transformed and normalized to HNRPM and TBP expression. (TIFF 26 KB)13058_2010_2713_MOESM4_ESM.TIFF
Additional file 5: Figure S4. Correlation of expression levels of FGFR2 and FGF10 mRNA in 44 breast fibroblast cultures. FGFR2 and FGF10 expression levels were normalized to HNRPM and TBP and log2-transformed. Each dot represents the expression levels of one patient (Spearman's ρ = 0.25, P = 0.11). (TIFF 20 KB)13058_2010_2713_MOESM5_ESM.TIFF
Additional file 6: Figure S5. Western blot analysis measuring FGFR2 protein levels in fibroblasts. (A) Western blot showing results for seven different fibroblast cultures. H: lanes with total protein from fibroblasts with high FGFR2 mRNA levels; L: lanes with total protein from fibroblasts with low FGFR2 mRNA levels; empty: empty lane; M: lane with length marker; recomb: lane with pure recombinant FGFR2. (B) FGFR2 mRNA levels and FGFR2 protein levels in the seven fibroblast samples. The FGFR2 mRNA results were normalized to HNRPM and TBP and log2-transformed. The FGFR2 protein results were normalized to α-tubulin. Each dot represents the results for one fibroblast sample. (TIFF 381 KB)13058_2010_2713_MOESM6_ESM.TIFF
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- Allele-specific regulation of FGFR2 expression is cell type-dependent and may increase breast cancer risk through a paracrine stimulus involving FGF10
Petra EA Huijts
Minka van Dongen
Moniek CM de Goeij
Adrian J van Moolenbroek
Maaike PG Vreeswijk
Esther M de Kruijf
Wilma E Mesker
Erik W van Zwet
Rob AEM Tollenaar
Vincent THBM Smit
Christi J van Asperen
- BioMed Central
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