Stromal fibroblasts with nuclear β-catenin are present within breast tumours and increase proliferation and invasion of epithelial breast cancer cells

Background

β-catenin, when located within the nucleus, acts as an oncoprotein by activating TCF/LEF transcription factors, which in turn regulate transcription of a wide range of growth and proliferation-associated genes. Nuclear β-catenin is frequently seen in epithelial cancer cells as a result of either inappropriate Wnt signalling or inactivating mutations in genes for key β-catenin regulators or for β-catenin itself. Breast tumours are unusual, however, in that nuclear β-catenin is relatively rare in epithelial breast cancer cells. On the other hand, nuclear β-catenin expression has been documented in fibroblasts within breast fibroadenomas and benign phyllodes tumours [1]. Preliminary observations within our laboratory indicated that stromal fibroblasts in and around breast carcinomas also frequently express nuclear β-catenin. Our aim in the present work was to validate this observation, and to determine how fibroblasts with nuclear β-catenin might influence cancer behaviour.

Methods

We performed immunohistochemistry for β-catenin on whole sections of breast cancers from 200 individual cases. A scoring system based on the number of fibroblasts expressing nuclear β-catenin was devised and fibroblasts around tumour and normal breast tissue were scored. To examine the potential influence of nuclear β-catenin-positive fibroblasts on breast tumour behaviour, we have developed a tissue culture model. With appropriate controls, fibroblasts (MRC5/immortalised primary breast fibroblasts) were transfected to overexpress β-catenin and the influence of these cells on breast cancer cells (MCF7/MDA-MB-231) in vitro was determined. First, proliferation rates of breast cancer cells treated with conditioned media from transfected fibroblasts were determined (MTT assays). Secondly, invasion assays were carried out in transwell plates; fibroblasts were β-catenin or control transfected in lower chambers and breast cancer cells were seeded into upper chambers onto membranes coated with extracellular matrix (Matrigel/ECMatrix). Epithelial cells invading through membranes were quantified (cell counting/fluorometric assay using CyQuant GR dye).

Results and conclusion

We found that fibroblasts expressing nuclear β-catenin are frequent in and around breast tumours, while they are very rare around normal breast. In our tissue culture model, β-catenin-transfected fibroblasts stimulated both proliferation and invasion of breast cancer cells. In conclusion, nuclear β-catenin within stromal fibroblasts may have a potent influence on breast cancer behaviour. This influence further highlights the importance of stromal–epithelial interactions in breast carcinogenesis.