Synthesis and secretion of transforming growth factor beta isoforms by primary cultures of human breast tumour fibroblasts in vitro and their modulation by tamoxifen

Tamoxifen may mediate its effect in early breast cancer in part via an oestrogen receptor (ER)-independent pathway by directly stimulating fibroblasts to produce the negative paracrine growth factor transforming growth Factor (TGF)-beta. We have previously shown that secretion of this factor is induced 3- to 30-fold in human fetal fibroblasts in vitro, and by stromal fibroblasts in vivo following tamoxifen treatment of ER-positive and ER-negative breast cancer patients. Primary cultures of breast tumour fibroblasts have been exposed to tamoxifen for 48 h, and rates of secretion of TGF-beta(1) and TGF-beta(2) measured using a quantitative immunoassay. Fibroblast strains derived from malignant and benign tumours produced and secreted similar amounts of TGF-beta(1), but benign breast tumour fibroblasts secreted significantly higher levels of TGF-beta(2) compared with fibroblasts of malignant origin. Tamoxifen did not induce any consistent increase in TGF-beta secretion into the conditioned medium, but immunofluorescence analysis for the intracellular form of TGF-beta(1) revealed evidence of increased immunoreactive protein in tamoxifen-treated fibroblasts, which is localised to the nucleus. Therefore synthesis of TGF-beta(1) appears to be stimulated by tamoxifen, but increased secretion may be abrogated in vitro. Furthermore, using immunocytochemistry and transient transfection with an ER-responsive reporter construct, no ER was demonstrable in these fibroblasts supporting the proposed ER-independent paracrine pathway.