Molecular mediators of mammographic density

Mammographic density (MD), created predominantly by increased stromal tissue, is a major breast cancer risk factor, though little is known about the biological mechanisms mediating it. Tamoxifen prevents breast cancer in a subset of high risk women via mechanisms that appear dependent on reduction of MD. Animal models suggest tamoxifen remodels the mammary stroma to a tumour-inhibitory phenotype. This study aims to analyse the effect of tamoxifen on human breast fibroblast function and identify pro-tumourigenic pathways contributing to density-associated risk. Methods Primary human breast fibroblasts from normal, high risk or breast cancer patients were treated with hydroxytamoxifen (100nM-5μM). Fibroblast function was analysed by measuring: proliferation, expression of stromal proteins fibronectin and collagen 1; effects on TGF-β signalling and up-regulation of myofibroblast marker SMA. Genome wide analysis was performed using RNA-Seq. Significantly deregulated pathways were validated by PCR, western blotting and mass spectrometry. Results Fibroblasts from 23 patients were treated with hydroxytamoxifen. All patients showed reduced proliferation with treatment. 62% of patients showed reduced fibronectin expression. TGF- β-mediated up-regulation of SMA and fibronectin were consistently inhibited by tamoxifen. RNA-Seq analysis revealed down-regulation of Wnt signalling, an established profibrogenic and pro-tumourigenic pathway. In addition, there was significant modulation of many metabolic pathways, including components of the microsomal anti-oestrogen binding site (AEBS). Binding of tamoxifen to the AEBS inhibits cholesterol epoxide hydrolase (ChEH) enzyme activity, promoting an anti-tumourigenic phenotype. The effects of tamoxifen on fibroblasts could be partly replicated using tesmilifene, a commercially available 5 inhibitor of ChEH. Mass spectrometry analysis confirmed an altered cholesterol metabolite profile in tamoxifen treated fibroblasts. Conclusion These data indicate that tamoxifen can directly remodel the mammary stromal microenvironment, generating a less 'reactive' stroma. Thus, tamoxifen impacts on multiple pathways, many independent of the oestrogen receptor, to create a tumourinhibitory microenvironment. This offers exciting potential for patient monitoring and alternative breast cancer prevention strategies.