Abstract P4-15-04: Molecular mediators of mammographic density

Abstract

Abstract Introduction Mammographic density (MD), created predominantly by increased stromal tissue, is a major risk factor for breast cancer, 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 the density-associated risk. Methods Primary human breast fibroblasts from normal, high risk or breast cancer patients were treated with hydroxytamoxifen (100nM-5uM), the active metabolite of tamoxifen. Fibroblast function was analysed by measuring: proliferation, expression of stromal proteins fibronectin and collagen 1; effects on TGF-β signalling via SMAD phosphorylation and upregulation of the myofibroblast marker SMA. Genome wide analysis was performed using RNA-Seq. Significantly deregulated pathways were validated by quantitative real time PCR, western blotting and mass spectrometry. Results Fibroblasts from 25 patients were treated with hydroxytamoxifen. All patients showed reduced proliferation with treatment. 62% of patients showed reduced fibronectin expression. Collagen 1 expression and TGF-β-mediated upregulation of SMA and fibronectin were consistently inhibited by tamoxifen. RNA-Seq analysis revealed downregulation of Wnt signalling, an established pro-fibrogenic and pro-tumourigenic pathway, and other stromal signalling pathways including matrix/receptor interaction, focal adhesion signalling and collagen formation. Intriguingly, 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 replicated using tesmilifene, a commercially available inhibitor of ChEH. Mass spectrometry analysis confirmed an altered cholesterol metabolite profile in the 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 tumour-inhibitory microenvironment. This offers exciting potential for patient monitoring and alternative breast cancer prevention strategies.Introduction Mammographic density (MD), created predominantly by increased stromal tissue, is a major risk factor for breast cancer, 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 the density-associated risk. Methods Primary human breast fibroblasts from normal, high risk or breast cancer patients were treated with hydroxytamoxifen (100nM-5uM), the active metabolite of tamoxifen. Fibroblast function was analysed by measuring: proliferation, expression of stromal proteins fibronectin and collagen 1; effects on TGF-β signalling via SMAD phosphorylation and upregulation of the myofibroblast marker SMA. Genome wide analysis was performed using RNA-Seq. Significantly deregulated pathways were validated by quantitative real time PCR, western blotting and mass spectrometry. Results Fibroblasts from 25 patients were treated with hydroxytamoxifen. All patients showed reduced proliferation with treatment. 62% of patients showed reduced fibronectin expression. Collagen 1 expression and TGF-β-mediated upregulation of SMA and fibronectin were consistently inhibited by tamoxifen. RNA-Seq analysis revealed downregulation of Wnt signalling, an established pro-fibrogenic and pro-tumourigenic pathway, and other stromal signalling pathways including matrix/receptor interaction, focal adhesion signalling and collagen formation. Intriguingly, 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 replicated using tesmilifene, a commercially available inhibitor of ChEH. Mass spectrometry analysis confirmed an altered cholesterol metabolite profile in the 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 tumour-inhibitory microenvironment. This offers exciting potential for patient monitoring and alternative breast cancer prevention strategies. Citation Format: Ironside A, Hawkesford K, Gomm J, Haywood L, Goulding I, Wang J, Lamaziere A, Poirot M, Silvente-Poirot S, Chelala C, Jones JL. Molecular mediators of mammographic density [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-15-04.