Characterizing SMAD-Dependent TGF-β Signaling in Estrogen Receptor-Positive Breast Cancer Cells that Metastasize to the Bone

Abstract

ER+ breast cancer is the most frequently diagnosed subtype of breast cancer, and also has the highest predilection to form clinically evident osteolytic bone metastases. Though the exact signaling pathways that drive ER+ breast cancer bone metastases are unknown, SMAD-dependent TGF-β signaling has been shown to be an important driver of ER- breast cancer bone metastases. Inhibition of this pathway by treatment with curcuminoids has been characterized in ER- breast cancer cells in vitro and in vivo in xenograft mouse models; however the role of TGF- β signaling in ER+ breast cancer bone metastasis prevention has not been well characterized. To investigate this, we used three bone-tropic ER+ cell lines (MCF-7, T47D, and ZR-75-1) and characterized their SMAD-dependent TGF-β signaling in the presence and absence of estrogen, as well as the effect of curcuminoid treatment. Increasing doses of curcuminoid treatment of cells grown in an estrogenic milieu showed a dose-dependent decrease of cell viability, while cells grown in an estrogen-deplete milieu did not show as marked a decrease. We show that only MCF-7 and ZR-75-1 cells phosphorylate SMAD2/3 upon TGF-β stimulation, and levels of pSMAD2/3 decrease in the presence of estrogen. We then treated MCF-7 and ZR-75-1 cells with an optimal dose of curcuminoids (30 uM) in media with or without estrogen and growth factors and evaluated the effect of SMAD phosphorylation. Our results show that, curcuminoids can downregulate TGF-β stimulated SMAD2/3 phosphorylation in MCF-7 cells (but not ZR-75-1 cells) grown in an estrogenic and growth factor-containing milieu in vitro. Our results indicate that curcuminoids may offer potential therapeutic benefit through inhibition of micrometastasis progression post-therapeutically in women with a history of ER+ breast cancer. Further studies of this pathway, as well as in vivo experiments, will be conducted to further characterize the potential therapeutic effect of curcuminoids.