PurposeThe aim of this study is to investigate the mechanisms of interactions between TGF-β and Wnt/β-catenin pathways that induce and regulate EMT and promote breast cancer cells to become resistant to treatment.MethodsThe effect of TGF-β on Wnt/β-catenin signaling pathway was examined by using a human Wnt/β-catenin-regulated cDNA plate array and western blot analysis. The interaction of Twist at promoter of Wnt3 was examined by chromatin immunoprecipitation (ChIP) assay. Secreted Wnt3 level was determined by ELISA assay.ResultsHER2-overexpressing breast cancer cells treated with TGF-β have a reduced response to trastuzumab and exhibited EMT-like phenotype. The TGF-β-induced EMT in HER2-cells was concordant with upregulation of Wnt3 and β-catenin pathways. The TGF-β-induced induction of Wnt3 during EMT was found to be Smad3-dependent. ChIP analysis identified occupancy of Twist at promoter region of Wnt3. Knock-down of Twist by shRNA confirmed the significance of Twist in response to TGF-β regulating Wnt3 during EMT. Subsequently, TGF-β-induced matrix metalloproteinases, MMP1, MMP7, MMP9, MMP26, Vascular endothelial growth factors (VEGF), and activation of Wnt/β-catenin signaling were repressed by the shRNA treatment. TGF-βR1 ALK5 kinase inhibitor, A83-01 can effectively prevent the TGF-β-induced Twist and Wnt3. Co-treating A83-01 and trastuzumab inhibited TGF-β-induced cell invasion significantly in both trastuzumab responsive and resistant cells.ConclusionsOur data demonstrated an important interdependence between TGF-β and Wnt/β-catenin pathways inducing EMT in HER2-overexpressing breast cancer cells. Twist served as a linkage between the two pathways during TGF-β-induced EMT. A83-01 could inhibit the TGF-β-initiated pathway interactions and enhance HER2-cells response to trastuzumab treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-017-4211-y) contains supplementary material, which is available to authorized users.

Interactions between Wnt and TGF-β (Transforming growth factor-β) signaling pathways play a critical role in promoting EMT (Epithelial to Mesenchymal Transition) and tumor metastasis. We previously demonstrated that activation of Wnt3/β-catenin signaling pathway promoting EMT is an important mechanism leading to HER2-cells becoming resistant to trastuzumab. In this study, we used HER2-overexpressing breast cancer cells to investigate the interactions between TGF-β and Wnt/β-catenin pathways in regulation of EMT and promoting breast cancer cells invasion. Our data showed that phosphorylation of Smad3 at ser423/425 by TGF-β led to nuclear localization of Smad3 and upregulation of Twist protein expression in HER2-overexpressing breast cancer cells. Cells treated with TGF-β showed poor response to trastuzumab and exhibited EMT-like phenotype; decreased E-cadherin and increased N-cadherin expression. A small molecule inhibitor, A83-01 which inhibits phosphorylation of Smad3 repressed TGF-β-induced nuclear accumulation of Smad3 and Twist expression. The A83-01 also inhibited TGF-β-induced cell migration and invasion and enhanced anti-tumor activity of trastuzumab. The TGF-β/Smad3 induced EMT in HER2-cells was found to be concordant with activation of Wnt3/β-catenin pathway. Following TGF-β treatment, Chromatin immunoprecipitation identified occupancy of Twist at promoter region of Wnt3. This data suggests that Twist in response to TGF-β activated Wnt3 gene transcription in HER2-overexpressing breast cancer cells. Knock-down of Twist by shRNA down-regulated Wnt3 expression and inhibited TGF-β-induced nuclear accumulation of β-catenin. Subsequently, TGF-β-induced matrix metalloproteinases, MMP1, MMP7, MMP9, VEGF (Vascular endothelial growth factors) and activation of Wnt/β-catenin signaling were repressed by the shRNA treatment. In summary, our data demonstrated for the first time that the TGF-β/Smad3 mediated Twist results in transcriptional upregulation of Wnt3/β-catenin pathway and promotes EMT and cell invasion. The data suggests that therapeutic targeting of Twist by A83-01 and similar small molecules could inhibit the interaction between TGF-β and Wnt pathways and prevent breast cancer progressing to EMT and metastases. Citation Format: Yanyuan Wu, Tran Trinh, Marianna Sarkissyan, Sami Dwabe, Juri Kim, Robin Farias-Eisner, Jay Vadgama. A83-01 inhibits TGF-β/Smad3 and Wnt/β-catenin-mediated EMT and tumor metastasis in HER2-overexpressing breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4602.