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.