Abstract B46: EMT-inducing transcription factors Twist1, Snail1 and Six1 increase metastasis of neighboring tumor cells via induction of Hedgehog-Gli signaling

Breast cancer is the second leading cause of cancer related deaths in women and more than 90% of these deaths result from metastatic disease rather than from the primary tumor burden. Recent studies have shown that intratumoral heterogeneity exists, and can affect chemotherapeutic and as well as metastatic outcome. To determine if crosstalk between metastatic and non-metastatic breast cancer cells can occur in the earlier stages of metastasis, where an epithelial-to-mesenchymal transition (EMT) is thought to occur in a small population of cells within a tumor, allowing them to metastasize, we examined whether different EMT-inducing pro-metastatic transcription factors (TFs) can influence the EMT status and metastatic properties of cells not expressing these factors (control cells). Twist1 and Snail1 are two EMT-inducing TFs that are known to increase metastasis through cell autonomous mechanisms. Our data demonstrate that these TFs can also non-cell autonomously alter the expression of numerous genes and proteins associated with EMT, as well as increase migration and invasion, when conditioned medium (CM) taken from HMLER-Twist1/Snail1 cells is placed on control cells. We show that the non-cell autonomous effects of Twist1 and Snail1 are in part induced via their ability to up-regulate another known EMT-TF, Six1. Loss of Six1 downstream of these TFs mitigates their ability to non-cell autonomously influence migration and invasion of non-metastatic, non-EMT TF expressing cells, indicating that Six1 is a key player downstream of Twist1 and Snail1. In multiple breast cancer models (including the MCF7 and 4t1 series of cell lines), CM taken from cells that overexpress Six1, when placed on control cells, leads to alterations in expression of EMT-related proteins, as well as increased migration and invasion. This does not occur when CM is taken from Six1 knockdown cells, showing that Six1 can non-cell autonomously cause an EMT in the control non-Six1 expressing cells. Using mouse models, we also demonstrate that highly metastatic, Six1 expressing cells can non-cell autonomously increase the metastatic incidence of weakly metastatic, non-Six1 expressing cells. We find that CM taken from cells expressing Twist1, Snail1, and Six1, when placed on control cells, leads to the induction of hedgehog (Hh) signaling as measured by Gli activity in the control recipient cells. Further, we demonstrate that activation of Hh signaling in control cells (when exposed to CM from metastatic-EMT cells) is critical for the induction of EMT-like characteristics. Interestingly, the mechanism of Gli activation in recipient cells differs depending on the EMT-TF expressed by the cells from which the CM is taken; such that upstream inhibitors of the pathway (cyclopamine and 5E1 monoclonal antibody against Hh ligands) only work in specific contexts, whereas more downstream inhibitors targeting Gli1 (Gant61) inhibit the effects in all contexts. These data suggest that EMT-TFs are able to activate the Gli signaling pathway non-cell autonomously via multiple mechanisms. We observe a strong correlation of Twist1, Snail1, and Six1 with Gli1 in numerous breast cancer datasets, whereas such a correlation is not observed with the ligands of Hh signaling, the more upstream activators of the pathway; an indication that the EMT-TFs may be converging in on increasing Gli activity and hence activating the Hh signaling pathway. Our data demonstrate that EMT-TFs, even if expressed in only a percentage of cells within a primary tumor, can increase metastasis of neighboring cells, and further suggest that inhibitors targeting downstream aspects of the Hedgehog pathway, such as Gli1, may be a novel means to target metastatic progression in heterogeneous breast cancers to increase patient survival. Citation Format: Deepika Neelakantan, Hengo Zhou, Joshua Cabrera, Heide Ford. EMT-inducing transcription factors Twist1, Snail1 and Six1 increase metastasis of neighboring tumor cells via induction of Hedgehog-Gli signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B46.