Abstract B084: EMT-inducing transcription factors Twist1 and Snail1 non-cell autonomously increase metastatic properties of non-Twist1 or Snail1 expressing cells via induction of Six1

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. The primary tumor itself is a heterogeneous mass of cells. Thus a better understanding of metastasis and cancer cell dynamics within the primary tumor will aid in developing efficient strategies to increase patient survival. Twist1 and Snail1 are two epithelial to mesenchymal transition (EMT)-inducing transcription factors (TF) that are known to increase metastasis through mechanisms that are cell autonomous. Our data demonstrate that these TFs can also non-cell autonomously increase in-vitro invasion and migration, as well as alter the expression of numerous genes and proteins associated with EMT, when conditioned medium (CM) taken from cells expressing these TFs is placed on cells not expressing the TFs. We show that the non-cell autonomous effects of Twist1 and Snail1 are in part induced via the ability of these TFs to up-regulate another known EMT related transcription factor, Six1. Indeed, we show that loss of Six1 downstream of these TFs mitigates their ability to non-cell autonomously influence EMT related characteristics including invasion and migration of non-metastatic non-TF expressing cells in-vitro, indicating that Six1 is a key player downstream of these TFs. Overexpression and knockdown of Six1 itself, in multiple mouse and human breast carcinoma cell lines non-cell autonomously influences invasion, migration, anoikis resistance, and alters the expression of EMT-related genes in the non-metastatic non-Six1 expressing cells in-vitro. We also show that Six1 expression in metastatic cells is sufficient to non-cell autonomously increase the metastatic potential of weakly metastatic, non Six1-expressing cells, when these cells are fluorescently tagged and co-injected into mice. We have identified several pro-metastatic cytokines, including IL-6 and Osteopontin, that are not only increased in CM from cells expressing Six1, but also have increased expression in cells expressing Twist1 and Snail1, in a manner that is dependent on Six1. We are currently assessing whether these cytokines play a role in the ability of Twist1, Snail1 and Six1 to mediate invasion, migration as well as metastasis of neighboring cells that do not express these EMT-inducing TFs. Importantly, EMT related transcription factors such as Six1, Twist1 and Snail1 are associated with poor prognosis and increased metastasis in breast cancer as well as in other cancers. Therefore, it is critical that we understand not only how these TFs mediate aggressive phenotypes in cells expressing these factors, but also how they mediate non-cell autonomous effects that contribute to increased metastasis of the neighboring non-TF expressing cells within the heterogeneous tumor. Citation Format: Deepika Neelakantan, Ritsuko Iwanaga, David J. Drasin, Heide L. Ford. EMT-inducing transcription factors Twist1 and Snail1 non-cell autonomously increase metastatic properties of non-Twist1 or Snail1 expressing cells via induction of Six1. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B084.