SUMMARY SIX1 interacts with EYA to form a bipartite transcription factor essential for development. Loss of function of this complex causes branchio-oto-renal syndrome (BOR), while re-expression of SIX1 or EYA promotes metastasis. Here we describe the 2.0 Å structure of SIX1 bound to EYA2, which suggests a novel DNA binding mechanism for SIX1 and provides a rationale for the effect of BOR syndrome mutations. The structure also reveals that SIX1 uses predominantly a single helix to interact with EYA. Substitution of a single amino acid in this helix is sufficient to disrupt the SIX1–EYA interaction, SIX1-mediated epithelial-mesenchymal transition and metastasis in mouse models. Given that SIX1 and EYA are co-overexpressed in many tumor types, our data indicate that targeting the SIX1–EYA complex may be a potent approach to inhibit tumor progression in multiple cancer types.

Epithelial-to-mesenchymal transition (EMT) is a dynamic process that relies on cellular plasticity. Recently, the process of an oncogenic EMT, followed by a reverse mesenchymal-to-epithelial transition (MET), has been implicated as critical in the metastatic colonization of carcinomas. Unlike governance of epithelial programming, regulation of mesenchymal programming is not well understood in EMT. Here, we describe and characterize the first microRNA that enhances exclusively mesenchymal programming. We demonstrate that microRNA-424 is upregulated early during a TWIST1 or SNAI1-induced EMT, and that it causes cells to express mesenchymal genes without affecting epithelial genes, resulting in a mixed/intermediate EMT. Furthermore, microRNA-424 increases motility, decreases adhesion and induces a growth arrest, changes associated with a complete EMT, that can be reversed when microRNA-424 expression is lowered, concomitant with an MET-like process. Breast cancer patient microRNA-424 levels positively associate with TWIST1/2 and EMT-like gene signatures, and miR-424 is increased in primary tumors versus matched normal breast. However, microRNA-424 is downregulated in patient metastases versus matched primary tumors. Correspondingly, microRNA-424 decreases tumor initiation and is post-transcriptionally downregulated in macrometastases in mice, suggesting the need for biphasic expression of miR-424 to transit the EMT-MET axis. Next-generation RNA sequencing revealed microRNA-424 regulates numerous EMT and cancer stemness-associated genes, including TGFBR3, whose downregulation promotes mesenchymal phenotypes, but not tumor-initiating phenotypes. Instead, we demonstrate that increased MAPK/ERK signaling is critical for miR-424-mediated decreases in tumor-initiating phenotypes. These findings suggest microRNA-424 plays distinct roles in tumor progression, potentially facilitating earlier, but repressing later, stages of metastasis by regulating an EMT-MET axis.

Background Although binge drinking peaks at age 21 to 25 years, there is limited research on the effects of serious games in this population, as well as on the process by which playing serious games impacts alcohol-related outcomes. Designed with both health behavioral theory and game theory, One Shot is an online serious game that aims to prevent binge drinking. Objective This study utilized a conceptual model for serious video game processes. Using One Shot, the model assessed the following process stages: (1) Alcohol Use Disorders Identification Test-Concise (AUDIT-C); (2) in-game factors of game time and risky alcohol decisions; (3) game enjoyment; and (4) postgame outcomes of intention to drink less and drinking refusal self-efficacy. Methods In a one-group pretest-posttest quasi-experimental design, a sample (N=550) of young adults (age 21-25 years) who reported recent binge drinking played the One Shot game. Intention to drink less and drinking refusal self-efficacy were measured at pregame and postgame, with their effects lagged in statistical analysis. Participants were presented with various scenarios in the game that pertained to risky alcohol decisions, which, along with game time, were unobtrusively recorded by the server. A structural equation model (SEM) was used to test the conceptual model, with assessments made to determine if enjoyment mediated the effects of game time and risky alcohol decisions on the 2 postgame alcohol-related outcomes. Results A well-fitting SEM demonstrated support for the multistep model, with AUDIT-C predicting risky alcohol decisions (β=.30). Risky alcohol decisions (β=−.22) and game time (β=.18) predicted enjoyment, which, in turn, predicted intention to drink less (β=.21) and drinking refusal self-efficacy (β=.16). Enjoyment significantly (P<.001) mediated the effects of game time and risky alcohol decision on intention to drink less and drinking refusal self-efficacy. Conclusions The results support a conceptual model in which staggered individual and in-game factors influence alcohol-related outcomes. Enjoyment is important for participants’ intentions to drink less and beliefs that they can refuse alcohol.

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.