ABSTRACT
The Eya family of proteins (consisting of Eyas1-4 in mammals) play vital roles in embryogenesis by regulating processes such as proliferation, migration/invasion, cellular survival and pluripotency/plasticity of epithelial and mesenchymal states. Eya proteins carry out such diverse functions through a unique combination of transcriptional co-factor, Tyr phosphatase, and PP2A/B55α-mediated Ser/Thr phosphatase activities. Since their initial discovery, re-expression of Eyas has been observed in numerous tumor types, where they are known to promote tumor progression through a combination of their transcriptional and enzymatic activities. Eya proteins thus reinstate developmental processes during malignancy and represent a compelling class of therapeutic targets for inhibiting tumor progression.
Subject(s)
Neoplasms , Protein Tyrosine Phosphatases , Humans , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/metabolism , Protein Tyrosine Phosphatases/metabolism , Protein Tyrosine Phosphatases/genetics , Animals , Gene Expression Regulation, Neoplastic , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Eye Proteins/metabolism , Eye Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/geneticsABSTRACT
Ewing sarcoma (ES), which is characterized by the presence of oncogenic fusion proteins such as EWS/FLI1, is an aggressive pediatric malignancy with a high rate of early dissemination and poor outcome after distant spread. Here we demonstrate that the SIX1 homeoprotein, which enhances metastasis in most tumor types, suppresses ES metastasis by co-regulating EWS/FLI1 target genes. Like EWS/FLI1, SIX1 promotes cell growth/transformation, yet dramatically inhibits migration and invasion, as well as metastasis in vivo. We show that EWS/FLI1 promotes SIX1 protein expression, and that the two proteins share genome-wide binding profiles and transcriptional regulatory targets, including many metastasis-associated genes such as integrins, which they co-regulate. We further show that SIX1 downregulation of integrins is critical to its ability to inhibit invasion, a key characteristic of metastatic cells. These data demonstrate an unexpected anti-metastatic function for SIX1, through coordinate gene regulation with the key oncoprotein in ES, EWS/FLI1.
Subject(s)
Sarcoma, Ewing , Humans , Child , Sarcoma, Ewing/pathology , Gene Regulatory Networks , Cell Line, Tumor , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS/genetics , Gene Expression Regulation , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Integrins/metabolism , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolismABSTRACT
Necroptosis is a form of programmed necrotic cell death in which a signaling cascade induces oligomerization of mixed lineage kinase domain-like (MLKL) protein, leading to plasma membrane rupture. Necroptotic cell death is recognized as important for protection against viral infection and has roles in a variety of diseases, including cancer and diabetes. Despite its relevance to health and disease states, many questions remain about the precise mechanism of necroptotic cell death, cellular factors that can protect cells from necroptosis, and the role of necroptosis in disease models. In this study, we engineered a light-activated version of MLKL that rapidly oligomerizes and is recruited to the plasma membrane in cells exposed to light, inducing rapid cell death. We demonstrate this tool can be controlled spatially and temporally, used in a chemical genetic screen to identify chemicals and pathways that protect cells from MLKL-induced cell death, and used to study signaling responses of non-dying bystander cells. In additional studies, we re-engineered MLKL to block its cell-killing capacity but retain light-mediated membrane recruitment, developing a new single-component optogenetic tool that allows modulation of protein function at the plasma membrane.
