ABSTRACT
The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.
Subject(s)
Autophagy , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Signal Transduction , Animals , CHO Cells , Cricetulus , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/ultrastructure , HEK293 Cells , Humans , Lysosomes/metabolism , Male , Mice , Models, Biological , Phenotype , Protein Binding , Protein Domains , Rats, Wistar , Regulatory-Associated Protein of mTOR/metabolism , Sequestosome-1 Protein/metabolismABSTRACT
Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal lining of the upper aerodigestive tract and display few treatment options in advanced stages. Despite increased knowledge of HNSCC molecular biology, the identification of new players involved in triggering HNSCC recurrence and metastatic disease is needed. We uncover that G-protein-coupled receptor kinase-2 (GRK2) expression is reduced in undifferentiated, high-grade human HNSCC tumors, whereas its silencing in model human HNSCC cells is sufficient to trigger epithelial-to-mesenchymal transition (EMT) phenotypic features, an EMT-like transcriptional program and enhanced lymph node colonization from orthotopic tongue tumors in mice. Conversely, enhancing GRK2 expression counteracts mesenchymal cells traits by mechanisms involving phosphorylation and decreased functionality of the key EMT inducer Snail1. Our results suggest that GRK2 safeguards the epithelial phenotype, whereas its downregulation contributes to the activation of EMT programs in HNSCC.
Subject(s)
G-Protein-Coupled Receptor Kinase 2/metabolism , Head and Neck Neoplasms/enzymology , Head and Neck Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/enzymology , Squamous Cell Carcinoma of Head and Neck/pathology , Animals , Cell Line, Tumor , Disease Progression , Down-Regulation , Epithelial Cells/enzymology , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition , G-Protein-Coupled Receptor Kinase 2/biosynthesis , G-Protein-Coupled Receptor Kinase 2/genetics , Head and Neck Neoplasms/genetics , Heterografts , Humans , Mice , Mice, Nude , Phosphorylation , Snail Family Transcription Factors/metabolism , Squamous Cell Carcinoma of Head and Neck/geneticsABSTRACT
PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.
Subject(s)
Breast Neoplasms/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Microtubule-Associated Proteins/biosynthesis , Neoplasm Proteins/metabolism , Protein Phosphatase 2/metabolism , Protein Serine-Threonine Kinases/biosynthesis , Animals , Breast Neoplasms/genetics , Breast Neoplasms/mortality , Breast Neoplasms/therapy , Cell Line, Tumor , Female , Gene Knockdown Techniques , Humans , Mice , Mice, Nude , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , Protein Phosphatase 2/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/geneticsABSTRACT
Greatwall is a protein kinase involved in the inhibition of protein phosphatase 2 (PP2A)-B55 complexes to maintain the mitotic state. Although its biochemical activity has been deeply characterized in Xenopus, its specific relevance during the progression of mitosis is not fully understood. By using a conditional knockout of the mouse ortholog, Mastl, we show here that mammalian Greatwall is essential for mouse embryonic development and cell cycle progression. Yet, Greatwall-null cells enter into mitosis with normal kinetics. However, these cells display mitotic collapse after nuclear envelope breakdown (NEB) characterized by defective chromosome condensation and prometaphase arrest. Intriguingly, Greatwall is exported from the nucleus to the cytoplasm in a CRM1-dependent manner before NEB. This export occurs after the nuclear import of cyclin B-Cdk1 complexes, requires the kinase activity of Greatwall, and is mediated by Cdk-, but not Polo-like kinase 1-dependent phosphorylation. The mitotic collapse observed in Greatwall-deficient cells is partially rescued after concomitant depletion of B55 regulatory subunits, which are mostly cytoplasmic before NEB. These data suggest that Greatwall is an essential protein in mammals required to prevent mitotic collapse after NEB.
