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Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that control a diverse range of biological processes during development and in adult tissues. We recently reported that somatic FGFR2 mutations are associated with shorter survival in endometrial cancer. However, little is known about how these FGFR2 mutations contribute to endometrial cancer metastasis. Here, we report that expression of the activating mutations FGFR2N550K and FGFR2Y376C in an endometrial cancer cell model induce Golgi fragmentation, and loss of polarity and directional migration. In mutant FGFR2-expressing cells, this was associated with an inability to polarise intracellular pools of FGFR2 towards the front of migrating cells. Such polarization defects were exacerbated in three-dimensional culture, where FGFR2 mutant cells were unable to form well-organised acini, instead undergoing exogenous ligand-independent invasion. Our findings uncover collective cell polarity and invasion as common targets of disease-associated FGFR2 mutations that lead to poor outcome in endometrial cancer patients.
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Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Neoplasias do Endométrio/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Biotinilação , Linhagem Celular Tumoral , Movimento Celular/genética , Polaridade Celular/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Quimiotaxia/genética , Quimiotaxia/fisiologia , Neoplasias do Endométrio/genética , Feminino , Imunofluorescência , Células HEK293 , Humanos , Immunoblotting , Lentivirus/genética , Mutação/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
Cells migrating through complex three-dimensional environments experience considerable physical challenges, including tensile stress and compression. To move, cells need to resist these forces while also squeezing the large nucleus through confined spaces. This requires highly coordinated cortical contractility. Microtubules can both resist compressive forces and sequester key actomyosin regulators to ensure appropriate activation of contractile forces. Yet, how these two roles are integrated to achieve nuclear transmigration in three dimensions is largely unknown. Here, we demonstrate that compression triggers reinforcement of a dedicated microtubule structure at the rear of the nucleus by the mechanoresponsive recruitment of cytoplasmic linker-associated proteins, which dynamically strengthens and repairs the lattice. These reinforced microtubules form the mechanostat: an adaptive feedback mechanism that allows the cell to both withstand compressive force and spatiotemporally organize contractility signalling pathways. The microtubule mechanostat facilitates nuclear positioning and coordinates force production to enable the cell to pass through constrictions. Disruption of the mechanostat imbalances cortical contractility, stalling migration and ultimately resulting in catastrophic cell rupture. Our findings reveal a role for microtubules as cellular sensors that detect and respond to compressive forces, enabling movement and ensuring survival in mechanically demanding environments.
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Voltage-gated sodium (NaV) channels are critical regulators of neuronal excitability and are targeted by many toxins that directly interact with the pore-forming α subunit, typically via extracellular loops of the voltage-sensing domains, or residues forming part of the pore domain. Excelsatoxin A (ExTxA), a pain-causing knottin peptide from the Australian stinging tree Dendrocnide excelsa, is the first reported plant-derived NaV channel modulating peptide toxin. Here we show that TMEM233, a member of the dispanin family of transmembrane proteins expressed in sensory neurons, is essential for pharmacological activity of ExTxA at NaV channels, and that co-expression of TMEM233 modulates the gating properties of NaV1.7. These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein, position TMEM233 and the dispanins as accessory proteins that are indispensable for toxin-mediated effects on NaV channel gating, and provide important insights into the function of NaV channels in sensory neurons.
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Toxinas Biológicas , Urtica dioica , Austrália , Dor , Peptídeos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismoRESUMO
BACKGROUND: Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprises four molecular subtypes with differing etiology, prognoses, and responses to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. As pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. METHODS: Here, we report whole exome or whole genome sequencing of 11 PDX models and their matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the copy number high/p53-mutant and mismatch-repair deficient molecular subtypes of EC. RESULTS: PDX models were successfully generated from grade 2/3 tumors, including three uterine carcinosarcomas. The models showed similar histomorphology to the primary tumors and represented all four molecular subtypes of EC, including five mismatch-repair deficient models. The different PDX lineages showed a wide range of inter-tumor and intra-tumor heterogeneity. However, for most PDX models, one arm recapitulated the molecular landscape of the primary tumor without major genomic drift. An in vivo response to talazoparib was detected in four copy number high models. Two models (carcinosarcomas) showed a response consistent with stable disease and two models (one copy number high serous EC and another carcinosarcoma) showed significant tumor growth inhibition, albeit one consistent with progressive disease; however, all lacked the HR deficiency genomic signature. CONCLUSIONS: EC PDX models represent the four molecular subtypes of disease and can capture intra-tumor heterogeneity of the original primary tumor. PDXs of the copy number high molecular subtype showed sensitivity to PARPi; however, deeper and more durable responses will likely require combination of PARPi with other agents.
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Antineoplásicos , Neoplasias do Endométrio , Antineoplásicos/uso terapêutico , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Feminino , Genômica , Xenoenxertos , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific elimination.
Assuntos
Melanoma , Monofenol Mono-Oxigenase , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Melanócitos/metabolismo , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologiaRESUMO
Epithelial networks are commonly generated by processes where multicellular aggregates elongate and branch. Here, we focus on understanding cellular mechanisms for elongation using an organotypic culture system as a model of mammary epithelial anlage. Isotropic cell aggregates broke symmetry and slowly elongated when transplanted into collagen 1 gels. The elongating regions of aggregates displayed enhanced cell proliferation that was necessary for elongation to occur. Strikingly, this locoregional increase in cell proliferation occurred where collagen 1 fibrils reorganized into bundles that were polarized with the elongating aggregates. Applying external stretch as a cell-independent way to reorganize the extracellular matrix, we found that collagen polarization stimulated regional cell proliferation to precipitate symmetry breaking and elongation. This required ß1-integrin and ERK signaling. We propose that collagen polarization supports epithelial anlagen elongation by stimulating locoregional cell proliferation. This could provide a long-lasting structural memory of the initial axis that is generated when anlage break symmetry.
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Proliferação de Células , Colágeno/fisiologia , Células Epiteliais/citologia , HumanosRESUMO
We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.
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Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Análise de Célula Única/métodos , Animais , Células Cultivadas , Humanos , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Plasmídeos , RatosRESUMO
Epithelia are active materials where mechanical tension governs morphogenesis and homeostasis. But how that tension is regulated remains incompletely understood. We now report that caveolae control epithelial tension and show that this is necessary for oncogene-transfected cells to be eliminated by apical extrusion. Depletion of caveolin-1 (CAV1) increased steady-state tensile stresses in epithelial monolayers. As a result, loss of CAV1 in the epithelial cells surrounding oncogene-expressing cells prevented their apical extrusion. Epithelial tension in CAV1-depleted monolayers was increased by cortical contractility at adherens junctions. This reflected a signaling pathway, where elevated levels of phosphoinositide-4,5-bisphosphate (PtdIns(4,5)P2) recruited the formin, FMNL2, to promote F-actin bundling. Steady-state monolayer tension and oncogenic extrusion were restored to CAV1-depleted monolayers when tension was corrected by depleting FMNL2, blocking PtdIns(4,5)P2, or disabling the interaction between FMNL2 and PtdIns(4,5)P2. Thus, caveolae can regulate active mechanical tension for epithelial homeostasis by controlling lipid signaling to the actin cytoskeleton.
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Cavéolas/metabolismo , Células Epiteliais/metabolismo , Proteínas Oncogênicas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células CACO-2 , Caveolina 1/metabolismo , Células Epiteliais/ultraestrutura , Forminas/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Proteínas Oncogênicas/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estresse MecânicoAssuntos
Sistema de Condução Cardíaco/fisiopatologia , Síndrome do QT Longo/fisiopatologia , Sindactilia/fisiopatologia , Potenciais de Ação , Transtorno Autístico , Canais de Cálcio Tipo L/genética , Pré-Escolar , Eletrocardiografia , Feminino , Predisposição Genética para Doença , Humanos , Síndrome do QT Longo/diagnóstico , Síndrome do QT Longo/genética , Mutação , Fenótipo , Sindactilia/diagnóstico , Sindactilia/genéticaAssuntos
Bloqueio de Ramo/etiologia , Neoplasias Cardíacas/complicações , Neoplasias Cardíacas/secundário , Neoplasias Pulmonares/patologia , Taquicardia Ventricular/etiologia , Septo Interventricular/fisiopatologia , Bloqueio de Ramo/diagnóstico , Bloqueio de Ramo/fisiopatologia , Bloqueio de Ramo/terapia , Desfibriladores Implantáveis , Ecocardiografia Transesofagiana , Cardioversão Elétrica/instrumentação , Eletrocardiografia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Prevenção Secundária/instrumentação , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/fisiopatologia , Taquicardia Ventricular/terapia , Septo Interventricular/diagnóstico por imagemRESUMO
Endometrial cancer is the most commonly diagnosed gynaecological malignancy. Unfortunately, 15-20% of women demonstrate persistent or recurrent tumours that are refractory to current chemotherapies. We previously identified activating mutations in fibroblast growth factor receptor 2 (FGFR2) in 12% (stage I/II) to 17% (stage III/IV) endometrioid ECs and found that these mutations are associated with shorter progression-free and cancer-specific survival. Although FGFR inhibitors are undergoing clinical trials for treatment of several cancer types, little is known about the mechanism by which they induce cell death. We show that treatment with BGJ398, AZD4547 and PD173074 causes mitochondrial depolarization, cytochrome c release and impaired mitochondrial respiration in two FGFR2-mutant EC cell lines (AN3CA and JHUEM2). Despite this mitochondrial dysfunction, we were unable to detect caspase activation following FGFR inhibition; in addition, the pan-caspase inhibitor Z-VAD-FMK was unable to prevent cell death, suggesting that the cell death is caspase-independent. Furthermore, while FGFR inhibition led to an increase in LC3 puncta, treatment with bafilomycin did not further increase lipidated LC3, suggesting that FGFR inhibition led to a block in autophagosome degradation. We confirmed that cell death is mitochondrial-dependent as it can be blocked by overexpression of Bcl-2 and/or Bcl-XL. Importantly, we show that combining FGFR inhibitors with the BH3 mimetics ABT737/ABT263 markedly increased cell death in vitro and is more effective than BGJ398 alone in vivo, where it leads to marked tumour regression. This work may have implications for the design of clinical trials to treat a wide range of patients with FGFR-dependent malignancies.
Assuntos
Apoptose , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Mitocôndrias/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Autofagossomos/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
The importance of studying cancer cell invasion is highlighted by the fact that 90% of all cancer-related mortalities are due to metastatic disease. Melanoma metastasis is driven fundamentally by aberrant cell motility within three-dimensional or confined environments. Within this realm of cell motility, cytokines, growth factors, and their receptors are crucial for engaging signaling pathways, which both mediate crosstalk between cancer, stromal, and immune cells in addition to interactions with the surrounding microenvironment. Recently, the study of the mechanical biology of tumor cells, stromal cells and the mechanics of the microenvironment have emerged as important themes in driving invasion and metastasis. While current anti-melanoma therapies target either the MAPK signaling pathway or immune checkpoints, there are no drugs available that specifically inhibit motility and thus invasion and dissemination of melanoma cells during metastasis. One of the reasons for the lack of so-called "migrastatics" is that, despite decades of research, the precise biology of metastatic disease is still not fully understood. Metastatic disease has been traditionally lumped into a single classification, however what is now emergent is that the biology of melanoma metastasis is highly diverse, heterogeneous and exceedingly dynamic-suggesting that not all cases are created equal. The following mini-review discusses melanoma heterogeneity in the context of the emergent theme of mechanobiology and how it influences the tumor-stroma crosstalk during metastasis. Thus, highlighting future therapeutic options for migrastatics and mechanomedicines in the prevention and treatment of metastatic melanoma.
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Improved therapeutic approaches are needed for the treatment of recurrent and metastatic endometrial cancer. Endometrial cancers display hyperactivation of the MAPK and PI3K pathways, the result of somatic aberrations in genes such as FGFR2, KRAS, PTEN, PIK3CA, and PIK3R1 The FGFR2 and PI3K pathways, have emerged as potential therapeutic targets in endometrial cancer. Activation of the PI3K pathway is seen in more than 90% of FGFR2mutant endometrial cancers. This study aimed to examine the efficacy of the pan-FGFR inhibitor BGJ398 with pan-PI3K inhibitors (GDC-0941, BKM120) and the p110α-selective inhibitor BYL719. We assessed synergy in three FGFR2mutant endometrial cancer cell lines (AN3CA, JHUEM2, and MFE296), and the combination of BGJ398 and GDC-0941 or BYL719 showed strong synergy. A significant increase in cell death and decrease in long-term survival was seen when PI3K inhibitors were combined with BGJ398. Importantly, these effects were seen at low concentrations correlating to only partial inhibition of AKT. The combination of BGJ398 and GDC-0941 showed tumor regressions in vivo, whereas each drug alone only showed moderate tumor growth inhibition. BYL719 alone resulted in increased tumor growth of AN3CA xenografts but in combination with BGJ398 resulted in tumor regression in both AN3CA- and JHUEM2-derived xenografts. These data provide evidence that subtherapeutic doses of PI3K inhibitors enhance the efficacy of anti-FGFR therapies, and a combination therapy may represent a superior therapeutic treatment in patients with FGFR2mutant endometrial cancer. Mol Cancer Ther; 16(4); 637-48. ©2017 AACR.