RESUMEN
In ovarian cancer, the prometastatic RTK AXL promotes motility, invasion and poor prognosis. Here, we show that reduced survival caused by AXL overexpression can be mitigated by the expression of the GPI-anchored tumour suppressor OPCML Further, we demonstrate that AXL directly interacts with OPCML, preferentially so when AXL is activated by its ligand Gas6. As a consequence, AXL accumulates in cholesterol-rich lipid domains, where OPCML resides. Here, phospho-AXL is brought in proximity to the lipid domain-restricted phosphatase PTPRG, which de-phosphorylates the RTK/ligand complex. This prevents AXL-mediated transactivation of other RTKs (cMET and EGFR), thereby inhibiting sustained phospho-ERK signalling, induction of the EMT transcription factor Slug, cell migration and invasion. From a translational perspective, we show that OPCML enhances the effect of the phase II AXL inhibitor R428 in vitro and in vivo We therefore identify a novel mechanism by which two spatially restricted tumour suppressors, OPCML and PTPRG, coordinate to repress AXL-dependent oncogenic signalling.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Tirosina Fosfatasas Clase 5 Similares a Receptores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Benzocicloheptenos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Pollos , Colesterol/metabolismo , Activación Enzimática/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Trompas Uterinas/patología , Femenino , Proteínas Ligadas a GPI/metabolismo , Silenciador del Gen/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Microdominios de Membrana/metabolismo , Invasividad Neoplásica , Neoplasias Ováricas/patología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Resultado del Tratamiento , Triazoles/farmacología , Tirosina Quinasa del Receptor AxlRESUMEN
Neutrophil migration is vital for immunity and precedes effector functions such as pathogen killing. Here, we report that this process is regulated by the Rab27a GTPase, a protein known to control granule exocytosis. Rab27a-deficient (Rab27a KO) neutrophils exhibit migration defects in vitro and in vivo, and live-cell microscopy suggests that delayed uropod detachment causes the migratory defect. Surface expression of CD11b, a key adhesion molecule, is increased in chemokine-stimulated Rab27a KO neutrophils compared with the control, suggesting a turnover delay caused by a defect in elastase secretion from azurophilic granules at the rear of bone marrow polymorphonuclear leukocytes (BM-PMNs). We suggest that Rab27a-dependent protease secretion regulates neutrophil migration through proteolysis-dependent de-adhesion of uropods, a mechanism that could be conserved in cell migration and invasion.
Asunto(s)
Neutrófilos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Movimiento Celular , Ratones , Ratones Noqueados , Ratones Transgénicos , Neutrófilos/citología , Proteínas de Unión al GTP rab/deficiencia , Proteínas rab27 de Unión a GTPRESUMEN
The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Anticuerpos Monoclonales/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rab GTPases are master regulators of intracellular trafficking and, in recent years, their role in the control of different aspects of tumour progression has emerged. In the present review, we show that Rab GTPases are disregulated in many cancers and have central roles in tumour cell migration, invasion, proliferation, communication with stromal cells and the development of drug resistance. As a consequence, Rab proteins may be novel potential candidates for the development of anticancer drugs and, in this context, the preliminary results obtained with an inhibitor of Rab function are also discussed.
Asunto(s)
Neoplasias/enzimología , Proteínas de Unión al GTP rab/fisiología , Animales , Ciclo Celular , Movimiento Celular , Progresión de la Enfermedad , Resistencia a Antineoplásicos , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Transporte de Proteínas , Transducción de SeñalRESUMEN
Proteolytic degradation of the extracellular matrix (ECM) is one intrinsic property of metastatic tumor cells to breach tissue barriers and to disseminate into different tissues. This process is initiated by the formation of invadopodia, which are actin-driven, finger-like membrane protrusions. Yet, little is known on how invadopodia are endowed with the functional machinery of proteolytic enzymes [1, 2]. The key protease MT1-MMP (membrane type 1-matrix metalloproteinase) confers proteolytic activity to invadopodia and thus invasion capacity of cancer cells [3-6]. Here, we report that MT1-MMP-dependent matrix degradation at invadopodia is regulated by the v-SNARE TI-VAMP/VAMP7, hence providing the molecular inventory mediating focal degradative activity of cancer cells. As observed by TIRF microscopy, MT1-MMP-mCherry and GFP-VAMP7 were simultaneously detected at proteolytic sites. Functional ablation of VAMP7 decreased the ability of breast cancer cells to degrade and invade in a MT1-MMP-dependent fashion. Moreover, the number of invadopodia was dramatically decreased in VAMP7- and MT1-MMP-depleted cells, indicative of a positive-feedback loop in which the protease as a cargo of VAMP7-targeted transport vesicles regulates maturation of invadopodia. Collectively, these data point to a specific role of VAMP7 in delivering MT1-MMP to sites of degradation, maintaining the functional machinery required for invasion.
Asunto(s)
Neoplasias de la Mama/patología , Extensiones de la Superficie Celular/enzimología , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas R-SNARE/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Humanos , Metaloproteinasa 14 de la Matriz/genéticaRESUMEN
OPCML is a highly conserved glycosyl phosphatidylinositol (GPI)-anchored protein belonging to the IgLON family of cell adhesion molecules. OPCML functions as a tumor suppressor and is silenced in over 80% of ovarian cancers by loss of heterozygosity and by epigenetic mechanisms. OPCML inactivation is also observed in many other cancers suggesting a conservation of tumor suppressor function. Although epigenetic silencing and subsequent loss of OPCML expression correlate with poor progression-free and overall patient survival, its mechanism of action is only starting to be fully elucidated. Recent discoveries have demonstrated that OPCML exerts its tumor suppressor effect by inhibiting several cancer hallmark phenotypes in vitro and abrogating tumorigenesis in vivo, by downregulating/inactivating a specific spectrum of Receptor Tyrosine Kinases (RTKs), including EphA2, FGFR1, FGFR3, HER2, HER4, and AXL. This modulation of RTKs can also sensitize ovarian and breast cancers to lapatinib, erlotinib, and anti-AXL therapies. Furthermore, OPCML has also been shown to function in synergy with the tumor suppressor phosphatase PTPRG to inactivate pro-metastatic RTKs such as AXL. Recently, the identification of inactivating point mutations and the elucidation of the crystal structure of OPCML have provided valuable insights into its structure-function relationships, giving rise to its potential as an anti-cancer therapeutic.
Asunto(s)
Moléculas de Adhesión Celular/efectos de los fármacos , Glicosilfosfatidilinositoles/uso terapéutico , Neoplasias/tratamiento farmacológico , Proteínas Ligadas a GPI/efectos de los fármacos , Glicosilfosfatidilinositoles/farmacología , HumanosRESUMEN
OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 Å resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers.
Asunto(s)
Moléculas de Adhesión Celular/genética , Epigénesis Genética , Neoplasias Ováricas/genética , Moléculas de Adhesión Celular/química , Transformación Celular Neoplásica , Cristalografía por Rayos X , Femenino , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Glicosilación , Humanos , Mutación Missense , Invasividad Neoplásica , Agregación Patológica de Proteínas/genética , Estructura Terciaria de ProteínaAsunto(s)
Endocitosis/fisiología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/metabolismo , Animales , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Modelos Biológicos , Transporte de Proteínas , Proteínas/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
Objective: Apolipoprotein A1 (ApoA1) is remarkably decreased in serum and ovarian tissues of ovarian cancer patients. ApoA1 and ApoA1 mimetic peptides can sequestrate pro-inflammatory phospholipids, some of which are known to activate a variety of oncogenic pathways. Besides, more intrinsic anti-tumorigenic properties, independent from interaction with lipids, have also been described for ApoA1. We aimed to disclose the effects of ApoA1 and a mimetic peptide on the malignant phenotype of ovarian cancer cells, particularly regarding cell viability, invasiveness and platinum sensitization. Methods: Cells viability was assessed by MTS assay. Extracellular matrix invasion was assessed by transwell and spheroid invasion assays. Western blotting was performed to evaluate the effect of test compounds on intracellular pathways. Sensitization assays were performed in vitro and in the biologically relevant in ovo chorioallantoic membrane model. Results: Both ApoA1 and the mimetic peptide, at a concentration of 100 µg/mL, were able to decrease the viability of SKOV3, CAOV3, and OVCAR3 cells (p < 0.05). The peptide at this concentration was not able to affect the viability of immortalized non-neoplastic ovarian cells (p > 0.05). ApoA1 decreased SKOV3 cells invasiveness at 300 µg/mL after 72 and 96 h of exposure (p < 0.05), while the ApoA1 mimetic peptide prevented cell invasion at 50 and 100 µg/mL (p < 0.01). Treatment with 100 µg/mL of ApoA1 mimetic peptide decreased Akt phosphorylation in SKOV3 cells (p < 0.01). Accordingly, treatment with increasing concentrations of the peptide sensitized SKOV3, OVCAR3 and CAOV3 cells to cisplatin. This synergistic effect was observed both in vitro and in ovo. Conclusions: These results support the role of ApoA1 and ApoA1 mimetics as suppressors of ovarian tumorigenesis and as chemo-sensitising agents.
RESUMEN
The Ras superfamily of small monomeric GTPases includes some of the most prominent cancer targets for which no selective therapeutic agent has yet been successfully developed. The turn of the millennium saw a resurgence of efforts to target these enzymes using new and improved biophysical techniques to overcome the perceived difficulties of insurmountably high affinity for guanosine nucleotides and flat, flexible topology lacking suitable pockets for small molecule inhibitors. Further, recent investigations have begun to probe the dynamic conformational status of GTP-bound Ras, opening up new mechanisms of inhibition. While much of the literature has focused on the oncogenic Ras proteins, particularly K-Ras, these represent only a small minority of therapeutically interesting targets within the superfamily; for example, the Rab GTPases are the largest subfamily of about 70 members, and present an as yet untapped class of potential targets. The present review documents the key methodologies employed to date in structure-guided attempts to drug the Ras GTPases, and forecasts their transferability to other similarly challenging proteins in the superfamily.
Asunto(s)
Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Proteínas ras/efectos de los fármacos , Estructura Molecular , Proteínas ras/químicaRESUMEN
Opioid-binding protein/cell adhesion molecule-like (OPCML) is a tumor-suppressor gene that is frequently inactivated in ovarian cancer and many other cancers by somatic methylation. We have previously shown that OPCML exerts its suppressor function by negatively regulating a spectrum of receptor tyrosine kinases (RTK), such as ErbB2/HER2, FGFR1, and EphA2, thus attenuating their related downstream signaling. The physical interaction of OPCML with this defined group of RTKs is a prerequisite for their downregulation. Overexpression/gene amplification of EGFR and HER2 is a frequent event in multiple cancers, including ovarian and breast cancers. Molecular therapeutics against EGFR/HER2 or EGFR only, such as lapatinib and erlotinib, respectively, were developed to target these receptors, but resistance often occurs in relapsing cancers. Here we show that, though OPCML interacts only with HER2 and not with EGFR, the interaction of OPCML with HER2 disrupts the formation of the HER2-EGFR heterodimer, and this translates into a better response to both lapatinib and erlotinib in HER2-expressing ovarian and breast cancer cell lines. Also, we show that high OPCML expression is associated with better response to lapatinib therapy in breast cancer patients and better survival in HER2-overexpressing ovarian cancer patients, suggesting that OPCML co-therapy could be a valuable sensitizing approach to RTK inhibitors. Mol Cancer Ther; 16(10); 2246-56. ©2017 AACR.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Moléculas de Adhesión Celular/genética , Receptores ErbB/genética , Neoplasias Ováricas/tratamiento farmacológico , Receptor ErbB-2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib/administración & dosificación , Femenino , Proteínas Ligadas a GPI/genética , Amplificación de Genes/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Lapatinib , Terapia Molecular Dirigida , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinazolinas/administración & dosificación , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab/administración & dosificaciónRESUMEN
Ovarian cancer is a complex disease with heterogeneity among the gene expression molecular subtypes (GEMS) between patients. Patients with tumors of a mesenchymal ("Mes") subtype have a poorer prognosis than patients with tumors of an epithelial ("Epi") subtype. We evaluated GEMS of ovarian cancer patients for molecular signaling profiles and assessed how the differences in these profiles could be leveraged to improve patient clinical outcome. Kinome enrichment analysis identified AXL as a particularly abundant kinase in Mes-subtype tumor tissue and cell lines. In Mes cells, upon activation by its ligand GAS6, AXL coclustered with and transactivated the receptor tyrosine kinases (RTKs) cMET, EGFR, and HER2, producing sustained extracellular signal-regulated kinase (ERK) activation. In Epi-A cells, AXL was less abundant and induced a transient activation of ERK without evidence of RTK transactivation. AXL-RTK crosstalk also stimulated sustained activation of the transcription factor FRA1, which correlated with the induction of the epithelial-mesenchymal transition (EMT)-associated transcription factor SLUG and stimulation of motility exclusively in Mes-subtype cells. The AXL inhibitor R428 attenuated RTK and ERK activation and reduced cell motility in Mes cells in culture and reduced tumor growth in a chick chorioallantoic membrane model. A higher concentration of R428 was needed to inhibit ERK activation and cell motility in Epi-A cells. Silencing AXL in Mes-subtype cells reversed the mesenchymal phenotype in culture and abolished tumor formation in an orthotopic xenograft mouse model. Thus, AXL-targeted therapy may improve clinical outcome for patients with Mes-subtype ovarian cancer.
Asunto(s)
Benzocicloheptenos/farmacología , Movimiento Celular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Triazoles/farmacología , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Embrión de Pollo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Sistema de Señalización de MAP Quinasas/genética , Ratones , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
The mechanism(s) by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1) as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP), is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.
Asunto(s)
Melanocitos/metabolismo , Melanosomas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Humanos , Immunoblotting , Ratones , Microscopía Confocal , Mutación , Unión Proteica , Interferencia de ARN , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
BACKGROUND: Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs) in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1ß/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1ß/IL-18, neutrophilia and subsequent disease exacerbations. METHODS: To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis. RESULTS: Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1ß/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia. CONCLUSIONS: This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.
Asunto(s)
Asma/metabolismo , Micropartículas Derivadas de Células/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Infecciones del Sistema Respiratorio/metabolismo , Adenosina Trifosfato/farmacología , Animales , Asma/complicaciones , Caspasas/metabolismo , Línea Celular Tumoral , Micropartículas Derivadas de Células/efectos de los fármacos , Micropartículas Derivadas de Células/ultraestructura , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Receptores Purinérgicos P2X7/metabolismo , Infecciones del Sistema Respiratorio/complicacionesRESUMEN
Mediator release from mast cells is a critical step in allergic and inflammatory disease. However, the processes regulating the latter stages of granule release are yet to be fully understood. Rab27 small GTPases regulate release of secretory lysosomes in a variety of cells, including mast cell granules. In the present study, using murine bone marrow-derived mast cells (BMMC) from Rab27-deficient mutant mice, we found that, in contrast to Rab27b, Rab27a primarily plays an inhibitory role in regulating degranulation. Immunofluorescence analysis revealed that resting Rab27a-deficient (ashen) BMMCs display abnormal cortical F-actin distribution. Actin disassembly prior to IgE cross-linking increased wild-type BMMC secretion to ashen levels, suggesting that changes in the integrity of cortical F-actin underlie the ashen phenotype. Comparison of the secretory impairment of Rab27b knockout and Rab27a/b double knockout BMMCs highlighted a secondary positive role for Rab27a in enhancing degranulation. Rab27 is known to interact with actin via its effectors melanophilin (Mlph) and myosin Va (MyoVa) in other cell types. To better understand the differing roles of Rab27 proteins, we analysed the secretory phenotype of BMMCs derived from mice lacking Rab27 effector proteins. These experiments revealed that the phenotype of BMMCs deficient in Mlph (leaden) and BMMCs deficient in MyoVa (dilute) resembles the hyper-secretion of ashen BMMCs, while Munc13-4-deficient (jinx) BMMCs phenocopy the Rab27b knockout and double Rab27a/b knockout secretory impairment. We conclude that Rab27a and Rab27b regulate distinct steps in the BMMC degranulation pathway, with Rab27a/Mlph/MyoVa regulating cortical actin stability upstream of Rab27a/b/Munc13-4-dependent granule exocytosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mastocitos/metabolismo , Proteínas de la Membrana/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Degranulación de la Célula/genética , Degranulación de la Célula/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Exocitosis/genética , Exocitosis/fisiología , Femenino , Immunoblotting , Masculino , Mastocitos/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/genética , Receptores de IgE/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context.
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Exosomas/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Microambiente Tumoral , Proteínas de Unión al GTP rab/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Quimiocinas/metabolismo , Citocinas/metabolismo , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Metaloproteinasas de la Matriz Secretadas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Neutrófilos/patología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Tumoral/genética , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTPRESUMEN
Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Línea Celular , Línea Celular Tumoral , Reactivos de Enlaces Cruzados/farmacología , Femenino , Humanos , Metaloproteinasa 14 de la Matriz/metabolismo , Modelos Biológicos , Proteínas Mutantes/metabolismo , Invasividad Neoplásica , Unión Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismo , Transporte de Proteínas/efectos de los fármacos , Seudópodos/efectos de los fármacos , Seudópodos/enzimología , Proteínas Activadoras de ras GTPasa/químicaRESUMEN
Staphylococcus aureus nuclease is a small, secreted protein which has been successfully used as a reporter system to identify exported products in Lactococcus lactis. Here, biochemical evidence is provided that the nuclease is exported by Mycobacterium smegmatis in the presence, but also in the absence of a signal sequence, and thus probably independently of the Sec translocation pathway. This implies that the nuclease should not be used as a reporter system in mycobacteria for the identification of exported products, despite what has been reported previously in the literature. The nuclease can be extended to create hybrid proteins that remain compatible with its secretion, whereas some other shorter fusions are not tolerated. This suggests that correct folding is required for efficient export. Extensive mutational analysis did not identify a specific secretion pathway. This suggests that the nuclease may be exported by different redundant systems or that components of this alternative Sec pathway are essential for bacterial survival.
Asunto(s)
Nucleasa Microcócica/metabolismo , Mycobacterium smegmatis/genética , Secuencia de Bases , ADN Bacteriano/genética , Genes Reporteros , Nucleasa Microcócica/química , Nucleasa Microcócica/genética , Mycobacterium smegmatis/fisiología , Pliegue de Proteína , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Rv1395 is annotated as a potential transcriptional regulator of the AraC family. The Rv1395 insertional mutant was identified in a signature tag mutagenesis study in Mycobacterium tuberculosis and was shown to be attenuated in the lungs of mice. Here, we used comparative genomics and biochemical methods to show that Rv1395 is unique to the M. tuberculosis complex and that it encodes a protein that binds the region between two divergent genes, a member of the cytochrome P450 family (Rv1394c or cyp132) and Rv1395 itself. Rv1395 binds to this DNA region by its helix-turn-helix-containing C-terminal domain, and it recognizes two sites with different affinity. We identified the transcriptional start points (TSP) of Rv1394c and Rv1395: both genes have two TSPs, three of which are located in the intergenic region. We constructed and compared various transcriptional fusions consisting of the promoter regions and a reporter gene in Mycobacterium smegmatis: this showed that Rv1395 induces the expression of the cytochrome P450 gene (Rv1394c) and represses its own transcription. This was confirmed in M. tuberculosis when the wild type and a Rv1395-overexpressing strain were used as hosts for the fusions. Site-directed mutagenesis showed that Rv1395 binds to the two sites in a co-operative manner and that binding to both sites is required for Rv1395 optimal activity. A model describing the potential mode of action of Rv1395 is discussed.