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Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.
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Neoplasias Hepáticas , Hígado , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/cirugía , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Masculino , Femenino , Persona de Mediana Edad , Anciano , Hígado/patología , Hígado/metabolismo , Biopsia , Estadificación de Neoplasias , Pancreatectomía , Trampas Extracelulares/metabolismo , PronósticoRESUMEN
OBJECTIVES: To assess the effect of Tideglusib and CHIR99021 small molecules on the odontogenic differentiation potential of human dental pulp stem cells (hDPSCs) via Wnt/ß-catenin pathway activation. METHODOLOGY: hDPSCs were isolated from impacted third molars indicated for extraction and were characterized by flow cytometry. hDPSCs were then induced to differentiate into odontogenic lineage in the presence of Tideglusib and CHIR99021. Odontogenic differentiation was evaluated using Alizarin Red stain and RT-PCR for expression of odontogenic specific differentiation markers: DSPP, DMP1, ALP, OPN, and RUNX2 in relation to undifferentiated cells. RT-PCR was also conducted to assess the expression of Wnt/ß-catenin pathway activation marker (AXIN2). One-way ANOVA Kruskal-Wallis test was used for statistical analysis. RESULTS: Wnt/ß-catenin pathway was successfully activated by Tideglusib and CHIR99021 in hDPSCs where AXIN2 was significantly upregulated. Successful odontogenic differentiation was confirmed by Alizarin Red staining of calcified nodules. RT-PCR for odontogenic differentiation markers DSPP, DMP1, and RUNX expression by hDPSCs induced by CHIR99021 was higher than that expressed by hDPSCs induced by Tideglusib, whereas expression of OPN and ALP was higher in Tideglusib-induced cells than in CHIR99021-induced cells. CONCLUSIONS: Both small molecules successfully induced odontogenic differentiation of hDPSCs through Wnt/ß-catenin pathway activation. CLINICAL RELEVANCE: These findings suggest that Tideglusib and CHIR99021 can be applied clinically in pulp regeneration to improve strategies for vital pulp regeneration and to promote dentine repair.
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Pulpa Dental , beta Catenina , Humanos , Regeneración , Antígenos de Diferenciación , Células MadreRESUMEN
Small molecules have demonstrated promising results as successful alternatives to growth factors. In this study, focus was drawn to CHIR99021 and tideglusib as GSK-3 inhibitors known for their anti-inflammatory and regenerative potential. The effect of both tideglusib and CHIR99021 on the proliferation, viability, and stemness of human dental pulp stem cells (hDPSCs) was investigated to assess their possible role in regenerative dentistry. Briefly, hDPSCs were isolated from sound premolars extracted for orthodontic purposes. Cytotoxicity and proliferation assessment were performed via cell counting kit-8 followed by flow cytometric analysis of apoptotic marker ANNEXIN V. The effect of both small molecules on the stemness of hDPSCs was analyzed by qRT-PCR. Both tideglusib and CHIR99021 were proven to be safe on hDPSCs. The tideglusib concentration that resulted in higher viable cells was 100 nM, while the concentration for CHIR99021 was 5 nM. Both small molecules successfully induced cellular proliferation and demonstrated minimal expression of ANNEXIN V, indicative of the absence of cellular apoptosis and further confirming their positive effect on proliferation. Finally, both small molecules enhanced stemness markers expression as evidenced by qRT-PCR, which, again, highlighted the positive effect of both tideglusib and CHIR99021 on safely promoting the proliferation of hDPSCs while maintaining their stemness.
RESUMEN
Metastasis remains the main challenge to overcome for treating ovarian cancers. In this study, we investigate the potential role of the Cdc42 GAP StarD13 in the modulation of cell motility, invasion in ovarian cancer cells. StarD13 depletion does not affect the 2D motility of ovarian cancer cells. More importantly, StarD13 inhibits matrix degradation, invadopodia formation and cell invasion through the inhibition of Cdc42. StarD13 does not localize to mature TKS4-labeled invadopodia that possess matrix degradation ability, while a Cdc42 FRET biosensor, detects Cdc42 activation in these invadopodia. In fact, StarD13 localization and Cdc42 activation appear mutually exclusive in invadopodial structures. Finally, for the first time we uncover a potential role of Cdc42 in the direct recruitment of TKS4 to invadopodia. This study emphasizes the specific role of StarD13 as a narrow spatial regulator of Cdc42, inhibiting invasion, suggesting the suitability of StarD13 for targeted therapy.
Asunto(s)
Adenocarcinoma , Proteínas Activadoras de GTPasa/genética , Podosomas , Proteínas Supresoras de Tumor/genética , Proteína de Unión al GTP cdc42/genética , Línea Celular Tumoral , Humanos , Invasividad NeoplásicaRESUMEN
Intercellular communication among immune cells is vital for the coordination of proper immune responses. Extracellular vesicles and particles (EVPs) act as messengers in intercellular communication, with important consequences for target cell and organ physiology in both health and disease. Under normal physiological conditions, immune cell-derived EVPs participate in immune responses by regulating innate and adaptive immune responses. EVPs play a major role in antigen presentation and immune activation. On the other hand, immune cell-derived EVPs exert immunosuppressive and regulatory effects. Consequently, EVPs may contribute to pathological conditions, such as autoimmune and inflammatory diseases, graft rejection, and cancer progression and metastasis. Here, we provide an overview of the role of EVPs in immune homeostasis and pathophysiology, with a particular focus on their contribution to innate and adaptive immunity and their potential use for immunotherapies.
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Inmunidad Adaptativa , Comunicación Celular/inmunología , Micropartículas Derivadas de Células/inmunología , Vesículas Extracelulares/inmunología , Inmunidad Innata , Animales , Humanos , InmunoterapiaRESUMEN
BACKGROUND: Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549. METHODS: We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation. RESULTS: We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation. CONCLUSION: In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases. Video abstract.
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Adenocarcinoma del Pulmón/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Neoplasias Pulmonares/metabolismo , Podosomas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Células A549 , Adenocarcinoma del Pulmón/patología , Movimiento Celular , Humanos , Neoplasias Pulmonares/patología , Invasividad Neoplásica/patología , Podosomas/patologíaRESUMEN
Vital pulp therapy (VPT) defined as "treatment which aims at preserving and maintaining the pulp tissue that has been compromised but not destroyed by extensive dental caries, dental trauma, and restorative procedures or for iatrogenic reasons", offers some beneficial advantages over the conventional root canal treatment such as protective resistance for mastication forces or to prevent the loss of environmental changes sensation ability, which can lead to unnoticeable progression of caries and later fracture. A wide range of materials are suggested in the literature to be used as pulp capping protective dressing materials that varies from ready-made synthetic materials to biological based scaffolds and composites. The aim of the present review is to provide a full understanding of currently used materials to clinicians in order to help in their decision-making process delivering the best available evidence-based treatments to their patients. An extensive search for recent available data regarding direct pulp capping materials and potential suggestions for future use have been made. Newly developed biological based scaffolds showed promising results in dentine regeneration therefore strengthening the tooth structure and overcoming potential drawbacks of use of currently available recommended materials.
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Caries Dental , Caries Dental/terapia , Humanos , Motivación , Tratamiento del Conducto RadicularRESUMEN
Purpose: Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study. Methods: Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs. Results: Adult neural retina released EVs at a rate of 1.42 +/- 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs. Conclusions: The adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.
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Vesículas Extracelulares/metabolismo , Proteínas del Ojo/metabolismo , Neuronas/metabolismo , Transporte de Proteínas/fisiología , Transporte de ARN/fisiología , Retina/metabolismo , Animales , Ratones , Microscopía Electrónica de Transmisión , ARN Mensajero/metabolismoRESUMEN
The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. In this study, we examined a novel mechanism of intercellular communication, namely membranous actin-based tunneling nanotubes (TNTs), that occurs between macrophages and tumor cells in the promotion of macrophage-dependent tumor cell invasion. The presence of heterotypic TNTs between macrophages and tumor cells induced invasive tumor cell morphology, which was dependent on EGF-EGFR signaling. Furthermore, reduction of a protein involved in TNT formation, M-Sec (TNFAIP2), in macrophages inhibited tumor cell elongation, blocked the ability of tumor cells to invade in 3D and reduced macrophage-dependent long-distance tumor cell streaming in vitro Using an in vivo zebrafish model that recreates macrophage-mediated tumor cell invasion, we observed TNT-mediated macrophage-dependent tumor cell invasion, distant metastatic foci and areas of metastatic spread. Overall, our studies support a role for TNTs as a novel means of interaction between tumor cells and macrophages that leads to tumor progression and metastasis.
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Neoplasias de la Mama/genética , Comunicación Celular/genética , Células Epiteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Macrófagos/metabolismo , Neoplasias Mamarias Animales/genética , Animales , Transporte Biológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Embrión no Mamífero , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Células Epiteliales/patología , Células Epiteliales/ultraestructura , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Xenoinjertos , Humanos , Macrófagos/ultraestructura , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Cultivo Primario de Células , Células RAW 264.7 , Ratas , Transducción de Señal , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismo , Pez CebraRESUMEN
Background: Metastasis is the cause of most cancer-related deaths. It is known that breast cancer cells in proximity to macrophages become more invasive in an Epidermal Growth Factor (EGF) dependent manner. Tunneling nanotubes (TNTs) are thin, F-actin containing, cellular protrusions that mediate intercellular communication and have been identified in many tumors. The mechanism of TNT formation varies between different cell types. M-Sec (TNFAIP2) has been demonstrated to be involved in TNT formation in some cell types including macrophages. Yet, the requirement of M-Sec in tumor cell TNT formation in response to macrophages has not been explored. Aim: The aim of this study was to determine whether EGF was required for macrophage induced tumor cell TNTs in an M-Sec dependent manner and what possible roles tumor cell TNTs play in tumor cell migration and invasion. Methods and Results: Macrophage Conditioned Media (CM) was used to induce an increase in TNTs in a number of breast cancer cell lines as measured by live cell microscopy. Tumor cell TNT formation by CM was dependent on the presence of EGF which was sufficient to induce TNT formation. CM treatment enhanced the level of M-Sec identified using western blot analysis. Reduction of endogenous M-Sec levels via shRNA in MTLn3 mammary adenocarcinoma cells inhibited the formation of TNTs. The role of tumor cell TNTs in cell behavior was tested using in vitro transwell and 3D invasion assays. No effect on chemotaxis was detected but 3D invasion was reduced following the knockdown of M-Sec in tumor cell TNTs. Conclusions: Our results show that EGF was necessary and sufficient for tumor cell TNT formation which was dependent on cellular M-Sec levels. While tumor cell TNTs may not play a role in individual cell behaviors like chemotaxis, they may be important in more complex tumor cell behaviors such as 3D invasion.
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Citoesqueleto de Actina/metabolismo , Neoplasias de la Mama/patología , Macrófagos/patología , Microtúbulos/metabolismo , Actinas/metabolismo , Animales , Comunicación Celular , Quimiotaxis , Medios de Cultivo Condicionados , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Intravital , Células MCF-7 , Macrófagos/citología , Ratones , Invasividad Neoplásica/patología , Cultivo Primario de Células , Células RAW 264.7 , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismoRESUMEN
Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.
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Actinas/metabolismo , Extensiones de la Superficie Celular/metabolismo , Macrófagos/metabolismo , Polimerizacion , Proteínas de Unión al GTP rho/metabolismo , Animales , Comunicación Celular , Línea Celular , Humanos , Macrófagos/citología , Ratones , Transducción de Señal , Imagen de Lapso de Tiempo/métodos , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Cell-cell communication is critical to coordinate the activity and behavior of a multicellular organism. The cells of the immune system not only must communicate with similar cells, but also with many other cell types in the body. Therefore, the cells of the immune system have evolved multiple ways to communicate. Exosomes and tunneling nanotubes (TNTs) are two means of communication used by immune cells that contribute to immune functions. Exosomes are small membrane vesicles secreted by most cell types that can mediate intercellular communication and in the immune system they are proposed to play a role in antigen presentation and modulation of gene expression. TNTs are membranous structures that mediate direct cell-cell contact over several cell diameters in length (and possibly longer) and facilitate the interaction and/or the transfer of signals, material and other cellular organelles between connected cells. Recent studies have revealed additional, but sometimes conflicting, structural and functional features of both exosomes and TNTs. Despite the new and exciting information in exosome and TNT composition, origin and in vitro function, biologically significant functions are still being investigated and determined. In this review, we discuss the current field regarding exosomes and TNTs in immune cells providing evaluation and perspectives of the current literature.
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Comunicación Celular/inmunología , Exosomas/metabolismo , HumanosRESUMEN
Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.
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Técnicas Biosensibles , Proteína de Unión al GTP cdc42/análisis , Animales , Células Cultivadas , Transferencia Resonante de Energía de Fluorescencia , Fluorometría , Ratones , Células 3T3 NIHRESUMEN
Breast cancer is one of the most commonly diagnosed cancers in women around the world. In general, the more aggressive the tumor, the more rapidly it grows and the more likely it metastasizes. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in breast cancer cell motility and metastasis. The switch between active GTP-bound and inactive GDP-bound state is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine-nucleotide dissociation inhibitors (GDIs). We studied the role of StarD13, a recently identified Rho-GAP that specifically inhibits the function of RhoA and Cdc42. We aimed to investigate its role in breast cancer proliferation and metastasis. The levels of expression of this Rho-GAP in tumor tissues of different grades were assayed using immunohistochemistry. We observed that, while the level of StarD13 expression decreases in cancer tissues compared to normal tissues, it increases as the grade of the tumor increased. This was consistent with the fact that although StarD13 was indeed a tumor suppressor in our breast cancer cells, as seen by its effect on cell proliferation, it was needed for cancer cell motility. In fact, StarD13 knockdown resulted in an inhibition of cell motility and cells were not able to detach their tail and move forward. Our study describes, for the first time, a tumor suppressor that plays a positive role in cancer motility.
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Neoplasias de la Mama/metabolismo , Invasividad Neoplásica/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Mama/patología , Adhesión Celular , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Proteínas Activadoras de GTPasa , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Células MCF-7 , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck(-/-) bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck(-/-) BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP(-/-) BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.
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Macrófagos/citología , Proteínas Proto-Oncogénicas c-hck/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/química , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Células de la Médula Ósea/citología , Línea Celular , Movimiento Celular , Quimiotaxis , Quimiotaxis de Leucocito , Colágeno/química , Cruzamientos Genéticos , Células Endoteliales/citología , Macrófagos/metabolismo , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Isoformas de Proteínas/química , Interferencia de ARN , Migración Transendotelial y Transepitelial , Tirosina/químicaRESUMEN
Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process.
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Astrocitoma/patología , Movimiento Celular , Adhesiones Focales/metabolismo , Proteínas Supresoras de Tumor/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Astrocitoma/genética , Astrocitoma/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/genética , Proteínas Activadoras de GTPasa , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño/farmacología , Distribución Tisular/efectos de los fármacos , Distribución Tisular/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Colon cancer is a cancer of the epithelial cells lining the colon. It is mainly divided into different stages according to the invasiveness and metastatic ability of the tumor. Many mutations are acquired which leads to the development of this malignancy. These occur in entities that greatly affect the cell cycle, cell signaling pathways and cell motility, which all involve the action of Rho GTPases. The protein of interest in the present study was DLC2, also known as StarD13 or START-GAP2, a GTPase-activating protein (GAP) for Rho and Cdc42. Literature data indicate that this protein is considered a tumor-suppressor in hepatocellular carcinoma. Previous research in our laboratory confirmed StarD13 as a tumor suppressor in astrocytoma and in breast cancer. In the present study, we investigated the role of StarD13 in colon cancer. When overexpressed, StarD13 was found to lead to a decrease in cell proliferation in colon cancer cells. Consistently, knockdown of StarD13 led to an increase in cell proliferation. This showed that, similarly to its role in astrocytoma and breast cancer, StarD13 appears to be a tumor suppressor in colon cancer as well. We also examined the role of StarD13 in cell motility. StarD13 knockdown resulted in the inhibition of 2D cell motility. This was due to the inhibition of Rho; consequently Rac-dependent focal complexes were not formed nor detached for the cells to move forward. However, StarD13 knockdown led to an increase in 3D cell motility. Although StarD13 was indeed a tumor suppressor in our colon cancer cells, as evidenced by its effect on cell proliferation, it was required for cancer cell invasion. The present study further describes the role of StarD13 as a tumor suppressor as well as a Rho GAP.
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Adhesión Celular/genética , Movimiento Celular/genética , Neoplasias Colorrectales/patología , Proteínas Supresoras de Tumor/genética , Células CACO-2 , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Neoplasias Colorrectales/metabolismo , Proteínas Activadoras de GTPasa , Genes Supresores de Tumor , Células HT29 , Humanos , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño , Transducción de Señal/genética , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador beta1/genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/inmunología , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The last decades have witnessed an exponential increase in our knowledge of Rho GTPase signaling network which further highlighted the cross talk between these proteins and the complexity of their signaling pathways. In this review, we summarize the upstream and downstream players from Rho GTPases that are mainly involved in actin polymerization leading to cell motility and potentially playing a role in cancer cell metastasis.
Asunto(s)
Actinas/metabolismo , Movimiento Celular/genética , Neoplasias/genética , Proteínas de Unión al GTP rho/genética , Actinas/química , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Astrocytomas are tumors occurring in young adulthood. Astrocytic tumors can be classified into four grades according to histologic features: grades I, II, III and grade IV. Malignant tumors, those of grades III and IV, are characterized by uncontrolled proliferation, which is known to be regulated by the family of Rho GTPases. StarD13, a GAP for Rho GTPases, has been described as a tumor suppressor in hepatocellular carcinoma. In the present study, IHC analysis on grades I-IV brain tissues from patients showed StarD13 to be overexpressed in grades III and IV astrocytoma tumors when compared to grades I and II. However, when we mined the REMBRANDT data, we found that the mRNA levels of StarD13 are indeed higher in the higher grades but much lower than the normal tissues. Knocking down StarD13 using siRNA led to a decrease in cell death and an increase in cell viability, proving that StarD13 is indeed a tumor suppressor in astrocytomas. This was found to be mainly through cell cycle arrest independently of apoptosis. Finally, we detected an increase in p-ERK in StarD13 knockdown cells, uncovering a potential link between Rho GTPases and ERK activation.
Asunto(s)
Astrocitoma/genética , Neoplasias Encefálicas/genética , Genes Supresores de Tumor , Proteínas Supresoras de Tumor/genética , Astrocitoma/metabolismo , Astrocitoma/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Activadoras de GTPasa , Técnicas de Silenciamiento del Gen , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Clasificación del Tumor , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Transfección , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.