RESUMO
Estrogen-related receptor alpha (ERRα) plays an important role in endometrial cancer (EC) progression. However, the biological roles of ERRα in EC invasion and metastasis are not clear. This study aimed to investigate the role of ERRα and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) in regulating intracellular cholesterol metabolism to promote EC progression. ERRα and HMGCS1 interactions were detected by co-immunoprecipitation, and the effects of ERRα/HMGCS1 on the metastasis of EC were investigated by wound-healing and transwell chamber invasion assays. Cellular cholesterol content was measured to verify the relationship between ERRα and cellular cholesterol metabolism. Additionally, immunohistochemistry was performed to confirm that ERRα and HMGCS1 were related to EC progression. Furthermore, the mechanism was investigated using loss-of-function and gain-of-function assays or treatment with simvastatin. High expression levels of ERRα and HMGCS1 promoted intracellular cholesterol metabolism for invadopodia formation. Moreover, inhibiting ERRα and HMGCS1 expression significantly weakened the malignant progression of EC in vitro and in vivo. Our functional analysis showed that ERRα promoted EC invasion and metastasis through the HMGCS1-mediated intracellular cholesterol metabolism pathway, which was dependent on the epithelial-mesenchymal transition pathway. Our findings suggest that ERRα and HMGCS1 are potential targets to suppress EC progression.
Assuntos
Neoplasias do Endométrio , Podossomos , Feminino , Humanos , Linhagem Celular Tumoral , Neoplasias do Endométrio/patologia , Hidroximetilglutaril-CoA Sintase , Podossomos/fisiologia , Receptores de Estrogênio/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Cancer cells degrade the extracellular matrix through actin-rich protrusions termed invadopodia. The formation of functional invadopodia requires polarized membrane trafficking driven by Rho GTPase-mediated cytoskeletal remodeling. We identify the Rho GTPase-activating protein deleted in liver cancer 3 (DLC3; also known as STARD8) as an integral component of the endosomal transport and sorting machinery. We provide evidence for the direct regulation of RhoB by DLC3 at endosomal membranes to which DLC3 is recruited by interacting with the sorting nexin SNX27. In TGF-ß-treated MCF10A breast epithelial cells, DLC3 knockdown enhanced metalloproteinase-dependent matrix degradation, which was partially rescued by RhoB co-depletion. This was recapitulated in MDA-MB-231 breast cancer cells in which early endosomes demonstrated aberrantly enriched F-actin and accumulated the metalloproteinase MT1-MMP (also known as MMP14) upon DLC3 knockdown. Remarkably, Rab4 (herein referring to Rab4A) downregulation fully rescued the enhanced matrix degradation of TGF-ß-treated MCF10A and MDA-MB-231 cells. In summary, our findings establish a novel role for DLC3 in the suppression of MT1-MMP-dependent matrix degradation by inactivating RhoB signaling at endosomal membranes. We propose that DLC3 function is required to limit endosomal actin polymerization, Rab4-dependent recycling of MT1-MMP and, consequently, matrix degradation mediated by invadopodial activity.
Assuntos
Endossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Actinas/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Matriz Extracelular/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Células HeLa , Humanos , Podossomos/fisiologia , Nexinas de Classificação/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Proteínas rab4 de Ligação ao GTP/metabolismoRESUMO
Compared with noninvasive tumor cells, glioma cells overexpress chemokine receptor type 4 (CXCR4), which exhibits significantly greater expression in invasive tumor cells than in noninvasive tumor cells. C-X-C motif chemokine ligand 12 (CXCL12, also known as stromal derived factor-1, SDF-1) and its cell surface receptor CXCR4 activate a signaling axis that induces the expression of membrane type-2 matrix metalloproteinase (MT2-MMP), which plays a pivotal role in the invasion and migration of various cancer cells; however, the specific mechanism involved in this is unclear. Recently, studies have shown that invadopodia can recruit and secrete related enzymes, such as matrix metalloproteinases (MMPs), to degrade the surrounding extracellular matrix (ECM), promoting the invasion and migration of tumor cells. Phosphorylated cortactin (pY421-cortactin) is required for the formation and maturation of invadopodia, but the upstream regulatory factors and kinases involved in phosphorylation have not been elucidated. In this study, we found that CXCL12/CXCR4 was capable of inducing glioma cell invadopodia formation, probably by regulating cortactin phosphorylation. The interaction of cortactin and Arg (also known as Abl-related nonreceptor tyrosine kinase, ABL2) in glioma cells was demonstrated. The silencing of Arg inhibited glioma cell invadopodia formation and invasion by blocking cortactin phosphorylation. Moreover, CXCL12 could not induce glioma cell invasion in Arg-knockdown glioma cells. Based on these results, it can be concluded that Arg mediates CXCL12/CXCR4-induced glioma cell invasion, and CXCL12/CXCR4 regulates invadopodia maturation through the Arg-cortactin pathway, which indicates that Arg could be a candidate therapeutic target to inhibit glioma cell invasion.
Assuntos
Neoplasias Encefálicas/patologia , Quimiocina CXCL12/fisiologia , Glioma/patologia , Podossomos/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores CXCR4/fisiologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Quimiocina CXCL12/metabolismo , Glioma/genética , Glioma/metabolismo , Humanos , Invasividade Neoplásica , Fosforilação , Podossomos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores CXCR4/metabolismo , Transdução de Sinais/genéticaRESUMO
Osteolytic skeletal disorders are caused by an imbalance in the osteoclast and osteoblast function. Suppressing the differentiation and resorptive function of osteoclast is a key strategy for treating osteolytic diseases. Dracorhodin perchlorate (D.P), an active component from dragon blood resin, has been used for facilitating wound healing and anti-cancer treatments. In this study, we determined the effect of D.P on osteoclast differentiation and function. We have found that D.P inhibited RANKL-induced osteoclast formation and resorbed pits of hydroxyapatite-coated plate in a dose-dependent manner. D.P also disrupted the formation of intact actin-rich podosome structures in mature osteoclasts and inhibited osteoclast-specific gene and protein expressions. Further, D.P was able to suppress RANKL-activated JNK, NF-κB and Ca2+ signalling pathways and reduces the expression level of NFATc1 as well as the nucleus translocation of NFATc1. Overall, these results indicated a potential therapeutic effect of D.P on osteoclast-related conditions.
Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Osteoclastos/citologia , Osteogênese/efeitos dos fármacos , Osteólise Essencial/tratamento farmacológico , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/antagonistas & inibidores , Osteólise Essencial/patologia , Podossomos/fisiologia , Ligante RANK/antagonistas & inibidores , Fator de Transcrição RelA/metabolismoRESUMO
Cellular migration, coupled with the degradation of the extracellular matrix (ECM), is a key step in tumor invasion and represents a promising therapeutic target in malignant tumors. Focal adhesions (FAs) and invadopodia, which are distinct actin-based cellular structures, play key roles in cellular migration and ECM degradation, respectively. The molecular machinery coordinating the dynamics between FAs and invadopodia is not fully understood, although several lines of evidence suggest that the disassembly of FAs is an important step in triggering the formation of invadopodia. In a previous study, we identified the ZF21 protein as a regulator of both FA turnover and invadopodia-dependent ECM degradation. ZF21 interacts with multiple factors for FA turnover, including focal adhesion kinase (FAK), microtubules, m-Calpain, and Src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2). In particular, the dephosphorylation of FAK by ZF21 is a key event in tumor invasion. However, the precise role of ZF21 binding to FAK remains unclear. We established a method to disrupt the interaction between ZF21 and FAK using the FAK-binding NH2 -terminal region of ZF21. Tumor cells expressing the ZF21-derived polypeptide had significantly decreased FA turnover, migration, invadopodia-dependent ECM degradation, and Matrigel invasion. Furthermore, the expression of the polypeptide inhibited an early step of experimental lung metastasis in mice. These findings indicate that the interaction of ZF21 with FAK is necessary for FA turnover as well as ECM degradation at the invadopodia. Thus, ZF21 is a potential regulator that coordinates the equilibrium between FA turnover and invadopodia activity by interacting with FAK.
Assuntos
Adesão Celular/fisiologia , Quinase 1 de Adesão Focal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Invasividade Neoplásica , Animais , Movimento Celular , Proliferação de Células , Matriz Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Podossomos/fisiologiaRESUMO
Scaffold proteins are typically thought of as multi-domain "bridging molecules." They serve as crucial regulators of key signaling events by simultaneously binding multiple participants involved in specific signaling pathways. In the case of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR) binding, the activated EGFR contacts cytosolic SRC tyrosine-kinase, which then becomes activated. This process leads to the phosphorylation of SRC-substrates, including the tyrosine kinase substrates (TKS) scaffold proteins. The TKS proteins serve as a platform for the recruitment of key players in EGFR signal transduction, promoting cell spreading and migration. The TKS4 and the TKS5 scaffold proteins are tyrosine kinase substrates with four or five SH3 domains, respectively. Their structural features allow them to recruit and bind a variety of signaling proteins and to anchor them to the cytoplasmic surface of the cell membrane. Until recently, TKS4 and TKS5 had been recognized for their involvement in cellular motility, reactive oxygen species-dependent processes, and embryonic development, among others. However, a number of novel functions have been discovered for these molecules in recent years. In this review, we attempt to cover the diverse nature of the TKS molecules by discussing their structure, regulation by SRC kinase, relevant signaling pathways, and interaction partners, as well as their involvement in cellular processes, including migration, invasion, differentiation, and adipose tissue and bone homeostasis. We also describe related pathologies and the established mouse models.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Diferenciação Celular , Movimento Celular , Homeostase , Podossomos/fisiologia , Humanos , Transdução de SinaisRESUMO
Invadopodia, cancer cell protrusions with proteolytic activity, are functionally associated with active remodeling of the extracellular matrix. Here, we show that the invadopodia-related protein TKS5 is expressed in human pancreatic adenocarcinoma lines, and demonstrate that pancreatic cancer cells depend on TKS5 for invadopodia formation and function. Immunofluorescence staining of human pancreatic cancer cells reveals that TKS5 is a marker of mature and immature invadopodia. We also analyze the co-staining patterns of TKS5 and the commonly used invadopodia marker Cortactin, and find only partial co-localization of these two proteins at invadopodia, with a large fraction of TKS5-positive invadopodia lacking detectable levels of Cortactin. Whereas compelling evidence exist on the role of invadopodia as mediators of invasive migration in cultured cells and in animal models of cancer, these structures have never been detected inside human tumors. Here, using antibodies against TKS5 and Cortactin, we describe for the first time structures strongly resembling invadopodia in various paraffin-embedded human tumor surgical specimens from pancreas and other organs. Our results strongly suggest that invadopodia are present inside human tumors, and warrants further investigation on their regulation and occurrence in surgical specimens, and on the value of TKS5 antibodies as pathological research and diagnostic tools.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Adenocarcinoma/patologia , Proteínas de Neoplasias/fisiologia , Neoplasias Pancreáticas/patologia , Podossomos/fisiologia , Adenocarcinoma/química , Adenocarcinoma/cirurgia , Adenocarcinoma/ultraestrutura , Adulto , Idoso , Linhagem Celular Tumoral , Cortactina/análise , Feminino , Técnica Direta de Fluorescência para Anticorpo , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias/química , Neoplasias/patologia , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/cirurgia , Neoplasias Pancreáticas/ultraestrutura , Inclusão em Parafina , Podossomos/química , Podossomos/ultraestrutura , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Podosomes are actin-rich adhesion structures that depend on Arp2/3-complex-based actin nucleation. We now report the identification of the formins FHOD1 and INF2 as novel components and additional actin-based regulators of podosomes in primary human macrophages. FHOD1 surrounds the podosome core and is also present at podosome-connecting cables, whereas INF2 localizes at the podosome cap structure. Using a variety of microscopy-based methods; including a semiautomated podosome reformation assay, measurement of podosome oscillations, FRAP analysis of single podosomes, and structured illumination microscopy, both formins were found to regulate different aspects of podosome-associated contractility, with FHOD1 mediating actomyosin contractility between podosomes, and INF2 regulating contractile events at individual podosomes. Moreover, INF2 was found to be a crucial regulator of podosome de novo formation and size. Collectively, we identify FHOD1 and INF2 as novel regulators of inter- and intra-structural contractility of podosomes. Podosomes thus present as one of the few currently identified structures which depend on the concerted activity of both Arp2/3 complex and specific formins and might serve as a model system for the analysis of complex actin architectures in cells.
Assuntos
Proteínas Fetais/fisiologia , Proteínas dos Microfilamentos/fisiologia , Proteínas Nucleares/fisiologia , Podossomos/fisiologia , Actinas/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Forminas , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Podossomos/ultraestruturaRESUMO
Cancer cells exploit different strategies to escape from the primary tumor, gain access to the circulation, disseminate throughout the body, and form metastases, the leading cause of death by cancer. Invadopodia, proteolytically active plasma membrane extensions, are essential in this escape mechanism. Cortactin is involved in every phase of invadopodia formation, and its overexpression is associated with increased invadopodia formation, extracellular matrix degradation, and cancer cell invasion. To analyze endogenous cortactin domain function in these processes, we characterized the effects of nanobodies that are specific for the N-terminal acidic domain of cortactin and expected to target small epitopes within this domain. These nanobodies inhibit cortactin-mediated actin-related protein (Arp)2/3 activation, and, after their intracellular expression in cancer cells, decrease invadopodia formation, extracellular matrix degradation, and cancer cell invasion. In addition, one of the nanobodies affects Arp2/3 interaction and invadopodium stability, and a nanobody targeting the Src homology 3 domain of cortactin enabled comparison of 2 functional regions in invadopodium formation or stability. Given their common and distinct effects, we validate cortactin nanobodies as an instrument to selectively block and study distinct domains within a protein with unprecedented precision, aiding rational future generation of protein domain-selective therapeutic compounds.-Bertier, L., Boucherie, C., Zwaenepoel, O., Vanloo, B., Van Troys, M., Van Audenhove, I., Gettemans, J. Inhibitory cortactin nanobodies delineate the role of NTA- and SH3-domain-specific functions during invadopodium formation and cancer cell invasion.
Assuntos
Cortactina/química , Invasividade Neoplásica , Podossomos/fisiologia , Anticorpos de Domínio Único/fisiologia , Linhagem Celular Tumoral , Clonagem Molecular , Cortactina/metabolismo , Epitopos , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Domínios ProteicosRESUMO
Dendritic cells (DCs) involved in proinflammatory immune responses derive mainly from peripheral monocytes, and the cells subsequently mature and migrate into the inflammatory micromilieu. Here we report that suppressing of 15-lipoxygenase-1 led to a substantial reduction in DC spreading and podosome formation in vitro. The surface expression of CD83 was significantly lower in both sh-15-lipoxygenase-1 (15-LOX-1)-transduced cells and DCs cultivated in the presence of a novel specific 15-LOX-1 inhibitor. The T-cell response against tetanus-pulsed DCs was only affected to a minor extent on inhibition of 15-LOX-1. In contrast, endocytosis and migration ability of DCs were significantly suppressed on 15-LOX-1 inhibition. The expression of 15-LOX-1 in DCs was also demonstrated in affected human skin in atopic and contact dermatitis, showing that the enzyme is indeed expressed in inflammatory diseases in vivo. This study demonstrated that inhibiting 15-LOX-1 led to an impaired podosome formation in DCs, and consequently suppressed antigen uptake and migration capacity. These results indicated that 15-LOX-1 is a potential target for inhibiting the trafficking of DCs to lymphoid organs and inflamed tissues and decreasing the inflammatory response attenuating symptoms of certain immunologic and inflammatory disorders such as dermatitis.-Han, H., Liang, X., Ekberg, M., Kritikou, J. S., Brunnström, Å., Pelcman, B., Matl, M., Miao, X., Andersson, M., Yuan, X., Schain, F., Parvin, S., Melin, E., Sjöberg, J., Xu, D., Westerberg, L. S., Björkholm, M., Claesson, H.-E. Human 15-lipoxygenase-1 is a regulator of dendritic-cell spreading and podosome formation.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Citocinas/metabolismo , Células Dendríticas/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Podossomos/fisiologia , Araquidonato 15-Lipoxigenase/genética , Movimento Celular/fisiologia , Citocinas/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Células de Langerhans/metabolismo , Monócitos , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismoRESUMO
Voltage-gated sodium channels (VGSCs) play very important roles in the generation and conduction of action potential in the excitable cells. Recent studies have showed that VGSCs are also expressed in the macrophages and regulate a variety of biological functions, including phagocytosis, endosomal acidification, podosome formation, polarization, and antiviral responses, etc. This paper will review the roles of VGSCs in regulating the biological functions of macrophages and the underlying mechanisms, which would provide clues for the studies of the functions of VGSCs in the other immune cells.
Assuntos
Macrófagos/fisiologia , Canais de Sódio Disparados por Voltagem/fisiologia , Potenciais de Ação , Animais , Endossomos/fisiologia , Humanos , Fagocitose , Podossomos/fisiologiaRESUMO
Invadopodium formation is a crucial early event of invasion and metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying regulation of invadopodia remain elusive. This study aimed to investigate the potential role of discs large homolog 5 (Dlg5) in invadopodium formation and function in HCC. We found that Dlg5 expression was significantly lower in human HCC tissues and cell lines than adjacent nontumor tissues and liver cells. Lower Dlg5 expression was associated with advanced stages of HCC, and poor overall and disease-free survival of HCC patients. Dlg5-silencing promoted epithelial-mesenchymal transition, invadopodium formation, gelatin degradation function, and invadopodium-associated invasion of HepG2 cells. In contrast, Dlg5 overexpression inhibited epithelial-mesenchymal transition, functional invadopodium formation, and invasion of SK-Hep1 cells. Both Girdin and Tks5, but not the Tks5 nonphosphorylatable mutant, were responsible for the enhanced invadopodium formation and invasion of Dlg5-silenced HepG2 cells. Furthermore, Dlg5 interacted with Girdin and interfered with the interaction of Girdin and Tks5. Dlg5 silencing promoted Girdin and Tks5 phosphorylation, which was abrogated by Girdin silencing and rescued by inducing shRNA-resistant Girdin expression. Moreover, Dlg5 overexpression significantly inhibited HCC intrahepatic and lung metastasis in vivo. Taken together, our data indicate that Dlg5 acts as a novel regulator of invadopodium-associated invasion via Girdin and by interfering with the interaction between Girdin and Tks5, which might be important for Tks5 phosphorylation in HCC cells. Conceivably, Dlg5 may act as a new biomarker for prognosis of HCC patients.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Podossomos/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , FosforilaçãoRESUMO
Podosomes are dynamic cytoskeletal membrane structures with local adhesive and proteolytic activity. They are critically involved in angiogenesis and vascular adaptive growth. Here, we studied in HUVECs and murine small vessels whether shear stress controls podosome assembly and local proteolytic activity. Podosomes were characterized by immunohistochemistry, and their proteolytic activity was assessed as degradation imprints in fluorescent gelatin that was used as growth substrate. Compared with controls (10 dyn/cm(2)), the number of podosomes formed per time was doubled when cells were exposed to low shear stress (0.3 dyn/cm(2)) or even increased 5-fold under static conditions. This was a result of an enhanced expression of VEGF after reduction of shear stress. Consequently, enhanced podosome formation could be prevented by a VEGF receptor antagonist as well by interruption of VEGF signaling via inhibition of PI3K, Src, or p38. Increase of podosome assembly went along with significantly augmented cell motility. In vivo experiments in mouse arteries confirmed increased endothelial podosome numbers when shear stress was abolished by vessel occlusion. We conclude that shear stress, by reducing VEGF release, inhibits podosome assembly. Hence, endothelial cell-mediated matrix proteolysis and migratory activity are inhibited, thereby stabilizing the structure of the vessel wall.-Fey, T., Schubert, K. M., Schneider, H., Fein, E., Kleinert, E., Pohl, U., Dendorfer, A. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.
Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Podossomos/fisiologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular , Regulação para Baixo , Humanos , Masculino , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Fisiológico , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
While it has been established that a number of microenvironment components can affect the likelihood of metastasis, the link between microenvironment and tumor cell phenotypes is poorly understood. Here we have examined microenvironment control over two different tumor cell motility phenotypes required for metastasis. By high-resolution multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor cells, different in locomotion speed. Only slower tumor cells exhibited protrusions with molecular, morphological, and functional characteristics associated with invadopodia. Each region in the primary tumor exhibited either fast- or slow-locomotion. To understand how the tumor microenvironment controls invadopodium formation and tumor cell locomotion, we systematically analyzed components of the microenvironment previously associated with cell invasion and migration. No single microenvironmental property was able to predict the locations of tumor cell phenotypes in the tumor if used in isolation or combined linearly. To solve this, we utilized the support vector machine (SVM) algorithm to classify phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility phenotype. We then demonstrated that varying one of the conditions may change tumor cell behavior only in a context-dependent manner. In addition, to establish the link between phenotypes and cell fates, we photoconverted and monitored the fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodium-rich microenvironments degraded extracellular matrix (ECM) and disseminated. The number of invadopodia positively correlated with degradation, while the inhibiting metalloproteases eliminated degradation and lung metastasis, consistent with a direct link among invadopodia, ECM degradation, and metastasis. We have detected and characterized two phenotypes of motile tumor cells in vivo, which occurred in spatially distinct microenvironments of primary tumors. We show how machine-learning analysis can classify heterogeneous microenvironments in vivo to enable prediction of motility phenotypes and tumor cell fate. The ability to predict the locations of tumor cell behavior leading to metastasis in breast cancer models may lead towards understanding the heterogeneity of response to treatment.
Assuntos
Movimento Celular , Neoplasias Mamárias Experimentais/patologia , Podossomos/fisiologia , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Humanos , Camundongos SCID , Invasividade Neoplásica , FenótipoRESUMO
Podosomes are submicron adhesive and mechanosensitive structures formed by macrophages, dendritic cells and osteoclasts that are capable of protruding into the extracellular environment. Built of an F-actin core surrounded by an adhesion ring, podosomes assemble in a network interconnected by acto-myosin cables. They have been shown to display spatiotemporal instability as well as protrusion force oscillations. To analyse the entire population of these unstable structures, we have designed an automated multi-particle tracking adapted to both topographical and fluorescence data. Here we describe in detail this approach and report the measurements of individual and collective characteristics of podosome ensembles, providing an integrated picture of their activity from the complementary angles of organisation, dynamics, mobility and mechanics. We believe that this will lead to a comprehensive view of podosome collective behaviour and deepen our knowledge about the significance of mechanosensing mediated by protrusive structures.
Assuntos
Macrófagos/fisiologia , Podossomos/fisiologia , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Macrófagos/ultraestrutura , Microscopia de Força Atômica , Microscopia de Fluorescência , Podossomos/ultraestruturaRESUMO
The study of cell behavior in constricted environment is particularly relevant to our understanding of the mechanisms of cell invasion. In this regard, microfluidic systems offer promising platforms as microfabricated fluidic chips provide well-controlled physical, chemical and confined environments to study cell phenotype and behavior. Here, we report a fast and effective manufacturing process of user-friendly microfluidic chips ideally suited for quantitative live cell analysis in combination with immunofluorescence microscopy. The chip body, made of polydimethylsiloxane, is composed of two incubation chambers connected by one rectangular intermediate entry channel which provides access to a series of transversal slits where the observation can be made. The height of the slit is designed to be slightly smaller than that of the cells under study. To validate the chip performance, we analyzed the reorganization of the cytoskeleton of endothelial cells under various degree of spatial confinement. We illustrate how the constricted environment affects endothelial cell behavior in inducing the formation of podosomes. Moreover, the process was stimulated further when the surface of the slit was coated with a thin layer of fibronectin. The study demonstrates the suitability of this technological process for cost-effective fabrication of custom-made single-use chips for biological applications.
Assuntos
Citoesqueleto de Actina/fisiologia , Células Endoteliais/fisiologia , Dispositivos Lab-On-A-Chip , Podossomos/fisiologia , Análise de Célula Única/instrumentação , Citoesqueleto de Actina/ultraestrutura , Animais , Bovinos , Células Cultivadas , Meios de Cultura , Células Endoteliais/ultraestrutura , Microscopia de FluorescênciaRESUMO
Cells interact with their environment through highly localized contact structures. Podosomes represent a subgroup of cell-matrix contacts, which is especially prominent in cells of the monocytic lineage such as monocytes, macrophages and dendritic cells, but also in a variety of other cell types. Comparable to other adhesion structures, podosomes feature a complex architecture, which forms the basis for their extensive repertoire of sensory and effector functions. These functions are mainly linked to interactions with the extracellular matrix and comprise well known properties such as cell-matrix adhesion and extracellular matrix degradation. A more recent discovery is the ability of podosomes to act as mechanosensory devices, by detecting rigidity and topography of the substratum. In this review, we focus especially on the molecular events involved in mechanosensing by podosomes, the structural elements of podosomes that enable this function, as well as the intra- and extracellular signals generated downstream of podosome mechanosensing.
Assuntos
Mecanotransdução Celular , Podossomos/fisiologia , Matriz Extracelular/metabolismo , Humanos , Modelos BiológicosRESUMO
BACKGROUND: Cell migration and invasion through interstitial tissues are dependent upon several specialized characteristics of the migratory cell notably generation of proteolytic membranous protrusions or invadopodia. Ameloblastoma is a benign odontogenic epithelial neoplasm with a locally infiltrative behaviour. Cortactin and MMT1-MMP are two invadopodia proteins implicated in its local invasiveness. Other invadopodia regulators, namely N-WASP, WIP and Src kinase remain unclarified. This study addresses their roles in ameloblastoma. MATERIALS AND METHOD: Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters. RESULTS: Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations. CONCLUSIONS: Present results suggest that local invasiveness of ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal dynamics.
Assuntos
Ameloblastoma/metabolismo , Cortactina/fisiologia , Proteínas do Citoesqueleto/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Neoplasias Maxilomandibulares/metabolismo , Podossomos/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Actinas/análise , Actinas/biossíntese , Actinas/fisiologia , Adolescente , Adulto , Idoso , Ameloblastoma/patologia , Movimento Celular/fisiologia , Criança , Cortactina/biossíntese , Proteínas do Citoesqueleto/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Neoplasias Maxilomandibulares/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/biossíntese , Adulto Jovem , Quinases da Família src/análise , Quinases da Família src/fisiologiaRESUMO
Invadosome is an umbrella term used to describe a family of cellular structures including podosomes and invadopodia. They serve as contact zones between the cell plasma membrane and extracellular matrix, contributing to matrix remodeling by locally enriched proteolytic enzymes. Invadosomes, which are actin-dependent, are implicated in cellular processes promoting adhesion, migration, and invasion. Invadosomes, which exist in various cell types, play crucial roles in physiological phenomena such as vascularization and bone resorption. Invadosomes are also implicated in pathological processes such as matrix tissue remodeling during metastatic tumor cell invasion. This review summarizes basic information and recent advances about mechanisms underlying podosome and invadopodia formation, their organization and function.
Title: Invadosomes - Entre mobilité et invasion, naviguer dans la dualité des fonctions cellulaires. Abstract: Le terme « invadosome ¼ désigne une famille de structures cellulaires, comprenant les podosomes et les invadopodes, qui constituent des zones de contact entre la membrane plasmique des cellules et la matrice extracellulaire. Ces structures contribuent au remodelage de la matrice grâce à un enrichissement local en enzymes protéolytiques qui dégradent ses constituants fibrillaires. Les invadosomes, présents dans des types cellulaires variés, contribuent à des processus physiologiques, tels que la vascularisation, ou pathologiques, comme l'invasion des tissus par les cellules métastatiques.
Assuntos
Movimento Celular , Matriz Extracelular , Invasividade Neoplásica , Neoplasias , Podossomos , Humanos , Podossomos/fisiologia , Podossomos/patologia , Movimento Celular/fisiologia , Animais , Neoplasias/patologia , Matriz Extracelular/fisiologia , Matriz Extracelular/patologiaRESUMO
Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min.