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1.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502140

RESUMO

Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options.


Assuntos
Genes Reporter , Luz , NF-kappa B/metabolismo , Optogenética/métodos , Receptor 4 Toll-Like/metabolismo , Adesão Celular , Movimento Celular , Células HEK293 , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oxigênio/metabolismo , Pâncreas/citologia , Podossomos/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/genética
2.
Exp Cell Res ; 407(2): 112806, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34487727

RESUMO

At present, there are still many poorly understood aspects of the mechanisms underlying hepatocellular carcinoma (HCC) invasion and metastasis. Invadopodia are important structures for cancer cell invasion and metastasis. We determined that high T-lymphoma invasion and metastasis 1 (Tiam1) expression is associated with HCC invasion and metastasis and poor patient prognosis after surgery. Gain- and loss-of-function studies confirmed that Tiam1 promotes invadopodia formation in HCC by activating Rac1. A series of biochemical experiments confirmed that this effect is regulated by the PI3K/Akt signaling pathway. We also confirmed that PIP2 facilitates this effect. In summary, these findings reveal that Tiam1 plays an important role in invadopodia formation in HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Podossomos/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Animais , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Podossomos/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Taxa de Sobrevida , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Int J Mol Sci ; 22(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34576260

RESUMO

Osteoclasts differentiate from hematopoietic cells and resorb the bone in response to various signals, some of which are received directly from noncellular elements of the bone. In vitro, adherence to the bone triggers the reduction of cell-cell fusion events between osteoclasts and the activation of osteoclasts to form unusual dynamic cytoskeletal and membrane structures that are required for degrading the bone. Integrins on the surface of osteoclasts are known to receive regulatory signals from the bone matrix. Regulation of the availability of these signals is accomplished by enzymatic alterations of the bone matrix by protease activity and phosphorylation/dephosphorylation events. Other membrane receptors are present in osteoclasts and may interact with as yet unidentified signals in the bone. Bone mineral has been shown to have regulatory effects on osteoclasts, and osteoclast activity is also directly modulated by mechanical stress. As understanding of how osteoclasts and other bone cells interact with the bone has emerged, increasingly sophisticated efforts have been made to create bone biomimetics that reproduce both the structural properties of the bone and the bone's ability to regulate osteoclasts and other bone cells. A more complete understanding of the interactions between osteoclasts and the bone may lead to new strategies for the treatment of bone diseases and the production of bone biomimetics to repair defects.


Assuntos
Matriz Óssea/metabolismo , Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Membrana Celular/metabolismo , Osteoclastos/metabolismo , Actinas/química , Animais , Biomimética , Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Imunoglobulinas/metabolismo , Integrinas/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Osteopontina/metabolismo , Fosforilação , Podossomos/metabolismo , Ligação Proteica , Estresse Mecânico , Sinaptotagminas/metabolismo
4.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360570

RESUMO

Cancer cell invasion through tissue barriers is the intrinsic feature of metastasis, the most life-threatening aspect of cancer. Detailed observation and analysis of cancer cell behaviour in a 3D environment is essential for a full understanding of the mechanisms of cancer cell invasion. The inherent limits of optical microscopy resolution do not allow to for in-depth observation of intracellular structures, such as invadopodia of invading cancer cells. The required resolution can be achieved using electron microscopy techniques such as FIB-SEM. However, visualising cells in a 3D matrix using FIB-SEM is challenging due to difficulties with localisation of a specific cell deep within the resin block. We have developed a new protocol based on the near-infrared branding (NIRB) procedure that extends the pattern from the surface grid deep inside the resin. This 3D burned pattern allows for precise trimming followed by targeted 3D FIB-SEM. Here we present detailed 3D CLEM results combining confocal and FIB-SEM imaging of cancer cell invadopodia that extend deep into the collagen meshwork.


Assuntos
Neoplasias da Mama/patologia , Fibrossarcoma/patologia , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura/métodos , Podossomos/patologia , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Invasividade Neoplásica , Células Tumorais Cultivadas
5.
Cells ; 10(8)2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34440749

RESUMO

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM-ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM-ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.


Assuntos
Actinas/metabolismo , Membrana Basal/metabolismo , Miosina Tipo II/metabolismo , Fibras de Estresse/metabolismo , Células Acinares/citologia , Células Acinares/metabolismo , Mama/citologia , Mama/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Microscopia Confocal , Podossomos/metabolismo , Pseudópodes/metabolismo , Fibras de Estresse/química
6.
Cells ; 10(8)2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34440617

RESUMO

Skin melanocytes reside on the basement membrane (BM), which is mainly composed of laminin, collagen type IV, and proteoglycans. For melanoma cells, in order to invade into the skin, melanocytes must cross the BM. It has been reported that changes in the composition of the BM accompany melanocytes tumorigenesis. Previously, we reported high gelsolin (GSN)-an actin-binding protein-levels in melanoma cell lines and GSN's importance for migration of A375 cells. Here we investigate whether melanoma cells migrate differently depending on the type of fibrous extracellular matrix protein. We obtained A375 melanoma cells deprived of GSN synthesis and tested their migratory properties on laminin, collagens type I and IV, fibronectin, and Matrigel, which resembles the skin's BM. We applied confocal and structured illuminated microscopy (SIM), gelatin degradation, and diverse motility assays to assess GSN's influence on parameters associated with cells' ability to protrude. We show that GSN is important for melanoma cell migration, predominantly on laminin, which is one of the main components of the skin's BM.


Assuntos
Membrana Basal/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Gelsolina/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Microambiente Tumoral , Membrana Basal/patologia , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Matriz Extracelular/patologia , Fibronectinas/metabolismo , Gelsolina/genética , Humanos , Laminina/metabolismo , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica , Podossomos/metabolismo , Podossomos/patologia , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
7.
Cells ; 10(8)2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34440806

RESUMO

Non-receptor tyrosine kinases (NRTKs) are crucial mediators of intracellular signaling and control a wide variety of processes such as cell division, morphogenesis, and motility. Aberrant NRTK-mediated tyrosine phosphorylation has been linked to various human disorders and diseases, among them cancer metastasis, to which no treatment presently exists. Invasive cancer cells leaving the primary tumor use invadopodia, feet-like structures which facilitate extracellular matrix (ECM) degradation and intravasation, to escape the primary tumor and disseminate into distant tissues and organs during metastasis. A major challenge in metastasis research is to elucidate the molecular mechanisms and signaling pathways underlying invadopodia regulation, as the general belief is that targeting these structures can potentially lead to the eradication of cancer metastasis. Non-receptor tyrosine kinases (NRTKs) play a central role in regulating invadopodia formation and function, but how they coordinate the signaling leading to these processes was not clear until recently. Here, we describe the major NRTKs that rule invadopodia and how they work in concert while keeping an accurate hierarchy to control tumor cell invasiveness and dissemination.


Assuntos
Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Podossomos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Humanos , Morfogênese , Transdução de Sinais , Quinases da Família src/metabolismo
8.
Cell Mol Life Sci ; 78(17-18): 6087-6104, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34296319

RESUMO

Different types of multinucleated giant cells (MGCs) of myeloid origin have been described; osteoclasts are the most extensively studied because of their importance in bone homeostasis. MGCs are formed by cell-to-cell fusion, and most types have been observed in pathological conditions, especially in infectious and non-infectious chronic inflammatory contexts. The precise role of the different MGCs and the mechanisms that govern their formation remain poorly understood, likely due to their heterogeneity. First, we will introduce the main populations of MGCs derived from the monocyte/macrophage lineage. We will then discuss the known molecular actors mediating the early stages of fusion, focusing on cell-surface receptors involved in the cell-to-cell adhesion steps that ultimately lead to multinucleation. Given that cell-to-cell fusion is a complex and well-coordinated process, we will also describe what is currently known about the evolution of F-actin-based structures involved in macrophage fusion, i.e., podosomes, zipper-like structures, and tunneling nanotubes (TNT). Finally, the localization and potential role of the key fusion mediators related to the formation of these F-actin structures will be discussed. This review intends to present the current status of knowledge of the molecular and cellular mechanisms supporting multinucleation of myeloid cells, highlighting the gaps still existing, and contributing to the proposition of potential disease-specific MGC markers and/or therapeutic targets.


Assuntos
Adesão Celular , Células Gigantes/metabolismo , Células Mieloides/metabolismo , Podossomos/metabolismo , Células Gigantes/citologia , Humanos , Integrinas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Células Mieloides/citologia , Células Mieloides/ultraestrutura , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Receptores Imunológicos/metabolismo
9.
Biochem Biophys Res Commun ; 571: 145-151, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34325130

RESUMO

Podosomes are actin-rich adhesion structures formed in a variety of cell types, such as monocytic cells or cancer cells, to facilitate attachment to and degradation of the extracellular matrix (ECM). Previous studies showed that dynamin 2, a large GTPase involved in membrane remodeling and actin organization, is required for podosome function. However, precise roles of dynamin 2 at the podosomes remain to be elucidated. In this study, we identified a BAR (Bin-Amphiphysin-Rvs167) domain protein pacsin 2 as a functional partner of dynamin 2 at podosomes. Dynamin 2 and pacsin 2 interact and co-localize to podosomes in Src-transformed NIH 3T3 (NIH-Src) cells. RNAi of either dynamin 2 or pacsin 2 in NIH-Src cells inhibited podosome formation and maturation, suggesting essential and related roles at podosomes. Consistently, RNAi of pacsin 2 prevented dynamin 2 localization to podosomes, and reciprocal RNAi of dynamin 2 prevented pacsin 2 localization to podosomes. Taking these results together, we conclude that dynamin 2 and pacsin 2 co-operatively regulate organization of podosomes in NIH-Src cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Dinamina II/metabolismo , Podossomos/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos
10.
Nat Commun ; 12(1): 3552, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117241

RESUMO

Important dynamic processes in mechanobiology remain elusive due to a lack of tools to image the small cellular forces at play with sufficient speed and throughput. Here, we introduce a fast, interference-based force imaging method that uses the illumination of an elastic deformable microcavity with two rapidly alternating wavelengths to map forces. We show real-time acquisition and processing of data, obtain images of mechanical activity while scanning across a cell culture, and investigate sub-second fluctuations of the piconewton forces exerted by macrophage podosomes. We also demonstrate force imaging of beating neonatal cardiomyocytes at 100 fps which reveals mechanical aspects of spontaneous oscillatory contraction waves in between the main contraction cycles. These examples illustrate the wider potential of our technique for monitoring cellular forces with high throughput and excellent temporal resolution.


Assuntos
Diagnóstico por Imagem/métodos , Mecanotransdução Celular/fisiologia , Microscopia de Interferência/métodos , Animais , Adesão Celular , Fibroblastos/citologia , Humanos , Macrófagos/citologia , Camundongos , Modelos Teóricos , Células NIH 3T3 , Podossomos/metabolismo
11.
ACS Biomater Sci Eng ; 7(6): 2255-2267, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-33938726

RESUMO

The degradation mechanism of human trabecular bone harvested from the central part of the femoral head of a patient with a fragility fracture of the femoral neck under conditions of senile osteoporosis was investigated by high-resolution electron microscopy. As evidenced by light microscopy, there is a disturbance of bone metabolism leading to severe and irreparable damages to the bone structure. These defects are evoked by osteoclasts and thus podosome activity. Podosomes create typical pit marks and holes of about 300-400 nm in diameter on the bone surface. Detailed analysis of the stress field caused by the podosomes in the extracellular bone matrix was performed. The calculations yielded maximum stress in the range of few megapascals resulting in formation of microcracks around the podosomes. Disintegration of hydroxyapatite and free lying collagen fibrils were observed at the edges of the plywood structure of the bone lamella. At the ultimate state, the disintegration of the mineralized collagen fibrils to a gelatinous matrix comes along with a delamination of the apatite nanoplatelets resulting in a brittle, porous bone structure. The nanoplatelets aggregate to big hydroxyapatite plates with a size of up to 10 x 20 µm2. The enhanced plate growth can be explained by the interaction of two mechanisms in the ruffled border zone: the accumulation of delaminated hydroxyapatite nanoplatelets near clusters of podosomes and the accelerated nucleation and random growth of HAP nanoplatelets due to a nonsufficient concentration of process-directing carboxylated osteocalcin cOC.


Assuntos
Osteoporose , Podossomos , Apatitas , Osso e Ossos/diagnóstico por imagem , Humanos , Osteoclastos
12.
J Biol Chem ; 296: 100790, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019873

RESUMO

Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src-/- osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src's functionality is, in part, derived from an SH3-proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src's unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.


Assuntos
Osteoclastos/metabolismo , Podossomos/metabolismo , Domínios de Homologia de src , Quinases da Família src/metabolismo , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Células HEK293 , Humanos , Camundongos , Osteoclastos/citologia , Quinases da Família src/genética
13.
Mol Cell Biochem ; 476(9): 3383-3391, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33954907

RESUMO

Although luminal breast cancer cells are typically highly cohesive epithelial cells and have low invasive ability, many eventually develop metastasis. Until now, the underlying mechanisms remain obscure. In this work, we showed that the level of hyaluronic acid synthase 2 (HAS2) was positively correlated with the malignant phenotype of breast cancer cells. Notably, the increased expression of HAS2 promoted the invasive and migratory abilities of luminal breast cancer cells in vitro, followed by a reduced expression of E-cadherin, ß-catenin, and ZO-1, and an elevated expression of N-cadherin and vimentin. Furthermore, overexpression of HAS2 promoted while knockdown of HAS2 impeded invadopodia formation, which subsequently increased or decreased the activation of cortactin, Tks5, and metalloproteinases (MMPs). Activation of these invadopodia-related proteins was prevented by inhibition of HAS2 or disruption of HA, which in turn attenuated the increased motility and invasiveness. Further, in vivo study showed that, HAS2 increased tumor growth and the rate of lung metastasis via driving transition to an invasive cell phenotype in SCID mice that were orthotopically transplanted with luminal breast cancer cells. Collectively, our results showed that HAS2 promoted cell invasion by inducing transition to an invasive phenotype and by enhancing invadopodia formation in luminal breast cancer cells, which may provide new mechanistic insights into its role in tumor metastasis.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Hialuronan Sintases/metabolismo , Podossomos/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Proliferação de Células , Feminino , Humanos , Hialuronan Sintases/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Podossomos/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
PLoS One ; 16(5): e0252428, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34043722

RESUMO

Diaphanous related formins are highly conserved proteins regulated by Rho-GTPases that act as actin nucleation and assembly factors. Here we report the functional characterization of a non-inherited heterozygous FMNL2 p.L136P mutation carried by a patient who presented with severe very early onset inflammatory bowel disease (IBD). We found that the FMNL2 L136P protein displayed subcellular mislocalization and deregulated protein autoinhibition indicating gain-of-function mechanism. Expression of FMNL2 L136P impaired cell spreading as well as filopodia formation. THP-1 macrophages expressing FMNL2 L136P revealed dysregulated podosome formation and a defect in matrix degradation. Our data indicate that the L136P mutation affects cellular actin dynamics in fibroblasts and immune cells such as macrophages.


Assuntos
Forminas/genética , Doenças Inflamatórias Intestinais/genética , Diferenciação Celular , Linhagem Celular , Doença Crônica , Forminas/química , Forminas/metabolismo , Humanos , Doenças Inflamatórias Intestinais/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Podossomos/metabolismo , Polimorfismo de Nucleotídeo Único , Pseudópodes/metabolismo , Pseudópodes/patologia
16.
Cells ; 10(4)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917849

RESUMO

It was reported that lumican inhibits the activity of metalloproteinase MMP-14 and melanoma cell migration in vitro and in vivo. Moreover, Snail triggers epithelial-to-mesenchymal transition and the metastatic potential of cancer cells. Therefore, the aim of this study was to examine the effect of lumican on Mock and Snail overexpressing melanoma B16F1 cells in vivo. Lung metastasis was analyzed after intravenous injections of Mock-B16F1 and Snail-B16F1 cells in Lum+/+ and Lum-/- mice. At day 14, mice were sacrificed, and lungs were collected. The number of lung metastatic nodules was significantly higher in mice injected with Snail-B16F1 cells as compared to mice injected with Mock-B16F1 cells confirming the pro-metastatic effect of Snail. This effect was stronger in Lum-/- mice as compared to Lum+/+, suggesting that endogenous lumican of wild-type mice significantly inhibits metastasis to lungs. Scanning electron and confocal microscopy investigations demonstrated that lumican inhibits the development of elongated cancer cell phenotypes which are known to develop invadopodia releasing MMPs. Moreover, lumican was shown to affect the expression of cyclin D1, cortactin, vinculin, hyaluronan synthase 2, heparanase, MMP-14 and the phosphorylation of FAK, AKT, p130 Cas and GSK3α/ß. Altogether, these data demonstrated that lumican significantly inhibits lung metastasis in vivo, as well as cell invasion in vitro, suggesting that a lumican-based strategy targeting Snail-induced metastasis could be useful for melanoma treatment.


Assuntos
Biomarcadores Tumorais/metabolismo , Lumicana/metabolismo , Melanoma/patologia , Podossomos/patologia , Neoplasias Cutâneas/patologia , Animais , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Forma Celular , Cortactina/metabolismo , Ciclina D1/metabolismo , Matriz Extracelular/metabolismo , Adesões Focais/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Melanoma/ultraestrutura , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Fosforilação , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/ultraestrutura , Fatores de Transcrição da Família Snail/metabolismo , Vinculina/metabolismo
17.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33904858

RESUMO

Podosomes and invadopodia, collectively termed invadosomes, are adhesive and degradative membrane structures formed in many types of cells and are well known for recruiting various proteases. However, another major class of degradative enzymes, deoxyribonuclease (DNase), remains unconfirmed and not studied in invadosomes. Here, using surface-immobilized nuclease sensor (SNS), we demonstrated that invadosomes recruit DNase to their core regions, which degrade extracellular double-stranded DNA. We further identified the DNase as GPI-anchored membrane-bound DNase X. DNase recruitment is ubiquitous and consistent in invadosomes of all tested cell types. DNase activity exhibits within a minute after actin nucleation, functioning concomitantly with protease in podosomes but preceding it in invadopodia. We further showed that macrophages form DNase-active podosome rosettes surrounding bacteria or micropatterned antigen islets, and the podosomes directly degrade bacterial DNA on a surface, exhibiting an apparent immunological function. Overall, this work reports DNase in invadosomes for the first time, suggesting a richer arsenal of degradative enzymes in invadosomes than known before.


Assuntos
Actinas/genética , DNA/genética , Desoxirribonucleases/genética , Fenômenos Fisiológicos Celulares/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Matriz Extracelular/genética , Humanos , Podossomos/genética
18.
Eur J Cell Biol ; 100(4): 151161, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33836409

RESUMO

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.


Assuntos
Macrófagos/metabolismo , Podossomos/metabolismo , Voluntários Saudáveis , Humanos , Fagocitose
20.
Diagn Pathol ; 16(1): 33, 2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879222

RESUMO

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the most relevant malignant neoplasm among all head and neck tumours due to its high prevalence and unfavourable prognosis. Tumour invasion and metastasis that affect prognosis are result of a set of complex events that cells with invasive potential use to spread to other regions. These cells use several mechanisms to invade tissues, including a type of finger-like membrane protrusion called invadopodia. This study aims to investigate the immunoexpression of invaopodia related-proteins TKs5, cortactin, TKs4 and MT1-MMP in OSCC and correlate it to clinicopathological data. METHODS: An immunohistochemical evaluation of fifty cases of OSCCs and 20 cases of oral mucosa (OM) were assessed. The expression of invadopodia proteins were analysed in comparison to normal tissue (OM) and correlated to different clinical-stage and histological grade of OSCC. RESULTS: TKs5, cortactin, TKs4 and MT1-MMP were significantly overexpressed in OSCC when compared to OM (p < 0.0001). Among tumour stages, TKs5 showed a statistical difference in immunolabelling between stage I and III (p = 0.026). Cortactin immunolabelling was statistically higher in grade I than in grade II and III. No differences were seen on TKs4 expression based on tumour staging or grading. MT1-MMP was higher expressed and showed statistical difference between stages I and III and grades I compared to II and III. CONCLUSIONS: The invadopodia related-proteins were found to be overexpressed in OSCC when compared to OM, suggesting invadopodia formation and activity. Besides overexpressed in OSCC, cortactin, TKs4 and TKs5 showed no or ambiguous differences in protein expression when compared among clinical-stages or histological grades groups. Conversely, the expression of MT1-MMP increased in advanced stages and less differentiated tumours, suggesting MT1-MMP expression as a promising prognostic marker in OSCC.


Assuntos
Biomarcadores Tumorais/análise , Metaloproteinase 14 da Matriz/análise , Neoplasias Bucais/enzimologia , Podossomos/enzimologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/enzimologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transporte Vesicular/análise , Cortactina/análise , Estudos Transversais , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Gradação de Tumores , Estadiamento de Neoplasias , Podossomos/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia
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