Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 70
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Exp Cell Res ; : 112939, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34813733

RESUMO

One of the hallmarks of cancer cells is their exceptional ability to migrate within the extracellular matrix (ECM) for gaining access to the circulatory system, a critical step of cancer metastasis. RhoA, a small GTPase, is known to be a key molecular switch that toggles between actomyosin contractility and lamellipodial protrusion during cell migration. Current understanding of RhoA activity in cell migration has been largely derived from studies of cells plated on a two-dimensional (2D) substrate using a FRET biosensor. There has been increasing evidence that cells behave differently in a more physiologically relevant three-dimensional (3D) environment. However, studies of RhoA activities in 3D have been hindered by low signal-to-noise ratio in fluorescence imaging. In this paper, we present a FRET technique in conjunction with a machine learning-assisted cell segmentation method to follow the spatiotemporal dynamics of RhoA activities of single breast tumor cells (MDA-MB-231) migrating in a 3D as well as a 2D environment using a RhoA biosensor. We found that RhoA activity is more polarized along the long axis of the cell for single cells migrating on 2D fibronectin-coated glass versus those embedded in 3D collagen matrices. In particular, RhoA activities of cells in 2D exhibit a distinct front-to-back and back-to-front movement during migration in contrast to those in 3D. Finally, regardless of dimensionality, RhoA polarization is found to be moderately correlated with cell shape.

3.
Commun Biol ; 4(1): 1091, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34531530

RESUMO

During breast cancer metastasis, cancer cell invasion is driven by actin-rich protrusions called invadopodia, which mediate the extracellular matrix degradation required for the success of the invasive cascade. In this study, we demonstrate that TC10, a member of a Cdc42 subfamily of p21 small GTPases, regulates the membrane type 1 matrix metalloproteinase (MT1-MMP)-driven extracellular matrix degradation at invadopodia. We show that TC10 is required for the plasma membrane surface exposure of MT1-MMP at these structures. By utilizing our Förster resonance energy transfer (FRET) biosensor, we demonstrate the p190RhoGAP-dependent regulation of spatiotemporal TC10 activity at invadopodia. We identified a pathway that regulates invadopodia-associated TC10 activity and function through the activation of p190RhoGAP and the downstream interacting effector Exo70. Our findings reveal the role of a previously unknown regulator of vesicular fusion at invadopodia, TC10 GTPase, in breast cancer invasion and metastasis.

4.
Mol Cancer Res ; 19(5): 862-873, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33495400

RESUMO

We demonstrate that inhibition of cyclin-dependent kinases 4/6 (CDK4/6) leads to senescence in human papillomavirus (HPV)-negative (-) head and neck squamous cell carcinoma (HNSCC), but not in HPV-positive (+) HNSCC. The BCL-2 family inhibitor, navitoclax, has been shown to eliminate senescent cells effectively. We evaluated the efficacy of combining palbociclib and navitoclax in HPV- HNSCC. Three HPV- HNSCC cell lines (CAL27, HN31, and PCI15B) and three HPV+ HNSCC cell lines (UPCI-SCC-090, UPCI-SCC-154, and UM-SCC-47) were treated with palbociclib. Treatment drove reduced expression of phosphorylated Rb (p-Rb) and phenotypic evidence of senescence in all HPV- cell lines, whereas HPV+ cell lines did not display a consistent response by Rb or p-Rb and did not exhibit morphologic changes of senescence in response to palbociclib. In addition, treatment of HPV- cells with palbociclib increased both ß-galactosidase protein expression and BCL-xL protein expression compared with untreated controls in HPV- cells. Co-expression of ß-galactosidase and BCL-xL occurred consistently, indicating elevated BCL-xL expression in senescent cells. Combining palbociclib with navitoclax led to decreased HPV- HNSCC cell survival and led to increased apoptosis levels in HPV- cell lines compared with each agent given alone. IMPLICATIONS: This work exploits a key genomic hallmark of HPV- HNSCC (CDKN2A disruption) using palbociclib to induce BCL-xL-dependent senescence, which subsequently causes the cancer cells to be vulnerable to the senolytic agent, navitoclax.

5.
Biophys Rev Lett ; 15(3): 131-141, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33033500

RESUMO

Tumor invasion, the process by which tumor cells break away from their primary tumor and gain access to vascular systems, is an important step in cancer metastasis. Most current 3D tumor invasion assays consisted of single tumor cells embedded within an extracellular matrix (ECM). These assays taught us much of what we know today on how key biophysical (e.g. ECM stiffness) and biochemical (e.g. cytokine gradients) parameters within the tumor microenvironment guided and regulated tumor invasion. One limitation of the single tumor cell invasion assay was that it did not account for cell-cell adhesion within the tumor. In this article, we developed a micrometer scale 3D co-culture spheroid invasion assay that was compatible with microscopic imaging. Micrometer scale co-culture spheroids (1:1 ratio of metastatic breast cancer MDA-MB-231 and non-tumorigenic epithelial MCF-10A cells) were made using an array of microwells, and then were embedded within a collagen matrix in a microfluidic platform. Real time imaging of tumor spheroid invasion revealed that the spatial distribution of the two cell types within the tumor spheroid critically regulated tumor invasion. This work linked tumor architecture with tumor invasion and highlighted the importance of the biophysical cues within the bulk of the tumor in tumor invasion.

6.
Sci Rep ; 10(1): 9648, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541776

RESUMO

Tumor invasion within the interstitial space is critically regulated by the force balance between cell-extracellular matrix (ECM) and cell-cell interactions. Interstitial flows (IFs) are present in both healthy and diseased tissues. However, the roles of IFs in modulating cell force balance and subsequently tumor invasion are understudied. In this article, we develop a microfluidic model in which tumor spheroids are embedded within 3D collagen matrices with well-defined IFs. Using co-cultured tumor spheroids (1:1 mixture of metastatic and non-tumorigenic epithelial cells), we show that IFs downregulate the cell-cell adhesion molecule E-cadherin on non-tumorigenic cells and promote tumor invasion. Our microfluidic model advances current tumor invasion assays towards a more physiologically realistic model using tumor spheroids instead of single cells under perfusion. We identify a novel mechanism by which IFs can promote tumor invasion through an influence on cell-cell adhesion within the tumor and highlight the importance of biophysical parameters in regulating tumor invasion.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Invasividade Neoplásica/patologia , Esferoides Celulares/citologia , Adesão Celular , Linhagem Celular , Técnicas de Cocultura , Colágeno/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos
7.
Methods Mol Biol ; 2108: 273-279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939188

RESUMO

Macrophages are known to play multiple roles in the breast cancer microenvironment including the promotion of tumor cell invasion that is dependent on soluble factors or through direct contact. Macrophages can also enhance the production of Tunneling Nanotubes (TNTs) in tumor cells which can be mimicked using macrophage-conditioned medium. TNTs are long thin F-actin structures that connect two or more cells together that have been found in many different cell types including macrophages and tumor cells and have been implicated in enhancing tumor cells functions, such as invasion. Here we describe basic procedures used to stimulate tumor cell TNT formation through macrophage-conditioned medium along with methods for quantifying TNTs.


Assuntos
Biomarcadores , Macrófagos/metabolismo , Macrófagos/patologia , Microscopia , Citoesqueleto de Actina , Actinas/metabolismo , Animais , Transporte Biológico , Comunicação Celular , Meios de Cultivo Condicionados/metabolismo , Imunofluorescência , Camundongos , Microscopia/métodos , Células RAW 264.7 , Microambiente Tumoral
8.
Integr Biol (Camb) ; 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31041443

RESUMO

Cancer metastasis is a physical process in which tumor cells break away from the primary tumor, enter, and then exit the blood or lymph vessels, and establish secondary tumors in distant organs. Current clinical studies report a higher risk of cancer metastasis for diabetics than non-diabetics. However, due to complex overlapping risk factors between diabetes and cancer, the mechanism underlying this correlation is largely unknown. Elevated lifetime blood sugar levels in diabetics are known to increase glycation of collagen, causing stiffening of the ECM and connective tissue. In this study, we explored the roles of glycation of 3D collagen matrices in tumor cell invasion and migration. Using time-lapse images, we quantitatively compared the motility behavior of malignant breast tumor cells (MDA-MB-231) and co-culture spheroids (1:1 ratio of MDA-MB-231 cells with normal epithelial MCF-10A cells) embedded in glycated and non-glycated collagen matrices of various concentrations. Experimental results demonstrated that glycation increased tumor invasion within collagen matrices. More specifically, the average speed of MDA-MB-231 cells was higher in glycated collagen gels than in non-glycated collagen gels for all three gel concentrations tested. Cell spreading characterized by its diffusion coefficient or the effective spheroid radii at various time points was significantly greater in glycated collagen than in non-glycated collagen at a concentration of 3.5 mg/mL. This enhancement was moderate and less evident at lower collagen concentrations of 1.0 and 2.0 mg/mL. These results suggest a possible biomechanical link that relates to the high blood sugar level in diabetic patients and the cancer metastatic outcome.

9.
Cancer Rep (Hoboken) ; 2(6): e1213, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-32467880

RESUMO

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.

10.
Breast Cancer Res ; 20(1): 131, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30367629

RESUMO

BACKGROUND: Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. METHODS: We crossed AREG-null (AREG-/-) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG-/- PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. RESULTS: Intriguingly, PyMT-induced lesions progress more rapidly in AREG-/- mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG-/- mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG-/- PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. CONCLUSIONS: Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.


Assuntos
Anfirregulina/metabolismo , Células Epiteliais/patologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Anfirregulina/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/virologia , Feminino , Humanos , Glândulas Mamárias Animais/citologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/virologia , Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Invasividade Neoplásica/patologia , Polyomavirus/genética , Polyomavirus/imunologia
11.
Breast Cancer Res ; 20(1): 24, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29636067

RESUMO

BACKGROUND: The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are influenced by these interactions. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. In this paper, we identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling. METHODS: The aim of this study was to determine the role of NRG1, a ligand of the ErbB3 receptor, in macrophage stimulation of tumor cell transendothelial migration and intravasation. We used fluorescence-activated cell sorting (FACS) and western blot to determine ErbB3 and NRG1 expression, respectively. An in vitro transendothelial migration (iTEM) assay was used to examine the effects of short hairpin (sh)RNA targeting NRG1 in tumor cells and clustered regularly interspaced short palindromic repeats (CRISPR) knockout of jagged 1 (JAG1) in macrophages. Orthotopic xenograft injections in mice were used to confirm results in vivo. RESULTS: In our system, macrophages were the primary cells showing expression of ErbB3, and a blocking antibody against ErbB3 resulted in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Stimulation of macrophages with NRG1 upregulated mRNA and protein expression of JAG1, a ligand of the Notch receptor, and JAG1 production by macrophages was important for transendothelial migration of tumor cells. CONCLUSIONS: This study demonstrates that stimulation of macrophages by tumor cell NRG1 can enhance transendothelial migration and intravasation. We also demonstrate that this effect is due to induction of macrophage JAG1, an important ligand of the Notch signaling pathway.


Assuntos
Neoplasias da Mama/genética , Proteína Jagged-1/genética , Neuregulina-1/genética , Migração Transendotelial e Transepitelial/genética , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Comunicação Parácrina/genética , Receptor ErbB-3/genética , Receptores Notch/genética , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Cell Biol ; 216(12): 4331-4349, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29061650

RESUMO

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and ßPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias da Mama/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Integrina beta1/genética , Neoplasias Mamárias Animais/genética , Proteínas rac de Ligação ao GTP/genética , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Adesão Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Células HEK293 , Humanos , Integrina beta1/metabolismo , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/deficiência
13.
Lab Chip ; 17(19): 3221-3233, 2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28805874

RESUMO

Tumor cell invasion, whether penetrating through the extracellular matrix (ECM) or crossing a vascular endothelium, is a critical step in the cancer metastatic cascade. Along the way from a primary tumor to a distant metastatic site, tumor cells interact actively with the microenvironment either via biomechanical (e. g. ECM stiffness) or biochemical (e.g. secreted cytokines) signals. Increasingly, it is recognized that the tumor microenvironment (TME) is a critical player in tumor cell invasion. A main challenge for the mechanistic understanding of tumor cell-TME interactions comes from the complexity of the TME, which consists of extracellular matrices, fluid flows, cytokine gradients and other cell types. It is difficult to control TME parameters in conventional in vitro experimental designs such as Boyden chambers or in vivo such as in mouse models. Microfluidics has emerged as an enabling tool for exploring the TME parameter space because of its ease of use in recreating a complex and physiologically realistic three dimensional TME with well-defined spatial and temporal control. In this perspective, we will discuss designing principles for modeling the biophysical microenvironment (biological flows and ECM) for tumor cells using microfluidic devices and the potential microfluidic technology holds in recreating a physiologically realistic tumor microenvironment. The focus will be on applications of microfluidic models in tumor cell invasion.


Assuntos
Microfluídica , Modelos Biológicos , Invasividade Neoplásica , Microambiente Tumoral , Animais , Permeabilidade Capilar , Desenho de Equipamento , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Neovascularização Patológica
14.
Am J Pathol ; 187(10): 2259-2272, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28751006

RESUMO

Oral squamous cell carcinoma (OSCC) patients generally have a poor prognosis, because of the invasive nature of these tumors. In comparing transcription profiles between OSCC tumors with a more invasive (worst pattern of tumor invasion 5) versus a less invasive (worst pattern of tumor invasion 3) pattern of invasion, we identified a total of 97 genes that were overexpressed at least 1.5-fold in the more invasive tumor subtype. The most functionally relevant genes were assessed using in vitro invasion assays with an OSCC cell line (UM-SCC-1). Individual siRNA knockdown of 15 of these 45 genes resulted in significant reductions in tumor cell invasion compared to a nontargeting siRNA control. One gene whose knockdown had a strong effect on invasion corresponded to apolipoprotein E (APOE). Both matrix degradation and the number of mature invadopodia were significantly decreased with APOE knockdown. APOE knockdown also resulted in increased cellular cholesterol, consistent with APOE's role in regulating cholesterol efflux. APOE knockdown resulted in decreased levels of phospho-extracellular signal-regulated kinase 1/2, phospho-c-Jun N-terminal kinase, and phospho-cJun, as well as decreased activator protein 1 (AP-1) activity. Expression of matrix metalloproteinase 7 (MMP7), an AP-1 target, was also significantly decreased. Our findings suggest that APOE protein plays a significant role in OSCC tumor invasion because of its effects on cellular cholesterol and subsequent effects on cell signaling and AP-1 activity, leading to changes in the expression of invasion-related proteins, including MMP7.


Assuntos
Apolipoproteínas E/metabolismo , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Apolipoproteínas E/genética , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Colesterol/metabolismo , Matriz Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genoma Humano , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Modelos Biológicos , Neoplasias Bucais/genética , Invasividade Neoplásica , Fosforilação , Podossomos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Fator de Transcrição AP-1/metabolismo , Transcriptoma/genética
15.
Am J Pathol ; 187(7): 1523-1536, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28499703

RESUMO

Invasion is a hallmark of advanced head and neck squamous cell carcinoma (HNSCC). We previously determined that low relative miR-375 expression was associated with poor patient prognosis. HNSCC cells with increased miR-375 expression have lower invasive properties and impaired invadopodium activity. Using stable isotope labeling with amino acids in cell culture and reverse-phase liquid chromatography mass spectrometry, we assessed the impact of miR-375 expression on protein levels in UM-SCC-1 cells. Increased miR-375 expression was associated with down-regulation of proteins involved in cellular assembly and organization, death and survival, and movement. Two invasion-associated proteins, vimentin and L-plastin, were strongly down-regulated by miR-375. Luciferase reporter assays demonstrated that high miR-375 expression reduced vimentin promoter activity, suggesting that vimentin is an indirect target of miR-375. Runt-related transcription factor 1 (RUNX1) is a potential miR-375 direct target, and its knockdown reduced vimentin and L-plastin expression. Data in The Cancer Genome Atlas HNSCC database showed a significant inverse correlation between miR-375 expression and RUNX1, vimentin, and L-plastin RNA expression. These clinical correlations validate our in vitro model findings and support a mechanism in which miR-375 suppresses RUNX1 levels, resulting in reduced vimentin and L-plastin expression. Furthermore, knockdown of RUNX1, L-plastin, and vimentin resulted in significant reductions in cell invasion in vitro, indicating the functional significance of miR-375 regulation of specific proteins involved in HNSCC invasion.


Assuntos
Carcinoma de Células Escamosas/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Neoplasias de Cabeça e Pescoço/genética , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genética , Vimentina/genética , Subunidade alfa 2 de Fator de Ligação ao Core/isolamento & purificação , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas dos Microfilamentos/isolamento & purificação , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Invasividade Neoplásica , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Proteômica , Carcinoma de Células Escamosas de Cabeça e Pescoço , Vimentina/isolamento & purificação , Vimentina/metabolismo
16.
J Vis Exp ; (116)2016 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-27805587

RESUMO

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Macrófagos , Microglia , Técnicas de Cocultura , Humanos , Invasividade Neoplásica
17.
Am J Physiol Cell Physiol ; 311(1): C1-C14, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27076614

RESUMO

The process of entering the bloodstream, intravasation, is a necessary step in the development of distant metastases. The focus of this review is on the pathways and molecules that have been identified as being important based on current in vitro and in vivo assays for intravasation. Properties of the vasculature which are important for intravasation include microvessel density and also diameter of the vasculature, with increased intravasation correlating with increased vessel diameter in some tumors. TGFB signaling can enhance intravasation at least in part through induction of EMT, and we discuss other TGFB target genes that are important for intravasation. In addition to TGFB signaling, a number of studies have demonstrated that activation of EGF receptor family members stimulates intravasation, with downstream signaling through PI3K, N-WASP, RhoA, and WASP to induce invadopodia. With respect to proteases, there is strong evidence for contributions by uPA/uPAR, while the roles of MMPs in intravasation may be more tumor specific. Other cells including macrophages, fibroblasts, neutrophils, and platelets can also play a role in enhancing tumor cell intravasation. The technology is now available to interrogate the expression patterns of circulating tumor cells, which will provide an important reality check for the model systems being used. With a better understanding of the mechanisms underlying intravasation, the goal is to provide new opportunities for improving prognosis as well as potentially developing new treatments.


Assuntos
Movimento Celular , Microvasos/patologia , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Neovascularização Patológica , Proteínas Angiogênicas/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Humanos , Microvasos/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Transdução de Sinais
18.
Arch Pathol Lab Med ; 139(11): 1349-61, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26172508

RESUMO

CONTEXT: Head and neck squamous cell carcinoma (HNSCC) is a highly invasive cancer with an association with locoregional recurrence and lymph node metastasis. We have previously reported that low microRNA-375 (miR-375) expression levels correlate with poor patient survival, increased locoregional recurrence, and distant metastasis. Increasing miR-375 expression in HNSCC cell lines to levels found in normal cells results in suppressed invasive properties. HNSCC invasion is mediated in part by invadopodia-associated degradation of the extracellular matrix. OBJECTIVE: To determine whether elevated miR-375 expression in HNSCC cell lines also affects invadopodia formation and activity. DESIGN: For evaluation of the matrix degradation properties of the HNSCC lines, an invadopodial matrix degradation assay was used. The total protein levels of invadopodia-associated proteins were measured by Western blot analyses. Immunoprecipitation experiments were conducted to evaluate the tyrosine phosphorylation state of cortactin. Human protease arrays were used for the detection of the secreted proteases. Quantitative real time-polymerase chain reaction measurements were used to evaluate the messenger RNA (mRNA) expression of the commonly regulated proteases. RESULTS: Increased miR-375 expression in HNSCC cells suppresses extracellular matrix degradation and reduces the number of mature invadopodia. Higher miR-375 expression does not reduce cellular levels of selected invadopodia-associated proteins, nor is tyrosine phosphorylation of cortactin altered. However, HNSCC cells with higher miR-375 expression had significant reductions in the mRNA expression levels and secreted levels of specific proteases. CONCLUSIONS: MicroRNA-375 regulates invadopodia maturation and function potentially by suppressing the expression and secretion of proteases.


Assuntos
Extensões da Superfície Celular/metabolismo , Endopeptidases/genética , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cortactina/metabolismo , Endopeptidases/metabolismo , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Análise Serial de Proteínas/métodos , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteoma/genética , Proteoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases
19.
Arch Pathol Lab Med ; 139(11): 1334-48, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26046491

RESUMO

CONTEXT: The highly invasive properties demonstrated by head and neck squamous cell carcinoma (HNSCC) are often associated with locoregional recurrence and lymph node metastasis in patients and is a key factor leading to an expected 5-year survival rate of approximately 50% for patients with advanced disease. It is important to understand the features and mediators of HNSCC invasion so that new treatment approaches can be developed. OBJECTIVES: To provide an overview of the characteristics, mediators, and mechanisms of HNSCC invasion. DATA SOURCES: A literature review of peer-reviewed articles in PubMed on HNSCC invasion. CONCLUSIONS: Histologic features of HNSCC tumors can help predict prognosis and influence clinical treatment decisions. Cell surface receptors, signaling pathways, proteases, invadopodia function, epithelial-mesenchymal transition, microRNAs, and tumor microenvironment are all involved in the regulation of the invasive behavior of HNSCC cells. Identifying effective HNSCC invasion inhibitors has the potential to improve outcomes for patients by reducing the rate of spread and increasing responsiveness to chemoradiation.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Quimiorradioterapia/métodos , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/terapia , Humanos , Metástase Linfática , MicroRNAs/genética , Invasividade Neoplásica , Transdução de Sinais , Resultado do Tratamento
20.
Cell Rep ; 4(3): 429-36, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23911287

RESUMO

A paracrine interaction between epidermal growth factor (EGF)-secreting tumor-associated macrophages (TAMs) and colony-stimulating factor 1 (CSF-1)-secreting breast carcinoma cells promotes invasion and metastasis. Here, we show that mice deficient in the hematopoietic-cell-specific Wiskott-Aldrich syndrome protein (WASp) are unable to support TAM-dependent carcinoma cell invasion and metastasis in both orthotopic and transgenic models of mammary tumorigenesis. Motility and invasion defects of tumor cells were recapitulated ex vivo upon coculture with WASp(-/-) macrophages. Mechanistically, WASp is required for macrophages to migrate toward CSF-1-producing carcinoma cells, as well as for the release of EGF through metalloprotease-dependent shedding of EGF from the cell surface of macrophages. Our findings suggest that WASp acts to support both the migration of TAMs and the production of EGF, which in concert promote breast tumor metastasis.


Assuntos
Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Ratos , Proteína da Síndrome de Wiskott-Aldrich/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...