RESUMO
Invadopodia are proteolytic membrane protrusions formed by highly invasive cancer cells, commonly observed on substrate(s) mimicking extracellular matrix. Although invadopodia are proposed to have roles in cancer invasion and metastasis, direct evidence has not been available. We previously reported that neural Wiskott-Aldrich syndrome protein (N-WASP), a member of WASP family proteins that regulate reorganization of the actin cytoskeleton, is an essential component of invadopodia. Here, we report that N-WASP-mediated invadopodium formation is essential in breast cancer invasion, intravasation and lung metastasis. We established stable cell lines based on MTLn3 rat mammary adenocarcinoma cells that either overexpressed a dominant-negative (DN) N-WASP construct or in which N-WASP expression was silenced by a pSuper N-WASP shRNA. Both the N-WASP shRNA and DN N-WASP cells showed a markedly decreased ability to form invadopodia and degrade extracellular matrix. In addition, formation of invadopodia in primary tumors and collagen I degradation were reduced in the areas of invasion (collagen-rich areas in the invasive edge of the tumor) and in the areas of intravasation (blood-vessel-rich areas). Our results suggest that tumor cells in vivo that have a decreased activity of N-WASP also have a reduced ability to form invadopodia, migrate, invade, intravasate and disseminate to lung compared with tumor cells with parental N-WASP levels.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Invasividade Neoplásica/fisiopatologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Mamárias Experimentais/genética , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Interferente Pequeno/genética , Ratos , Ratos Endogâmicos F344 , Proteína Neuronal da Síndrome de Wiskott-Aldrich/antagonistas & inibidores , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genéticaRESUMO
We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena(INV)) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena(INV) increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena(INV) is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena(INV) and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Migração Transendotelial e Transepitelial , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fator Estimulador de Colônias de Macrófagos/biossíntese , Macrófagos/metabolismo , Camundongos , Camundongos SCID , Proteínas dos Microfilamentos , Invasividade Neoplásica , Metástase Neoplásica , Isoformas de Proteínas/metabolismo , RatosRESUMO
INTRODUCTION: The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development. METHODS: To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice. RESULTS: Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching. CONCLUSIONS: Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.
Assuntos
Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/fisiologia , Neoplasias Mamárias Experimentais/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Proteínas do Citoesqueleto/genética , Progressão da Doença , Fator de Crescimento Epidérmico/metabolismo , Feminino , Expressão Gênica , Neoplasias Pulmonares/secundário , Macrófagos , Glândulas Mamárias Animais/anatomia & histologia , Glândulas Mamárias Animais/embriologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos SCID , Camundongos Transgênicos , Proteínas dos Microfilamentos , Invasividade Neoplásica , Metástase Neoplásica , Reação em Cadeia da Polimerase , Microambiente TumoralRESUMO
The C57BLKS/J (BKS) inbred mouse strain is a widely used animal model of type 2 diabetes. In the presence of the diabetes (db) mutation, obese BKS-db mice develop severe diabetes. Genetic studies of diabetes-susceptibility in this strain are facilitated by the fact that BKS is a genetic composite between the diabetes-resistant C57BL/6J (B6) and susceptible DBA/2J (DBA) strains. On this basis, it has been hypothesized that diabetes-susceptibility in BKS is conferred by DBA-derived alleles. However, recent studies revealed non-B6/non-DBA genetic material in BKS. To identify the origin of this genetic component, we generated a genomic map of BKS using 537 microsatellite markers. Our results demonstrate that, in addition to B6 and DBA, BKS contains alleles from at least three other strains, including 129, C57BL/10 and an unidentified mouse strain. We also analyzed two congenic strains, B6-db and BKS-db, which are widely used for the genetic mapping of diabetes-susceptibility loci. We identified several donor-derived genomic regions introduced during the generation of these congenic strains. In summary, our study reveals novel aspects of the genetic fine-structure of BKS and related strains and facilitates the identification of diabetes-susceptibility loci in this mouse model.
Assuntos
Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Camundongos Endogâmicos/genética , Camundongos Mutantes/genética , Alelos , Animais , Cruzamentos Genéticos , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Endogâmicos DBA/genética , Camundongos Obesos/genética , Repetições de Microssatélites , MutaçãoRESUMO
Chemotaxis of tumour cells and stromal cells in the surrounding microenvironment is an essential component of tumour dissemination during progression and metastasis. This Review summarizes how chemotaxis directs the different behaviours of tumour cells and stromal cells in vivo, how molecular pathways regulate chemotaxis in tumour cells and how chemotaxis choreographs cell behaviour to shape the tumour microenvironment and to determine metastatic spread. The central importance of chemotaxis in cancer progression is highlighted by discussion of the use of chemotaxis as a prognostic marker, a treatment end point and a target of therapeutic intervention.
Assuntos
Quimiotaxia , Neoplasias/fisiopatologia , Animais , Movimento Celular , Progressão da Doença , Humanos , Prognóstico , Células Tumorais CultivadasRESUMO
Characterizing biological mechanisms dependent upon the interaction of many cell types in vivo requires both multiphoton microscope systems capable of expanding the number and types of fluorophores that can be imaged simultaneously while removing the wavelength and tunability restrictions of existing systems, and enhanced software for extracting critical cellular parameters from voluminous 4D data sets. We present a procedure for constructing a two-laser multiphoton microscope that extends the wavelength range of excitation light, expands the number of simultaneously usable fluorophores and markedly increases signal to noise via 'over-clocking' of detection. We also utilize a custom-written software plug-in that simplifies the quantitative tracking and analysis of 4D intravital image data. We begin by describing the optics, hardware, electronics and software required, and finally the use of the plug-in for analysis. We demonstrate the use of the setup and plug-in by presenting data collected via intravital imaging of a mouse model of breast cancer. The procedure may be completed in â¼24 h.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Software , Animais , Feminino , Processamento de Imagem Assistida por Computador , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentaçãoRESUMO
Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena invasive (Mena(INV)) and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-Mena(INV) expressing tumor cells are significantly less cohesive and have discontinuous cell-cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more Mena(INV) mRNA than early non-metastatic ones. Furthermore, Mena(INV) expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that Mena(INV) is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that Mena(INV) expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.
Assuntos
Neoplasias da Mama/patologia , Proteínas do Citoesqueleto/metabolismo , Neoplasias Mamárias Experimentais , Metástase Neoplásica/patologia , Isoformas de Proteínas/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/secundário , Adesão Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Proteínas dos Microfilamentos , Metástase Neoplásica/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Células Tumorais CultivadasRESUMO
Epithelial-mesenchymal transition (EMT) is a developmental program implicated in cancer progression and was the subject of the 2010 AACR meeting on the topic of EMT and Cancer Progression and Treatment held on February 28 to March 2 in Arlington, Virginia. A review of the involvement of EMT in gastrulation, organogenesis, carcinogenesis, and metastatic progression elucidated the overlap of EMT in these physiologic and pathologic conditions. Both novel and traditional markers of cells undergoing EMT were discussed and compared with features used to define cancer stem cells. Importantly, these defining characteristics of cells undergoing EMT were discussed in the context of therapeutic and prognostic developments.
Assuntos
Neoplasias/patologia , Neoplasias/terapia , Animais , Progressão da Doença , Células Epiteliais/patologia , Humanos , Mesoderma/patologia , Camundongos , Células-Tronco Neoplásicas/patologiaRESUMO
Class IA (p85/p110) phosphoinositide 3-kinases play a major role in regulating cell growth, survival, and motility. Activating mutations in the p110alpha isoform of the class IA catalytic subunit (PIK3CA) are commonly found in human cancers. These mutations lead to increased proliferation and transformation in cultured cells, but their effects on cell motility and tumor metastasis have not been evaluated. We used lentiviral-mediated gene transfer and knockdown to produce stable MDA-MB-231 cells in which the endogenous human p110alpha is replaced with either wild-type bovine p110alpha or the two most common activating p110alpha mutants, the helical domain mutant E545K and the kinase domain mutant H1047R. The phosphoinositide 3-kinase/Akt pathway was hyperactivated in cells expressing physiologic levels of helical or kinase domain mutants. Cells expressing either mutant showed increased motility in vitro, but only cells expressing the helical domain mutant showed increased directionality in a chemotaxis assay. In severe combined immunodeficient mice, xenograft tumors expressing either mutant showed increased rates of tumor growth compared with tumors expressing wild-type p110alpha. However, tumors expressing the p110alpha helical domain mutant showed a marked increase in both tumor cell intravasation into the blood and tumor cell extravasation into the lung after tail vein injection compared with tumors expressing wild-type p110alpha or the kinase domain mutant. Our observations suggest that, when compared with kinase domain mutations in a genetically identical background, expression of helical domain mutants of p110alpha produce a more severe metastatic phenotype.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Movimento Celular/genética , Mutação , Fosfatidilinositol 3-Quinases/genética , Animais , Neoplasias da Mama/genética , Domínio Catalítico , Bovinos , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Secundária de Proteína , Subunidades ProteicasRESUMO
The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, Mena(INV). Here we show that Mena and Mena(INV) promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and Mena(INV) potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, Mena(INV) is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of Mena(INV) could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels.