RESUMO
Lysyl oxidase is a multifunctional enzyme required for collagen biosynthesis. Various growth factors regulate lysyl oxidase during osteoblast differentiation, subject to modulation by cytokines such as TNF-α in inflammatory osteopenic disorders including diabetic bone disease. Canonical Wnt signaling promotes osteoblast development. Here we investigated the effect of Wnt3a and TNF-α on lysyl oxidase expression in pluripotent C3H10T1/2 cells, bone marrow stromal cells, and committed osteoblasts. Lysyl oxidase was up-regulated by a transcriptional mechanism 3-fold in C3H10T1/2 cells, and 2.5-fold in bone marrow stromal cells. A putative functional TCF/LEF element was identified in the lysyl oxidase promoter. Interestingly, lysyl oxidase was not up-regulated in committed primary rat calvarial- or MC3T3-E1 osteoblasts. TNF-α down-regulated lysyl oxidase both in Wnt3a-treated and in non-treated C3H10T1/2 cells by a post-transcriptional mechanism mediated by miR203. Non-differentiated cells do not produce a collagen matrix; thus, a novel biological role for lysyl oxidase in pluripotent cells was investigated. Lysyl oxidase shRNAs effectively silenced lysyl oxidase expression, and suppressed the growth of C3H10T1/2 cells by 50%, and blocked osteoblast differentiation. We propose that interference with lysyl oxidase expression under excess inflammatory conditions such as those that occur in diabetes, osteoporosis, or rheumatoid arthritis can result in a diminished pool of pluripotent cells which ultimately contributes to osteopenia.
Assuntos
Doenças Ósseas Metabólicas/enzimologia , Doenças Ósseas Metabólicas/patologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/enzimologia , Proteína-Lisina 6-Oxidase/metabolismo , Animais , Sequência de Bases , Doenças Ósseas Metabólicas/epidemiologia , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Inflamação/patologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Osteoblastos/citologia , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Osteogênese , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Proteína-Lisina 6-Oxidase/genética , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , Proteína Wnt3A/farmacologiaRESUMO
The peritoneal metastatic route of cancer dissemination is shared by cancers of the ovary and gastrointestinal tract. Once initiated, peritoneal metastasis typically proceeds rapidly in a feed-forward manner. Several factors contribute to this efficient progression. In peritoneal metastasis, cancer cells exfoliate into the peritoneal fluid and spread locally, transported by peritoneal fluid. Inflammatory cytokines released by tumor and immune cells compromise the protective, anti-adhesive mesothelial cell layer that lines the peritoneal cavity, exposing the underlying extracellular matrix to which cancer cells readily attach. The peritoneum is further rendered receptive to metastatic implantation and growth by myofibroblastic cell behaviors also stimulated by inflammatory cytokines. Individual cancer cells suspended in peritoneal fluid can aggregate to form multicellular spheroids. This cellular arrangement imparts resistance to anoikis, apoptosis, and chemotherapeutics. Emerging evidence indicates that compact spheroid formation is preferentially accomplished by cancer cells with high invasive capacity and contractile behaviors. This review focuses on the pathological alterations to the peritoneum and the properties of cancer cells that in combination drive peritoneal metastasis.
Assuntos
Matriz Extracelular/metabolismo , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Actomiosina/antagonistas & inibidores , Actomiosina/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ascite , Adesão Celular , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Humanos , Inflamação/metabolismo , Miofibroblastos/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Peritônio/metabolismo , Peritônio/patologia , ProteóliseRESUMO
Ovarian cancer cells are present in malignant ascites both as individual cells and as multicellular spheroid aggregates. Although spheroid formation affords protection of cancer cells against some chemotherapeutic agents, it has not been established whether a relationship exists between invasive behavior and predisposition to spheroid formation. Aspects of spheroid formation, including cell-matrix adhesion, remodeling and contractility are characteristic myofibroblast-like behaviors associated with fibrosis that contribute to tumor growth and dissemination. We explored the possibility that cell behaviors that promote spheroid formation also facilitate invasion. Our analysis of 6 human ovarian cancer cell lines indicated that ovarian cancer cells possessing myofibroblast-like properties formed compact spheroids and invaded 3D matrices. These cells readily contracted collagen I gels, possessed a spindle-like morphology, and had elevated expression of genes associated with the TGFbeta-mediated fibrotic response and/or beta1 integrin function, including fibronectin (FN), connective tissue growth factor (CTGF/CCN2), lysyl oxidase (LOX1), tissue transglutaminase 2 (TGM2) and urinary plasminogen activator receptor (uPAR). Whereas cell aggregation was induced by TGFbeta, and by beta1-integrin overexpression and activation, these treatments did not stimulate the contractile activity required for spheroid compaction. The positive relationship found between compact spheroid formation and invasive behavior implies a preferential survival of an invasive subpopulation of ovarian cancer cells, as cells in spheroids are more resistant to several chemotherapeutics. Preventing the formation of ovarian cancer spheroids may represent a novel strategy to improve the efficacy of existing therapeutics.
Assuntos
Adesão Celular , Neoplasias Ovarianas/patologia , Esferoides Celulares/patologia , Movimento Celular , Proliferação de Células , Colágeno/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Invasividade Neoplásica , Neoplasias Ovarianas/metabolismo , Fenótipo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Proteína 2 Glutamina gama-Glutamiltransferase , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Esferoides Celulares/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP) activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. METHODS: The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. RESULTS: The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only approximately 30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol), which lacks telopeptide regions. CONCLUSION: Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis.
Assuntos
Colágeno/química , Laminina/química , Metaloproteinases da Matriz/química , Neoplasias Ovarianas/patologia , Proteoglicanas/química , Animais , Linhagem Celular Tumoral , Movimento Celular , Combinação de Medicamentos , Feminino , Humanos , Microscopia Confocal , Invasividade Neoplásica , Neoplasias Ovarianas/metabolismo , Pepsina A/química , Peptídeos/química , Ratos , CicatrizaçãoRESUMO
Proteomic profiling has emerged as a useful tool for identifying tissue alterations in disease states including malignant transformation. The aim of this study was to reveal expression profiles associated with the highly motile/invasive ovarian cancer cell phenotype. Six ovarian cancer cell lines were subjected to proteomic characterization using multidimensional protein identification technology (MudPIT), and evaluated for their motile/invasive behavior, so that these parameters could be compared. Within whole cell extracts of the ovarian cancer cells, MudPIT identified proteins that mapped to 2245 unique genes. Western blot analysis for selected proteins confirmed the expression profiles revealed by MudPIT, demonstrating the fidelity of this high-throughput analysis. Unsupervised cluster analysis partitioned the cell lines in a manner that reflected their motile/invasive capacity. A comparison of protein expression profiles between cell lines of high (group 1) versus low (group 2) motile/invasive capacity revealed 300 proteins that were differentially expressed, of which 196 proteins were significantly upregulated in group 1. Protein network and KEGG pathway analysis indicated a functional interplay between proteins up-regulated in group 1 cells, with increased expression of several key members of the actin cytoskeleton, extracellular matrix (ECM) and focal adhesion pathways. These proteomic expression profiles can be utilized to distinguish highly motile, aggressive ovarian cancer cells from lesser invasive ones, and could prove to be essential in the development of more effective strategies that target pivotal cell signaling pathways used by cancer cells during local invasion and distant metastasis.