RESUMO
During epithelial-mesenchymal transition (EMT), epithelial cells are converted into isolated motile and invasive mesenchymal cells. In model systems, EMT is induced most often by the activation of tyrosine kinase receptors through signaling pathways involving translational and post-translational regulation. In this study, we have used the NBT-II bladder carcinoma cell system to investigate in vitro Fibroblast Growth Factor-1 (FGF-1)-induced EMT. Transcriptome analyses were performed on NBT-II cells stimulated for 2, 6, 24, and 48 h with FGF-1. As some phenotypic changes occurred around 6 h post-stimulation, a supervised analysis was designed to identify transcript variations across defined time-periods. Our results clearly indicate that immediately after FGF-1 stimulation a set of genes assigned to transcriptional regulation (e.g., jun-B and v-ets) and to EMT induction (e.g., Notch 1) is transiently up-regulated. A set of genes involved in proteolytic systems (e.g., MMP-13 and uPAR) is immediately up-regulated but subsequently maintained throughout FGF-1 stimulation. Then follows a second wave of gene expression that includes a strong but transient up-regulation of ephrin B1 and arginase I. Finally, a third group of genes is stably modulated over 48 h which consists primarily of down-regulated genes specifically associated with the EMT-based loss of the epithelial phenotype and maintenance of the mesenchymal and invasive phenotype of carcinoma cells. Using genome-wide oligoarray technology, we have identified novel expressions of immediate, immediate-early and later EMT biomarkers that are specifically activated downstream of the FGF/FGFR pathway and which might be significant prognostic factors for tumor progression of carcinoma.
Assuntos
Carcinoma/metabolismo , Transformação Celular Neoplásica , Epitélio/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Mesoderma/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Modelos Estatísticos , Fenótipo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Fibroblast growth factor (FGF)-1 and -2 have potent biological activities implicated in malignant tumor development. Their autocrine and nonautocrine activity in tumor progression of carcinoma was investigated in the NBT-II cell system. Cells were manipulated to either produce and be autocrine for FGF-1 or -2 or to only produce but not respond to these factors. The autocrine cells are highly invasive and tumorigenic and the determination of specific targets of FGF/fibroblast growth factor receptor (FGFR) signaling was assessed. In vitro studies showed that nonautocrine cells behave like epithelial parental cells, whereas autocrine cells have a mesenchymal phenotype correlated with the overexpression of urokinase plasminogen activator receptor (uPAR), the internalization of E-cadherin, and the redistribution of beta-catenin from the cell surface to the cytoplasm and nucleus. uPAR was defined as an early target, whereas E-cadherin and the leukocyte common antigen-related protein-tyrosine phosphatase (LAR-PTP) were later targets of FGF signaling, with FGFR1 activation more efficient than FGFR2 at modulating these targets. Behavior of autocrine cells was consistent with a decrease of tumor-suppressive activities of both E-cadherin and LAR-PTP. These molecular analyses show that the potential of these two growth factors in tumor progression is highly dependent on specific FGFR signaling and highlights its importance as a target for antitumor therapy.
Assuntos
Carcinoma/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Animais , Comunicação Autócrina , Caderinas/metabolismo , Linhagem Celular Tumoral , Forma Celular , Proteínas do Citoesqueleto/metabolismo , Desmoplaquinas , Invasividade Neoplásica , Ratos , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transativadores/metabolismo , beta CateninaRESUMO
FGF-1 and FGF-2 are pleiotropic growth factors for many cell types, operating through the activation of specific transmembrane FGF receptors (FGFRs). The role of these factors in tumor progression was investigated, with specific discrimination between their autocrine and non autocrine cellular activity. The rat bladder carcinoma NBT-II cells were engineered to produce FGF-1 or 18 kDa FGF-2 in the presence or absence of their specific receptor. Non-autocrine cells that produced FGF-1 or FGF-2 but lacked FGFRs were epithelial and reminiscent of the parental NBT-II cells. Whilst autocrine cells, which both constitutively produced and secreted the growth factor and expressed FGFRs, had a highly invasive mesenchymal phenotype. Correspondingly, the autocrine cells were highly tumorigenic in vivo compared to the parental and non-autocrine cells, which correlated with the increased production of uPAR and active uPA and increased in vitro invasive potential. Although all cells produced VEGF, only tumors derived from cells that produced FGF-1 or FGF-2 were highly vascularized, suggesting that these two growth factors could be involved in the angiogenic process by activating host endothelial cells. As a result of activation of the FGFR in autocrine cells, changes in cell morphology and an increase in the invasive and tumorigenic properties were observed, however no in vitro or in vivo differential functions between FGF-1 and FGF-2 could be identified in this system. In conclusion, our data demonstrates that rapid tumor development is not dependent upon increased tumor vascularization, suggesting that 'basal' angiogenesis, probably mediated by VEGF, is sufficient to support tumor growth.
Assuntos
Carcinoma/patologia , Fator 1 de Crescimento de Fibroblastos/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Proteínas de Neoplasias/fisiologia , Neovascularização Patológica/fisiopatologia , Neoplasias da Bexiga Urinária/patologia , Animais , Comunicação Autócrina , Carcinoma/irrigação sanguínea , Carcinoma/genética , Carcinoma/metabolismo , Progressão da Doença , Fatores de Crescimento Endotelial/metabolismo , Células Epiteliais/patologia , Feminino , Fator 1 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Linfocinas/metabolismo , Metaloproteinases da Matriz/biossíntese , Metaloproteinases da Matriz/genética , Mesoderma , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Neovascularização Patológica/genética , Fenótipo , Ratos , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/fisiologia , Deleção de Sequência , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-2/genética , Transfecção , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/transplante , Neoplasias da Bexiga Urinária/irrigação sanguínea , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/genética , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The hypothesis that tumor growth is angiogenesis-dependent has been documented by a considerable body of direct and indirect experimental data. Since the discovery of the vascular endothelial growth factor (VEGF), most attention has been focused on the VEGF system. Although fibroblast growth factors 1 and 2 (FGF-1 and FGF-2) can exert a strong angiogenic activity when they are supplied as a single pharmacological agent, their role in pathological angiogenesis in preclinical models remains controversial. To decipher the contribution of FGF receptors in various models of angiogenesis, we took advantage of the anti-idiotypic strategy to obtain circulating agonists specific for FGFR-1 and FGFR-2 (AIdF-1 and AIdF-2). They mimicked FGF-1 and FGF-2 for receptor binding, signal transduction, proliferation of endothelial cells and differentiation of the bladder carcinoma cell NBT-II which expresses FGFR-2b but not FGFR-1. The constitutive expression of FGFR-1 allowed binding of FGF-2 and AIdF-2 and inhibition of the proliferation of NBT-II cells. AIdF-1 and AIdF-2 induced angiogenesis in the corneal pocket assay. Although FGFR-1 dimerization achieved by AIdF-2 injection led to highly differentiated and smaller NBT-II tumors, no sign of reduction of tumor angiogenesis was observed, thus suggesting that endothelial cells are resistant to FGF.
Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Neoplasias da Bexiga Urinária/patologia , Córtex Suprarrenal/irrigação sanguínea , Animais , Anticorpos Anti-Idiotípicos/imunologia , Capilares , Divisão Celular , Linhagem Celular Tumoral , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Fosforilação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Transdução de Sinais/fisiologia , Transplante Heterólogo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/imunologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Nuclear-targeted high molecular weight 24 kDa fibroblast growth factor 2 (FGF-2) may induce specific cell functions through intracrine mechanisms. The role of nuclear FGF-2 on the metastatic potential of carcinoma cells was examined by conditional FGF-2 expression, which demonstrated that spontaneous metastasis in nude mice is a direct consequence of its expression. The lung colonizing capacities of fluorescent nuclear FGF-2-expressing cells following intravenous injection was also investigated. All cells reaching the lung extravasated as soon as 5 min following injection with similar in vivo behavior during the first 24 h. However, after 2 days, dramatic differences were observed between the FGF-2 and parental cells: most control cells underwent apoptosis, while the FGF-2-producing cells instigated a survival program and proliferated. Therefore, sustained apoptosis in vivo prevents growth of metastatic foci, while nuclear FGF-2 induction of a survival program is responsible for growth of the lung metastases. In vitro serum deprivation assays also established that 24 kDa FGF-2 expression improves carcinoma cell survival. This study provides both in vitro and in vivo evidence that the role of the nuclear 24 kDa FGF-2 isoform in carcinoma is the promotion of cell survival, thereby defining its association with poor prognosis in some human carcinomas.
Assuntos
Sobrevivência Celular/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neoplasias Pulmonares/secundário , Metástase Neoplásica , Neoplasias da Bexiga Urinária/genética , Animais , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/química , Células Clonais , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Injeções Subcutâneas , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Peso Molecular , Transplante de Neoplasias , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Transplante Heterólogo , Neoplasias da Bexiga Urinária/patologiaRESUMO
Engineered retroviruses are widely used vectors for cancer gene therapy approaches. However, the ability to target cells of therapeutic interest while controlling the expression of the transferred genes would improve both the efficiency and the safety of viral vectors. In this study, we investigated the ability of a retroviral amphotropic envelope displaying single-chain variable-fragment (scFv) directed against the c-Met receptor, to target the entry of recombinant retroviruses to human hepatocarcinoma cells. Four single-chain antibody fragments directed against the c-Met receptor were generated and inserted into the viral envelope protein as an N-terminal fusion. The modified envelopes were incorporated into virus particles and one of the chimeric viruses, 3D6-Env, transduced preferentially human hepatoma cells rather than proliferating human hepatocytes. In another construct, the urokinase cleavage site was inserted between the scFv moiety and the envelope. Chimeric scFv-urokinase-Env viruses transduced hepatoma cells with a similar efficiency to that of the control virus and their infectivity in human hepatocytes remained low. These results indicate that amphotropic retroviruses with engineered envelopes to display scFv directed against the c-Met receptor can efficiently and selectively deliver genes into hepatoma cells.
Assuntos
Carcinoma Hepatocelular/genética , Técnicas de Transferência de Genes , Região Variável de Imunoglobulina/genética , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-met/imunologia , Anticorpos/genética , Anticorpos/imunologia , Carcinoma Hepatocelular/virologia , Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Hepatócitos/fisiologia , Hepatócitos/virologia , Humanos , Engenharia de Proteínas/métodos , Retroviridae/genética , Retroviridae/patogenicidade , Transdução Genética , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/genética , VírionRESUMO
It is widely accepted that the development of carcinoma is not only due to somatic mutations in epithelial cells but also is influenced by the tumor microenvironment ie the stroma. The different stroma components produced growth factors, cytokines, the extra cellular matrix and also participated to the recruitment of the endothelial cells necessary for the tumor neovascularisation. The stroma favored the oncogenesis through synergistic reciprocal paracrine signals with the tumor cells. The stroma is determinant for the tumor progression and therefore is an important therapeutic target.
Assuntos
Carcinoma/patologia , Comunicação Celular , Neoplasias/patologia , Células Estromais/patologia , Carcinoma/fisiopatologia , Membrana Celular/fisiologia , Progressão da Doença , Fatores de Crescimento Endotelial/metabolismo , Inibidores Enzimáticos/uso terapêutico , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Neoplasias/irrigação sanguínea , Neoplasias/metabolismo , Neovascularização Patológica/etiologia , Células Estromais/fisiologia , Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Combretastatin A4 phosphate (CA4P) causes rapid disruption of the tumor vasculature and is currently being evaluated for antivascular therapy. We describe the initial results obtained with a noninvasive multiparametric magnetic resonance imaging (MRI) approach to assess the early effects of CA4P on rat bladder tumors implanted on nude mice. MRI (4.7 T) comprised a fast spin-echo sequence for growth curve assessment; a multislice multiecho sequence for T2 measurement before, 15 minutes after, and 24 hours after CA4P (100 mg/kg); and a fast T2w* gradient-echo sequence to assess MR signal modification under carbogen breathing before, 35 minutes after, and 24 hours after CA4P. The tumor fraction with increased T2w* signal intensity under carbogen (T+) was used to quantify CA4P effect on functional vasculature. CA4P slowed tumor growth over 24 hours and accelerated necrosis development. T+ decrease was observed already at 35 minutes post-CA4P. Early T2 increase was observed in regions becoming necrotic at 24 hours post-CA4P, as confirmed by high T2 and histology. These regions exhibited, under carbogen, a switch from T2w* signal increase before CA4P to a decrease postCA4P. The combination of carbogen-based functional MRI and T2 measurement may be useful for the early follow-up of antivascular therapy without the administration of contrast agents.