RESUMO
BACKGROUND: CRIPTO-1 (CR-1) is involved in the pathogenesis and progression of human carcinoma of different histological origin. In this study we addressed the expression and the functional role of CR-1 in cutaneous melanoma. METHODS: Expression of CR-1 protein in melanomas and melanoma cell lines was assessed by immunohistochemistry, western blotting and/or flow cytometry. Levels of mRNA were evaluated by real-time PCR. Invasion assays were performed in Matrigel-coated modified Boyden chambers. RESULTS: Expression of CR-1 protein and/or mRNA was found in 16 out of 37 primary human cutaneous melanomas and in 12 out of 21 melanoma cell lines. Recombinant CR-1 protein activated in melanoma cells c-Src and, at lesser extent, Smad signalling. In addition, CR-1 significantly increased the invasive ability of melanoma cells that was prevented by treatment with either the ALK4 inhibitor SB-431542 or the c-Src inhibitor saracatinib (AZD0530). Anti-CR-1 siRNAs produced a significant inhibition of the growth and the invasive ability of melanoma cells. Finally, a close correlation was found in melanoma cells between the levels of expression of CR-1 and the effects of saracatinib on cell growth. CONCLUSION: These data indicate that a significant fraction of cutaneous melanoma expresses CR-1 and that this growth factor is involved in the invasion and proliferation of melanoma cells.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Receptores de Ativinas Tipo I/antagonistas & inibidores , Receptores de Ativinas Tipo I/metabolismo , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Western Blotting , Proteína Tirosina Quinase CSK , Adesão Celular , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Dioxóis/farmacologia , Citometria de Fluxo , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Humanos , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Melanoma/genética , Invasividade Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Quinazolinas/farmacologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Proteínas Smad/metabolismo , Células Tumorais Cultivadas , Quinases da Família srcRESUMO
Epithelial-mesenchymal transition (EMT) is a process occurring during both embryogenesis and early stages of invasive cancer. Epithelial cells that undergo EMT become more migratory and invasive with a mesenchymal morphology. Herein we assess EMT induction in a mouse mammary epithelial cell line driven by Msx2, a homeobox-containing transcription factor important during mammary gland development. NMuMG cells, a normal mouse mammary epithelial cell line, stably transfected with a Msx2 cDNA showed downregulation of an epithelial marker E-cadherin and upregulation of the mesenchymal markers vimentin and N-cadherin. Furthermore, overexpression of Cripto-1, a member of the epidermal growth factor-CFC protein family already known to be involved in EMT, was detected in Msx2-transfected cells. The expression of Cripto-1 was accompanied by activation of the tyrosine kinase c-Src pathway and an increase in the invasive ability of the cells. Functional assays also demonstrated inhibition of the invasive behavior of the Msx2-transfected cells by a c-Src specific inhibitor. Moreover, immunohistochemistry of human infiltrating breast carcinomas showed positive staining for Msx2 only in the infiltrating tumor cells while the non-infiltrating tumor cells were negative. These results suggest that Msx2 may play a significant role in promoting EMT in epithelial cells that acquire properties involved in tumor invasion. J. Cell. Physiol. 219: 659-666, 2009. Published 2009 Wiley-Liss, Inc.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Proteínas de Homeodomínio/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteína Tirosina Quinase CSK , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Linhagem Celular , Primers do DNA/genética , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Proteínas de Homeodomínio/genética , Humanos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/fisiopatologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinases/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Regulação para Cima , Quinases da Família srcRESUMO
Transgenic mice overexpressing Notch4 intracellular domain (Int3) under the control of the whey acidic protein (WAP) or mouse mammary tumor virus-long terminal repeat promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneously implanted Alzet pumps. Phosphorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed small interfering RNA (siRNA) to knock down c-Kit, PDGFRs and c-Abl alone or in combination and observed the effects on soft agar growth of HC11 cells overexpressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrate an inhibitory effect of Gleevec on Int3-induced transformation of HC11 cells and mammary tumors and indicate an oncogenic role for c-Kit and PDGFR-alpha tyrosine kinases in the context of Int3 signaling.
Assuntos
Transformação Celular Neoplásica , Glândulas Mamárias Animais/crescimento & desenvolvimento , Neoplasias Mamárias Experimentais/patologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Notch/fisiologia , Animais , Antineoplásicos/uso terapêutico , Benzamidas , Northern Blotting , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Mesilato de Imatinib , Imunoprecipitação , Neoplasias Mamárias Experimentais/genética , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Fosforilação , Piperazinas/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-kit/química , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/uso terapêutico , RNA Interferente Pequeno/farmacologia , Receptor Notch4 , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This review article provides an overview on the most recent advances on the role of ErbB receptors and growth factors of the epidermal growth factor (EGF)-family of peptides in cancer pathogenesis and progression. The ErbB tyrosine kinases and the EGF-like peptides form a complex system. In fact, the interactions occurring between receptors and ligands of these families affect the type and the duration of the intracellular signals that derive from receptor activation. Interestingly, activation of ErbB receptors is also driven by different classes of membrane receptor, suggesting that ErbB kinases can amplify growth promoting signals carried by different pathways. The importance of ErbB receptors and EGF-like peptides in development of organs and tissues has been demonstrated by using different mouse models. In vitro and in vivo studies have also shown that ErbB receptors and their ligands can act as transforming genes. However, evidence suggests that cooperation of different receptors and ligands is necessary to induce a fully transformed phenotype. Indeed, co-expression of different ErbB receptors and EGF-like growth factors is a common phenomenon in human primary carcinomas. This observation suggests that the growth and the survival of carcinoma cells is sustained by a network of receptors/ligands of the ErbB family. In this respect, the contemporary expression of different ErbB tyrosine kinases and/or EGF-like growth factors in human carcinomas might also affect tumor response to target based agents directed against the ErbB receptor/ligand system.
Assuntos
Receptores ErbB/fisiologia , Neoplasias/etiologia , Receptor ErbB-2/fisiologia , Receptor ErbB-3/fisiologia , Animais , Transformação Celular Neoplásica , Dimerização , Receptores ErbB/análise , Humanos , Ligantes , Neoplasias/química , Neoplasias/patologia , Receptor ErbB-2/análise , Receptor ErbB-3/análise , Receptor ErbB-4 , Transdução de Sinais , Ativação TranscricionalRESUMO
Activated platelets may engage in dynamic interplay with other blood cells. We examined the evidence for platelet activation and the formation of platelet-erythrocyte aggregates in chronic hemodialysis patients. Circulating activated platelets (P-selectin/CD63-positive platelets) were higher than normal controls (p < 0.001) and further increased during hemodialysis sessions, the increase being higher when patients were dialyzed with cellulosic than with synthetic membranes. We found direct evidence of uremic platelet-erythrocyte adherence in vitro and increased levels of circulating platelet-erythrocyte aggregates in dialysis patients, which represents a new observation in uremia. Platelet-erythrocyte aggregates were subject to further increase during hemodialysis, and again higher levels were found with cellulosic than synthetic membranes. This phenomenon was reproduced in vitro by both ADP and PAF, but not by either complement factor C3a or by heparin concentrations corresponding to those used for clinical hemodialysis. We conclude that platelet-erythrocyte aggregates occur in hemodialysis patients probably owing to a primary platelet activation mechanism.
Assuntos
Plaquetas/patologia , Eritrócitos/patologia , Falência Renal Crônica/sangue , Ativação Plaquetária , Diálise Renal , Difosfato de Adenosina/farmacologia , Idoso , Plaquetas/efeitos dos fármacos , Agregação Celular , Complemento C3a/farmacologia , Feminino , Heparina/farmacologia , Humanos , Falência Renal Crônica/terapia , Masculino , Membranas Artificiais , Microscopia Eletrônica , Pessoa de Meia-Idade , Fator de Ativação de Plaquetas/farmacologia , Ativação Plaquetária/efeitos dos fármacosRESUMO
The expression of angiogenic factors may represent useful markers for diagnosis and prediction of disease outcome. Basic fibroblast growth factor (b-FGF) is a potent angiogenic factor which promotes in vitro growth of endothelial cells and in vivo vessel formation. We investigated the expression of b-FGF in patients affected with malignant and non-malignant pleural diseases and presenting clinically with non-specific signs and symptoms. We also studied the relationships between the expression of b-FGF in patients with malignant pleural mesothelioma (MM) and tumour aggressiveness, assessed as tumour vessel density (TVD), or patient survival. Basic-FGF was measured by immunoassay in the serum and pleural effusions (PE) of 37 patients. Of these, MM was diagnosed in 15/37 patients while the remaining patients had either peripheral lung adenocarcinoma (PLA) or benign inflammatory pleural disease (BPD). The mean b-FGF level measured 8.5+/-6.1 pg/ml in the PE of the malignant group (MM + PLA) and 23.9+/-19.8 in the PE of the non-malignant group (BPD) (p=0.001). The mean b-FGF level was significantly lower in the PE of MM patients (6.9+/-5.2 pg/ml) compared to BPD patients (p=0.004). Linear regression analysis showed a significant inverse correlation (r=-0.59; p=0.041) between b-FGF levels found in MM PE and patient survival. A noteworthy relationship between high serum b-FGF levels and reduced survival was also observed (r=-0.57; p=0.052). Interestingly, both serum (r=0.48; p=0.114) and PE (r=0.26; p=0.413) b-FGF levels in MM patients correlated poorly with TVD. Our data indicate that b-FGF is significantly more expressed in non-malignant compared to malignant PE, this difference being particularly evident between MM and BPD. Our results also suggest that high b-FGF levels correlate with poor MM patient survival through mechanisms which may be independent of b-FGF angiogenic potential.
Assuntos
Biomarcadores Tumorais/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Mesotelioma/mortalidade , Neoplasias Mesoteliais/mortalidade , Neovascularização Patológica/metabolismo , Derrame Pleural Maligno/metabolismo , Neoplasias Pleurais/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Fator VIII/metabolismo , Feminino , Humanos , Masculino , Mesotelioma/irrigação sanguínea , Mesotelioma/metabolismo , Microscopia Eletrônica , Pessoa de Meia-Idade , Neoplasias Mesoteliais/irrigação sanguínea , Neoplasias Mesoteliais/metabolismo , Neoplasias Pleurais/irrigação sanguínea , Neoplasias Pleurais/metabolismo , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
The prognosis of patients with malignant pleural mesothelioma (MM) is dependent more on tumor extension and differentiation than on therapeutic effects. Reduplication of the basal lamina (RBL) is an ultrastructural feature of some benign and malignant tumors that has been inversely correlated with aggressiveness and was recently described in MM. To investigate whether RBL is important for predicting the survival of patients with MM, transmission electron microscopy was used to identify the presence of basal lamina or RBL in biopsy specimens obtained by thoracoscopy from 35 patients. Cox's regression analysis was used to study the relation of these ultrastructural features to survival. Better outcomes were found for patients whose tumors expressed either basal lamina (HR 0.48; 95% CI, 0.09-2.47) or RBL (HR 0.38; 95% CI 0.12-1.22) compared with the reference category, where basal lamina or RBL was not found. The expression of basal lamina and RBL is an important novel prognostic factors in MM. HUM PATHOL 31:1341-1345.
Assuntos
Membrana Basal/ultraestrutura , Mesotelioma/patologia , Neoplasias Pleurais/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Feminino , Humanos , Imuno-Histoquímica , Masculino , Mesotelioma/química , Mesotelioma/mortalidade , Microscopia Eletrônica , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Estadiamento de Neoplasias , Neoplasias Pleurais/química , Neoplasias Pleurais/mortalidade , Prognóstico , Análise de Sobrevida , Taxa de SobrevidaRESUMO
Pleural malignant mesothelioma (MM) shows poor survival, regardless of tumour stage at diagnosis. MM is unresponsive to present treatment regimens and new protocols are desperately needed. The localised nature, the potential accessibility, and the relative lack of distant metastases make MM a particularly attractive candidate for somatic gene therapy. A common target for cancer gene therapy is the tumour suppressor protein p53. p53 does not seem to be mutated or deleted in MM, but it can be inactivated by binding to other proteins, like mdm2 and SV40 large T antigen. We tested the effects of a replication-deficient adenoviral vector carrying wild-type p53 cDNA in human MM cells. Our results show that >95% of MM cells were efficiently infected with 25 multiplicity of infection (MOI) of vector. Wild-type p53 was effectively expressed resulting in >80% inhibition of proliferation in MM cells. AdCMV.p53 infection induced apoptosis while controls did not show any evident morphological alterations. Ex vivo p53 gene transfer experiments inhibited tumourigenesis in nude mice. In vivo, direct intratumour injection of AdCMV.p53 arrested tumour growth and prolonged survival of treated mice. These results indicate that p53-gene therapy should be strongly exploited for clinical trials in MM patients.
Assuntos
Adenoviridae , Apoptose , Vetores Genéticos , Mesotelioma , Neoplasias Pleurais , Proteína Supressora de Tumor p53/metabolismo , Animais , Testes de Carcinogenicidade , Expressão Gênica , Humanos , Camundongos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Polyomaviruses are expressed in both human tumors and immunodepressed patients. Malignant and nonmalignant pleural effusions create an environment that could favor the expression of opportunistic viral infections. We studied if SV40, JC, and BK viral DNA can be amplified from biopsies obtained from different pleural diseases. MATERIALS AND METHODS: DNA was extracted from mesotheliomas (MM), nonspecific inflammatory and tubercular pleural biopsies, blood and urinary sediments from patients with MM, and pleural effusion cytological specimens. SV40, JC and BK viral early regions were amplified by PCR and analyzed by Southern Blot hybridization with specific probes. RESULTS: SV40 was positive in 9/23 MM, 5/18 tubercular and 1/7 nonspecific inflammatory biopsies, and 5/12 pleural effusion cytological specimens. JC was positive in 2/23 MM and in 7/15 urinary sediments. All blood samples were negative and BK was also negative in all samples. CONCLUSIONS: Tissue specific factors, characteristic of MM and TB, may contribute to expression of SV40 in these diseases.
Assuntos
Vírus BK/isolamento & purificação , Vírus JC/isolamento & purificação , Mesotelioma/virologia , Doenças Pleurais/virologia , Neoplasias Pleurais/virologia , Vírus 40 dos Símios/isolamento & purificação , Southern Blotting , DNA Viral/análise , Humanos , Mesotelioma/sangue , Mesotelioma/patologia , Mesotelioma/urina , Doenças Pleurais/sangue , Doenças Pleurais/patologia , Doenças Pleurais/urina , Derrame Pleural/virologia , Derrame Pleural Maligno/virologia , Neoplasias Pleurais/sangue , Neoplasias Pleurais/patologia , Neoplasias Pleurais/urina , Reação em Cadeia da PolimeraseRESUMO
The ability to immortalize human mesothelioma cells in vitro with simian virus (SV) 40 and the fact that SV40 induces mesotheliomas in hamsters prompted us to look for SV40 deoxyribonucleic acid (DNA) sequences in human mesotheliomas. In a previous study, we found that over half (29/48) of human malignant pleural mesotheliomas contained SV40-like sequences whereas only a few (3/47) control samples contained the same detectable sequences. The SV40 genome encodes the 90 KD nuclear large T-antigen (Tag) and the 17 KD small-t antigen (tag), responsible for SV40's transforming and oncogenic properties. These antigens block tumour suppressor gene products, such as p53. We considered the possibility of reverting this effect by adding exogenous wild-type p53 and thus restoring normal cell functions. For this purpose, we developed a recombinant adenovirus carrying complementary DNA (cDNA) for wild type p53 (AdCMV.p53) and infected mesothelioma cell lines with this virus. Inhibition of proliferation, halting of the cell cycle and massive apoptosis was observed in all mesothelioma cell lines tested. In addition, proliferation of human mesothelioma tumours into nude mice was inhibited by in vivo adenovirus-mediated p53 transgene expression. We also report preliminary evidence of expression, by immunoreactivity, of the extracellular matrix protein tenascin in human malignant pleural mesotheliomas. It was interesting to find predominant tenascin positivity at the tumour's invasive edge and in areas of tumour vascularization. This preliminary report suggests that adenovirus-mediated p53 hyperexpression counteracts transforming properties of the large T-antigen and suggests that gene therapy may be useful in treating human malignant mesothelioma.
Assuntos
Vírus de DNA Tumorais/isolamento & purificação , Mesotelioma/diagnóstico , Mesotelioma/terapia , Neoplasias Pleurais/diagnóstico , Neoplasias Pleurais/terapia , Animais , Cricetinae , Proteínas de Ligação a DNA/análise , Humanos , Mesotelioma/virologia , Camundongos , Neoplasias Pleurais/virologia , Prognóstico , Infecções Tumorais por Vírus/diagnóstico , Infecções Tumorais por Vírus/terapiaRESUMO
Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stemcell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cellinduced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a nonrandom fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.
Assuntos
Neuroblastoma/terapia , Células-Tronco Pluripotentes/citologia , Teratoma/patologia , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Humanos , Mesoderma/citologia , Camundongos , Neuroblastoma/embriologia , Neuroblastoma/patologia , Células-Tronco/patologia , Transplante Heterólogo , Tropismo/genéticaRESUMO
Transgenic mice expressing the Notch 4 intracellular domain (ICD) (Int3) in the mammary gland have two phenotypes: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. We have conditionally ablated the Rbpj gene in the mammary glands of mice expressing whey acidic protein (Wap)-Int3. Interestingly, Rbpj knockout mice (Wap-Cre(+)/Rbpj(-/-)/Wap-Int3) have normal mammary gland development, suggesting that the effect of endogenous Notch signaling on mammary gland development is complete by day 15 of pregnancy. RBP-J heterozygous (Wap-Cre(+)/Rbpj(-/+)/Wap-Int3) and Rbpj control (Rbpj(flox/flox)/Wap-Int3) mice are phenotypically the same as Wap-Int3 mice with respect to mammary gland development and tumorigenesis. In addition, the Wap-Cre(+)/Rbpj(-/-)/Wap-Int3-knockout mice also developed mammary tumors at a frequency similar to Rbpj heterozygous and Wap-Int3 control mice but with a slightly longer latency. Thus, the effect on mammary gland development is dependent on the interaction of the Notch ICD with the transcription repressor/activator Rbpj, and Notch-induced mammary tumor development is independent of this interaction.
Assuntos
Glândulas Mamárias Animais/embriologia , Neoplasias Mamárias Experimentais/genética , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Notch/fisiologia , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/patologia , Ágar , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Viral/genética , Feminino , Proteínas de Homeodomínio/metabolismo , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Knockout , Camundongos Nus , Proteínas do Leite/genética , Proteínas de Neoplasias/genética , Gravidez , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptor Notch4 , Receptores Notch/química , Receptores Notch/deficiência , Receptores Notch/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/genética , Sequências Repetidas Terminais/genética , Fatores de Transcrição HES-1 , Células Tumorais Cultivadas/citologiaRESUMO
Overexpression of Cripto-1 (CR-1) in FVB/N mice using the MMTV-LTR promoter results in increased mammary tumourigenesis in these female transgenic mice (MMTV-CR-1). Here, we characterize uterine tumours that developed in 15/76 (19.7%) of MMTV-CR-1 female nulliparous or multiparous mice during 24 months of observation compared with 0/33 (0%) of FVB/N normal control mice observed during the same time period (p < 0.01). The uterine tumours collected from the MMTV-CR-1 mice were classified as leiomyosarcomas and found to express the CR-1 transgene by polymerase chain reaction analysis and immunohistochemistry. Detection by western blot analysis showed higher levels of phosphorylated (P) forms of c-src, Akt, GSK-3beta, and dephosphorylated (DP)-beta-catenin in lysates from MMTV-CR-1 uterine leiomyosarcomas in comparison with lysates from normal control FVB/N uteri. Immunostaining showed increased nuclear localization of beta-catenin in the MMTV-CR-1 uterine leiomyosarcomas. Increased immunostaining for CR-1 was detected in 9/13 (69.2%) cases of human leiomyosarcoma compared with staining in 3/15 (20%) human leiomyoma sections. Stronger immunostaining for P-src, P-Akt, P-GSK-3beta and increased nuclear localization of beta-catenin was also seen in human leiomyosarcomas in comparison with leiomyomas. Normal human uterine smooth muscle (UtSM) cells treated with exogenous soluble rhCR-1 showed increased levels of P-src, P-Akt, P-GSK-3beta and dephosphorylated (DP)-beta-catenin and increased proliferation (p < 0.05) and migration (p < 0.01) in comparison with untreated control UtSM cells. Inhibitors against c-src, Akt or beta-catenin, individually or in combination, significantly reduced CR-1-induced migration. These results suggest a role for CR-1 during uterine tumourigenesis either directly by activating c-src and Akt and/or via cross-talk with the canonical Wnt signalling pathway, as suggested by the increased expression of P-GSK-3beta, DP-beta-catenin, and increased nuclear localization of beta-catenin in human and MMTV-CR-1 mice leiomyosarcomas.
Assuntos
Fator de Crescimento Epidérmico/genética , Regulação Neoplásica da Expressão Gênica , Leiomiossarcoma/patologia , Glicoproteínas de Membrana/genética , Proteínas de Neoplasias/genética , Neoplasias Uterinas/patologia , Animais , Western Blotting/métodos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Feminino , Proteínas Ligadas por GPI , Humanos , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intercelular , Leiomiossarcoma/química , Leiomiossarcoma/genética , Vírus do Tumor Mamário do Camundongo/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Neoplasias Uterinas/química , Neoplasias Uterinas/genética , Proteína Wnt1/análise , Proteína Wnt1/genética , Proteína Wnt1/metabolismoRESUMO
Methionine aminopeptidase-2 (MetAP2) is the molecular target of the angiogenesis inhibitors, fumagillin and ovalacin. Fumagillin can also inhibit cancer cell proliferation, implying that MetAP2 may play a quite complex role in tumor progression. Here, we examined the expression and function of MetAP2 in an in vitro model of human mesothelioma. We found that mesothelioma cells expressed higher MetAP2 mRNA levels than primary normal mesothelial cells. Consistently, fumagillin induced apoptosis, owing to early mitochondrial damage, in malignant, but not in normal mesothelial cells. Transfection of mesothelioma cells with a MetAP2 anti-sense oligonucleotide determined a time-dependent inhibition of cell survival and induced nucleosome formation. Interestingly, mRNA and protein levels of the anti-apoptotic gene bcl-2 as well as telomerase activity were selectively reduced after MetAP2 inhibition in mesothelioma cells, whereas bcl-2 overexpression counteracted the effect of MetAP2 inhibition on telomerase activity and apoptosis. MetAP2 inhibition also increased caspase activity and the caspase inhibitor, zVAD-fmk, prevented fumagillin-induced apoptosis, but it did not alter telomerase activity. These results indicate that MetAP2 is a main regulator of proliferative and apoptotic pathways in mesothelioma cells and suggest that MetAP2 inhibition may represent a potential target for therapeutic intervention in human mesothelioma.
Assuntos
Aminopeptidases/genética , Aminopeptidases/metabolismo , Inibidores da Angiogênese/farmacologia , Apoptose/fisiologia , Mesotelioma/enzimologia , Mesotelioma/patologia , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Telomerase/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Colágeno/farmacologia , Cicloexanos , Inibidores de Cisteína Proteinase/farmacologia , Endostatinas , Ácidos Graxos Insaturados/farmacologia , Genes bcl-2 , Humanos , Mesotelioma/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , Sesquiterpenos , Suramina/farmacologia , Talidomida/farmacologia , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Vascular endothelial growth factor (VEGF), a potent mitogen for vascular endothelium, is expressed in malignant pleural mesothelioma (MM). The present report examines the effect of VEGF on MM growth. Four MM cell lines produced significantly higher VEGF levels than normal mesothelial cells (1946+/-14 pg/ml vs. 180+/-17 pg/ml; p<0.001). In addition, MM cells expressed the tyrosine kinase-related VEGF receptors Flt-1 and KDR. Recombinant human VEGF phosphorylated both Flt-1 and KDR and increased proliferation of all four MM cell lines in a dose-dependent fashion. Neutralizing antibodies against either VEGF, Flt-1 or KDR significantly reduced MM cellular proliferation. In addition, expression of VEGF, Flt-1, and KDR was observed in MM biopsies. Moreover, higher VEGF levels were found in the pleural effusions of MM patients than in the effusions of patients with non-malignant pleural disease (1885.7+/-894.9 pg/ml vs. 266.9+/-180.5 pg/ml; p<0.001). Linear regression analysis showed a significant inverse correlation between serum VEGF levels and MM patient survival (r=0.72; p<0.01). No correlation was found between tumour vessel density and either serum (r=0.26; p=0.42) or pleural effusion (r=0.35; p=0.26) VEGF levels. These results indicate that VEGF, via activation of its tyrosine kinase receptors, may be a key regulator of MM growth. In addition, VEGF production could have an impact on patient survival, not only by promoting tumour angiogenesis but also by directly stimulating tumour growth.
Assuntos
Comunicação Autócrina/fisiologia , Biomarcadores Tumorais/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fatores de Crescimento Endotelial/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Linfocinas/farmacologia , Masculino , Mesotelioma/irrigação sanguínea , Pessoa de Meia-Idade , Neovascularização Patológica/patologia , Fosforilação/efeitos dos fármacos , Derrame Pleural Maligno/metabolismo , Neoplasias Pleurais/irrigação sanguínea , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes/farmacologia , Estudos Retrospectivos , Taxa de Sobrevida , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The expression of the tumour-associated glycoprotein 90K in patients with malignant pleural mesothelioma (MM) has not been described. This study used enzyme-linked immunoassay (ELISA) to measure 90K in pleural effusions (PEs) and sera from patients with MM (n=28), lung cancer (LC) (n=14) and benign pleural disease (BPD) (n=15). Immunohistochemistry was used to investigate 90K expression in MM and LC tissue sections. The expression of 90K was further evaluated in vitro by ELISA and western blot analysis of conditioned media and cellular extracts of MM, LC and normal human mesothelial (NHM) cell cultures. Finally, the relationships between 90K expression in MM and patient age and survival were studied. The mean 90K level was significantly higher (p<0.05) in PEs of MM patients (11.0+/-6.6 microg/ml) than in LC (6.1+/-3.2 microg/ml) or BPD (6.2+/-5.0 microg/ml) patients. Immunohistochemistry showed a positive reaction for 90K in MM biopsy sections and positive staining limited to inflammatory infiltrates in LC sections. The level of 90K was significantly higher in cell culture media of MM than of LC or NHM (p<0.001). Bands representing proteins with molecular weight of approximately 90 kDa were detected by western blot in MM cellular extracts. An inverse correlation between PE 90K levels and MM patient age (r=-0.45; p=0.017) and a positive correlation between serum 90K levels and MM patient survival (r=0.62; p=0.006) were detected by linear regression analysis. Kaplan-Meier univariate analysis showed increased survival probability for MM patients with serum 90K level >7.3 microg/ml (log rank, p<0.05). This is the first report in MM of the expression of 90K and of its potential diagnostic and prognostic application.
Assuntos
Biomarcadores Tumorais/análise , Glicoproteínas/análise , Mesotelioma/diagnóstico , Neoplasias Pleurais/diagnóstico , Adulto , Idoso , Antígenos de Neoplasias , Western Blotting , Proteínas de Transporte , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Mesotelioma/química , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Derrame Pleural Maligno/química , Derrame Pleural Maligno/diagnóstico , Neoplasias Pleurais/química , Prognóstico , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Several biochemical and clinical factors have been shown to correlate with survival in human malignant pleural mesothelioma (MM). Nevertheless, average survival of 4 to 10 months from diagnosis is sometimes not sufficient for full expression of these factors. Several studies have reported SV40 sequences in MM, suggesting a possible pathogenic role. We investigated whether the presence of these sequences had any effect on MM patient survival. For this study, we used polymerase chain reaction and Southern blot analysis to search for and identify SV40 DNA in biopsy samples from 83 MM patients. These cases were divided according to histology: 62/83 (74. 7%) had epithelioid morphology (EMM) and 21/83 (25.3%) had either biphasic or sarcomatous morphology (B/SMM). SV40 positivity was significantly associated with B/SMM growth pattern (chi-squared test = 5.03, P = 0.025). Kaplan-Meier univariate analysis confirmed the independent effect of histology on MM survival (log-rank test = 13.9, P < 0.001) and showed a trend for increased survival in SV40-negative patients (log-rank test = 2.83, P = 0.09). Most importantly, Cox's regression model showed that SV40-positive status affected the predictive value of histology on patient survival. In particular, when SV40 expression was added to the B/SMM histotype, Cox's regression model showed a significant increase in hazard ratio (HR) with respect to SV40-negative B/SMM (HR = 4.25, 95% CI = 2.00-9. 00, likelihood ratio test = 14.31, P < 0.001). We conclude that SV40 expression is significantly associated with B/SMM histology and represents an important prognostic cofactor when associated with the tumor subtype in MM patients.
Assuntos
Mesotelioma/genética , Mesotelioma/virologia , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/virologia , Vírus 40 dos Símios/genética , Adulto , Idoso , Southern Blotting , Feminino , Humanos , Masculino , Mesotelioma/mortalidade , Pessoa de Meia-Idade , Derrame Pleural Maligno/mortalidade , Reação em Cadeia da Polimerase , Prognóstico , Análise de Sequência de DNA , Análise de SobrevidaRESUMO
Malignant mesothelioma of the pleura is divided in three morphological variants: epithelioid, sarcomatous, and biphasic. Histological similarities between epithelioid malignant mesothelioma (EMM) and lung adenocarcinoma are responsible for the difficult differential diagnosis. Monoclonal antibodies are useful for distinguishing the two neoplasms through immunohistochemical phenotyping, although many cases require ultrastructural characterization for definitive diagnosis. In this study, transmission electron microscopic observations of EMM were compared with those of peripheral adenocarcinoma of the lung (PAL). More specifically, the morphology of the basal lamina is described in 23 cases of EMM and 12 cases of PAL. Reduplication of the basal lamina (RBL) was found in 11 cases (48%) of EMM and in none of the PAL cases. The same cases were immunostained for type IV collagen and the localization of this basement membrane component corresponded to the areas where basal lamina was observed. Since RBL has been associated with neoplastic differentiation in other tumors, this novel feature in EMM needs to be evaluated in future prognostic studies in malignant mesothelioma of the pleura. Moreover, RBL expression in EMM may be an additional ultrastructural parameter used in the differential diagnosis between EMM and adenocarcinoma.
Assuntos
Membrana Basal/ultraestrutura , Mesotelioma/patologia , Neoplasias Pleurais/patologia , Adenocarcinoma/química , Adenocarcinoma/patologia , Anticorpos Monoclonais/análise , Biomarcadores Tumorais/análise , Colágeno/análise , Diagnóstico Diferencial , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patologia , Mesotelioma/química , Neoplasias Pleurais/químicaRESUMO
Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.