RESUMO
OBJECTIVE/BACKGROUND: Protein S (PS; encoded by the PROS1 gene), a key vitamin K-dependent anticoagulant protein, is emerging as a key structural and functional protein that is overexpressd in various malignancies, but how PS signals to promote lung cancer progression is unclear. METHODS: We used immortalized, nontumorigenic human lung epithelial cell line NL-20, A549 cells as experimental cellular models for lung cancer, and human microvascular endothelial cells (HMEC-1) as a model system for angiogenesis. A loss- and gain-of-function approach was then used to analyze the role of tumor-derived PS and their natural TAM receptors Tyro3 and MerTK in regulating cell proliferation, migration, anchorage-independent growth, and capillary-like tube formation, all prominent attributes of the metastatic phenotype of tumor cells. RESULTS: Evidence is now provided that regulation of PROS1 gene expression using either stable cell lines expressing lentiviral-short hairpin RNA (shRNAs) or a replication-incompetent adenovirus alters the phosphorylation of several major signaling pathways, including Erk, PKB/Akt, p38, and focal adhesion kinase (FAK), and modulates PS-dependent Tyro3- and MerTK-mediated cell migration, proliferation, and anchorage-independent growth of lung cancer cells, and endothelial cell capillary-like tube formation. CONCLUSION: These finding suggest that the PS-Tyro3 and -MerTK axis mediates important signaling pathways to promote lung cancer progression. Genetic inhibition of endogenous PS may serve as a promising target for anticancer drug development.
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Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, is a novel gene that positively regulates cancer cell motility, invasion, and metastasis through distinct biochemical and signaling pathways, but how MDA-9/syntenin is regulated in response to signals with the extracellular environment and promotes tumor progression is unclear. We now demonstrate that MDA-9/syntenin is dramatically up-regulated by a combination of rFVIIa and factor F(X) in malignant melanoma. Induction of MDA-9/syntenin in melanoma was found to occur in a thrombin-independent signaling pathway and involves the PAR-1/c-Src/Rho GTPases Rac1 and Cdc42/c-Jun N-terminal kinase axis resulting in the activation of paxillin, NF-κB, and matrix metalloproteinase-2 (MMP-2). MDA-9/syntenin physically interacts with c-Src through its PDZ binding motif following stimulation of melanoma cells with rFVIIa and FX. We also document that induction of this signaling pathway is required for TF·FVIIa·Xa-induced cell migration, invasion, and metastasis by melanoma cells. The present finding uncovers a novel role of MDA-9/syntenin as an important TF·FVIIa·Xa/PAR-1-regulated gene that initiates a signaling circuit essential for cell motility and invasion of metastatic melanoma. In these contexts, targeting TF·FVIIa·Xa and its relevant downstream targets such as MDA-9/syntenin, may represent a novel therapeutic strategy to control the evolution of neoplastic cells.
Assuntos
Movimento Celular , Fator VIIa/metabolismo , Melanoma/metabolismo , Transdução de Sinais , Sinteninas/metabolismo , Animais , Linhagem Celular Tumoral , Fator X/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/patologia , Camundongos , NF-kappa B/metabolismo , Células NIH 3T3 , Metástase Neoplásica , Domínios PDZ , Paxilina/metabolismo , Ligação Proteica , Receptor PAR-1/metabolismo , Sinteninas/química , Sinteninas/genética , Regulação para Cima , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
This study investigated the underlying mechanism of 4-hydroxy-3-methoxycinnamic acid (ACCA), on the growth of breast cancer cells and normal immortal epithelial cells, and compared their cytotoxic effects responses. Treatment of breast cancer cells (MCF-7, T47D, and MDA-231) with ACCA resulted in dose- and time-dependent decrease of cell proliferation, viability in colony formation assay, and programmed cell death (apoptosis) with minimal effects on non-tumoral cells. The ability of ACCA to suppress growth in cancer cells not expressing or containing defects in p53 gene indicates a lack of involvement of this critical tumor suppressor element in mediating ACCA-induced growth inhibition. Induction of apoptosis correlated with an increase in Bax protein, an established inducer of programmed cell death, and the ratio of Bax to Bcl-2, an established inhibitor of apoptosis. We also documented the ability of ACCA to inhibit the migration and invasion of MDA-231 cells with ACCA in vitro. Additionally, tumor growth of MDA-231 breast cancer cells in vivo was dramatically affected with ACCA. On the basis of its selective anticancer inhibitory activity on tumor cells, ACCA may represent a promising therapeutic drug that should be further evaluated as a chemotherapeutic agent for human breast cancer.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Cinamatos/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cinamatos/administração & dosagem , Cinamatos/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Células MCF-7 , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/metabolismoRESUMO
Since its discovery in 1970, protein S (PS) has emerged as a key vitamin K-dependent natural anticoagulant protein at the crossroads of multiple biological processes, including coagulation, apoptosis, atherosclerosis, angiogenesis/vasculogenesis, and cancer progression. Following the binding to a unique family of protein tyrosine kinase receptors referred to as Tyro-3, Axl and Mer (TAM) receptors, PS can lead to regulation of coagulation, phagocytosis of apoptotic cells, cell survival, activation of innate immunity, vessel integrity and angiogenesis, and local invasion and metastasis. Because of these dynamics and multiple functions of PS, which are largely lost following invalidation of the mouse PROS1 gene, this molecule is currently intensively studied in biomedical research. The purpose of this review is to provide a brief chronicle of the discovery and current understanding of the mechanisms of PS signaling, and how PS and their signaling partners regulate various cellular functions, with a particular focus on TAM receptors.
Assuntos
Proteína S/genética , Proteína S/metabolismo , Animais , Coagulação Sanguínea/fisiologia , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Fisiológica/fisiologia , Regiões Promotoras Genéticas , Proteína S/química , Vitamina K/metabolismoRESUMO
Melanoma differentiation associated gene-9 (MDA-9), also known as syntenin, functions as a positive regulator of melanoma progression and metastasis. In contrast, the Raf kinase inhibitor, RKIP, a negative modulator of RAF-stimulated MEKK activation, is strongly downregulated in metastatic melanoma cells. In this study, we explored a hypothesized inverse relationship between MDA-9 and RKIP in melanoma. Tumor array and cell line analyses confirmed an inverse relationship between expression of MDA-9 and RKIP during melanoma progression. We found that MDA-9 transcriptionally downregulated RKIP in support of a suggested cross-talk between these two proteins. Furthermore, MDA-9 and RKIP physically interacted in a manner that correlated with a suppression of FAK and c-Src phosphorylation, crucial steps necessary for MDA-9 to promote FAK/c-Src complex formation and initiate signaling cascades that drive the MDA-9-mediated metastatic phenotype. Finally, ectopic RKIP expression in melanoma cells overrode MDA-9-mediated signaling, inhibiting cell invasion, anchorage-independent growth, and in vivo dissemination of tumor cells. Taken together, these findings establish RKIP as an inhibitor of MDA-9-dependent melanoma metastasis, with potential implications for targeting this process therapeutically.
Assuntos
Melanoma/metabolismo , Melanoma/patologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Sinteninas/antagonistas & inibidores , Quinases raf/antagonistas & inibidores , Animais , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Embrião de Galinha , Regulação para Baixo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Imuno-Histoquímica , Melanoma/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Proteína de Ligação a Fosfatidiletanolamina/biossíntese , Proteína de Ligação a Fosfatidiletanolamina/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Sinteninas/biossíntese , Sinteninas/metabolismo , Quinases raf/genética , Quinases raf/metabolismoRESUMO
Melanoma differentiation associated gene-9 (MDA-9), synonymous with syntenin, is an adapter protein that provides a central role in regulating cell-cell and cell-matrix adhesion. MDA-9/syntenin transduces signals from the cell-surface to the interior through its interaction with a plethora of additional proteins and actively participates in intracellular trafficking and cell-surface targeting, synaptic transmission, and axonal outgrowth. Recent studies demarcate a seminal role of MDA-9/syntenin in cancer metastasis. In the context of melanoma, MDA-9/syntenin functions as a positive regulator of melanoma progression and metastasis through interactions with c-Src and promotes the formation of an active FAK/c-Src signaling complex leading to NF-k B and matrix metalloproteinase (MMP) activation. The present review provides a current perspective of our understanding of the important features of MDA-9/syntenin and its significant role in tumor cell metastasis with special focus on molecular mechanism of action.
Assuntos
Melanoma/secundário , Sinteninas/fisiologia , Precursores Enzimáticos/metabolismo , Quinase 1 de Adesão Focal/química , Quinase 1 de Adesão Focal/metabolismo , Gelatinases/metabolismo , Humanos , Melanoma/patologia , Melanoma/fisiopatologia , Modelos Biológicos , Complexos Multiproteicos/química , Sistema Nervoso/fisiopatologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Sindecanas/metabolismo , Sinteninas/química , Sinteninas/genética , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
Astrocyte-elevated gene-1 (AEG-1) expression is increased in multiple cancers and plays a central role in Ha-ras-mediated oncogenesis through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Additionally, overexpression of AEG-1 protects primary and transformed human and rat cells from serum starvation-induced apoptosis through activation of PI3K/Akt signaling. These findings suggest, but do not prove, that AEG-1 may function as an oncogene. We now provide definitive evidence that AEG-1 is indeed a transforming oncogene and show that stable expression of AEG-1 in normal immortal cloned rat embryo fibroblast (CREF) cells induces morphological transformation and enhances invasion and anchorage-independent growth in soft agar, two fundamental biological events associated with cellular transformation. Additionally, AEG-1-expressing CREF clones form aggressive tumors in nude mice. Immunohistochemistry analysis of tumor sections demonstrates that AEG-1-expressing tumors have increased microvessel density throughout the entire tumor sections. Overexpression of AEG-1 increases expression of molecular markers of angiogenesis, including angiopoietin-1, matrix metalloprotease-2, and hypoxia-inducible factor 1-alpha. In vitro angiogenesis studies further demonstrate that AEG-1 promotes tube formation in Matrigel and increases invasion of human umbilical vein endothelial cells via the PI3K/Akt signaling pathway. Tube formation induced by AEG-1 correlates with increased expression of angiogenesis markers, including Tie2 and hypoxia-inducible factor-alpha, and blocking AEG-1-induced Tie2 with Tie2 siRNA significantly inhibits AEG-1-induced tube formation in Matrigel. Overall, our findings demonstrate that aberrant AEG-1 expression plays a dominant positive role in regulating oncogenic transformation and angiogenesis. These findings suggest that AEG-1 may provide a viable target for directly suppressing the cancer phenotype.
Assuntos
Moléculas de Adesão Celular/genética , Transformação Celular Neoplásica , Neovascularização Patológica/etiologia , Oncogenes/fisiologia , Animais , Moléculas de Adesão Celular/fisiologia , Transformação Celular Neoplásica/genética , Células Cultivadas , Fibroblastos , Humanos , Proteínas de Membrana , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Neoplasias Experimentais , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Proteínas de Ligação a RNA , Ratos , Transplante HeterólogoRESUMO
The scaffold PDZ-domain containing protein mda-9/syntenin functions as a positive regulator of cancer cell progression in human melanoma and other tumors. mda-9/Syntenin regulates cell motility and invasion by altering defined biochemical and signaling pathways, including focal adhesion kinase (FAK), p38 mitogen-activated protein kinase (MAPK) and NF-kappaB, but precisely how mda-9/syntenin organizes these multiprotein signaling complexes is not well understood. Using a clinically relevant human melanoma model, we demonstrate that mda-9/syntenin physically interacts with c-Src and this communication correlates with an increase in FAK/c-Src complex formation and c-Src activation. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin or addition of c-Src siRNA, suppresses melanoma cell migration, anchorage-independent growth, and spontaneous tumor cell dissemination in vivo in a human melanoma animal metastasis model. These data are compatible with a model wherein interaction of MDA-9/syntenin with c-Src promotes the formation of an active FAK/c-Src signaling complex, leading to enhanced tumor cell invasion and metastatic spread. These provocative findings highlight mda-9/syntenin and its interacting partners as promising therapeutic targets for intervention of metastasis.
Assuntos
Melanoma Experimental/patologia , Metástase Neoplásica/patologia , Proteínas Tirosina Quinases/metabolismo , Sinteninas/fisiologia , Animais , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Ligação Proteica , Ratos , Ratos Wistar , Sinteninas/metabolismo , Quinases da Família srcRESUMO
Tumor cells evade adaptive immunity by a variety of mechanisms, including selection of variants that are resistant to specific cytotoxic T lymphocyte (CTL) pressure. Recently, we have reported that the reorganization of the actin cytoskeleton can be used by tumor cells as a strategy to promote their resistance to CTL-mediated lysis. In this study, we further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways. Our data indicate that although the resistant cells do not display an increased migratory potential, an alteration of adhesion to the extracellular matrix was observed. When Rho-GTPases were activated in cells by the bacterial CNF1 (cytotoxic necrotizing factor 1), striking changes in the cell morphology, including actin cytoskeleton, focal adhesions, and membrane extensions, were observed. More importantly, such activation also resulted in a significant attenuation of resistance to CTL-induced cell death. Furthermore, we demonstrate that FAK signaling pathways were constitutively defective in the resistant cells. Silencing of FAK in the sensitive target cells resulted in the inhibition of immune synapse formation with specific CTLs and their subsequent lysis. Expression of the FAK mutant (Y397F) resulted in an inhibition of IGR-Heu cell adhesion and of their susceptibility to specific lysis. These results suggest that FAK activation plays a role in the control of tumor cell susceptibility to CTL-mediated lysis.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias/enzimologia , Neoplasias/imunologia , Linfócitos T Citotóxicos/enzimologia , Linfócitos T Citotóxicos/imunologia , Proteínas rho de Ligação ao GTP/metabolismo , Adesão Celular , Linhagem Celular , Forma Celular , Ativação Enzimática , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Regulação Enzimológica da Expressão Gênica , Macrófagos/enzimologia , Mutação/genética , Neoplasias/genética , Ligação ProteicaRESUMO
Cancer is a progressive disease that, in many instances, if untreated, can culminate in metastatic spread of primary tumor cells to distant sites in the body. Metastasis frequently confers virulence and therapy resistance to cancer cells, and defining the molecular events that control metastasis will be mandatory to develop rational, targeted therapies for effective intervention, prevention of recurrence, and the "holy grail" of engendering a cure. Adapter proteins are physiologically pertinent molecules that, through interactions with key regulatory proteins via specific conserved domains, control important cellular events. Melanoma differentiation associated gene-9 (mda-9), also known as syntenin, is a PDZ domain-containing adapter protein that is involved in organization of protein complexes in the plasma membranes, regulation of B-cell development, intracellular trafficking and cell-surface targeting, synaptic transmission, and axonal outgrowth. Recent studies now define a seminal role for mda-9/syntenin in cancer metastasis. The present review provides a current perspective of our understanding of this important aspect of mda-9/syntenin, suggesting that this gene and its encoded protein and interacting protein partners may provide viable targets for intervening in the final and invariably the most lethal stage of cancer progression, namely, cancer metastasis.
Assuntos
Metástase Neoplásica/genética , Sinteninas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Adesão Celular/genética , Clonagem Molecular , Genes Supressores de Tumor/fisiologia , Ácido Glutâmico/metabolismo , Humanos , Modelos Biológicos , Neuritos/metabolismo , Neuritos/fisiologia , Ligação Proteica , Transdução de Sinais/genética , Sinteninas/genética , Sinteninas/metabolismo , Distribuição TecidualRESUMO
Subtraction hybridization applied to a 'differentiation therapy' model of cancer employing human melanoma cells resulted in the cloning of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). Initial studies confirm an inverse correlation between mda-7 expression and melanoma development and progression. Forced expression of mda-7 by means of a plasmid or via a replication incompetent adenovirus (Ad.mda-7) promotes growth suppression and induces apoptosis in a broad array of human cancers. In contrast, mda-7 does not induce growth suppressive or toxic effects in normal cells. Based on structure (containing an IL-10 signature motif), secretion by cells (including subsets of T-cells) and location on chromosome 1q (in an area containing IL-10-family genes), mda-7 has now been renamed mda-7/IL-24. Studies by several laboratories have uncovered many of mda-7/IL-24's unique properties, including cancer-specific apoptosis-induction, cell cycle regulation, an ability to inhibit angiogenesis, potent 'bystander antitumor activity' and a capacity to enhance the sensitivity of tumor cells to radiation, chemotherapy and monoclonal antibody therapy. Moreover, based on its profound cancer tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant clinical activity. In these contexts, mda-7/IL-24 represents a unique cytokine gene with potential for therapy of human cancers. The present review focuses on three unique properties of mda-7/IL-24, namely its potent 'bystander antitumor activity', ability to sensitize tumor cells to radiation, and its antiangiogenesis properties. Additionally, an overview of the phase I clinical trial is provided. These studies affirm that mda-7/IL-24 has promise for the management of diverse cancers.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Interleucinas/farmacologia , Radiossensibilizantes/farmacologia , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Interleucinas/genética , Interleucinas/uso terapêutico , Invasividade Neoplásica , Transdução de Sinais/efeitos dos fármacos , TransgenesRESUMO
mda-9/Syntenin is a scaffolding PDZ domain-containing protein overexpressed in multiple human cancers that functions as a positive regulator of melanoma metastasis. Using a normal immortal human melanocyte cell line and weakly and highly metastatic human melanoma cell lines, we presently show that mda-9/syntenin initiates a signaling cascade that activates nuclear factor-kappaB (NF-kappaB) in human melanoma cells. As a consequence of elevated mda-9/syntenin expression, tumor cell growth and motility, fundamental components of tumor cell invasion and metastatic spread of melanoma cells, are enhanced through focal adhesion kinase (FAK)-induced and p38 mitogen-activated protein kinase (MAPK)-induced activation of NF-kappaB. Inhibiting mda-9/syntenin, using an adenovirus expressing antisense mda-9/syntenin, NF-kappaB, using an adenovirus expressing a mutant super-repressor of IkappaBalpha, or FAK, and using a dominant-negative mutant of FAK (FRNK), blocks melanoma cell migration, anchorage-independent growth, and invasion. Downstream signaling changes mediated by mda-9/syntenin, which include activation of FAK, p38 MAPK, and NF-kappaB, promote induction of membrane-type matrix metalloproteinase-1 that then activates pro-MMP-2-promoting migration and extracellular matrix invasion of melanoma cells. These results highlight the importance of mda-9/syntenin as a key component of melanoma metastasis providing a rational molecular target for potentially intervening in the metastatic process.
Assuntos
Melanoma/genética , Melanoma/patologia , NF-kappa B/metabolismo , Sinteninas/biossíntese , Adenoviridae/genética , Adesão Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Movimento Celular/fisiologia , Precursores Enzimáticos/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos/genética , Humanos , Metaloproteinase 14 da Matriz/biossíntese , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/metabolismo , NF-kappa B/biossíntese , NF-kappa B/genética , Metástase Neoplásica , Fenótipo , Sinteninas/antagonistas & inibidores , Sinteninas/genética , Fator de Transcrição RelA/metabolismo , Transdução Genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Differential gene expression represents the entry point for comprehending complex biological processes. In this context, identification and cloning of differentially expressed genes represent critical elements in this process. Many techniques have been developed to facilitate achieving these objectives. Although effective in many situations, most currently described approaches are not trouble-free and have limitations, including complexity of performance, redundancy of gene identification (reflecting cloning biases) and false-positive gene identification. A detailed methodology to perform a rapid and efficient cloning approach, called rapid subtraction hybridization is described in this chapter. This strategy has been applied successfully to a number of cell culture systems and biological processes, including terminal differentiation and cancer progression in human melanoma cells, resistance or sensitivity to HIV-1 in human T cells and gene expression changes following infection of normal human fetal astrocytes with HIV-1 or treatment with neutrotoxic agents. Based on its simplicity of performance and high frequency of genuine differential gene identification, the rapid subtraction hybridization (RaSH) approach will allow wide applications in diverse systems and biological contexts.
Assuntos
Clonagem Molecular/métodos , Perfilação da Expressão Gênica/métodos , Genômica/métodos , Northern Blotting , DNA Complementar , Hibridização de Ácido NucleicoRESUMO
Astrocyte elevated gene-1 (AEG-1) was initially identified as an HIV-1- and tumor necrosis factor alpha (TNF-alpha)-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells and AEG-1 cooperates with Ha-ras to promote transformation of immortalized melanocytes. Activation of the transcription factor nuclear factor kappaB (NF-kappaB), a TNF-alpha downstream signaling component, is associated with several human illnesses, including cancer, and NF-kappaB controls the expression of multiple genes involved in tumor progression and metastasis. We now document that AEG-1 is a significant positive regulator of NF-kappaB. Enhanced expression of AEG-1 via a replication-incompetent adenovirus (Ad.AEG-1) in HeLa cells markedly increased binding of the transcriptional activator p50/p65 complex of NF-kappaB. The NF-kappaB activation induced by AEG-1 corresponded with degradation of IkappaBalpha and nuclear translocation of p65 that resulted in the induction of NF-kappaB downstream genes. Infection with an adenovirus expressing the mt32IkappaBalpha superrepressor (Ad.IkappaBalpha-mt32), which prevents p65 nuclear translocation, inhibited AEG-1-induced enhanced agar cloning efficiency and increased matrigel invasion of HeLa cells. We also document that TNF-alpha treatment resulted in nuclear translocation of both AEG-1 and p65 wherein these two proteins physically interacted, suggesting a potential mechanism by which AEG-1 could activate NF-kappaB. Our findings suggest that activation of NF-kappaB by AEG-1 could represent a key molecular mechanism by which AEG-1 promotes anchorage-independent growth and invasion, two central features of the neoplastic phenotype.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , NF-kappa B/metabolismo , Adenoviridae/genética , Sequência de Aminoácidos , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Adesão Celular/fisiologia , Moléculas de Adesão Celular , Processos de Crescimento Celular/fisiologia , Progressão da Doença , Células HeLa , Humanos , Proteínas I-kappa B/metabolismo , Interleucina-8/biossíntese , Interleucina-8/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , NF-kappa B/antagonistas & inibidores , Subunidade p50 de NF-kappa B/metabolismo , Ligação Proteica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Fator de Transcrição RelA/metabolismo , Transfecção , Regulação para CimaRESUMO
Metastasis is a significant event in cancer progression and continues to pose the greatest challenge for a cancer cure. Defining genes that control metastasis in vivo may provide new targets for intervening in this process with profound therapeutic implications. Melanoma differentiation associated gene-9 (mda-9) was initially identified by subtraction hybridization as a novel gene displaying biphasic expression during terminal differentiation in human melanoma cells. Mda-9, also known as syntenin, is a PDZ-domain protein overexpressed in many types of human cancers, where it is believed to function in tumor progression. However, a functional role of mda-9/syntenin in tumor growth and metastasis and the signaling pathways involved in mediating these biological activities remain to be defined. Evidence is now provided, using weakly and highly metastatic isogenic melanoma variants, that mda-9/syntenin regulates metastasis. Expression of mda-9/syntenin correlates with advanced stages of melanoma progression. Regulating mda-9/syntenin expression using a replication-incompetent adenovirus expressing either sense or antisense mda-9/syntenin modifies the transformed phenotype and alters metastatic ability in immortal human melanocytes and metastatic melanoma cells in vitro and in vivo in newborn rats. A direct relationship is observed between mda-9/syntenin expression and increased phosphorylation of focal adhesion kinase, c-Jun-NH2-kinase, and p38. This study provides the first direct link between mda-9/syntenin expression and tumor cell dissemination in vivo and indicates that mda-9/syntenin expression activates specific signal transduction pathways, which may regulate melanoma tumor progression. Based on its ability to directly alter metastasis, mda-9/syntenin provides a promising new focus for melanoma cancer research with potential therapeutic applications for metastatic diseases.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Melanoma/secundário , Proteínas de Membrana/fisiologia , Adenoviridae/genética , Animais , Adesão Celular/genética , Movimento Celular/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Melanócitos/metabolismo , Melanócitos/fisiologia , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Invasividade Neoplásica , Ratos , Ratos Wistar , Sinteninas , Transdução Genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Progression elevated gene-3 (PEG-3) is a novel rodent gene, identified and cloned by subtraction hybridization, that associates with transformation progression in virus- and oncogene-transformed rat embryo (RE) cells. Previous reports document that ectopic expression of PEG-3 in rodent or human tumor cells produces an aggressive transformed/tumorigenic phenotype. Moreover, PEG-3 expression in rodent tumor cells correlates directly with genomic instability, as indicated by chromosomal alterations and gene amplification, and it promotes angiogenesis. The present studies were designed to further elucidate the functional significance and role of PEG-3 in cancer progression with a specific focus on genomic instability and cancer invasion. Genomic instability was assessed by micronucleus assays and staining of centrosomes to define centrosomal amplification. Immunocytochemical observations revealed that overexpression of PEG-3 in transformed rodent cells induced a loss of chromosomes as established by the appearance of micronuclei and staining of the centrosomes with gamma-tubulin antibody, thereby confirming centrosome amplification. Overexpression of PEG-3 modulated the expression of several genes involved in centrosomal duplication, such as p21CIP1/WAF1/MDA-6, nucleophosmin (NPM), and aurora-A kinase. In vitro invasion of transformed rodent cells was augmented by PEG-3, which correlated with an increase in the transcription and activity of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9), which play important roles in local invasion during cancer progression. These findings demonstrate that PEG-3 plays a central role in augmenting tumor progression by modulating several critical parameters of the carcinogenic process, such as genomic stability and local tumor cell invasion.
Assuntos
Antígenos de Diferenciação/metabolismo , Transformação Celular Neoplásica/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Animais , Antígenos de Diferenciação/genética , Aurora Quinase A , Aurora Quinases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Proliferação de Células , Centrossomo/metabolismo , Aberrações Cromossômicas , Inibidor de Quinase Dependente de Ciclina p21 , Regulação Neoplásica da Expressão Gênica/genética , Genes Reguladores/genética , Instabilidade Genômica/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Micronúcleos com Defeito Cromossômico , Invasividade Neoplásica/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Ratos , Regulação para Cima/genética , Proteínas de XenopusRESUMO
PDZ (an acronym representing three proteins--postsynaptic density protein PSD95/SAP90, drosophila tumor suppressor DLGA, and tight junction protein ZO-1) domain containing proteins are adapter proteins that play indispensable roles in regulating cell growth, development, and differentiation, predominantly through their capacity to serve as central organizers of protein complexes at the plasma membrane. A recently identified member of this protein family is melanoma differentiation associated gene-9 (mda-9), also known as syntenin, which was first identified as a gene down-regulated during human melanoma differentiation as mda-9 and subsequently recognized as an interacting partner of the cell-surface heparan sulfate syndecans, syntenin. Interest in mda-9/syntenin is intensifying because of its involvement in organization of protein complexes in the plasma membranes, regulation of B cell development, intracellular trafficking and cell surface targeting, cancer metastasis, synaptic transmission, and axonal outgrowth. In this review, we discuss the identification, structure and function of mda-9/syntenin and delineate future studies to address its role in regulating key physiological and pathological processes.
Assuntos
Histona Desacetilases/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Repressoras/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Sinteninas , TransativadoresRESUMO
Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-alpha (TNF-alpha). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na+/K+-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Melanoma/genética , Metástase Neoplásica/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Clonagem Molecular , Biblioteca Gênica , HIV-1/fisiologia , Humanos , Melanoma/patologia , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Ratos , ATPase Trocadora de Sódio-Potássio/genética , Transplante HeterólogoRESUMO
We recently identified a novel 55-kDa cell-cell adhesion protein (p55) whose expression is upregulated in primary melanomas in the transition from radial growth phase to vertical growth phase. However, the functional role of p55 in various steps of the metastatic process had not been investigated. We provide evidence that subcutaneous injection of metastatic melanoma variant T1P26 in immunosuppressed newborn rats rapidly caused spontaneous metastatic lung lesions that could be readily detected by histochemical analysis with the anti-p55 monoclonal antibody (MAb) LY1. Subsequently, we were able to demonstrate that multiple subcutaneous injections of the LY1 MAb starting on the same day after tumor cell inoculation of T1P26 cells specifically blocked the formation of spontaneous lung metastases, yet had no effects on primary tumor growth, suggesting a critical role of p55 in the earlier steps of the intravasation process. To study later stages in spontaneous metastasis, we investigated the role of p55 in organ-specific cell adhesion of tumor cells in vitro. We showed that the T1P26 variant attached preferentially to lung frozen sections compared with other organs, reflecting the pattern of organ involvement of metastasis in vivo and that LY1 significantly blocked this interaction. However, no significant differences in attachment to lung sections were observed between the parental melanoma cell line M(4)Beu and its derived variant, although cellular topography analysis indicated a preferential attachment of a T1P26 variant on specific compartments of the lungs such as the perialveolar components, the endothelium and the vessel lumen of pulmonary venules. Attachment of the T1P26 variant to lung sections is not due to alterations of tumor cell adherence to basement membrane matrix by the LY1 MAb, suggesting that p55 is involved in cellular adhesion with cellular elements of the lung. p55 could represent a new functional constituent that contributes to the metastatic spread of melanoma cells by promoting the intravasation process and subsequent specific interactions between tumor cells and the target lung organ.