RESUMO
Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3ß, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.
Assuntos
Complexo Mediador/genética , Melanoma/patologia , Neoplasias Cutâneas/patologia , Tetraspaninas/genética , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Masculino , Complexo Mediador/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Tetraspaninas/metabolismo , Transfecção , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
This corrects the article DOI: 10.1038/onc.2016.219.
RESUMO
Hormone therapy is often used in association with chemotherapy in the treatment of estrogen-responsive breast cancers. By using breast adenocarcinoma cell lines, we show that antiestrogen treatment leads to a dramatic decrease of p53 protein levels. This effect leads to a loss of wild-type p53 response to genotoxic treatment. This inhibition is assessed by the lack of p53 protein accumulation and the loss of the p53-dependent induction of p21(WAF1/CIP1) expression. Given that the effects of several anticancer agents are mediated through DNA damage, these observations suggest that antiestrogen treatment could modulate cellular response to chemotherapeutic agents.
Assuntos
Antagonistas de Estrogênios/farmacologia , Tamoxifeno/farmacologia , Proteína Supressora de Tumor p53/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Estradiol/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.
Assuntos
Hormônio Luteinizante/imunologia , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos/imunologia , Cromatografia em Gel , Epitopos/análise , Humanos , Soros Imunes/biossíntese , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Relação Estrutura-AtividadeRESUMO
To characterize the membrane changes associated with cisplatin resistance, we raised monoclonal antibodies (MAbs) against a cisplatin-resistant subline (OV1/DDP) derived from a human ovarian carcinoma cell line (OV1/p). An MAb, designated OCP02, was selected for its particularly high affinity for the resistant cell line. It bound 3.1-fold higher to OV1/DDP cells than to OV1/p cells and recognized an M(r) 45K antigen. This antigen appeared to be present in several normal and tumorous tissues. Its distribution in normal tissues was mainly detected in tissues involved in secretory processes, suggesting that this antigen could be related to a transport mechanism in normal cells as well as in drug-resistant cells.
Assuntos
Antígenos de Neoplasias/biossíntese , Cisplatino/farmacologia , Neoplasias Ovarianas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Resistência a Medicamentos/imunologia , Feminino , Humanos , Hibridomas/imunologia , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Ovarianas/tratamento farmacológico , Células Tumorais Cultivadas/imunologiaRESUMO
The ΔNp63 protein, a product of the TP63 gene that lacks the N-terminal domain, has a critical role in the maintenance of self renewal and progenitor capacity in several types of epithelial tissues. ΔNp63 is frequently overexpressed in squamous cell carcinoma (SCC) and in some other epithelial tumours. This overexpression may contribute to tumour progression through dominant-negative effects on the transcriptionally active (TA) isoforms of the p53 family (TAp63, TAp73 and p53), as well as through independent mechanisms. However, the molecular basis of ΔNp63 overexpression is not fully understood. Here, we show that the expression of ΔNp63 is regulated by the Wnt/ß-catenin pathway in human hepatocellular carcinoma (HCC) and SCC cell lines. This regulation operates in particular through TCF/LEF sites present in the P2 promoter of TP63. In addition, we show that ΔNp63 and ß-catenin are frequently coexpressed and accumulated in oesophageal SCC, but not in HCC. These results suggest that activation of the ß-catenin pathway may contribute to overexpression of ΔNp63 during tumour progression, in a cell type-specific manner.
Assuntos
Carcinoma Hepatocelular/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Neoplasias Hepáticas/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , beta Catenina/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismo , beta Catenina/metabolismoRESUMO
In order to characterize the membrane changes related to Vinca alkaloid resistance, we raised monoclonal antibodies (mAbs) against a Vincristine resistant subline (OV1/VCR) derived from a human ovarian adenocarcinoma cell line (OV1/p). Among three monoclonal antibodies selected for a higher binding to OV1/VCR than to OV1/p cells, one designated OVR09, recognized a Mr 92,000 protein. This protein appears to be gradually overexpressed along the drug resistance establishment in vitro, and to decrease slowly in absence of drug. Further, mAb OVR09 showed a much higher binding to the vinblastine resistant epidermoid tumor cell line KbV1 than to its parental counterpart. The Mr 92,000 protein was also detected in various tumor cell lines and in an ovarian carcinoma surgical sample.
Assuntos
Antígenos de Neoplasias/imunologia , Células Tumorais Cultivadas/imunologia , Vincristina/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Carcinoma/imunologia , Membrana Celular/imunologia , Resistência a Medicamentos , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Neoplasias Ovarianas/imunologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
We recently reported the characterization of an antigen designated VRA09, identified by a monoclonal antibody and overexpressed on the surface of vincristine-resistant human ovarian carcinoma cells. In the present study, we analyze the distribution of this antigen in normal and tumor tissues. Its pattern of expression appears to differ from that described for other drug-resistance- and/or tumor-associated antigens. In normal tissues, the antigen has a restricted histological distribution and appears to be localized in mesoderm-derived tissues. In tumor tissues, VRA09 expression was mainly detected in serous ovarian tumors. Indeed, VRA09 is strongly expressed in papillary serous cystadenocarcinomas and their metastases, and more specifically in the basement membranes of serous tumors of borderline malignancy. In contrast, no immunostaining was observed in normal ovarian tissue or benign tumors. The detection of this antigen may help to identify serous ovarian tumors by distinguishing tumors of low malignancy from cystadenocarcinomas.
Assuntos
Antígenos de Neoplasias/análise , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/imunologia , Anticorpos Monoclonais , Diagnóstico Diferencial , Endométrio/química , Epitélio/química , Epitélio/patologia , Feminino , Glicoproteínas/química , Humanos , Imuno-Histoquímica , Radioisótopos do Iodo , Glomérulos Renais/química , Neoplasias Pulmonares/química , Membranas/imunologia , Neoplasias Ovarianas/patologia , Peritônio/química , Peritônio/patologia , Radioimunoensaio , Trofoblastos/químicaRESUMO
We have reported previously the physical interaction of B-cell translocation gene proteins (BTG)1 and BTG2 with the mouse protein CAF1 (CCR4-associated factor 1) and suggested that these proteins may participate, through their association with CAF1, in transcription regulation. Here we describe the in vitro and in vivo association of these proteins with hPOP2, the human paralog of hCAF1. The physical and functional relationships between the BTG proteins and their partners hCAF1 and hPOP2 were investigated to find out how these interactions affect cellular processes, and in particular transcription regulation. We defined their interaction regions and examined their expression in various human tissues. We also show functional data indicating their involvement in estrogen receptor alpha (ERalpha)-mediated transcription regulation. We found that BTG1 and BTG2, probably through their interaction with CAF1 via a CCR4-like complex, can play both positive or negative roles in regulating the ERalpha function. In addition, our results indicate that two LXXLL motifs, referred to as nuclear receptor boxes, present in both BTG1 and BTG2, are involved in the regulation of ERalpha-mediated activation.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Fúngicas/metabolismo , Genes Supressores de Tumor , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Estrogênio/metabolismo , Ribonucleases , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/biossíntese , Northern Blotting , Western Blotting , Divisão Celular , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA Complementar/metabolismo , Ensaio de Imunoadsorção Enzimática , Receptor alfa de Estrogênio , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Proteínas Imediatamente Precoces/biossíntese , Luciferases/metabolismo , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Distribuição Tecidual , Transcrição Gênica , Transfecção , Proteínas Supressoras de TumorRESUMO
BTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9.Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9.Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Genes Supressores de Tumor , Proteínas de Homeodomínio/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Neoplasias/metabolismo , Ativação Transcricional , Sítios de Ligação , Inibidores do Crescimento , Proteínas de Homeodomínio/genética , Proteínas Imediatamente Precoces/genética , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor , Técnicas do Sistema de Duplo-HíbridoRESUMO
The p53 protein can adopt several conformations in cells--"latent," "active," or mutant--depending on cellular stress or mutations of the TP53 gene. Today, only a few antibodies discriminating these conformations are available. We produced three new anti-p53 monoclonal antibodies (MAbs) directed against epitopes of human p53. The H53C1 MAb recognizes an epitope located at the N-terminal part of the central region of p53 and can discriminate mutant from wild-type conformation. The H53C2 and H53C3 MAbs are against different epitopes within the proline-rich region of p53. Moreover, the H53C2 epitope is located in the second negative regulatory domain of p53 between residues 80 and 93. These MAbs can be used as new tools to study and modulate the cellular functions of p53.