RESUMO
L1-CAM (L1 cell-adhesion molecule), or more simply L1, plays an important role in the progression of human carcinoma. Overexpression promotes tumour-cell invasion and motility, growth in nude mice and tumour metastasis. It is feasible that L1-dependent signalling contributes to these effects. However, little is known about its mechanism in tumour cells. We reported previously that L1 is cleaved by ADAM (a disintegrin and metalloprotease) and that the cytoplasmic part is essential for L1 function. Here we analysed more closely the role of proteolytic cleavage in L1-mediated nuclear signalling. Using OVMz carcinoma cells and L1-transfected cells as a model, we found that ADAM10-mediated cleavage of L1 proceeds in lipid raft and non-raft domains. The cleavage product, L1-32, is further processed by PS (presenilin)/gamma-secretase to release L1-ICD, an L1 intracellular domain of 28 kDa. Overexpression of dominant-negative PS1 or use of a specific gamma-secretase inhibitor leads to an accumulation of L1-32. Fluorescence and biochemical analysis revealed a nuclear localization for L1-ICD. Moreover, inhibition of ADAM10 and/or gamma-secretase blocks nuclear translocation of L1-ICD and L1-dependent gene regulation. Overexpression of recombinant L1-ICD mediates gene regulation in a similar manner to full-length L1. Our results establish for the first time that regulated proteolytic processing by ADAM10 and PS/gamma-secretase is essential for the nuclear signalling of L1 in human carcinoma cell lines.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Presenilinas/metabolismo , Transdução de Sinais , Proteínas ADAM/genética , Proteína ADAM10 , Transporte Ativo do Núcleo Celular , Secretases da Proteína Precursora do Amiloide/genética , Animais , Sítios de Ligação/genética , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Citometria de Fluxo , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Microscopia de Fluorescência , Molécula L1 de Adesão de Célula Nervosa/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Presenilinas/genética , RNA Interferente Pequeno/genética , Receptores do Ácido Retinoico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , TransfecçãoRESUMO
Recent work has identified L1CAM (CD171) as a novel marker for human carcinoma progression. Functionally, L1CAM promotes tumor cell invasion and motility, augments tumor growth in nude mice, and facilitates experimental tumor metastasis. These functional features qualify L1 as an interesting target molecule for tumor therapy. Here, we generated a series of novel monoclonal antibodies (mAb) to the L1CAM ectodomain that were characterized by biochemical and functional means. All novel mAbs reacted specifically with L1CAM and not with the closely related molecule CHL1, whereas antibodies to the COOH terminal part of L1CAM (mAb2C2, mAb745H7, pcytL1) showed cross-reactivity. Among the novel mAbs, L1-9.3 was selected and its therapeutic potential was analyzed in various isotype variants in a model of SKOV3ip cells growing i.p. in CD1 nude mice. Only therapy with the IgG2a variant efficiently prolonged survival and reduced tumor burden. This was accompanied by an increased infiltration of F4/80-positive monocytic cells. Clodronate pretreatment of tumor-bearing animals led to the depletion of monocytes and abolished the therapeutic effect of L1-9.3/IgG2a. Expression profiling of tumor-derived mRNA revealed that L1-9.3/IgG2a therapy induced altered expression of cellular genes associated with apoptosis and tumor growth. Our results establish that anti-L1 mAb therapy acts via immunologic and nonimmunologic effector mechanism to block tumor growth. The novel antibodies to L1CAM could become helpful tools for the therapy of L1-positive human carcinomas.
Assuntos
Anticorpos Monoclonais/farmacologia , Molécula L1 de Adesão de Célula Nervosa/imunologia , Neoplasias Ovarianas/terapia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Reações Cruzadas , Epitopos/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Camundongos , Camundongos Nus , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Exosomes are membrane vesicles that are released from many different cell types. Tumor derived-exosomes play a role in immune suppression. We hypothesized that in ovarian carcinoma patients exosomes initially produced at the local abdominal site may become systemic. We examined paired samples of ascites and blood from ovarian carcinoma patients for the presence of exosomes. We also studied the requirements for exosomal uptake by immune cells, the role of phosphatidyl-serine (PS) as uptake signal and the effect of exosome application on tumor growth. We used exosomes from ovarian carcinoma cell lines, malignant ascites and sera from ovarian carcinoma patients isolated by ultracentrifugation. PS-displayed by exosomes was detected by Anexin-V-FITC staining of latex beads adsorbed exosomes. For uptake experiments, labeled exosomes were exposed to cells in the presence or absence of cold Annexin-V as competitor. Uptake was examined by fluorescent microscopy and cytofluorographic analysis. Effects of exosomes on tumor growth were studied using SKOV3ip ovarian carcinoma cells in CD1 nu/nu mice. We found that malignant ascites-derived exosomes cargo tumor progression related proteins such as L1CAM, CD24, ADAM10, and EMMPRIN. We observed that exosomes become systemic via the blood stream. Uptake of ovarian carcinoma exosomes by NK cells was found to require PS at the exosomal surface but the presence of PS was not sufficient. Application of malignant ascites-derived exosomes to tumor bearing mice resulted in augmented tumor growth. Exosomes from the serum of tumor patients could be isolated from only one ml of blood and this analysis could serve for diagnostic purposes. We propose that tumor-derived exosomes could play a role in tumor progression.