RESUMO
Clinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis. Methods: We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses. We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence. Results: The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. In vivo, the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation. Conclusions: These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization.
Assuntos
Neoplasias Mamárias Experimentais , Proteína Tirosina Fosfatase não Receptora Tipo 13/fisiologia , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal , Feminino , Humanos , Junções Intercelulares , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Transplante de Neoplasias , Receptor ErbB-2/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors.
Assuntos
Antígenos de Neoplasias/fisiologia , Endocitose/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/metabolismo , Apoptose , Disbindina/metabolismo , Proteína Ligante Fas/análise , Proteína Ligante Fas/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/análise , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/fisiologia , Transdução de Sinais , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/metabolismo , Receptor fas/análise , Receptor fas/metabolismoRESUMO
Chronic myeloid leukemia (CML) is characterized by expression of Bcr-abl, a tyrosine kinase oncogene. Clinical outcomes in CML were revolutionized by development of Bcr-abl-targeted tyrosine kinase inhibitors (TKIs), but CML is not cured by these agents. CML leukemia stem cells (LSCs) are relatively TKI insensitive and persist even in remission. LSC persistence results in relapse upon TKI discontinuation, or drug resistance or blast crisis (BC) during prolonged treatment. We hypothesize that increased expression of Fas-associated phosphatase 1 (Fap1) in CML contributes to LSC persistence and BC. As Fap1 substrates include Fas and glycogen synthase kinase-3ß (Gsk3ß), increased Fap1 activity in CML is anticipated to induce Fas resistance and stabilization of ß-catenin protein. Resistance to Fas-induced apoptosis may contribute to CML LSC persistence, and ß-catenin activity increases during BC. In the current study, we directly tested the role of Fap1 in CML LSC persistence using in an in vivo murine model. In TKI-treated mice, we found that inhibiting Fap1, using a tripeptide or small molecule, prevented TKI resistance, BC and relapse after TKI discontinuation; all events observed with TKI alone. In addition, Fap1 inhibition increased Fas sensitivity and decreased ß-catenin activity in CD34(+) bone marrow cells from human subjects with CML. Therapeutic Fap1 inhibition may permit TKI discontinuation and delay in progression in CML.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteína Tirosina Fosfatase não Receptora Tipo 13/fisiologia , Animais , Apoptose/efeitos dos fármacos , Crise Blástica/etiologia , Crise Blástica/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Domínios PDZ , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 13/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Recidiva , Receptor fas/metabolismoRESUMO
The aim of the present work was to identify protein tyrosine phosphatases (PTPs) as novel, candidate tumor suppressor genes in lung cancer. Among the 38 PTPs in the human genome that show specificity for phosphotyrosine, we identified six PTPs by quantitative RT-PCR whose mRNA expression levels were significantly down-regulated in lung cancer-derived cell lines (ie, PTPRE, PTPRF, PTPRU, PTPRK, PTPRD, and PTPN13). After validation in primary samples of non-small cell lung cancer (NSCLC), we selected PTPN13 for further studies. The results presented here demonstrate that PTPN13 is a candidate tumor suppressor gene that is frequently inactivated in NSCLC through the loss of either mRNA and protein expression (64/87, 73%) or somatic mutation (approximately 8%). Loss of PTPN13 expression was apparently due to the loss of one or both copies of the PTPN13 locus at 4q (approximately 26% double deletion and approximately 37% single deletion) but not to promoter methylation. Finally, the manipulation of PTPN13 expression in lung cancer cells (ie, NCI-H292, A549) demonstrated that PTPN13 negatively regulates anchorage-dependent and anchorage-independent growth in vitro and restrains tumorigenicity in vivo, possibly through the control of the tyrosine phosphorylation of both EGFR and HER2. In conclusion, the expression screening of PTPs in lung cancer reported here has identified PTPN13 as a novel candidate tumor suppressor in NSCLC whose loss increases signaling from epidermal growth factor receptor and HER2 tyrosine kinase receptors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Deleção de Genes , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Proteína Tirosina Fosfatase não Receptora Tipo 13/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/fisiologia , RNA/metabolismo , RNA Interferente Pequeno/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Emerging evidence suggests that Ret oncoproteins expressed in medullary thyroid cancer (MTC) might evade the pro-apoptotic function of the dependence receptor proto-Ret by directly impacting the apoptosis machinery. Identification of the molecular determinants of the interplay between Ret signaling and apoptosis might provide a relevant contribution to the optimization of Ret-targeted therapies. Here, we describe the cross-talk between Ret-M918T oncogenic mutant responsible for type 2B multiple endocrine syndrome (MEN2B), and components of death receptor-mediated extrinsic apoptosis pathway. In the human MEN2B-type MTC cell line MZ-CRC-1 expressing Ret-M918T, Ret was found associated with Fap-1, known as inhibitor of the CD95 death receptor trafficking to the cell membrane, and with procaspase-8, the initiator pro-form caspase in the extrinsic apoptosis pathway. Cell treatment with the anti-tumor Ret kinase inhibitor RPI-1 inhibited tyrosine phosphorylation of procaspase-8, likely inducing its local activation, followed by downregulation of both Ret and Fap-1, and translocation of CD95 into lipid rafts. According to the resulting increase of CD95 cell surface expression, the CD95 agonist antibody CH11 enhanced RPI-1-induced cell growth inhibition and apoptosis. RET RNA interference downregulated Fap-1 protein in MZ-CRC-1 cells, whereas exogenous RET-M918T upregulated Fap-1 in HEK293 cells. Overall, these data indicate that the Ret oncoprotein exerts opposing controls on Fap-1 and CD95, increasing Fap-1 expression and decreasing CD95 cell surface expression. The functional interplay of the Ret mutant with the extrinsic apoptosis pathway provides a mechanism possibly contributing to MTC malignant phenotype and a rational basis for novel therapeutic strategies combining Ret inhibitors and CD95 agonists.
Assuntos
Apoptose , Carcinoma Medular/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 13/fisiologia , Proteínas Proto-Oncogênicas c-ret/fisiologia , Neoplasias da Glândula Tireoide/patologia , Receptor fas/fisiologia , Animais , Antineoplásicos/farmacologia , Carcinoma Medular/metabolismo , Carcinoma Neuroendócrino , Caspase 8/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Indóis/farmacologia , Camundongos , Camundongos SCID , Células NIH 3T3 , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Neoplasias da Glândula Tireoide/metabolismo , Transplante Heterólogo , Carga TumoralRESUMO
Protein tyrosine phosphatase, PTPL1, (also known as PTPN13, FAP-1, PTP-BAS, PTP1E) is a non-receptor type PTP and, at 270 kDa, is the largest phosphatase within this group. In addition to the well-conserved PTP domain, PTPL1 contains at least 7 putative macromolecular interaction domains. This structural complexity indicates that PTPL1 may modulate diverse cellular functions, perhaps exerting both positive and negative effects. In accordance with this idea, while certain studies suggest that PTPL1 can act as a tumor-promoting gene other experimental studies have suggested that PTPL1 may function as a tumor suppressor. The role of PTPL1 in the cancer cell is therefore likely to be both complex and context dependent with possible roles including the modulation of growth, stress-response, and cytoskeletal remodeling pathways. Understanding the nature of molecular complexes containing PTPL1, its interaction partners, substrates, regulation and subcellular localization are key to unraveling the complex personality of this protein phosphatase.