RESUMO
Systemic anaplastic large cell lymphoma is a category of T-cell non-Hodgkin's lymphoma which can be further subdivided into two distinct entities (ALK(+) and ALK(-)) based on the presence or absence of ALK gene rearrangements. Among several pathways triggered by ALK signaling, constitutive activation of STAT3 is strictly required for ALK-mediated transformation and survival. Here we performed genome-wide microRNA profiling and identified 48 microRNA concordantly modulated by the inducible knock-down of ALK and STAT3. To evaluate the functional role of differentially expressed miRNA, we forced their expression in ALK(+) anaplastic large cell lymphoma cells, and monitored their influence after STAT3 depletion. We found that the expression of the microRNA-17~92 cluster partially rescues STAT3 knock-down by sustaining proliferation and survival of ALK(+) cells. Experiments in a xenograft mouse model indicated that forced expression of microRNA-17~92 interferes with STAT3 knock-down in vivo. High expression levels of the microRNA-17~92 cluster resulted in down-regulation of BIM and TGFßRII proteins, suggesting that their targeting might mediate resistance to STAT3 knock-down in anaplastic large cell lymphoma cells. We speculate that the microRNA-17~92 cluster is involved in lymphomagenesis of STAT3(+) ALCL and that its inhibition might represent an alternative avenue to interfere with ALK signaling in anaplastic large cell lymphomas.
Assuntos
Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , MicroRNAs/genética , Família Multigênica , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Ativação Transcricional , Quinase do Linfoma Anaplásico , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Linfoma Anaplásico de Células Grandes/mortalidade , Interferência de RNARESUMO
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
The identification of proliferation/survival pathways constitutively activated by genetic alterations in multiple myeloma (MM), or sustained by the bone marrow (BM) microenvironment, provides novel opportunities for the development of targeted therapies. The deregulated function of protein tyrosine kinases plays a critical role in driving MM malignant phenotype. We investigated the effects of the multi-target tyrosine kinase inhibitor RPI-1 in a panel of human MM cell lines, including t(4;14) positive cell lines expressing the TK receptor FGF-R3. Cells harboring FGF-R3 activating mutations (KMS11 and OPM2) displayed the highest sensitivity to RPI-1 antiproliferative effect. The stimulating effect of the aFGF ligand was abrogated in cells harboring a non-constitutively active receptor. Drug treatment inhibited activation and expression of the FGF-R3(Y373C) mutant as well as aFGF-dependent signaling involving AKT and ERKs. Inhibition of JAK2, an additional RPI-1 target, resulted in STAT3 inactivation. Blockade of these proliferation/survival pathways was associated with caspase-dependent apoptosis. Moreover, drug treatment abrogated proliferative and pro-invasive stimuli provided by conditioned medium from mesenchymal stromal cells. Gene expression profile of KMS11 cells showed 22 upregulated and 52 downregulated genes upon RPI-1 treatment, with an early modulation of genes implicated in MM pathobiology such as SAT-1, MYC, MIP-1alpha/beta, FGF-R3, and the growth factor receptor B-cell maturation antigen (BCMA). Thus, concomitant blockade of FGF-R3 and JAK2 results in inhibition of several MM-promoting pathways, including BCMA-regulated signaling, and downregulation of disease-associated proteins. These data may have therapeutic implications in the design of treatment strategies resulting in the concomitant inhibition of FGF-R3 and JAK2 signaling pathways in t(4;14) MM.
Assuntos
Antígeno de Maturação de Linfócitos B/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Janus Quinase 2/metabolismo , Mieloma Múltiplo/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Apoptose , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismoRESUMO
A novel series of 3-arylureidobenzylidene-indolin-2-ones was synthesized and their inhibitory activity against Ret tyrosine kinase investigated in comparison with the Ret inhibitor RPI-1 as a reference compound for this series. A few compounds were able to revert the RETC634R oncogene-transformed morphologic phenotype of NIH3T3(MEN2A) cells and showed a selective antiproliferative activity against these cells as compared to parental NIH3T3 cells or NIH3T3 cells transformed with a non-tyrosine kinase oncogene (NIH3T3(H-RAS)). Inhibition of Ret enzyme activity by effective derivatives was confirmed in a kinase assay. Structure-activity relationship indicated a favourable activity for 5,6-dimethoxyindolinone derivatives with H, OH, or OMe in the para position of the distal aryl ring.
Assuntos
Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Animais , Western Blotting , Indóis/síntese química , Camundongos , Células NIH 3T3 , Inibidores de Proteínas Quinases/síntese química , Relação Estrutura-AtividadeRESUMO
The proto-oncogene c-myc is involved in multiple cell pathways with opposite effects on cell outcome of death or proliferation. It has been proposed that these different roles depend on the sequestration of c-Myc protein in cellular compartments and/or its phosphorylation. We speculated that subcellular localization of c-Myc protein and of its phosphorylated form (P-c-Myc) could have a role in the different response to paclitaxel (PTX) in two prostate carcinoma cell lines, PC3 and DU145, which undergo either multinucleation or c-myc-dependent apoptosis, respectively. c-myc is amplified only in PC3, but a similar extent of c-Myc phosphorylation was observed in both cell lines after PTX treatment. We found that PTX-induced upregulation of c-myc in DU145 cells, not occurring in PC3 cells, cannot be ascribed to a different protein localization, and that a comparable c-Myc and P-c-Myc nuclear translocation occurs in both cell lines after drug treatment. Thus, subcellular localization of c-Myc and P-c-Myc is not crucial in determining the mode of cell death in these prostate carcinoma cell lines.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma/tratamento farmacológico , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Paclitaxel/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Proto-Oncogene MasRESUMO
Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (-75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination.
Assuntos
Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Inibidores da Angiogênese/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/irrigação sanguínea , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/irrigação sanguínea , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Adesão Celular/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent evidence indicates that the success of molecular targeted therapies may depend on the identification of drug targets which are essential for the survival of subsets of tumors. RET oncogenes that have been implicated in the development of thyroid carcinomas are emerging as potential therapeutic targets. In the present study, we investigated the efficacy and the cellular bases of antitumor activity of the indolinone Ret tyrosine kinase inhibitor RPI-1 against large established s.c. TT tumor xenograft, a human medullary thyroid carcinoma (MTC) harboring oncogenic MEN-2A-type RET mutation. Oral treatment with RPI-1 caused growth arrest or regression in 81% treated tumors. Following treatment suspension, tumor inhibition was maintained (51%, P<0.05, 100 days) and cures were achieved in 2/11 mice. In treated tumors, Ret was tyrosine dephosphorylated. Moreover, compared to control tumors, a significant increase in apoptotic cells (210%, P<0.0001), loss of cellularity (47%, P<0.0001) and reduction of microvessel density (36%, P<0.0005) were detected. In vivo effects of RPI-1 were reflected in activation of BAD, cleavage of caspases, apoptotic DNA fragmentation and inhibition of VEGF production observed in in vitro RPI-1-treated TT cells. These findings thus indicate that RPI-1 antitumor effect on the MTC was characterized by apoptosis induction and angiogenesis inhibition. The results, consistent with a dependence on RET oncogene activation for maintenance and survival of MEN2A-type MTC, provide further preclinical rationale for a pharmacological RET-targeted intervention in thyroid cancer.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Medular/tratamento farmacológico , Indóis/farmacologia , Neovascularização Patológica/prevenção & controle , Neoplasias da Glândula Tireoide/tratamento farmacológico , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Medular/patologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Indóis/administração & dosagem , Indóis/uso terapêutico , Camundongos , Camundongos Nus , Mutação , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The RET proto-oncogene encodes a receptor tyrosine kinase. RET oncogenes arise through sporadic and inherited gene mutations and are involved in the etiopathogenesis of medullary thyroid carcinoma, a cancer that responds poorly to conventional chemotherapy. Medullary thyroid carcinoma is a component of multiple endocrine neoplasia type 2 or MEN2 syndromes. METHODS: We investigated the cellular effects of RPI-1, a novel 2-indolinone Ret tyrosine kinase inhibitor on cells that express RET C634 oncogenic mutants common in the MEN2A syndrome: NIH3T3 fibroblasts transfected with RET(C634R) and human medullary thyroid carcinoma TT cells that express endogenous RET(C634W). RPI-1 antiproliferative activity was determined by cell proliferation and anchorage-independent growth assays. Expression and phosphorylation of Ret and of proteins involved in downstream signaling pathways were examined by immunoblotting. Antitumor activity of oral RPI-1 treatment was tested by using two dosing levels in nude mice bearing subcutaneous TT xenograft tumors. All statistical tests were two-sided. RESULTS: The RPI-1 IC50 value for cell proliferation was 3.6 microM (95% confidence interval [CI] = 1.8 to 5.4 microM) in NIH3T3 cells expressing the Ret mutant compared with 16 microM (95% CI = 12.3 to 19.7 microM) in non-transfected NIH3T3 cells, and that for colony formation in soft agar was 2.4 microM (95% CI = 0.8 to 4.0 microM) and 26 microM (95% CI = 17 to 35 microM) in RET mutant-transfected and H-RAS-transfected NIH3T3 cells, respectively. In NIH3T3 cells expressing the Ret mutant, Ret protein and tyrosine phosphorylation were undetectable after 24 hours of RPI-1 treatment. In TT cells, RPI-1 inhibited proliferation, Ret tyrosine phosphorylation, Ret protein expression, and the activation of PLCgamma, ERKs and AKT. In mice, oral daily RPI-1 treatment inhibited the tumor growth of TT xenografts by 81% (P<.001 versus control mice) and reduced the plasma levels of the specific biomarker calcitonin (P =.01 versus control mice). Twenty-five percent of RPI-1-treated mice were tumor-free. CONCLUSIONS: Ret oncoproteins represent exploitable targets for therapeutic intervention in MEN2A-associated medullary thyroid carcinoma. The antitumor efficacy and oral bioavailability of RPI-1 support its therapeutic potential.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Medular/tratamento farmacológico , Indóis/farmacologia , Neoplasia Endócrina Múltipla Tipo 2a/complicações , Proteínas Oncogênicas/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Neoplasias da Glândula Tireoide/tratamento farmacológico , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Western Blotting , Carcinoma Medular/etiologia , Carcinoma Medular/metabolismo , Divisão Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indóis/administração & dosagem , Camundongos , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Oncogênicas/metabolismo , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Glândula Tireoide/etiologia , Neoplasias da Glândula Tireoide/metabolismo , Transplante Heterólogo , Fosfolipases Tipo C/metabolismoRESUMO
Ret oncoproteins expressed in thyroid carcinomas represent possible targets for therapeutic intervention. Oncogenic activation of the receptor tyrosine kinase encoding RET gene occurs typically by gene rearrangement in papillary thyroid carcinomas (PTC) and by missense mutation in medullary thyroid carcinomas (MTC). These genetic alterations lead to the expression of deregulated products characterized by ligand-independent activation of the intrinsic tyrosine kinase of Ret. Such features suggest the possibility of using specific tyrosine kinase inhibitors to block the Ret oncoproteins signaling. The present report summarizes the cellular effects of the arylidene 2-indolinone Ret inhibitor RPI-1 (formerly Cpd1) on the human PTC cell line TPC-1 which spontaneously harbors the RET/PTC1 oncogene. The results provide evidence that RPI-1 is able to inhibit cell growth and to interfere with Ret/ptc1-driven signaling. These findings support a role for Ret oncoproteins as therapeutic targets and the pharmacological interest of RPI-1 as a candidate drug for preclinical evaluation on thyroid tumors expressing RET oncogenes.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Papilar/tratamento farmacológico , Indóis/farmacologia , Proteínas Oncogênicas/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Glândula Tireoide/efeitos dos fármacos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Carcinoma Papilar/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismoRESUMO
Taxane-based therapies appear to have a significant efficacy in clinical trials on hormone-refractory prostate carcinoma. In the present study, we investigated the cellular response of androgen-independent prostate carcinoma cell lines to the novel taxane IDN 5109 (BAY 59-8862) and evaluated its antitumor activity. In previous preclinical studies, this new paclitaxel (PTX) analogue was characterized by high tolerability and antitumor efficacy, ability to overcome multidrug resistance, and activity by oral administration. Upon treatment, DU145 and PC3 prostate carcinoma cell lines underwent a transient mitotic arrest. This was followed by G1 arrest and rapid occurrence of apoptosis in DU145 cells, whereas in PC3 cells, which are defective for the postmitotic checkpoint, a slow cell death was preceded by DNA endoreduplication. At the biochemical level, such events were associated with tubulin polymerization, activation of the mitosis-promoting factor, and phosphorylation of Bcl-X(L)/Bcl-2/Raf-1. In addition, IDN 5109 shared with PTX the ability to down-regulate the expression of the two potent angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. These findings indicated that IDN 5109 affected the same pathways involved in the cellular response to PTX and suggested that an antiangiogenic effect mediated by inhibition of paracrine stimulation of endothelial cells might contribute to the antitumor effect of both drugs. In in vivo experiments, the new taxane displayed a superior and more persistent effect compared with PTX against DU145 tumor xenografts. Such an effect was associated with pronounced reduction of the tumor microvessel density, superior to that achieved by PTX. These results support a potential therapeutic advantage of IDN 5109 over PTX against hormone-refractory prostate carcinoma.