RESUMO
The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H2O2, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/patologia , Neuroblastoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptor trkA/metabolismo , Superóxido Dismutase/genética , Carcinogênese/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Mitocôndrias/efeitos dos fármacos , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/metabolismo , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor trkA/antagonistas & inibidores , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Superóxido Dismutase/deficiênciaRESUMO
The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs) and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α -tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC.
Assuntos
Processamento Alternativo/genética , Neuroblastoma/genética , Receptor trkA/genética , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Centrossomo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Microtúbulos/metabolismo , Microtúbulos/patologia , Estadiamento de Neoplasias , Neuroblastoma/patologia , Fosforilação , Ligação Proteica , Receptor trkA/metabolismo , Transdução de Sinais/genéticaRESUMO
The motility, angiogenesis and metastasis-stimulating factor Autotaxin (Atx), over expressed by human neuroblastomas (NB), is constitutively expressed by human Nmyc-amplified SK-N-BE and non-Nmyc-amplified SH-SY5Y NB cells. Here, we characterise a novel Atx transcriptional mechanism, utilised by both cell lines, that is restricted to the first 285bp of the Atx promoter and involves AP-1 and SP transcription factors, acting through a CRE/AP-1-like element at position -142 to -149 and a GAbox at position -227 to -235 relative to the Atx translational start site. This novel transcriptional mechanism can be inhibited by internally initiated SP-3 and the natural phenol curcumin.
Assuntos
Curcumina/farmacologia , Neuroblastoma/patologia , Diester Fosfórico Hidrolases/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição Sp/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , AMP Cíclico/genética , Deleção de Genes , Genes Reporter/genética , Humanos , Diester Fosfórico Hidrolases/deficiência , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
Irreversible MMP-9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP-9 but also promoted irreversible plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin-degradation sites within the MMP-9 catalytic-domain and producing an inhibitory hemopexin-domain fragment. This effect was also observed using MDA-MB-231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP-9 in the presence of Alendronate or EDTA. Degradation-mediated inactivation of proMMP-9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proMMP-9 and direct MMP-9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP-9-dependent pathology characterised by plasminogen activation.
Assuntos
Alendronato/farmacologia , Fibrinolisina/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Catálise , Domínio Catalítico , Cátions , Linhagem Celular Tumoral , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Ácido Edético/química , Hemopexina/química , Humanos , Plasminogênio/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
Increased expression of thioredoxin (Trx)-1 and matrix metalloproteinase (MMP)-9 associates with malignant breast cancer progression. Here, we describe a functional relationship between Trx-1 and MMP-9 in promoting MDA-MB-231 breast cancer cell invasive behaviour. Trx-1 overexpression stimulated MMP-9 expression, de-regulated the MMP-9/TIMP-1 equilibrium and augmented MMP-9 involvement in a more invasive phenotype. Trx-1 augmented MMP-9 transcription through NF-κB, AP-1 and SP1 elements; stimulated p50/p65 NF-κB activity and recruitment to the MMP-9 promoter; and facilitated MMP-9 promoter-accessibility to NF-κB by preventing HDAC recruitment and maintaining MMP-9 promoter histone acetylation. Our data provide a functional basis for Trx-1 and MMP-9 association in malignant breast cancer and identify Trx-1 and NF-κB as potentially druggable targets for reducing MMP-9 involvement in malignant behaviour.