RESUMO
BACKGROUND: Patients with unresectable recurrent rectal cancer (RRC) or colorectal cancer (CRC) with liver metastases, refractory to at least two lines of traditional systemic therapy, may receive third line intraarterial chemotherapy (IC) and targeted therapy (TT) using drugs selected by chemosensitivity and tumor gene expression analyses of liquid biopsy-derived circulating tumor cells (CTCs). METHODS: In this retrospective study, 36 patients with refractory unresectable RRC or refractory unresectable CRC liver metastases were submitted for IC and TT with agents selected by precision oncotherapy chemosensitivity assays performed on liquid biopsy-derived CTCs, transiently cultured in vitro, and by tumor gene expression in the same CTC population, as a ratio to tumor gene expression in peripheral mononuclear blood cells (PMBCs) from the same individual. The endpoint was to evaluate the predictive accuracy of a specific liquid biopsy precision oncotherapy CTC purification and in vitro culture methodology for a positive RECIST 1.1 response to the therapy selected. RESULTS: Our analyses resulted in evaluations of 94.12% (95% CI 0.71-0.99) for sensitivity, 5.26% (95% CI 0.01-0.26) for specificity, a predictive value of 47.06% (95% CI 0.29-0.65) for a positive response, a predictive value of 50% (95% CI 0.01-0.98) for a negative response, with an overall calculated predictive accuracy of 47.22% (95% CI 0.30-0.64). CONCLUSIONS: This is the first reported estimation of predictive accuracy derived from combining chemosensitivity and tumor gene expression analyses on liquid biopsy-derived CTCs, transiently cultured in vitro which, despite limitations, represents a baseline and benchmark which we envisage will be improve upon by methodological and technological advances and future clinical trials.
Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , Células Neoplásicas Circulantes , Neoplasias Retais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Expressão Gênica , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Células Neoplásicas Circulantes/patologia , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/genética , Estudos RetrospectivosRESUMO
Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM.
Assuntos
Melanoma , Neuroblastoma , Humanos , Neuroblastoma/genética , Receptor trkA/genética , Receptor trkA/metabolismo , Recidiva Local de Neoplasia , Processamento Alternativo/genética , Melanoma/genética , Melanoma Maligno CutâneoRESUMO
We identify a novel alternative TrkA splice variant, TrkAIII, with deletion of exons 6, 7, and 9 and functional extracellular IG-C1 and N-glycosylation domains, that exhibits expression restricted to undifferentiated early neural progenitors, human neuroblastomas (NBs), and a subset of other neural crest-derived tumors. This NGF-unresponsive isoform is oncogenic in NIH3T3 cells and promotes tumorigenic NB cell behavior in vitro and in vivo (cell survival, xenograft growth, angiogenesis) resulting from spontaneous tyrosine kinase activity and IP3K/Akt/NF-kappaB but not Ras/MAPK signaling. TrkAIII antagonizes NGF/TrkAI signaling, which is responsible for NB growth arrest and differentiation through Ras/MAPK, and its expression is promoted by hypoxia at the expense of NGF-responsive receptors, providing a mechanism for converting NGF/TrkA/Ras/MAPK antioncogenic signals to TrkAIII/IP3K/Akt/NF-kappaB tumor-promoting signals during tumor progression.
Assuntos
Processamento Alternativo/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Receptor trkA/genética , Receptor trkA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Hipóxia/genética , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Neovascularização Patológica , Fator de Crescimento Neural/metabolismo , Fator de Crescimento Neural/farmacologia , Neuroblastoma/irrigação sanguínea , Neuroblastoma/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Ligação Proteica , Receptor trkA/antagonistas & inibidores , Receptor trkA/química , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fosfolipases Tipo C/metabolismoRESUMO
BACKGROUND: Hepatic artery infusion (HAI) and drug selection by liquid biopsy precision oncotherapy are under investigation for the multidisciplinary treatment of unresectable colorectal liver metastases (CRCLM) in progression after systemic therapy. Here, we compare the safety and efficacy of third-line HAI followed by target therapy with drug regimes selected by liquid biopsy precision oncotherapy to third-line systemic therapy with drug regimes selected partly by tissue biopsy precision oncotherapy, in a retrospective real-life study of 106 unresectable CRCLM patients. METHODS: Drug regimens for HAI/target therapy were selected by assessing the sensitivity of purified circulating tumor cell (CTCs) to 5-fluorouracil, carboplatin, cisplatin, oxaliplatin, irinotecan, doxorubicin, mitomycin, raltitrexed, and melphalan in-vitro and by real-time qRT-PCR gene expression assays, and for the Systemic therapy cohort were selected by age, comorbidity, performance status, and absence of RAS mutations. Therapeutic responses, adverse events, and quality of life were evaluated by RECIST 1.1, CTCAE 4.03, and ECOG criteria, respectively, and chemo-filtration performed following HAI to reduce systemic toxic effects. RESULTS: HAI/target therapy with drugs selected by liquid biopsy precision oncotherapy (44 patients), resulted in 2.27% CRs, 38.63% PRs, 56.81% SD,s and 2.27% PDs; ECOG 2 to 1 improvement, but no infusion-related technical or vascular complications, or deaths. Systemic therapy (62 patients) resulted in 1.6% CRs, 17.74% PRs, 37.09% SDs, and 45.16% PDs; more grade 1-2 adverse events and 4.84% ECOG 1 to 2 worsening. The median 5 month PFS in the HAI/target therapy cohort was significantly longer than 3 months in the systemic cohort (P < 0.007) and the median 14 month survival in the HAI/target therapy cohort was longer than 8.5 months in the systemic therapy cohort but not statistically significant. Multivariate analysis identified ECOG grade 2 as the most unfavourable survival prognostic factor in both cohorts. CONCLUSIONS: HAI plus chemo-filtration followed by target therapy, with drug regimens selected by liquid biopsy precision oncotherapy, is a safe and efficacious alternative therapeutic strategy for unresectable CRCLM in progression after two lines of systemic therapy and should be considered for a multicentre prospective phase III study, to fully confirm this potential.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Idoso , Antineoplásicos/efeitos adversos , Estudos de Coortes , Neoplasias Colorretais/patologia , Feminino , Artéria Hepática , Humanos , Infusões Intra-Arteriais , Biópsia Líquida , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/efeitos dos fármacos , Medicina de Precisão , Intervalo Livre de Progressão , Qualidade de VidaRESUMO
Expansion and fate choice of pluripotent stem cells along the neuroectodermal lineage is regulated by a number of signals, including EGF, retinoic acid, and NGF, which also control the proliferation and differentiation of central nervous system (CNS) and peripheral nervous system (PNS) neural progenitor cells. We report here the identification of a novel gene, REN, upregulated by neurogenic signals (retinoic acid, EGF, and NGF) in pluripotent embryonal stem (ES) cells and neural progenitor cell lines in association with neurotypic differentiation. Consistent with a role in neural promotion, REN overexpression induced neuronal differentiation as well as growth arrest and p27Kip1 expression in CNS and PNS neural progenitor cell lines, and its inhibition impaired retinoic acid induction of neurogenin-1 and NeuroD expression. REN expression is developmentally regulated, initially detected in the neural fold epithelium of the mouse embryo during gastrulation, and subsequently throughout the ventral neural tube, the outer layer of the ventricular encephalic neuroepithelium and in neural crest derivatives including dorsal root ganglia. We propose that REN represents a novel component of the neurogenic signaling cascade induced by retinoic acid, EGF, and NGF, and is both a marker and a regulator of neuronal differentiation.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Biomarcadores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Células Cultivadas , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p27 , DNA Complementar , Fator de Crescimento Epidérmico/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Dados de Sequência Molecular , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco/metabolismo , Transferases , Tretinoína/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The alternative TrkAIII splice variant is expressed by murine and human thymus. Alternative TrkAIII splicing predominates in postembryonic day E13 (E17 and E18), postnatal murine (3 week and 3 month) and human thymuses, with TrkAIII mRNA expressed by selected thymocyte subsets and thymic epithelial cells (TECs) and a 100 kDa immunoprecipitable TrkAIII-like protein detected in purified thymocyte and whole thymus extracts. FACS and immunohistochemical analysis indicate a non-cell surface localisation for the TrkAIII-like protein in cortical CD4+/CD8+ double positive and, to a lesser extent, single positive thymocyte subsets at the cortex/medulla boundary and in Hassle's corpuscles, reticular epithelial and dendritic cells of the thymic medulla. TrkA(I/II) expression, on the other hand, predominates in sub-capsular regions of the thymus. TrkAIII-like immunoreactivity at the cortex/medulla boundary associates with regions of thymocyte proliferation and not apoptosis. A potential role for thymic hypoxia in thymocyte alternative TrkAIII splicing is supported by reversal to TrkAI splicing by normoxic but not hypoxic culture and induction of Jurkat T cell alternative TrkAIII splicing by the hypoxia mimic CoCl2. In contrast, TEC expression of TrkAIII predominates in both normoxic and hypoxic culture conditions. The data support a potential role for TrkAIII in thymic development and function, of particular relevance to intermediate stage CD4+/CD8+ thymocyte subsets and TECs, which potentially reflects a reversible thymocyte and more permanent TEC adaptation to thymic environment. Since intracellular TrkAIII neither binds nor responds to NGF and can impede regular NGF/TrkA signalling (Tacconelli et al., Cancer Cell, 2004), its expression would be expected to provide an alternative and/or impediment to regular NGF/TrkA signalling within the developing and developed thymus of potential functional importance.
Assuntos
Receptor trkA/genética , Receptor trkA/metabolismo , Timo/citologia , Timo/metabolismo , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Células Cultivadas , Embrião de Mamíferos , Células Epiteliais/fisiologia , Citometria de Fluxo/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica/métodos , Células Jurkat , Camundongos , Neuroblastoma , Extratos do Timo/metabolismoRESUMO
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
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
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
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.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/biossíntese , Proteínas de Neoplasias/metabolismo , Tiorredoxinas/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Metaloproteinase 9 da Matriz/genética , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Elementos de Resposta/genética , Tiorredoxinas/genética , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/genéticaRESUMO
Nerve growth factor receptor TrkA is critical for development and maturation of central and peripheral nervous systems, regulating proliferation, differentiation and apoptosis. In cancer, TrkA frequently exhibits suppressor activity in nonmutated form and oncogenic activity upon mutation. Our identification of a novel hypoxia-regulated alternative TrkAIII splice variant, expressed by neural crest-derived neuroblastic tumors, that exhibits neuroblastoma tumor promoting activity, adds significantly to our understanding of potential TrkA involvement in cancer. Our observation that hypoxia, which characterizes the tumor micro-environment, stimulates alternative TrkAIII splicing, provides a way by which TrkA tumor suppressing signals may convert to tumor promoting signals during progression and is consistent with conservation and pathological subversion by neural crest-derived neuroblastic tumors of a mechanism of potential physiological importance to normal neural stem/neural crest progenitors.
Assuntos
Processamento Alternativo , Hipóxia Celular , Variação Genética , Receptor trkA/genética , Receptor trkA/fisiologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Crista Neural/citologia , Crista Neural/fisiologia , Neuroblastoma/genética , Neuroblastoma/patologia , Neuroblastoma/fisiopatologia , Transdução de Sinais/genéticaRESUMO
An association between elevated tyrosine kinase receptor (Trk)-A expression and better prognosis; the absence of mutation-activated TrkA oncogenes; the induction of apoptosis, growth arrest, morphological differentiation and inhibition of xenograft growth; and angiogenesis by TrkA gene transduction, provide the basis for the current concept of an exclusively tumor-suppressor role for TrkA in the aggressive pediatric tumor, neuroblastoma. This concept, however, has recently been challenged by the discovery of a novel hypoxia-regulated alternative TrkAIII splice variant, initial data for which suggest predominant expression in advanced-stage neuroblastoma. TrkAIII exhibits neuroblastoma xenograft tumor-promoting activity associated with the induction of a more angiogenic and stress-resistant neuroblastoma phenotype and antagonises nerve growth factor/TrkAI antioncogenic signaling. In this short review, the authors integrate this novel information into a modified concept that places alternative TrkA splicing as a potential pivotal regulator of neuroblastoma behavior and identifies the TrkAIII alternative splice variant as a potential biomarker of patient prognosis and novel therapeutic target.
Assuntos
Processamento Alternativo/genética , Neuroblastoma , Receptor trkA/genética , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Receptor trkA/antagonistas & inibidores , Receptor trkA/metabolismo , Transdução de SinaisRESUMO
Thioredoxin (Trx) inhibited human HMEC-1 dermal microvascular endothelial cell capillary tubule forming capacity in a Matrigel based assay in vitro. Inhibition of capillary tubule formation was Trx catalytic site and thioredoxin reductase (TrxR) dependent, mediated at the Matrigel matrix level, and associated with a shift from morphological differentiation to continuous proliferation, with enhanced cell spreading resulting in eventual monolayer formation. Soluble complex carbohydrates, which inhibited capillary tubule formation on Matrigel without induction of cell spreading or monolayer formation, failed to impair Trx promotion of cell spreading and mono-layer formation, suggesting a shift away from carbohydrate-mediated cell/matrix adhesive interactions. Laminin peptides YIGRS and SIKVAV, which impaired tubule formation on Matrigel without inducing cell spreading or monolayer formation, partially impaired cell spreading upon Trx-treated Matrigel without restoring tubule formation, consistent with a potential role for laminin in Trx-mediated effects. Trx reduced laminin and destabilised laminin/galectin-3 complexes within Matrigel. Native purified EHS Laminin (also containing galectin-3), but not recombinant galectin-3, restored HMEC-1 capillary tubule formation on Trx-treated Matrigel. These data highlight a novel deregulatory effect of extracellular Trx upon morphological capillary differentiation that appears to depend upon the reduction of laminin and destabilisation of its interaction with galectin-3, possibly leading to galectin-3 neutralisation that shifts cell/matrix adhesive interactions away from being carbohydrate mediated and results in loss of proliferation-inhibiting and differentiation promoting cues from this tumor basement membrane matrix.
Assuntos
Capilares/crescimento & desenvolvimento , Endotélio Vascular/citologia , Neovascularização Fisiológica/efeitos dos fármacos , Tiorredoxinas/farmacologia , Carboidratos/farmacologia , Domínio Catalítico , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Colágeno , Combinação de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Galectina 3/metabolismo , Humanos , Laminina/metabolismo , Laminina/farmacologia , Proteoglicanas , Tiorredoxina Dissulfeto RedutaseRESUMO
Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.
Assuntos
Neoplasias da Mama/enzimologia , Metaloproteinase 3 da Matriz/metabolismo , Plasminogênio/metabolismo , Angiostatinas , Neoplasias da Mama/tratamento farmacológico , Membrana Celular/metabolismo , Feminino , Humanos , Laminina/metabolismo , Metaloproteinase 3 da Matriz/farmacologia , Invasividade Neoplásica/fisiopatologia , Fragmentos de Peptídeos/metabolismo , Células Tumorais CultivadasRESUMO
A comparison between retinoic acid (RA) differentiation-resistant and differentiation-sensitive SK-N-BE neuroblastoma (NB) cell lines revealed an association between resistance to differentiation, exhibited by N-myc stable transfected SK-N-BE 9N cells, with sensitivity to RA induction of p50/p65 nuclear factor kappaB (NF-kappaB) transcription factor activity and induction of matrix metalloproteinase (MMP)-9 expression leading to enhanced invasive behavior in vitro. These effects were not observed in differentiation-sensitive parental SK-N-BE or control-transfected SK-N-BE 2N counterparts. RA activated a MMP-9 promoter reporter gene construct in SK-N-BE 9N but not parental SK-N-BE or SK-N-BE 2N cells through a NF-kappaB element (-600) in association with enhanced p50 mRNA expression, reduced cytoplasmic inhibitor of nuclear factor kappaBalpha protein levels, and the induction of nuclear p50/p65 containing MMP-9 NF-kappaB site binding activity. RA activation of the MMP-9 promoter was inhibited by transient overexpression of a dominant-negative inhibitor of nuclear factor kappaBalpha protein and stimulated by transient p50 but not p65 overexpression in the absence of RA. A limited, nonessential function for activator protein 1 (-74), Ets (-540), and SP1 (-560) elements within the MMP-9 promoter was revealed by point mutation but was not associated with changes in the binding or position of complexes constitutive to differentiation-sensitive or -resistant cells. Our data indicates that in this model of NB resistance to differentiation that results from uncoupled RA regulation of N-myc expression, RA stimulates malignant NB cell behavior by inducing nuclear NF-kappaB transcription factor activity, which in turn induces MMP-9 expression and stimulation of basement membrane invasive capacity involving MMP-9 activity.