RESUMO
Recently, clinical trial results have established inhibitors of B-cell receptor (BCR)-associated kinase (BAKi), with or without CD20 moniclonal antibodies (mAbs), as the preferred first-line treatment for most chronic lymphocytic leukaemia (CLL) patients. Using phosphospecific flow cytometry, we showed that in leukaemic cells from CLL patients the CD20 therapeutic antibodies - rituximab, ofatumumab, and obinutuzumab - inhibited BCR signalling pathways targeting preferentially pBTKY551 - but not BTKY223 - and pAKT. On the contrary, ibrutinib and idelalisib reduced pBTKY223 to a higher extent than pBTKY551 . The strong reduction of pAKT induced by idelalisib was enhanced by its combination with rituximab or ofatumumab. Moreover, CD20 mAbs and BAKi induced the death of leukaemia cells that was significantly potentiated by their combination. Analysis of the enhancement of cell death in these combinations revealed an approximately additive enhancement induced by rituximab or obinutuzumab combined with ibrutinib or idelalisib. Taken together, our data identified negative regulatory effects of CD20 mAbs and their combinations with BAKi on BCR signalling and cell survival in CLL. In conclusion, this study advances our understanding of mechanisms of action of CD20 mAbs as single agents or in combination with BAKi and could inform on the potential of combined therapies in ongoing and future clinical trials in patients with CLL.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Antígenos de Linfócitos B/metabolismo , Rituximab/uso terapêutico , Adenina/análogos & derivados , Adenina/uso terapêutico , Antígenos CD20/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Piperidinas/uso terapêutico , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Transdução de Sinais/efeitos dos fármacosRESUMO
Tumour repopulation is recognized as a crucial event in tumour relapse where therapy-sensitive dying cancer cells influence the tumour microenvironment to sustain therapy-resistant cancer cell growth. Recent studies highlight the role of the oncometabolites succinate, fumarate, and 2-hydroxyglutarate in the aggressiveness of cancer cells and in the worsening of the patient's clinical outcome. These oncometabolites can be produced and secreted by cancer and/or surrounding cells, modifying the tumour microenvironment and sustaining an invasive neoplastic phenotype. In this review, we report recent findings concerning the role in cancer development of succinate, fumarate, and 2-hydroxyglutarate and the regulation of their related enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase. We propose that oncometabolites are crucially involved in tumour repopulation. The study of the mechanisms underlying the relationship between oncometabolites and tumour repopulation is fundamental for identifying efficient anti-cancer therapeutic strategies and novel serum biomarkers in order to overcome cancer relapse.
Assuntos
Fumarato Hidratase/metabolismo , Isocitrato Desidrogenase/metabolismo , Neoplasias/patologia , Succinato Desidrogenase/metabolismo , Animais , Humanos , Neoplasias/enzimologia , Recidiva , Microambiente TumoralRESUMO
Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The synthetic compound (S)-benzyl-2-amino-2-(S)-3-bromo-4,5-dihydroisoxazol-5-yl-acetate (AXP3009), which binds GAPDH at its redox-sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase-mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3-methyladenine revealing a protective role for Beclin1-mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Everolimo/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Iridoides/farmacologia , Neoplasias Pancreáticas/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proteína Desacopladora 2/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/enzimologia , Carcinoma Ductal Pancreático/fisiopatologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Everolimo/uso terapêutico , Feminino , Humanos , Iridoides/uso terapêutico , Masculino , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/fisiopatologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteína Desacopladora 2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated. To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins. Pathway analysis revealed activation of glycolysis, the pentose phosphate pathway, the pyruvate-malate cycle, and lipid metabolism as well as downregulation of the Krebs cycle, the splicesome and non-homologous end joining. These findings were supported by metabolomics and immunoblotting analysis. It was also found that inhibition of fatty acid synthase by cerulenin and of mevalonate pathways by atorvastatin have a greater anti-proliferative effect on cancer stem cells than parental cells. Taken together, these results clarify some important aspects of the metabolic network signature of pancreatic cancer stem cells, shedding light on key and novel therapeutic targets and suggesting that fatty acid synthesis and mevalonate pathways play a key role in ensuring their viability. BIOLOGICAL SIGNIFICANCE: To better understand the altered metabolic pathways of pancreatic cancer stem cells (CSCs), a comprehensive proteomic analysis and metabolite profiling investigation of Panc1 and Panc1 CSCs were carried out. The findings obtained indicate that Panc1 CSCs are characterized by upregulation of glycolysis, pentose phosphate pathway, pyruvate-malate cycle, and lipid metabolism and by downregulation of Krebs cycle, spliceosome and non-homologous end joining. Moreover, fatty acid synthesis and mevalonate pathways are shown to play a critical contribution to the survival of pancreatic cancer stem cells. This study is helpful for broadening the knowledge of pancreatic cancer stem cells and could accelerate the development of novel therapeutic strategies.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Ácidos Graxos/metabolismo , Redes e Vias Metabólicas/fisiologia , Ácido Mevalônico/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteômica/métodos , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Ácido Graxo Sintases/metabolismo , Humanos , Metaboloma , Metabolômica/métodos , Células-Tronco Neoplásicas/química , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/patologiaRESUMO
Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.