RESUMO
The process of carcinogenesis and its progression involves an intricate interplay between a number of signaling networks, metabolic pathways and the microenvironment. These include an alteration in the cellular redox metabolism and deregulation of cell cycle checkpoints. Similar to the dichotomy of redox signaling in cancer cell fate and state determination, a diverging effect of an irreversible cell cycle arrest or senescence on carcinogenesis has been demonstrated. In this regard, while overwhelming oxidative stress has a damaging effect on tissue architecture and organ function and promotes death execution, a mild "pro-oxidant" environment is conducive for cell proliferation, growth and survival. Similarly, cellular senescence has been shown to elicit both a tumor suppressor and an oncogenic effect in a context-dependent manner. Notably, there appears to be a crosstalk between these two critical regulators of cell fate and state, particularly from the standpoint of the divergent effects on processes that promote or abate carcinogenesis. This review aims to provide an overview of these overarching themes and attempts to highlight critical intersection nodes, which are emerging as potential diagnostic and/or therapeutic targets for novel anticancer strategies.
Assuntos
Antineoplásicos/farmacologia , Senescência Celular/fisiologia , Neoplasias/patologia , Neoplasias/terapia , Proliferação de Células , Humanos , Imunoterapia/métodos , Neoplasias/prevenção & controle , Oxirredução , Estresse Oxidativo , Fenótipo Secretor Associado à Senescência/efeitos dos fármacos , Fenótipo Secretor Associado à Senescência/fisiologia , Telomerase/antagonistas & inibidoresRESUMO
RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS) generation, which we previously demonstrated as a trigger for autophagy-associated execution of mutant KRAS-expressing cancer cells. Here we report that drug (merodantoin; C1)-induced activation of mutant KRAS promotes phospho-AKT S473-dependent ROS-mediated S616 phosphorylation and mitochondrial localization of DNM1L/DRP1 (dynamin 1 like) and cleavage of the fusion-associated protein OPA1 (OPA1 mitochondrial dynamin like GTPase). Interestingly, accumulation of the outer mitochondrial membrane protein VDAC1 (voltage dependent anion channel 1) is observed in mutant KRAS-expressing cells upon exposure to C1. Conversely, silencing VDAC1 abolishes C1-induced mitophagy, and gene knockdown of either KRAS, AKT or DNM1L rescues ROS-dependent VDAC1 accumulation and stability, thus suggesting an axis of mutant active KRAS-phospho-AKT S473-ROS-DNM1L-VDAC1 in mitochondrial morphology change and cancer cell execution. Importantly, we identified MTOR (mechanistic target of rapamycin kinsase) complex 2 (MTORC2) as the upstream mediator of AKT phosphorylation at S473 in our model. Pharmacological or genetic inhibition of MTORC2 abrogated C1-induced phosphorylation of AKT S473, ROS generation and mitophagy induction, as well as rescued tumor colony forming ability and migratory capacity. Finally, increase in thermal stability of KRAS, AKT and DNM1L were observed upon exposure to C1 only in mutant KRAS-expressing cells. Taken together, our work has unraveled a novel mechanism of selective targeting of mutant KRAS-expressing cancers via MTORC2-mediated AKT activation and ROS-dependent mitofission, which could have potential therapeutic implications given the relative lack of direct RAS-targeting strategies in cancer.Abbreviations: ACTB/ß-actin: actin beta; AKT: AKT serine/threonine kinase; C1/merodantoin: 1,3-dibutyl-2-thiooxo-imidazoldine-4,5-dione; CAT: catalase; CETSA: cellular thermal shift assay; CHX: cycloheximide; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2O2: hydrogen peroxide; HSPA1A/HSP70-1: heat shock protein family A (Hsp70) member 1A; HSP90AA1/HSP90: heat shock protein 90 alpha family class A member 1; KRAS: KRAS proto-oncogene, GTPase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; LC3B-I: unlipidated form of LC3B; LC3B-II: phosphatidylethanolamine-conjugated form of LC3B; MAPKAP1/SIN1: MAPK associated protein 1; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MFI: mean fluorescence intensity; MiNA: Mitochondrial Network Analysis; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; O2.-: superoxide; OMA1: OMA1 zinc metallopeptidase; OPA1: OPA1 mitochondrial dynamin like GTPase; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR/raptor: regulatory associated protein of MTOR complex 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; VDAC1: voltage dependent anion channel 1; VDAC2: voltage dependent anion channel 2.
Assuntos
Neoplasias Colorretais , Alvo Mecanístico do Complexo 2 de Rapamicina , Mitocôndrias , Mitofagia , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas p21(ras) , Espécies Reativas de Oxigênio , Humanos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Mitofagia/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mutação/genética , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Fosforilação/efeitos dos fármacosRESUMO
Molecular chaperons stabilize protein folding and play a vital role in maintaining tissue homeostasis. To this intent, mitochondrial molecular chaperons may be involved in the regulation of oxidative phosphorylation and apoptosis during stress events such as infections. However, specific human infectious diseases relatable to defects in molecular chaperons have yet to be identified. To this end, we performed whole exome sequencing and functional immune assessment in a previously healthy Asian female, who experienced severe respiratory failure due to Pneumocystis jiroveci pneumonia and non-HIV-related CD4 lymphocytopenia. This revealed that a chaperon, the mitochondrial paralog of HSP90, TRAP1, may have been involved in the patient's susceptibility to an opportunistic infection. Two rare heterozygous variants in TRAP1, E93Q, and A64T were detected. The patient's peripheral blood mononuclear cells displayed diminished TRAP1 expression, but had increased active, cleaved caspase-3, caspase-7, and elevated IL-1ß production. Transfection of A64T and E93Q variants in cell lines yielded decreased TRAP1 compared to transfected wildtype TRAP1 and re-capitulated the immunotypic phenotype of enhanced caspase-3 and caspase-7 activity. When infected with live P. jiroveci, the E93Q or A64T TRAP1 mutant expressing cells also exhibited reduced viability. Patient cells and cell lines transfected with the TRAP1 E93Q/A64T mutants had impaired respiration, glycolysis, and increased ROS production. Of note, co-expression of E93Q/A64T double mutants caused more functional aberration than either mutant singly. Taken together, our study uncovered a previously unrecognized role of TRAP1 in CD4+ lymphocytopenia, conferring susceptibility to opportunistic infections.
Assuntos
Apoptose , Proteínas de Choque Térmico HSP90 , Pneumocystis carinii , Pneumonia por Pneumocystis , Humanos , Pneumonia por Pneumocystis/imunologia , Pneumonia por Pneumocystis/genética , Feminino , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Pneumocystis carinii/genética , Apoptose/genética , Predisposição Genética para Doença , Mitocôndrias/metabolismo , Sequenciamento do Exoma , Suscetibilidade a Doenças , Pessoa de Meia-Idade , Caspase 3/metabolismo , Caspase 7/metabolismo , Caspase 7/genéticaRESUMO
Epitranscriptomic RNA modifications are crucial for the maintenance of glioma stem cells (GSCs), the most malignant cells in glioblastoma (GBM). 3-methylcytosine (m3C) is a new epitranscriptomic mark on RNAs and METTL8 represents an m3C writer that is dysregulated in cancer. Although METTL8 has an established function in mitochondrial tRNA (mt-tRNA) m3C modification, alternative splicing of METTL8 can also generate isoforms that localize to the nucleolus where they may regulate R-loop formation. The molecular basis for METTL8 dysregulation in GBM, and which METTL8 isoform(s) may influence GBM cell fate and malignancy remain elusive. Here, we investigated the role of METTL8 in regulating GBM stemness and tumorigenicity. In GSC, METTL8 is exclusively localized to the mitochondrial matrix where it installs m3C on mt-tRNAThr/Ser(UCN) for mitochondrial translation and respiration. High expression of METTL8 in GBM is attributed to histone variant H2AZ-mediated chromatin accessibility of HIF1α and portends inferior glioma patient outcome. METTL8 depletion impairs the ability of GSC to self-renew and differentiate, thus retarding tumor growth in an intracranial GBM xenograft model. Interestingly, METTL8 depletion decreases protein levels of HIF1α, which serves as a transcription factor for several receptor tyrosine kinase (RTK) genes, in GSC. Accordingly, METTL8 loss inactivates the RTK/Akt axis leading to heightened sensitivity to Akt inhibitor treatment. These mechanistic findings, along with the intimate link between METTL8 levels and the HIF1α/RTK/Akt axis in glioma patients, guided us to propose a HIF1α/Akt inhibitor combination which potently compromises GSC proliferation/self-renewal in vitro. Thus, METTL8 represents a new GBM dependency that is therapeutically targetable.
Assuntos
Glioblastoma , Subunidade alfa do Fator 1 Induzível por Hipóxia , Metiltransferases , Células-Tronco Neoplásicas , Proteínas Proto-Oncogênicas c-akt , Metilação de RNA , Animais , Humanos , Camundongos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/genética , Carcinogênese/genética , Carcinogênese/patologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Camundongos Nus , Mitocôndrias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Transdução de Sinais , Metilação de RNA/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis in leukemia cells by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We report here that silencing and functional inhibition of Rac1 block Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. Using confocal, electron microscopy and coimmunoprecipitation, as well as glutathione S-transferase-fusion proteins, we provide evidence for a colocalization and physical interaction between the 2 proteins. This interaction is blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia cells and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anticancer drug design.
Assuntos
Apoptose , Neuropeptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Superóxidos/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Inativação Gênica , Células HeLa , Humanos , Células Jurkat , Camundongos , Células NIH 3T3 , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neuropeptídeos/genética , Fragmentos de Peptídeos/farmacologia , Peptidomiméticos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
We report a novel topoisomerase IIα inhibitor, mercaptopyridine oxide (MPO), which induces G2/M arrest and senescence with distinctly different cell cycle regulators (p21 or p14ARF) in HCT116p 53WT and HCT116 p53-/- cells, respectively. MPO treatment induced defective topoisomerase IIα-mediated decatenation process and inhibition of the enzyme's catalytic activity that stalled entry into mitosis. Topoisomerase IIα inhibition was associated with ROS-mediated activation of ATM-Chk2 kinase axis in HCT116 p53WT cells, but not in HCT116 p53-/- cells displaying early Chk1 activation. Results suggest that E2F1 stabilization might link MPO-induced p53 phospho-activation in HCT116 p53WT cells or p14ARF induction in HCT116 p53-/- cells. Also, interaction between topoisomerase IIα and Chk1 was induced in both cell lines, which could be important for decatenation checkpoint activation, even upon p53 ablation. Notably, TCGA dataset analyses revealed topoisomerase IIα upregulation across a wide array of cancers, which was associated with lower overall survival. Corroborating that increased topoisomerase IIα expression might offer susceptibility to the novel inhibitor, MPO (5 µM) induced strong inhibition in colony forming ability of pancreatic and hepatocellular cancer cell lines. These data highlight a novel topoisomerase IIα inhibitor and provide proof-of-concept for its therapeutic potential against cancers even with loss-of-function of p53.
Assuntos
Proteínas de Ciclo Celular/genética , DNA Topoisomerases Tipo II/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteína Supressora de Tumor p53/metabolismo , HumanosRESUMO
AIMS: Preferential expression of receptors for TNF-family related apoptosis inducing ligand (TRAIL), DR4 and DR5 makes TRAIL an attractive anti-cancer therapeutic. However, the efficacy of targeting death receptors has not been extensively studied in nasopharyngeal cancer (NPC). Here we investigated TRAIL sensitivity and its underlying mechanism in NPC cell lines, and assessed the potential of TRAIL as a therapeutic option against NPC. RESULTS: Using two established NPC cell lines, we report the expression of DR4 and DR5, which respond to TRAIL ligation by triggering efficient Type II apoptosis. Mechanistically, early activation of caspase-3 and its membrane recruitment is identified in NPC cell lines, which is associated with, hitherto unreported, interaction with transmembrane and tetratricopeptide repeat containing 2 (TMTC2) in the lipid raft domains. TMTC2 expression is induced upon exposure to TRAIL and involves intracellular increase in peroxynitrite (ONOO-) production. While ONOO- increase is downstream of caspase-8 activation, it is involved in the upregulation of TMTC2, gene knockdown of which abrogated TRAIL-induced apoptotic execution. Bioinformatics analyses also provide evidence for a strong correlation between TMTC2 and DR4 or caspase-3 as well as a significantly better disease-free survival in patients with high TMTC2 expression. INNOVATION AND CONCLUSION: Collectively, redox-dependent execution of NPC cells upon ligation of TRAIL receptors reintroduces the possible therapeutic use of TRAIL in NPC as well as underscores the potential of using TMTC2 as a biomarker of TRAIL sensitivity.
RESUMO
Mitochondria are the main powerhouse of the cell, generating ATP through the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS), which drives myriad cellular processes. In addition to their role in maintaining bioenergetic homeostasis, changes in mitochondrial metabolism, permeability, and morphology are critical in cell fate decisions and determination. Notably, mitochondrial respiration coupled with the passage of electrons through the electron transport chain (ETC) set up a potential source of reactive oxygen species (ROS). While low to moderate increase in intracellular ROS serves as secondary messenger, an overwhelming increase as a result of either increased production and/or deficient antioxidant defenses is detrimental to biomolecules, cells, and tissues. Since ROS and mitochondria both regulate cell fate, attention has been drawn to their involvement in the various processes of carcinogenesis. To that end, the link between a prooxidant milieu and cell survival and proliferation as well as a switch to mitochondrial OXPHOS associated with recalcitrant cancers provide testimony for the remarkable metabolic plasticity as an important hallmark of cancers. In this review, the regulation of cell redox status by mitochondrial metabolism and its implications for cancer cell fate will be discussed followed by the significance of mitochondria-targeted therapies for cancer.
Assuntos
Metabolismo Energético , Mitocôndrias/metabolismo , Neoplasias/patologia , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismo , Animais , Respiração Celular , Sobrevivência Celular , Humanos , Neoplasias/metabolismo , OxirreduçãoRESUMO
Stabilization of c-Myc oncoprotein is dependent on post-translational modifications, especially its phosphorylation at serine-62 (S62), which enhances its tumorigenic potential. Herein we report that increase in intracellular superoxide induces phospho-stabilization and activation of c-Myc in cancer cells. Importantly, sustained phospho-S62 c-Myc was necessary for promoting superoxide dependent chemoresistance as non-phosphorylatable S62A c-Myc was insensitive to the redox impact when subjected to chemotherapeutic insults. This redox-dependent sustained S62 phosphorylation occurs through nitrative inhibition of phosphatase, PP2A, brought about by peroxynitrite, a reaction product of superoxide and nitric oxide. We identified a conserved tyrosine residue (Y238) in the c-Myc targeting subunit B56α of PP2A, which is selectively amenable to nitrative inhibition, further preventing holoenzyme assembly. In summary, we have established a novel mechanism wherein the pro-oxidant microenvironment stimulates a pro-survival milieu and reinforces tumor maintenance as a functional consequence of c-Myc activation through its sustained S62 phosphorylation via inhibition of phosphatase PP2A. SIGNIFICANCE STATEMENT: Increased peroxynitrite signaling in tumors causes sustained S62 c-Myc phosphorylation by PP2A inhibition. This is critical to promoting c-Myc stabilization and activation which promotes chemoresistance and provides significant proliferative and growth advantages to osteosarcomas.
Assuntos
Proteínas Proto-Oncogênicas c-myc , Serina , Proteínas Oncogênicas , Ácido Peroxinitroso , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The death inhibitory proteins, cFLIP and Bcl-2, canonically act at different steps to regulate receptor-mediated apoptosis in cancer cells. Here we report that pharmacological or genetic means to effect an increase in intracellular superoxide result in cFLIP upregulation. Interestingly, Bcl-2 overexpression is associated with a concomitant increase in cFLIP, and reducing superoxide sensitizes Bcl-2 overexpressing cancer cells to receptor-mediated apoptosis via downregulation of cFLIP. Moreover, inhibiting glycolytic flux overcomes apoptosis resistance by superoxide-dependent downregulation of cFLIP. Superoxide-induced upregulation of cFLIP is a function of enhanced transcription, as evidenced by increases in cFLIP promoter activity and mRNA abundance. The positive effect of superoxide on cFLIP is mediated through its reaction with nitric oxide to generate peroxynitrite. Corroborating these findings in cell lines, subjecting primary cells derived from lymphoma patients to glucose deprivation ex vivo, as a means to decrease superoxide, not only reduced cFLIP expression but also significantly enhanced death receptor sensitivity. Based on this novel mechanistic insight into the redox regulation of cancer cell fate, modulation of intracellular superoxide could have potential therapeutic implications in cancers in which these two death inhibitory proteins present a therapeutic challenge.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Linfoma/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Superóxidos/metabolismo , Regulação para Cima , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Linfoma/genética , Óxido Nítrico/metabolismo , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
The experimental validation of the existence of cancer stem cells (CSC) has had a significant impact on our understanding of the cellular mechanisms and signaling networks involved in the process of carcinogenesis and its progression. These findings provide insights into the critical role that tumor microenvironment and metabolism play in the acquisition of the drug resistance phenotype as well as provide potential targets for therapeutic exploitation. Here we briefly review the literature on the involvement of key signaling pathways such as Wnt/ß-catenin, Notch, Hedgehog and STAT3 in the appearance of cancer cells with stem cells-like characteristics. In addition, we also highlight some of the recent therapeutic strategies used to target these pathways as well as approaches aiming to specifically target CSCs through their distinctive metabolic features.
Assuntos
Antineoplásicos/uso terapêutico , Gerenciamento Clínico , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/fisiologia , Fenótipo , Microambiente Tumoral/fisiologia , Animais , Antineoplásicos/farmacologia , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologiaRESUMO
Drug resistance invariably limits the response of oncogene-addicted cancer cells to targeted therapy. The upregulation of signal transducer and activator of transcription 3 (STAT3) has been implicated as a mechanism of drug resistance in a range of oncogene-addicted cancers. However, the development of inhibitors against STAT3 has been fraught with challenges such as poor delivery or lack of specificity. Clinical experience with small molecule STAT3 inhibitors has seen efficacy signals, but this success has been tempered by drug limiting toxicities from off-target adverse events. It has emerged in recent years that, contrary to the Warburg theory, certain tumor types undergo metabolic reprogramming towards oxidative phosphorylation (OXPHOS) to satisfy their energy production. In particular, certain drug-resistant oncogene-addicted tumors have been found to rely on OXPHOS as a mechanism of survival. Multiple cellular signaling pathways converge on STAT3, hence the localization of STAT3 to the mitochondria may provide the link between oncogene-induced signaling pathways and cancer cell metabolism. In this article, we review the role of STAT3 and OXPHOS as targets of novel therapeutic strategies aimed at restoring drug sensitivity in treatment-resistant oncogene-addicted tumor types. Apart from drugs which have been re-purposed as OXPHOS inhibitors for-anti-cancer therapy (e.g., metformin and phenformin), several novel compounds in the drug-development pipeline have demonstrated promising pre-clinical and clinical activity. However, the clinical development of OXPHOS inhibitors remains in its infancy. The further identification of compounds with acceptable toxicity profiles, alongside the discovery of robust companion biomarkers of OXPHOS inhibition, would represent tangible early steps in transforming the therapeutic landscape of cancer cell metabolism.
Assuntos
Neoplasias/genética , Oncogenes , Fosforilação Oxidativa , Fator de Transcrição STAT3/metabolismo , Animais , Resistencia a Medicamentos Antineoplásicos , Humanos , Mitocôndrias/metabolismoRESUMO
We have recently reported that accumulation of misfolded nuclear hormone receptor corepressor (N-CoR) as insoluble protein aggregates in acute promyelocytic leukemia (APL) cells induces endoplasmic reticulum (ER) stress and activates unfolded protein response (UPR). Although accumulation of misfolded proteins is known to trigger UPR-induced cytotoxic cell death in several neurodegenerative disorders, APL cells are notably resistant to UPR-induced apoptosis. The molecular basis for the paradoxical response of APL cells to UPR is not known. Here, we report that a glycoprotease, selectively expressed in APL cells, regulates the response of APL cells to UPR-induced apoptosis through processing of misfolded N-CoR protein. Results show that misfolded N-CoR is cleaved selectively in APL cells, and cellular extracts of APL cells and human primary APL cells contain activity that cleaves N-CoR protein. Purification and spectrometric analysis of N-CoR cleaving activity from an APL cell line reveals that it is a glycoprotein endopeptidase known as OSGEP. Furthermore, the cleavage of N-CoR in APL cells could be blocked by the broad-spectrum protease inhibitor AEBSF and by RNA interference-mediated down-regulation of OSGEP expression. AEBSF selectively inhibits growth and promotes apoptosis of APL cells possibly through a mechanism involving AEBSF-induced accumulation of insoluble N-CoR protein and by triggering ER stress. Taken together, these findings suggest that selective induction of protease activity in APL cells may represent a novel cytoprotective component of UPR, which could be exploited by tumor cells to survive the toxic insult of misfolded protein(s).
Assuntos
Apoptose/fisiologia , Leucemia Promielocítica Aguda/metabolismo , Metaloendopeptidases/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/fisiologia , Ácido Edético/farmacologia , Retículo Endoplasmático/metabolismo , Células HL-60 , Humanos , Células K562 , Leucemia Promielocítica Aguda/enzimologia , Leucemia Promielocítica Aguda/patologia , Proteínas Nucleares/antagonistas & inibidores , Correpressor 1 de Receptor Nuclear , Inibidores de Proteases/farmacologia , Dobramento de Proteína , Proteínas Repressoras/antagonistas & inibidores , Sulfonas/farmacologia , Células U937RESUMO
Absence of the proapoptotic protein Bax renders tumor cells resistant to drug-induced apoptosis. We have shown that hydrogen peroxide (H(2)O(2))-mediated cytosolic acidification is an effector mechanism during drug-induced apoptosis of tumor cells. Here, we report that Bax is critical in determining the sensitivity of tumor cells to H(2)O(2)-induced apoptosis. More importantly, exposure of colorectal carcinoma (HCT116) and leukemia cells (HL60 and CEM) to H(2)O(2) or its intracellular production during drug-induced apoptosis is a signal for mitochondrial translocation of Bax. Furthermore, we provide evidence that drug-induced H(2)O(2)-mediated Bax translocation in tumor cells is caspase independent but involves cytosolic acidification. Inhibiting cytosolic acidification prevents Bax translocation, and contrarily enforced acidification of the intracellular milieu results in mitochondrial recruitment of Bax, even in the absence of a trigger. These findings provide a novel mechanism for mitochondrial translocation of Bax and directly implicate H(2)O(2)-mediated cytosolic acidification in the recruitment of the mitochondrial pathway during drug-induced apoptosis of tumor cells.
Assuntos
Apoptose/efeitos dos fármacos , Citosol/metabolismo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células HL-60 , Humanos , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2RESUMO
[This corrects the article DOI: 10.1371/journal.pone.0009996.].
RESUMO
Resistance to chemotherapy remains a challenge in the clinical management of diffuse B cell lymphomas despite aggressive chemotherapy such as CHOP and monoclonal CD20. Here we provide evidence that the apoptosome adaptor protein, Apaf-1, is mislocalized in primary cells derived from patients with diffuse large B cell lymphomas (DLBCL). Whereas, the total expression of Apaf-1 did not change, its sub-cellular localization was significantly different in DLBCL, compared to T cell lymphomas as well as cells derived from reactive lymphadenopathy biopsies. As expected, Apaf-1 was detected in the cytosolic fractions of non-B cell lymphomas and non-cancerous tissues; however, in B cell derived lymphomas the protein was detected in membrane raft sub-domains rather than the cytosol. Disruption of lipid raft structures resulted in the redistribution of Apaf-1 to the cytosol and restored apoptosis sensitivity of DLBCL. Furthermore, we identified novel small molecule compounds that target DLBCL by promoting Apaf-1 release form lipid rafts via mechanisms that involve an increase in intracellular reactive oxygen species production. Taken together, our results implicate Apaf-1 mislocalization as a potential diagnostic and prognostic marker for DLBCL, and provide a novel therapeutic strategy for circumventing the drug refractory nature of this sub-class of B cell lymphoma.
Assuntos
Apoptose , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Microdomínios da Membrana/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Células Jurkat , Peroxidação de Lipídeos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/patologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
AIM: Epithelial-mesenchymal transition (EMT) is characterized by the acquisition of invasive fibroblast-like morphology by epithelial cells that are highly polarized. EMT is recognized as a crucial mechanism in cancer progression and metastasis. In this study, we sought to assess the involvement of manganese superoxide dismutase (MnSOD) during the switch between epithelial-like and mesenchymal-like phenotypes in breast carcinoma. RESULTS: Analysis of breast carcinomas from The Cancer Genome Atlas database revealed strong positive correlation between tumors' EMT score and the expression of MnSOD. This positive correlation between MnSOD and EMT score was significant and consistent across all breast cancer subtypes. Similarly, a positive correlation of EMT score and MnSOD expression was observed in established cell lines derived from breast cancers exhibiting phenotypes ranging from the most epithelial to the most mesenchymal. Interestingly, using phenotypically distinct breast cancer cell lines, we provide evidence that constitutively high or induced expression of MnSOD promotes the EMT-like phenotype by way of a redox milieu predominantly driven by hydrogen peroxide (H2O2). Conversely, gene knockdown of MnSOD results in the reversal of EMT to a mesenchymal-epithelial transition (MET)-like program, which appears to be a function of superoxide (O2(-â¢))-directed signaling. INNOVATION AND CONCLUSION: These data underscore the involvement of MnSOD in regulating the switch between the EMT and MET-associated phenotype by influencing cellular redox environment via its effect on the intracellular ratio between O2(-â¢) and H2O2. Strategies to manipulate MnSOD expression and/or the cellular redox milieu vis-a-vis O2(-â¢):H2O2 could have potential therapeutic implications. Antioxid. Redox Signal. 25, 283-299.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Fenótipo , Superóxido Dismutase/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Superóxido Dismutase/metabolismoRESUMO
We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O2â¢-). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O2â¢- blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O2â¢- production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.