RESUMO
Ferroptosis is a form of regulated cell death that is induced by inhibiting glutathione peroxidase 4 (GPX4), which eliminates lipid peroxidation. Ferroptosis induction is influenced by the cell environment. However, the cellular states altering ferroptosis susceptibility remain largely unknown. We found that melanoma cell lines became resistant to ferroptosis as cell density increased. Comparative transcriptome and metabolome analyses revealed that cell density-dependent ferroptosis resistance was coupled with a shift toward a lipogenic phenotype accompanied by strong induction of stearoyl-CoA desaturase (SCD). Database analysis of gene dependency across hundreds of cancer cell lines uncovered a negative correlation between GPX4 and SCD dependency. Importantly, SCD inhibition, either pharmacologically or through genetic knockout, sensitized melanoma cells to GPX4 inhibition, thereby attenuating ferroptosis resistance in cells at high density. Our findings indicate that transition to an SCD-inducing, lipogenic cell state produces density-dependent resistance to ferroptosis, which may provide a therapeutic strategy against melanoma.
Assuntos
Ferroptose , Melanoma , Estearoil-CoA Dessaturase , Humanos , Contagem de Células , Morte Celular/genética , Melanoma/genética , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Estearoil-CoA Dessaturase/genéticaRESUMO
Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.
RESUMO
The unfolded protein response (UPR) is an adaptive stress response pathway that is essential for cancer cell survival under endoplasmic reticulum stress such as during glucose starvation. In this study, we identified spautin-1, an autophagy inhibitor that suppresses ubiquitin-specific peptidase 10 (USP10) and USP13, as a novel UPR inhibitor under glucose starvation conditions. Spautin-1 prevented the induction of UPR-associated proteins, including glucose-regulated protein 78, activating transcription factor 4, and a splicing variant of x-box-binding protein-1, and showed preferential cytotoxicity in glucose-starved cancer cells. However, USP10 and USP13 silencing and treatment with other autophagy inhibitors failed to result in UPR inhibition and preferential cytotoxicity during glucose starvation. Using transcriptome and chemosensitivity-based COMPARE analyses, we identified a similarity between spautin-1 and mitochondrial complex I inhibitors and found that spautin-1 suppressed the activity of complex I extracted from isolated mitochondria. Our results indicated that spautin-1 may represent an attractive mitochondria-targeted seed compound that inhibits the UPR and cancer cell survival during glucose starvation.
Assuntos
Glucose , Resposta a Proteínas não Dobradas , Benzilaminas , Sobrevivência Celular , Estresse do Retículo Endoplasmático , Glucose/metabolismo , Quinazolinas/farmacologiaRESUMO
ATF4 is a crucial transcription factor in the integrated stress response, a major adaptive signaling pathway activated by tumor microenvironment and therapeutic stresses. BRAF inhibitors, such as vemurafenib, induce ATF4 in BRAF-mutated melanoma cells, but the mechanisms of ATF4 induction are not fully elucidated. Here, we show that ATF4 expression can be upregulated by eukaryotic initiation factor 4B (eIF4B) in BRAF-mutated A375 cells. Indeed, eIF4B knockout (KO) prevented ATF4 induction and activation of the uORF-mediated ATF4 translation mechanism during vemurafenib treatment, which were effectively recovered by the rescue of eIF4B. Transcriptome analysis revealed that eIF4B KO selectively influenced ATF4-target gene expression among the overall gene expression changed by vemurafenib. Interestingly, eIF4B supported cellular proliferation under asparagine-limited conditions, possibly through the eIF4B-ATF4 pathway. Our findings indicate that eIF4B can regulate ATF4 expression, thereby contributing to cellular stress adaptation, which could be targeted as a therapeutic approach against malignancies, including melanoma.
Assuntos
Fator 4 Ativador da Transcrição/genética , Asparagina/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Melanoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Fator 4 Ativador da Transcrição/metabolismo , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Fatores de Iniciação em Eucariotos/deficiência , Humanos , Melanoma/tratamento farmacológico , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Células Tumorais Cultivadas , Vemurafenib/farmacologiaRESUMO
As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.
Assuntos
Estresse do Retículo Endoplasmático , Receptor ErbB-3/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , eIF-2 Quinase/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Linhagem Celular Tumoral , Desoxiglucose/farmacologia , Regulação para Baixo/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glicosilação , Meia-Vida , Proteínas de Choque Térmico/metabolismo , Humanos , Indóis/farmacologia , Mucoproteínas/metabolismo , Proteínas Oncogênicas/metabolismo , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Resposta a Proteínas não Dobradas , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genéticaRESUMO
It has been difficult to elucidate the structure of gene regulatory networks under anticancer drug treatment. Here, we developed an algorithm to highlight the hub genes that play a major role in creating the upstream and downstream relationships within a given set of differentially expressed genes. The directionality of the relationships between genes was defined using information from comprehensive collections of transcriptome profiles after gene knockdown and overexpression. As expected, among the drug-perturbed genes, our algorithm tended to derive plausible hub genes, such as transcription factors. Our validation experiments successfully showed the anticipated activity of certain hub gene in establishing the gene regulatory network that was associated with cell growth inhibition. Notably, giving such top priority to the hub gene was not achieved by ranking fold change in expression and by the conventional gene set enrichment analysis of drug-induced transcriptome data. Thus, our data-driven approach can facilitate to understand drug-induced gene regulatory networks for finding potential functional genes.
Assuntos
Antifúngicos/uso terapêutico , Ascomicetos/isolamento & purificação , Linfoma de Células T/complicações , Infecções Oportunistas/complicações , Feoifomicose/complicações , Idoso de 80 Anos ou mais , Ascomicetos/genética , DNA Fúngico/isolamento & purificação , Humanos , Japão , Masculino , Infecções Oportunistas/diagnóstico , Infecções Oportunistas/microbiologia , Infecções Oportunistas/terapia , Feoifomicose/diagnóstico , Feoifomicose/microbiologia , Feoifomicose/terapia , Tela Subcutânea/microbiologia , Tela Subcutânea/patologia , Tela Subcutânea/cirurgia , Resultado do TratamentoRESUMO
Insulin resistance is due to the reduced cellular response to insulin in peripheral tissues. The interaction of insulin with its receptor is the first step in insulin action and thus the identified target of insulin resistance. It has been well established that defects or mutations in the insulin receptor (IR) cause insulin resistance. Therefore, an IR activator might be a novel therapeutic approach for insulin resistance. Our previous report showed that mild electrical stress (MES) enhanced the insulin-induced signaling pathway. However, the molecular mechanism of the effect of MES remains unclear. We assessed the effect of MES, which is characterized by low-intensity direct current, on insulin signaling in vitro and in vivo. Here, we showed that MES activated the insulin signaling in an insulin-independent manner and improved insulin resistance in peripheral tissues of high fat-fed mice. Moreover, we found that MES increased the localization of IR in lipid rafts and enhanced the level of phosphorylated Akt in insulin-resistant hepatic cells. Ablation of lipid rafts disrupted the effect of MES on Akt activation. Our findings indicate that MES has potential as an activator of IR in an insulin-independent manner, and might be beneficial for insulin resistance in type 2 diabetes.
Assuntos
Estimulação Elétrica , Microdomínios da Membrana/metabolismo , Receptor de Insulina/metabolismo , Estresse Fisiológico , Animais , Dieta Hiperlipídica , Células Hep G2 , Humanos , Hiperglicemia/complicações , Insulina/farmacologia , Resistência à Insulina/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Primary effusion lymphoma (PEL) is a subtype of B-cell lymphoma caused by human herpes virus 8/Kaposi sarcoma-associated herpes virus (HHV-8/KSHV), which is mostly found in patients with AIDS and has poor prognosis. Nuclear factor (NF)-κB pathway is constitutively activated in HHV-8-infected PEL cells and plays a crucial role in tumorigenesis. Recently, it has been shown that diethyldithiocarbamate (DDTC), an active metabolite of disulfiram, has apoptotic activity in cancer cells. Here, we investigated the effect of DDTC on PEL using a PEL mouse model generated by intraperitoneal injection of BC-3 cells, a PEL cell line. DDTC ameliorated the symptoms of PEL in these mice, such as development of ascites, splenomegaly and increase of body weight, in comparison with PBS-treated controls. Moreover, we determined in vitro that DDTC suppressed the constitutively activated NF-κB pathway in BC-3 cells. Methylthiotetrazole assay revealed that the cell proliferation of various PEL cell lines was significantly suppressed by the treatment of DDTC. DDTC also induced the expression of cleaved caspase-3, an apoptosis marker, whereas the addition of Q-VD-OPh, a pan-caspase inhibitor, inhibited cell apoptosis induced by DDTC treatment. Together, our results indicated that DDTC induces apoptosis via inhibition of the NF-κB signaling pathway in HHV-8-infected PEL cells. This study suggests the potential use of DDTC as a therapeutic approach for PEL.
Assuntos
Apoptose/efeitos dos fármacos , Ditiocarb/farmacologia , Infecções por Herpesviridae/tratamento farmacológico , Herpesvirus Humano 8/fisiologia , Linfoma de Efusão Primária/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Animais , Caspase 3/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Humanos , Linfoma de Efusão Primária/metabolismo , Linfoma de Efusão Primária/patologia , Linfoma de Efusão Primária/virologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A-NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.