RESUMO
Type III IFNs are important mediators of antiviral immunity. IFN-λ4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-λ4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-λ4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our findings in HepG2 cell line models. Endogenous IFN-λ4 protein was detectable only in Sendai virus-infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-λ4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1 Transient treatment of PHHs with IFN-λ4, but not IFN-λ3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-λ3. Our results suggest unique functional properties of IFN-λ4 that can be important in viral clearance and other clinical conditions.
Assuntos
Alelos , Hepatócitos/imunologia , Interferons/genética , Interleucinas/genética , Infecções por Respirovirus/imunologia , Vírus Sendai/imunologia , Adolescente , Adulto , Idoso , Endopeptidases/genética , Feminino , Células Hep G2 , Hepacivirus/imunologia , Hepatite C/genética , Hepatite C/imunologia , Hepatócitos/virologia , Humanos , Imunidade , Interferons/metabolismo , Interleucinas/metabolismo , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteína 1 Supressora da Sinalização de Citocina/genética , Ubiquitina Tiolesterase , Regulação para Cima , Carga Viral , Adulto JovemRESUMO
Pancreatic Cancer (PC) is a deadly disease in need of new therapeutic options. We recently developed a novel tricarbonylmethane agent (CMC2.24) as a therapeutic agent for PC, and evaluated its efficacy in preclinical models of PC. CMC2.24 inhibited the growth of various human PC cell lines in a concentration and time-dependent manner. Normal human pancreatic epithelial cells were resistant to CMC2.24, indicating selectivity. CMC2.24 reduced the growth of subcutaneous and orthotopic PC xenografts in mice by up to 65% (P < 0.02), and the growth of a human patient-derived tumor xenograft by 47.5% (P < 0.03 vs vehicle control). Mechanistically, CMC2.24 inhibited the Ras-RAF-MEK-ERK pathway. Based on Ras Pull-Down Assays, CMC2.24 inhibited Ras-GTP, the active form of Ras, in MIA PaCa-2 cells and in pancreatic acinar explants isolated from Kras mutant mice, by 90.3% and 89.1%, respectively (P < 0.01, for both). The inhibition of active Ras led to an inhibition of c-RAF, MEK, and ERK phosphorylation by 93%, 91%, and 87%, respectively (P < 0.02, for all) in PC xenografts. Furthermore, c-RAF overexpression partially rescued MIA PaCa-2 cells from the cell growth inhibition by CMC2.24. In addition, downstream of ERK, CMC2.24 inhibited STAT3 phosphorylation levels at the serine 727 residue, enhanced the levels of superoxide anion in mitochondria, and induced intrinsic apoptosis as shown by the release of cytochrome c from the mitochondria to the cytosol and the further cleavage of caspase 9 in PC cells. In conclusion, CMC2.24, a potential Ras inhibitor, is an efficacious agent for PC treatment in preclinical models, deserving further evaluation.
Assuntos
Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Curcumina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Curcumina/farmacologia , Curcumina/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
IFNL3/IFNL4 polymorphisms are inversely associated with the risk of chronic hepatitis C virus (HCV) infection and cirrhosis, two major risk factors for developing hepatocellular carcinoma (HCC). To further explore these inverse associations and their molecular underpinnings, we analyzed IFNL3/IFNL4 polymorphisms represented by the IFNL4 genotype (presence of rs368234815-dG or rs12979860-T alleles) in HCV patients: 2969 from Japan and 2931 from Taiwan. IFNL4 genotype was associated with an increased risk of HCV-related HCC (OR=1.28, 95%CI=1.07-1.52, P=0.0058) in the general population of Japanese patients, but not in Taiwanese patients who achieved treatment-induced viral clearance. IFNL4 genotype was also associated with a decreased risk of cirrhosis (OR=0.66, 95%CI=0.46-0.93, P=0.018, in Taiwanese patients). We then engineered HepG2 cells to inducibly express IFN-λ4 in the presence or absence of interferon lambda receptor 1 (IFNLR1). Induction of IFN-λ4 resulted in its intracellular accumulation, mainly in lysosomes and late endosomes, and increased ER stress, leading to apoptosis and reduced proliferation. We identified the very-low-density lipoprotein receptor (VLDLR), which facilitates HCV entry into hepatocytes, as a transcript induced by IFN-λ4 but not IFN-λ3. Our results suggest that the molecular mechanisms underlying the anti-cirrhotic but pro-HCV associations observed for IFNL3/IFNL4 polymorphisms are, at least in part, contributed by intracellular accumulation of IFN-λ4 causing ER stress in hepatic cells.
Assuntos
Carcinoma Hepatocelular/metabolismo , Estresse do Retículo Endoplasmático , Hepatite C/metabolismo , Interleucinas/metabolismo , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Adulto , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Estudos de Casos e Controles , Proliferação de Células , Bases de Dados Factuais , Feminino , Predisposição Genética para Doença , Células Hep G2 , Hepatite C/genética , Hepatite C/virologia , Humanos , Interferons/genética , Interleucinas/genética , Japão , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/prevenção & controle , Cirrose Hepática/virologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo Genético , Fatores de Proteção , Medição de Risco , Fatores de Risco , TaiwanRESUMO
APOBEC3A (A3A) and APOBEC3B (A3B) enzymes drive APOBEC-mediated mutagenesis. Identification of factors affecting the activity of these enzymes could help modulate mutagenesis and associated clinical outcomes. Here, we show that canonical and alternatively spliced A3A and A3B isoforms produce corresponding mutagenic and non-mutagenic enzymes. Increased expression of the mutagenic A3B isoform predicted shorter progression-free survival in bladder cancer. We demonstrate that the production of mutagenic vs. non-mutagenic A3B protein isoforms was considerably affected by inclusion/skipping of exon 5 in A3B. Furthermore, exon 5 skipping, resulting in lower levels of mutagenic A3B enzyme, could be increased in vitro. Specifically, we showed the effects of treatment with an SF3B1 inhibitor affecting spliceosome interaction with a branch point site in intron 4, or with splice-switching oligonucleotides targeting exon 5 of A3B. Our results underscore the clinical role of A3B and implicate alternative splicing of A3B as a mechanism that could be targeted to restrict APOBEC-mediated mutagenesis.
Assuntos
Processamento Alternativo , Biomarcadores Tumorais/genética , Citidina Desaminase/genética , Antígenos de Histocompatibilidade Menor/genética , Mutagênese , Proteínas/genética , Neoplasias da Bexiga Urinária/genética , Biomarcadores Tumorais/metabolismo , Citidina Desaminase/metabolismo , Compostos de Epóxi/farmacologia , Éxons , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Isoenzimas , Macrolídeos/farmacologia , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Intervalo Livre de Progressão , Proteínas/metabolismo , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/metabolismo , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/terapiaRESUMO
Genomic regions have been associated with COVID-19 susceptibility and outcomes, including the chr12q24.13 locus encoding antiviral proteins OAS1-3. Here, we report genetic, functional, and clinical insights into genetic associations within this locus. In Europeans, the risk of hospitalized vs. non-hospitalized COVID-19 was associated with a single 19Kb-haplotype comprised of 76 OAS1 variants included in a 95% credible set within a large genomic fragment introgressed from Neandertals. The risk haplotype was also associated with impaired spontaneous but not treatment-induced SARS-CoV-2 clearance in a clinical trial with pegIFN-λ1. We demonstrate that two exonic variants, rs10774671 and rs1131454, affect splicing and nonsense-mediated decay of OAS1 . We suggest that genetically-regulated loss of OAS1 expression contributes to impaired spontaneous clearance of SARS-CoV-2 and elevated risk of hospitalization for COVID-19. Our results provide the rationale for further clinical studies using interferons to compensate for impaired spontaneous SARS-CoV-2 clearance, particularly in carriers of the OAS1 risk haplotypes.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, transcriptionally independent truncated isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In The Cancer Genome Atlas, the expression of dACE2 was enriched in squamous tumors of the respiratory, gastrointestinal and urogenital tracts. In vitro, dACE2, which lacks 356 amino-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, an inflammatory tumor microenvironment or viral co-infections is unlikely to increase the cellular entry of SARS-CoV-2 and promote infection.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Interferons/metabolismo , Vírus de RNA/fisiologia , Receptores de Coronavírus/metabolismo , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/genética , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/genética , Linhagem Celular , Indução Enzimática , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Receptores de Coronavírus/genética , Mucosa Respiratória/metabolismo , Mucosa Respiratória/virologia , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.