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The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacteroides , Carbapenémicos/farmacología , Carbapenémicos/uso terapéutico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/etiología , Meropenem , Ratones , Mucinas/metabolismo , Moco/metabolismo , Polisacáridos/metabolismo , XilosaRESUMEN
Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.
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Sinapsis Inmunológicas , Receptores de Antígenos de Linfocitos T , Linfocitos T , Humanos , Células Presentadoras de Antígenos/inmunología , Línea Celular Tumoral , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 16/genética , Sinapsis Inmunológicas/inmunología , Factores de Transcripción NFATC/metabolismo , Factores de Transcripción NFATC/inmunología , Proteínas E7 de Papillomavirus/inmunología , Proteínas E7 de Papillomavirus/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunologíaRESUMEN
Acute lower gastrointestinal GVHD (aLGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. Although the intestinal microbiota is associated with the incidence of aLGI-GVHD, how the intestinal microbiota impacts treatment responses in aLGI-GVHD has not been thoroughly studied. In a cohort of patients with aLGI-GVHD (n = 37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and a disrupted fecal microbiome characterized by reduced abundances of Bacteroides ovatus. In a murine GVHD model aggravated by carbapenem antibiotics, introducing B. ovatus reduced GVHD severity and improved survival. These beneficial effects of Bacteroides ovatus were linked to its ability to metabolize dietary polysaccharides into monosaccharides, which suppressed the mucus-degrading capabilities of colonic mucus degraders such as Bacteroides thetaiotaomicron and Akkermansia muciniphila, thus reducing GVHD-related mortality. Collectively, these findings reveal the importance of microbiota in aLGI-GVHD and therapeutic potential of B. ovatus.
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The diversity of bacteria associated with the fermentation of inyu, also known as black soy sauce, was studied through the nested PCR-denaturing gradient gel electrophoresis (DGGE) of samples collected from the fermentation stages of the inyu production process. The DGGE profiles targeted the bacterial 16S rDNA and revealed the presence of Citrobacter farmeri, Enterobacter cloacae, Enterobacter hormaechei, Enterococcus faecium, Klebsiella pneumoniae, Pantoea agglomerans, Salmonella enterica, Serratia marcescens, Staphylococcus sciuri and Weissella confusa. The bacterial compositions of 4 fermented samples were further elucidated using the plate count method. The bacteria isolated from the koji-making stage exhibited the highest diversity; Brachybacterium rhamnosum, E. hormaechei, K. pneumoniae, Kurthia gibsonii, Pantoea dispersa, Staphylococcus gallinarum, Staphylococcus kloosii and S. sciuri were identified. Koji collected during the preincubation stage presented the largest cell counts, and E. hormaechei, K. pneumoniae, E. cloacae and Enterobacter pulveris were identified. In brine samples aged for 7 and 31 days, the majority of the bacteria isolated belonged to 4 Bacillus species, but 4 Staphylococcus species and Delftia tsuruhatensis were also detected. This study demonstrates the benefits of using a combined approach to obtain a more complete picture of microbial populations and provides useful information for the control or development of bacterial flora during inyu fermentation.
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Bacterias/aislamiento & purificación , Bacterias/metabolismo , Biodiversidad , Recuento de Colonia Microbiana/métodos , Electroforesis en Gel de Gradiente Desnaturalizante/métodos , Glycine max/microbiología , Reacción en Cadena de la Polimerasa/métodos , Alimentos de Soja/microbiología , Bacterias/clasificación , Bacterias/genética , Técnicas de Tipificación Bacteriana/métodos , Fermentación , Datos de Secuencia Molecular , Filogenia , Glycine max/metabolismo , TemperaturaRESUMEN
Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.
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Copper is an essential micronutrient for cell growth but is toxic in excess. Copper transporter (Ctr1) plays an important role in regulating adequate copper levels in mammalian cells. We have shown previously that expression of the human high-affinity copper transporter (hCtr1) was transcriptionally up-regulated under copper-depleted conditions and down-regulated under replete conditions; moreover, elevated hCtr1 levels suppress hCtr1 expression. Specificity protein 1 (Sp1) regulates expression of hCtr1 under copper-stressed conditions. In this study, we made the following important observations: 1) Sp1 expression is down-regulated under copper-replete conditions but up-regulated under copper-depleted conditions. These up- and down-regulations of Sp1 in turn regulate hCtr1 expression to control copper homeostasis. 2) Copper-regulated Sp1 expression involved Sp1 binding to its own promoter as demonstrated by the chromatin immunoprecipitation assay; therefore, Sp1 is also transcriptionally self-regulated via hCtr1/copper intermediation. 3) Both zinc finger and glutamine-rich transactivation domains of Sp1 are involved in the Sp1-mediated hCtr1 and Sp1 regulation by copper stresses. 4) Although Sp3 expression is also regulated by copper availability, Sp3 does not regulate hCtr1 homeostasis. Collectively, our results demonstrated that mammalian cells use Sp1 oscillation in response to copper availability to regulate copper homeostasis through hCtr1 expression in a tripartite inter-regulatory relationship. These findings have important implications in mammalian copper physiology regulation.
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Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Homeostasis/fisiología , Factor de Transcripción Sp1/fisiología , Western Blotting , Proteínas de Transporte de Catión/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Transportador de Cobre 1 , Regulación hacia Abajo , Humanos , Inmunohistoquímica , Transcripción Genética , Regulación hacia Arriba , Dedos de ZincRESUMEN
Boosting anticancer immunity by blocking immune checkpoints such as the programmed death-1 (PD-1) or its ligand (PD-L1) is a breakthrough anticancer therapy. However, many cancer patients do not respond well to immune checkpoint blockades (ICBs) alone. Here we show that low-dose pharmacological immunoactivators (e.g., SN38, topotecan, sorafenib, etc.) notably downregulate PD-L1 and upregulate FOXO3 expression in various human and murine cancer cell lines. In a mouse tumor model, low-dose SN38 treatment markedly suppresses tumor growth, reduces PD-L1 expression, and enhances FOXO3 expression in primary tumor specimens. SN38 therapy engages the tumor-infiltrating mouse NK1.1/CD49b/NKG2D-positive natural killer (NK) cells to attack tumor cells by inducing mouse IFN-γ and granzyme-B secretion in the tumor microenvironment (TME) in vivo. SN38 treatment also promotes tumor cell apoptosis in the TME. SN38 treatment significantly decreases STAT3-pY705 and IL-6 protein levels; FOXO3 is essential for SN38-mediated PD-L1 downregulation. Collectively, these findings may contribute to future translational or clinical investigations tackling difficult-to-treat cancers with immune-activating medicines or combined with ICB immunotherapy.
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Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.
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Microbioma Gastrointestinal , Trasplante de Células Madre Hematopoyéticas , Neoplasias , Neutropenia , Ratones , Animales , Propionatos , Verrucomicrobia , Moco/metabolismo , Mucinas/metabolismo , Dieta , Neutropenia/metabolismo , Neoplasias/metabolismoRESUMEN
BACKGROUND: Stimulating antitumor immunity by blocking programmed death-1 (PD-1) or its ligand (programmed death-ligand 1 (PD-L1) is a promising antitumor therapy. However, numerous patients respond poorly to PD-1/PD-L1 blockade. Unresponsiveness to immune-checkpoint blockade (ICB) can cast significant challenges to the therapeutic options for patients with hard-to-treat tumors. There is an unmet clinical need to establish new therapeutic approaches for mitigating ICB unresponsiveness in patients. In this study, we investigated the efficacy and role of low-dose antineoplastic agent SN-38 or metformin in sensitizing unresponsive tumors to respond to ICB therapy. METHODS: We assessed the significant pathological relationships between PD-L1 and FOXO3 expression and between PD-L1 and c-Myc or STAT3 expression in patients with various tumors. We determined the efficacy of low-dose SN-38 or metformin in sensitizing unresponsive tumors to respond to anti-PD-1 therapy in a syngeneic tumor system. We deciphered novel therapeutic mechanisms underlying the SN-38 and anti-PD-1 therapy-mediated engagement of natural killer (NK) or CD8+ T cells to infiltrate tumors and boost antitumor immunity. RESULTS: We showed that PD-L1 protein level was inversely associated with FOXO3 protein level in patients with ovarian, breast, and hepatocellular tumors. Low-dose SN-38 or metformin abrogated PD-L1 protein expression, promoted FOXO3 protein level, and significantly increased the animal survival rate in syngeneic mouse tumor models. SN-38 or metformin sensitized unresponsive tumors responding to anti-PD-1 therapy by engaging NK or CD8+ T cells to infiltrate the tumor microenvironment (TME) and secret interferon-γ and granzyme B to kill tumors. SN-38 suppressed the levels of c-Myc and STAT3 proteins, which controlled PD-L1 expression. FOXO3 was essential for SN38-mediated PD-L1 suppression. The expression of PD-L1 was compellingly linked to that of c-Myc or STAT3 in patients with the indicated tumors. CONCLUSION: We show that SN-38 or metformin can boost antitumor immunity in the TME by inhibiting c-Myc and STAT3 through FOXO3 activation. These results may provide novel insight into ameliorating patient response to overarching immunotherapy for tumors.
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Neoplasias de la Mama/tratamiento farmacológico , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/tratamiento farmacológico , Proteína Forkhead Box O3/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Células Asesinas Naturales/inmunología , Neoplasias Ováricas/tratamiento farmacológico , Animales , Apoptosis , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proliferación Celular , Femenino , Proteína Forkhead Box O3/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoterapia , Irinotecán/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Inhibidores de Topoisomerasa I/farmacología , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
Many human malignancies require extracellular arginine (Arg) for survival because the key enzyme for de novo Arg biosynthesis, argininosuccinate synthetase 1 (ASS1), is silenced. Recombinant arginine deiminase (ADI-PEG20), which digests extracellular Arg, has been in clinical trials for treating ASS1-negative tumors. Reactivation of ASS1 is responsible for the treatment failure. We previously demonstrated that ASS1 reactivation is transcriptionally regulated by c-Myc via the upstream Gas6-Axl tyrosine kinase (RTK) signal. Here, we report that another RTK EphA2 is coactivated via PI3K-ERK/RSK1 pathway in a ligand-independent mechanism. EphA2 is also regulated by c-Myc. Moreover, we found that knockdown Axl upregulates EphA2 expression, demonstrating cross-talk between these RTKs. ADIR cell lines exhibits enhanced sensitivities to nutrient deprivation such as charcoal-stripped FBS and multiple RTK inhibitor foretinib but resistance to EGFR inhibitors. Knockdown EphA2, and to lesser extent, Axl, overcomes EGFRi resistance. c-Myc inhibitor JQ1 can also sensitize ADIR cells to ADI-PEG20. This study elucidates molecular interactions of multiple RTKs in Arg-stress response and offers approaches for developing strategies of overcoming ADI-PEG20 resistance.
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INTRODUCTION: Estrogen receptors (ERs) play key roles in breast cancer development and influence treatment outcome in breast cancer patients. Identification of molecules that regulate ER function may facilitate development of breast cancer treatment strategies. The forkhead box class O (FOXO) transcription factor FOXO3a has been suggested to function as a tumor suppressor in breast cancer. Using protein-protein interaction screening, we found that FOXO3a interacted with ER-alpha and ER-beta proteins in the human breast carcinoma cell line MCF-7, suggesting that there exists a crosstalk between the FOXO3a and ER signaling pathways in estrogen-dependent breast cancer cells. METHODS: The interaction between FOXO3a and ER was investigated by using co-immunoprecipitation and immunoblotting assays. Inhibition of ER-alpha and ER-beta transactivation activity by FOXO was determined by luciferase reporter assays. Cell proliferation in culture was evaluated by counting cell numbers. Tumorigenesis was assessed in athymic mice that were injected with MCF-7 cell lines over-expressing FOXO3a. Protein expression levels of cyclin-dependent kinase inhibitors, cyclins, ERs, FOXM1, and the proteins encoded by ER-regulated genes in MCF-7 cell lines and breast tumors were examined by immunoblotting analysis and immunohistochemical staining. RESULTS: We found that FOXO3a interacted with ER-alpha and ER-beta proteins and inhibited 17beta-estradiol (E2)-dependent, ER-regulated transcriptional activities. Consistent with these observations, expression of FOXO3a in the ER-positive MCF-7 cells decreased the expression of several ER-regulated genes, some of which play important roles in cell proliferation. Moreover, we found that FOXO3a upregulated the expression of the cyclin-dependent kinase inhibitors p21Cip1, p27Kip1, and p57Kip2. These findings suggest that FOXO3a induces cell growth arrest to effect tumor suppression. FOXO3a repressed the growth and survival of MCF-7 cells in cell culture. In an orthotopic breast cancer xenograft model in athymic mice, over-expression of FOXO3a in MCF-7 cells suppressed their E2-induced tumorigenesis, whereas knockdown of FOXO3a in MCF-7 resulted in the E2-independent growth. CONCLUSION: Functional interaction between FOXO3a and ER plays a critical role in suppressing estrogen-dependent breast cancer cell growth and tumorigenesis in vivo. This suggests that agents that activate FOXO3a may be novel therapeutic agents that can inhibit and prevent tumor proliferation and development in breast cancer.
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Neoplasias de la Mama/metabolismo , Factores de Transcripción Forkhead/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Estrógenos/metabolismo , Femenino , Proteína Forkhead Box O3 , Humanos , Ratones , Neoplasias Hormono-Dependientes/metabolismo , Transducción de SeñalRESUMEN
Argininosuccinate synthetase 1 (ASS1) is the key enzyme that controls biosynthesis of arginine (Arg). ASS1 is silenced in many human malignancies therefore, these tumors require extracellular Arg for growth. The Arg-degrading recombinant protein, pegylated arginine deiminase (ADI-PEG20), has been in clinical trials for targeting Arg auxotrophic tumors by Arg starvation therapy. Resistance to Arg starvation is often developed through reactivation of ASS1 expression. We previously demonstrated that ASS1 silencing is controlled by HIF-1α and Arg starvation-reactivated ASS1 is associated with HIF-1α downregulation. However, mechanisms underlying ASS1 repression and HIF-1α turnover are not known. Here, we demonstrate that interplay of p300-HDAC2-Sin3A in the chromatin remodeling system is involved in HIF-1α degradation at the ASS1 promoter. The histone acetyltransferase p300 is normally associated with the ASS1 promoter to maintain acetylated H3K14ac and H3K27ac for ASS1 silencing. Arg starvation induces p300 dissociation, allowing histone HDAC2 and cofactor Sin3A to deacetylate these histones at the ASS1 promoter, thereby facilitating HIF-1α-proteasomal complex, driven by PHD2, to degrade HIF-1α in situ. Arg starvation induces PHD2 and HDAC2 interaction which is sensitive to antioxidants. This is the first report describing epigenetic regulation of chromosomal HIF-1α turnover in gene activation that bears important implication in cancer therapy.
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Arginina/metabolismo , Argininosuccinato Sintasa/metabolismo , Ensamble y Desensamble de Cromatina , Proteína p300 Asociada a E1A/metabolismo , Histona Desacetilasa 2/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Represoras/metabolismo , Línea Celular , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Regiones Promotoras Genéticas , Proteolisis , Complejo Correpresor Histona Desacetilasa y Sin3RESUMEN
Argininosuccinate synthetase 1 (ASS1) is the rate-limiting enzyme that catalyzes the biosynthesis of arginine (Arg). Many malignant human tumors are auxotrophic for Arg because ASS1 is silenced. ASS1 has been established as a sensor of Arg auxotrophic response and a chemosensitivity marker for Arg starvation therapy. Here, we report that ASS1 is also a sensor for glutamine (Gln)-deprivation response, and that upregulation of ASS1 expression is associated with resistance to Gln-starvation treatments. Knockdown of ASS1 expression resulted in increased sensitivity to both Arg- and Gln-starvation, whereas increased ASS1 expression by ectopic transfection is associated with resistance to both Arg- and Gln-starvation. The addition of permeable fumarate, a metabolite that bridges the tricarboxylic acid and urea cycles, resulted in downregulation of ASS1 expression and increased sensitivity to both Arg- and Gln-deprivation treatments. Mechanistically, the Gln-deprivation response, like the arginine-auxotrophic response, downregulates HIF-1α resulting in de-silencing of ASS1. Our results demonstrate that ASS1 is a common biosensor for Arg and Gln deprivation response and a shared target for Arg- and Gln-starvation therapies which have been in several current clinical trials.
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Arginina/metabolismo , Argininosuccinato Sintasa/metabolismo , Glutamina/metabolismo , Línea Celular Tumoral , Humanos , TransfecciónRESUMEN
The adenoviral protein E1A associates with multiple anticancer activities, including stabilization of p53 tumor suppressor, and has been tested through gene therapy approaches in clinical trials. To identify potential E1A-binding proteins involved in E1A's anticancer activities, we screened a yeast two-hybrid library and identified Mdm4, an Mdm2-related p53-binding protein, as a novel E1A-binding protein. The NH(2)-terminal region of Mdm4 and the CR1 domain of E1A were required for the interaction between E1A and Mdm4. E1A preferentially bound to Mdm4 rather than Mdm2 and formed a complex with p53 in the presence of Mdm4, resulting in the stabilization of p53 in a p14(ARF)-independent manner. E1A failed to stabilize p53 in the absence of Mdm4, showing that Mdm4 was required for p53 stabilization by E1A. Moreover, E1A-mediated stabilization of p53 occurred in nucleus. Although it had no effect on the p53-Mdm2 interaction, E1A facilitated Mdm4 binding to p53 and inhibited Mdm2 binding to Mdm4, resulting in decreased nuclear exportation of p53. Thus, our findings highlighted a novel mechanism, whereby E1A stabilized the p53 tumor suppressor through Mdm4.
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Proteínas E1A de Adenovirus/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2 , Proteína p14ARF Supresora de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Many human tumors require extracellular arginine (Arg) for growth because the key enzyme for de novo biosynthesis of Arg, argininosuccinate synthetase 1 (ASS1), is silenced. These tumors are sensitive to Arg-starvation therapy using pegylated arginine deiminase (ADI-PEG20) which digests extracellular Arg. Many previous studies reported that ASS1 silencing is due to epigenetic inactivation of ASS1 expression by DNA methylation, and that the demethylation agent 5-aza-deoxycytidine (Aza-dC) can induce ASS1 expression. Moreover, it was reported that cisplatin suppresses ASS1 expression through ASS1 promoter methylation, leading to synthetic lethality to ADI-PEG20 treatment. We report here that cisplatin supppresses ASS1 expression is due to upregulation of HIF-1α and downregulation of c-Myc, which function as negative and positive regulators of ASS1 expression, respectively, by reciprocal bindings to the ASS1 promoter. In contrast, we found that Aza-dC induces ASS1 expression by downregulation of HIF-1α but upregulation of c-Myc. We further demonstrated that the clock protein DEC1 is the master regulator of HIF-1α and c-Myc that regulate ASS1. cDDP upregulates DEC1, whereas Aza-dC suppresses its expression. Using two proteasomal inhibitors bortezomib and carfilzomib which induce HIF-1α accumulation, we further demonstrated that HIF-1α is involved in ASS1 silencing for the maintenance of Arg auxotrophy for targeted Arg-starvation therapy.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Arginina/deficiencia , Argininosuccinato Sintasa/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cisplatino/farmacología , Metilación de ADN , Proteínas de Homeodominio/metabolismo , Hidrolasas/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/tratamiento farmacológico , Polietilenglicoles/farmacología , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética/efectos de los fármacos , Argininosuccinato Sintasa/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Proteínas de Homeodominio/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Inhibidores de Proteasoma/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , TransfecciónRESUMEN
INTRODUCTION: Platinum (Pt)-based antitumor agents remain important chemotherapeutic agents for treating many human malignancies. Elevated expression of the human high-affinity copper transporter 1 (hCtr1), resulting in enhanced Pt drug transport into cells, has been shown to be associated with improved treatment efficacy. Thus, targeting hCtr1 upregulation is an attractive strategy for improving the treatment efficacy of Pt-based cancer chemotherapy. AREA COVERED: Regulation of hCtr1 expression by cellular copper homeostasis is discussed. Association of elevated hCtr1 expression with intrinsic sensitivity of ovarian cancer to Pt drugs is presented. Mechanism of copper-lowering agents in enhancing hCtr1-mediated cis-diamminedichloroplatinum (II) (cisplatin, cDDP) transport is reviewed. Applications of copper chelation strategy in overcoming cDDP resistance through enhanced hCtr1 expression are evaluated. EXPERT OPINION: While both transcriptional and post-translational mechanisms of hCtr1 regulation by cellular copper bioavailability have been proposed, detailed molecular insights into hCtr1 regulation by copper homeostasis remain needed. Recent clinical study using a copper-lowering agent in enhancing hCtr1-mediated drug transport has achieved incremental improvement in overcoming Pt drug resistance. Further improvements in identifying predictive measures in the subpopulation of patients that can benefit from the treatment are needed.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Proteínas de Transporte de Catión/genética , Neoplasias/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Transporte Biológico , Quelantes/farmacología , Cobre/metabolismo , Transportador de Cobre 1 , Resistencia a Antineoplásicos , Humanos , Neoplasias/patología , Compuestos de Platino/administración & dosificación , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Many malignant human tumors, including melanomas, are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase-1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine, resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of resistance, we established several ADI-PEG20-resistant (ADI(R)) variants from A2058 and SK-Mel-2 melanoma cells. Compared with the parental lines, these ADI(R) variants showed the following characteristics: (i) all ADI(R) cell lines showed elevated ASS1 expression, resulting from the constitutive binding of the transcription factor c-Myc on the ASS1 promoter, suggesting that elevated ASS1 is the major mechanism of resistance; (ii) the ADI(R) cell lines exhibited enhanced AKT signaling and were preferentially sensitive to PI3K/AKT inhibitors, but reduced mTOR signaling, and were preferentially resistant to mTOR inhibitor; (iii) these variants showed enhanced expression of glucose transporter-1 and lactate dehydrogenase-A, reduced expression of pyruvate dehydrogenase, and elevated sensitivity to the glycolytic inhibitors 2-deoxy-glucose and 3-bromopyruvate, consistent with the enhanced glycolytic pathway (the Warburg effect); (iv) the resistant cells showed higher glutamine dehydrogenase and glutaminase expression and were preferentially vulnerable to glutamine inhibitors. We showed that c-Myc, not elevated ASS1 expression, is involved in upregulation of many of these enzymes because knockdown of c-Myc reduced their expression, whereas overexpressed ASS1 by transfection reduced their expression. This study identified multiple targets for overcoming ADI-PEG resistance in cancer chemotherapy using recombinant arginine-degrading enzymes.
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Resistencia a Antineoplásicos , Glucosa/metabolismo , Glutamina/metabolismo , Hidrolasas/farmacología , Melanoma/metabolismo , Polietilenglicoles/farmacología , Transducción de Señal/efectos de los fármacos , Arginina/metabolismo , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glucosa/genética , Glutamina/genética , Humanos , Lípidos/biosíntesis , Melanoma/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , TransfecciónRESUMEN
Melanomas and other cancers that do not express argininosuccinate synthetase (AS), the rate-limiting enzyme for arginine biosynthesis, are sensitive to arginine depletion with pegylated arginine deiminase (ADI-PEG20). However, ADI resistance eventually develops in tumors because of AS upregulation. Although it has been shown that AS upregulation involves c-Myc, the underlying mechanisms remain unknown. Here we show that ADI-PEG20 activates Ras signaling and the effector extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT/GSK-3ß kinase cascades, resulting in phosphorylation and stabilization of c-Myc by attenuation of its ubiquitin-mediated protein degradation mechanism. Inhibition of the induced cell signaling pathways using PI3K/AKT inhibitors suppressed c-Myc induction and enhanced ADI-mediated cell killing. Notably, in an animal model of AS-negative melanoma, combination therapy using a PI3K inhibitor plus ADI-PEG20 yielded additive antitumor effects as compared with either agent alone. Taken together, our findings offer mechanistic insight into arginine deprivation metabolism and ADI resistance, and they illustrate how combining inhibitors of the Ras/ERK and PI3K/AKT signaling pathways may improve ADI-PEG20 anticancer responses.
Asunto(s)
Argininosuccinato Sintasa/biosíntesis , Resistencia a Antineoplásicos , Hidrolasas/farmacología , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Polietilenglicoles/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Genes ras , Humanos , Hidrolasas/uso terapéutico , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Fosfatidilinositol 3-Quinasa , Polietilenglicoles/uso terapéutico , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Transducción de Señal , Ubiquitina , Regulación hacia ArribaRESUMEN
DNA damage as a result of environmental stress is recognized by sensor proteins that trigger repair mechanisms, or, if repair is unsuccessful, initiate apoptosis. Defects in DNA damage-induced apoptosis promote genomic instability and tumourigenesis. The protein ataxia-telangiectasia mutated (ATM) is activated by DNA double-strand breaks and regulates apoptosis via p53. Here we show that FOXO3 interacts with the ATM-Chk2-p53 complex, augments phosphorylation of the complex and induces the formation of nuclear foci in cells on DNA damage. FOXO3 is essential for DNA damage-induced apoptosis and conversely FOXO3 requires ATM, Chk2 and phosphorylated p53 isoforms to trigger apoptosis as a result of DNA damage. Under these conditions FOXO3 may also have a role in regulating chromatin retention of phosphorylated p53. These results suggest an essential link between FOXO3 and the ATM-Chk2-p53-mediated apoptotic programme following DNA damage.
Asunto(s)
Apoptosis , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa de Punto de Control 2 , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Humanos , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Platinum-based antitumor agents are widely used in cancer chemotherapy. Drug resistance is a major obstacle to the successful use of these agents because once drug resistance develops, other effective treatment options are limited. Recently, we conducted a clinical trial using a copper-lowering agent to overcome platinum drug resistance in ovarian cancer patients and the preliminary results are encouraging. In supporting this clinical study, using three pairs of cisplatin (cDDP)-resistant cell lines and two ovarian cancer cell lines derived from patients who had failed in platinum-based chemotherapy, we showed that cDDP resistance associated with reduced expression of the high-affinity copper transporter (hCtr1), which is also a cDDP transporter, can be preferentially resensitized by copper-lowering agents because of enhanced hCtr1 expression, as compared with their drug-sensitive counterparts. Such a preferential induction of hCtr1 expression in cDDP-resistant variants by copper chelation can be explained by the mammalian copper homeostasis regulatory mechanism. Enhanced cell-killing efficacy by a copper-lowering agent was also observed in animal xenografts bearing cDDP-resistant cells. Finally, by analyzing a public gene expression dataset, we found that ovarian cancer patients with elevated levels of hCtr1 in their tumors, but not ATP7A and ATP7B, had more favorable outcomes after platinum drug treatment than those expressing low hCtr1 levels. This study reveals the mechanistic basis for using copper chelation to overcome cDDP resistance in clinical investigations.