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Interleukin (IL)-2 and IL-21 dichotomously shape CD8+ T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (TSCM) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1 While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21.
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Linfócitos T CD8-Positivos/imunologia , Inibidores Enzimáticos/farmacologia , Interleucinas/metabolismo , L-Lactato Desidrogenase/antagonistas & inibidores , Melanoma Experimental/terapia , Células-Tronco/imunologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem Celular Tumoral/transplante , Humanos , Memória Imunológica , Imunoterapia Adotiva/métodos , Interleucina-2/imunologia , Interleucina-2/metabolismo , Interleucinas/imunologia , L-Lactato Desidrogenase/metabolismo , Melanoma Experimental/imunologia , Camundongos , Cultura Primária de Células , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
MJC13, a novel FKBP52 targeting agent, has potential use for the treatment of castration-resistant prostate cancer. The purpose of this work was to develop a solution formulation of MJC13, and obtain its efficacy profile in a human prostate cancer xenograft mouse model. Preformulation studies were conducted to evaluate the physicochemical properties. Co-solvent systems were evaluated for aqueous solubility and tolerance. A human prostate cancer xenograft mouse model was established by growing 22Rv1 prostate cancer cells in C.B-17 SCID mice. The optimal formulation was used to study the efficacy of MJC13 in this preclinical model of castrate-resistant prostate cancer. We found that MJC13 was stable (at least for 1 month), highly lipophilic (logP = 6.49), poorly soluble in water (0.28 µg/mL), and highly plasma protein bound (>98%). The optimal formulation consisting of PEG 400 and Tween 80 (1:1, v/v) allowed us to achieve a MJC13 concentration of 7.5 mg/mL, and tolerated an aqueous environment. After twice weekly intratumoral injection with 10 mg/kg MJC13 in this formulation for four consecutive weeks, tumor volumes were significantly reduced compared to vehicle-treated controls.
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Anilidas/síntese química , Antineoplásicos/síntese química , Química Farmacêutica/métodos , Cicloexanos/síntese química , Modelos Animais de Doenças , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Anilidas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cicloexanos/uso terapêutico , Humanos , Injeções Intralesionais , Masculino , Camundongos , Camundongos SCID , Soluções Farmacêuticas/síntese química , Soluções Farmacêuticas/uso terapêutico , Ratos , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The design of a single drug molecule that is able to simultaneously and specifically interact with multiple biological targets is gaining major consideration in drug discovery. However, the rational design of drugs with a desired polypharmacology profile is still a challenging task, especially when these targets are distantly related or unrelated. In this work, we present a computational approach aimed at the identification of suitable target combinations for multitarget drug design within an ensemble of biologically relevant proteins. The target selection relies on the analysis of activity annotations present in molecular databases and on ligand-based virtual screening. A few target combinations were also inspected with structure-based methods to demonstrate that the identified dual-activity compounds are able to bind target combinations characterized by remote binding site similarities. Our approach was applied to the heat shock protein 90 (Hsp90) interactome, which contains several targets of key importance in cancer. Promising target combinations were identified, providing a basis for the computational design of compounds with dual activity. The approach may be used on any ensemble of proteins of interest for which known inhibitors are available.
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Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Polifarmacologia , Sítios de Ligação , Bases de Dados de Compostos Químicos , Receptor alfa de Estrogênio/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mapas de Interação de Proteínas , Receptor ErbB-2/metabolismo , Relação Estrutura-AtividadeRESUMO
Hsp90 is an essential and highly conserved modular molecular chaperone whose N and middle domains are separated by a disordered region termed the charged linker. Although its importance has been previously disregarded, because a minimal linker length is sufficient for Hsp90 activity, the evolutionary persistence of extensive charged linkers of divergent sequence in Hsp90 proteins of most eukaryotes remains unexplained. To examine this question further, we introduced human and plasmodium native and length-matched artificial linkers into yeast Hsp90. After evaluating ATPase activity and biophysical characteristics in vitro, and chaperone function in vivo, we conclude that linker sequence affects Hsp90 function, cochaperone interaction, and conformation. We propose that the charged linker, in addition to providing the flexibility necessary for Hsp90 domain rearrangements--likely its original purpose--has evolved in eukaryotes to serve as a rheostat for the Hsp90 chaperone machine.
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Células Eucarióticas/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Deutério/metabolismo , Humanos , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Solventes , Relação Estrutura-AtividadeRESUMO
Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.
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Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteínas de Ligação a Tacrolimo/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Ensaio de Imunoadsorção Enzimática , Fluorescência , Humanos , Immunoblotting , Imunoprecipitação , Masculino , Camundongos , Simulação de Dinâmica Molecular , Estrutura Molecular , Receptores Androgênicos/química , Proteínas de Ligação a Tacrolimo/metabolismo , Leveduras , beta-GalactosidaseRESUMO
INTRODUCTION: Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy. METHODS: Transcriptomes and DNA methylomes of 14 SCLC and 10 NSCLC lines were compared with normal human small airway epithelial cells (SAECs) and induced pluripotent stem cell (iPSC) clones derived from SAEC. SCLC lines, lung iPSC (Lu-iPSC), and SAEC were further evaluated by DNase I hypersensitive site sequencing (DHS-seq). Changes in chromatin accessibility and depths of transcription factor (TF) footprints were quantified using Bivariate analysis of Genomic Footprint. Standard techniques were used to evaluate growth, tumorigenicity, and changes in transcriptomes and glucose metabolism of SCLC cells after NFIC knockdown and to evaluate NFIC expression in SCLC cells after exposure to BET inhibitors. RESULTS: Considerable commonality of transcriptomes and DNA methylomes was observed between Lu-iPSC and SCLC; however, this analysis was uninformative regarding pathways unique to lung cancer. Linking results of DHS-seq to RNA sequencing enabled identification of networks not previously associated with SCLC. When combined with footprint depth, NFIC, a transcription factor not previously associated with SCLC, had the highest score of occupancy at open chromatin sites. Knockdown of NFIC impaired glucose metabolism, decreased stemness, and inhibited growth of SCLC cells in vitro and in vivo. ChIP-seq analysis identified numerous sites occupied by BRD4 in the NFIC promoter region. Knockdown of BRD4 or treatment with Bromodomain and extra-terminal domain (BET) inhibitors (BETis) markedly reduced NFIC expression in SCLC cells and SCLC PDX models. Approximately 8% of genes down-regulated by BETi treatment were repressed by NFIC knockdown in SCLC, whereas 34% of genes repressed after NFIC knockdown were also down-regulated in SCLC cells after BETi treatment. CONCLUSIONS: NFIC is a key TF and possible mediator of transcriptional regulation by BET family proteins in SCLC. Our findings highlight the potential of genome-wide chromatin accessibility analysis for elucidating mechanisms of pulmonary carcinogenesis and identifying novel targets for lung cancer therapy.
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Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Animais , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/metabolismo , Fatores de Transcrição NFI/genética , Fatores de Transcrição NFI/metabolismo , Estudo de Associação Genômica Ampla , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The androgen receptor is a key regulator of prostate cancer and the principal target of current prostate cancer therapies collectively termed androgen deprivation therapies. Insensitivity to these drugs is a hallmark of progression to a terminal disease state termed castration-resistant prostate cancer. Therefore, novel therapeutic options that slow progression of castration-resistant prostate cancer and combine effectively with existing agents are in urgent need. We show that JG-98, an allosteric inhibitor of HSP70, re-sensitizes castration-resistant prostate cancer to androgen deprivation drugs by targeting mitochondrial HSP70 (HSPA9) to suppress aerobic respiration. Rather than impacting androgen receptor stability as previously described, JG-98's primary effect is inhibition of mitochondrial translation, leading to disruption of electron transport chain activity. Although functionally distinct from HSPA9 inhibition, direct inhibition of the electron transport chain with a complex I or II inhibitor creates a similar physiological state capable of re-sensitizing castration-resistant prostate cancer to androgen deprivation therapies. These data identify a significant role for HspA9 in mitochondrial ribosome function and highlight an actionable metabolic vulnerability of castration-resistant prostate cancer.
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A metabolic hallmark of cancer identified by Warburg is the increased consumption of glucose and secretion of lactate, even in the presence of oxygen. Although many tumors exhibit increased glycolytic activity, most forms of cancer rely on mitochondrial respiration for tumor growth. We report here that Hürthle cell carcinoma of the thyroid (HTC) models harboring mitochondrial DNA-encoded defects in complex I of the mitochondrial electron transport chain exhibit impaired respiration and alterations in glucose metabolism. CRISPR-Cas9 pooled screening identified glycolytic enzymes as selectively essential in complex I-mutant HTC cells. We demonstrate in cultured cells and a patient-derived xenograft model that small-molecule inhibitors of lactate dehydrogenase selectively induce an ATP crisis and cell death in HTC. This work demonstrates that complex I loss exposes fermentation as a therapeutic target in HTC and has implications for other tumors bearing mutations that irreversibly damage mitochondrial respiration. SIGNIFICANCE: HTC is enriched in somatic mtDNA mutations predicted to affect complex I of the electron transport chain (ETC). We demonstrate that these mutations impair respiration and induce a therapeutically tractable reliance on aerobic fermentation for cell survival. This work provides a rationale for targeting fermentation in cancers harboring irreversible genetically encoded ETC defects. See related article by Gopal et al., p. 1904. This article is highlighted in the In This Issue feature, p. 1749.
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Adenocarcinoma , Adenoma Oxífilo , Carcinoma , Neoplasias da Glândula Tireoide , Humanos , Fermentação , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adenoma Oxífilo/genética , DNA Mitocondrial/genéticaRESUMO
PURPOSE: Deregulated metabolism in cancer cells represents a vulnerability that may be therapeutically exploited to benefit patients. One such target is nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage pathway. NAMPT is necessary for efficient NAD+ production and may be exploited in cells with increased metabolic demands. We have identified NAMPT as a dependency in rhabdomyosarcoma (RMS), a malignancy for which novel therapies are critically needed. Here we describe the effect of NAMPT inhibition on RMS proliferation and metabolism in vitro and in vivo. EXPERIMENTAL DESIGN: Assays of proliferation and cell death were used to determine the effects of pharmacologic NAMPT inhibition in a panel of ten molecularly diverse RMS cell lines. Mechanism of the clinical NAMPTi OT-82 was determined using measures of NAD+ and downstream NAD+-dependent functions, including energy metabolism. We used orthotopic xenograft models to examine tolerability, efficacy, and drug mechanism in vivo. RESULTS: Across all ten RMS cell lines, OT-82 depleted NAD+ and inhibited cell growth at concentrations ≤1 nmol/L. Significant impairment of glycolysis was a universal finding, with some cell lines also exhibiting diminished oxidative phosphorylation. Most cell lines experienced profound depletion of ATP with subsequent irreversible necrotic cell death. Importantly, loss of NAD and glycolytic activity were confirmed in orthotopic in vivo models, which exhibited complete tumor regressions with OT-82 treatment delivered on the clinical schedule. CONCLUSIONS: RMS is highly vulnerable to NAMPT inhibition. These findings underscore the need for further clinical study of this class of agents for this malignancy.
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NAD , Rabdomiossarcoma , Humanos , NAD/metabolismo , Citocinas/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Pirazóis , Necrose , Rabdomiossarcoma/tratamento farmacológico , Linhagem Celular TumoralRESUMO
INTRODUCTION: Although human epidermal growth factor receptor 2 (HER2) positive or estrogen receptor (ER) positive breast cancers are treated with clinically validated anti-HER2 or anti-estrogen therapies, intrinsic and acquired resistance to these therapies appears in a substantial proportion of breast cancer patients and new therapies are needed. Identification of additional molecular factors, especially those characterized by aggressive behavior and poor prognosis, could prioritize interventional opportunities to improve the diagnosis and treatment of breast cancer. METHODS: We compiled a collection of 4,010 breast tumor gene expression data derived from 23 datasets that have been posted on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. We performed a genome-scale survival analysis using Cox-regression survival analyses, and validated using Kaplan-Meier Estimates survival and Cox Proportional-Hazards Regression survival analyses. We conducted a genome-scale analysis of chromosome alteration using 481 breast cancer samples obtained from The Cancer Genome Atlas (TCGA), from which combined expression and copy number data were available. We assessed the correlation between somatic copy number alterations and gene expression using analysis of variance (ANOVA). RESULTS: Increased expression of each of the heat shock protein (HSP) 90 isoforms, as well as HSP transcriptional factor 1 (HSF1), was correlated with poor prognosis in different subtypes of breast cancer. High-level expression of HSP90AA1 and HSP90AB1, two cytoplasmic HSP90 isoforms, was driven by chromosome coding region amplifications and were independent factors that led to death from breast cancer among patients with triple-negative (TNBC) and HER2-/ER+ subtypes, respectively. Furthermore, amplification of HSF1 was correlated with higher HSP90AA1 and HSP90AB1 mRNA expression among the breast cancer cells without amplifications of these two genes. A collection of HSP90AA1, HSP90AB1 and HSF1 amplifications defined a subpopulation of breast cancer with up-regulated HSP90 gene expression, and up-regulated HSP90 expression independently elevated the risk of recurrence of TNBC and poor prognosis of HER2-/ER+ breast cancer. CONCLUSIONS: Up-regulated HSP90 mRNA expression represents a confluence of genomic vulnerability that renders HER2 negative breast cancers more aggressive, resulting in poor prognosis. Targeting breast cancer with up-regulated HSP90 may potentially improve the effectiveness of clinical intervention in this disease.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Receptor ErbB-2/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Proteínas de Ligação a DNA/genética , Feminino , Amplificação de Genes , Fatores de Transcrição de Choque Térmico , Humanos , Estimativa de Kaplan-Meier , Fenótipo , Prognóstico , Modelos de Riscos Proporcionais , Análise de Sobrevida , Fatores de Transcrição/genética , Regulação para CimaRESUMO
A noninvasive test to discriminate indolent prostate cancers from lethal ones would focus treatment where necessary while reducing overtreatment. We exploited the known activity of heat shock protein 90 (Hsp90) as a chaperone critical for the function of numerous oncogenic drivers, including the androgen receptor and its variants, to detect aggressive prostate cancer. We linked a near-infrared fluorescing molecule to an HSP90 binding drug and demonstrated that this probe (designated HS196) was highly sensitive and specific for detecting implanted prostate cancer cell lines with greater uptake by more aggressive subtypes. In a phase I human study, systemically administered HS196 could be detected in malignant nodules within prostatectomy specimens. Single-cell RNA sequencing identified uptake of HS196 by malignant prostate epithelium from the peripheral zone (AMACR+ERG+EPCAM+ cells), including SYP+ neuroendocrine cells that are associated with therapeutic resistance and metastatic progression. A theranostic version of this molecule is under clinical testing.
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Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/genética , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias da Próstata/patologiaRESUMO
Lipids comprise diverse classes of compounds that are important for the structure and properties of membranes, as high-energy fuel sources and as signaling molecules. Therefore, the turnover rates of these varied classes of lipids are fundamental to cellular function. However, their enormous chemical diversity and dynamic range in cells makes detailed analysis very complex. Furthermore, although stable isotope tracers enable the determination of synthesis and degradation of complex lipids, the numbers of distinguishable molecules increase enormously, which exacerbates the problem. Although LC-MS-MS (Liquid Chromatography-Tandem Mass Spectrometry) is the standard for lipidomics, NMR can add value in global lipid analysis and isotopomer distributions of intact lipids. Here, we describe new developments in NMR analysis for assessing global lipid content and isotopic enrichment of mixtures of complex lipids for two cell lines (PC3 and UMUC3) using both 13C6 glucose and 13C5 glutamine tracers.
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As a conserved molecular chaperone, heat shock protein 90 (Hsp90) maintains the stability and homeostasis of oncoproteins and helps cancer cells survive. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a pivotal role in the non-homologous end joining pathway for DNA double-strand breaks (DSB) repair. Tumor cells contain higher levels of DNA-PKcs to survive by the hostile tumor microenvironment and various antitumor therapies. Here, we showed that increased levels of Hsp90α, Hsp90ß, and DNA-PKcs correlated with a poor overall survival in hepatocellular carcinoma (HCC). We revealed that Hsp90 N-terminal domain and C-terminal domain have different effects on DNA-PKcs protein and mRNA levels. The stability of DNA-PKcs depended on Hsp90α N-terminal nucleotide binding domain. Transcription factor SP1 regulates the transcription of PRKDC (gene name of DNA-PKcs) and is a client protein of Hsp90. Inhibition of Hsp90 N-terminal by STA9090 decreased the location of Hsp90α in nucleus, Hsp90α-SP1 interaction, SP1 level, and the binding of Hsp90α/SP1 at the proximal promoter region of PRKDC Because hyperthermia induces DSBs with increases level of DNA-PKcs, combined STA9090 treatment with hyperthermia effectively delayed the tumor growth and significantly decreased DNA-PKcs levels in xenografts model. Consistently, inhibition of Hsp90 increased the number of heat shock-induced γ-H2AX foci and delayed the repair of DSBs. Altogether, our results suggest that Hsp90 inhibitor STA9090 decreases DNA-PKcs protein stability and PRKDC mRNA level, which provide a theoretical basis for the promising combination therapy of hyperthermia and Hsp90 inhibitor in HCC.
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Carcinoma Hepatocelular/tratamento farmacológico , Dano ao DNA , Proteína Quinase Ativada por DNA/química , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Hipertermia Induzida/efeitos adversos , RNA Mensageiro/genética , Animais , Apoptose , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células , Reparo do DNA , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Prognóstico , Estabilidade Proteica , Taxa de Sobrevida , Triazóis , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors of Hsp90 have entered clinical trials for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms, which may lead to adverse effects. The development of Hsp90 isoform-selective inhibitors represents an alternative approach toward the treatment of cancer and may limit some of these detriments. Described herein, is a structure-based approach to develop isoform-selective inhibitors of Hsp90ß, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90ß-selective inhibitors as a method for overcoming the detriments associated with pan-inhibition.
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Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inativação Gênica , Proteínas de Choque Térmico HSP90/genética , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Modelos Moleculares , Conformação Molecular , Neoplasias/tratamento farmacológico , Dobramento de Proteína , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Especificidade por Substrato , Neoplasias da Bexiga Urinária/tratamento farmacológicoRESUMO
OBJECTIVE: Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival. METHODS: DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor. RESULTS: HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the ß-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses. CONCLUSIONS: HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC.
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BACKGROUND: Platelets play a pivotal role in hemostasis, wound healing, and inflammation, and are thus implicated in a variety of diseases, including cancer. Platelet function is associated with release of granule content, cellular shape change, and upregulation of receptors that promote establishment of a thrombus and maintenance of hemostasis. OBJECTIVES: The role of heat shock proteins (Hsps) in modulating platelet function has been studied for a number of years, but comparative roles of individual Hsps have not been thoroughly examined. METHODS: We utilized a panel of specific inhibitors of Hsp40, Hsp70, Hsp90, and Grp94 (the endoplasmic reticulum homolog of Hsp90) to assess their impact on several aspects of platelet function. RESULTS: Inhibition of each of the aforementioned Hsps reduced alpha granule release. In contrast, there was some selectivity in impacts on dense granule release. Thromboxane synthesis was impaired after exposure to inhibitors of Hsp40, Hsp90, and Grp94, but not after inhibition of Hsp70. Both expression of active glycoprotein IIb/IIIa (GPIIb/IIIa) and fibrinogen-induced platelet shape change were diminished by our inhibitors. In contrast, aggregation was selectively abrogated after inhibition of Hsp40 or Hsp90. Lastly, activated platelet-cancer cell interactions were reduced by inhibition of both Hsp70 and Grp94. CONCLUSIONS: These data suggest the importance of Hsp networks in regulating platelet activity.
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Proteínas de Choque Térmico , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Plaquetas , Proteínas de Choque Térmico/farmacologia , Hemostasia , Humanos , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
The reliance of many cancers on aerobic glycolysis has stimulated efforts to develop lactate dehydrogenase (LDH) inhibitors. However, despite significant efforts, LDH inhibitors (LDHi) with sufficient specificity and in vivo activity to determine whether LDH is a feasible drug target are lacking. We describe an LDHi with potent, on-target, in vivo activity. Using hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI), we demonstrate in vivo LDH inhibition in two glycolytic cancer models, MIA PaCa-2 and HT29, and we correlate depth and duration of LDH inhibition with direct anti-tumor activity. HP-MRSI also reveals a metabolic rewiring that occurs in vivo within 30 min of LDH inhibition, wherein pyruvate in a tumor is redirected toward mitochondrial metabolism. Using HP-MRSI, we show that inhibition of mitochondrial complex 1 rapidly redirects tumor pyruvate toward lactate. Inhibition of both mitochondrial complex 1 and LDH suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo.
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Quimioterapia Combinada/métodos , L-Lactato Desidrogenase/antagonistas & inibidores , Neoplasias/imunologia , Animais , Humanos , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glicólise/efeitos dos fármacos , L-Lactato Desidrogenase/antagonistas & inibidores , Sarcoma de Ewing/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Sarcoma de Ewing/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Castration-resistant prostate cancer (CRPC) is characterized by reactivation of androgen receptor (AR) signaling, in part by elevated expression of AR splice variants (ARv) including ARv7, a constitutively active, ligand binding domain (LBD)-deficient variant whose expression has been correlated with therapeutic resistance and poor prognosis. In a screen to identify small-molecule dual inhibitors of both androgen-dependent and androgen-independent AR gene signatures, we identified the chalcone C86. Binding studies using purified proteins and CRPC cell lysates revealed C86 to interact with Hsp40. Pull-down studies using biotinylated-C86 found Hsp40 present in a multiprotein complex with full-length (FL-) AR, ARv7, and Hsp70 in CRPC cells. Treatment of CRPC cells with C86 or the allosteric Hsp70 inhibitor JG98 resulted in rapid protein destabilization of both FL-AR and ARv, including ARv7, concomitant with reduced FL-AR- and ARv7-mediated transcriptional activity. The glucocorticoid receptor, whose elevated expression in a subset of CRPC also leads to androgen-independent AR target gene transcription, was also destabilized by inhibition of Hsp40 or Hsp70. In vivo, Hsp40 or Hsp70 inhibition demonstrated single-agent and combinatorial activity in a 22Rv1 CRPC xenograft model. These data reveal that, in addition to recognized roles of Hsp40 and Hsp70 in FL-AR LBD remodeling, ARv lacking the LBD remain dependent on molecular chaperones for stability and function. Our findings highlight the feasibility and potential benefit of targeting the Hsp40/Hsp70 chaperone axis to treat prostate cancer that has become resistant to standard antiandrogen therapy.Significance: These findings highlight the feasibility of targeting the Hsp40/Hsp70 chaperone axis to treat CRPC that has become resistant to standard antiandrogen therapy. Cancer Res; 78(14); 4022-35. ©2018 AACR.