RESUMO
Phospholipase D (PLD) hydrolyzes membrane lipids to produce phosphatidic acid (PA), a lipid mediator involved in various cellular and physiological processes. Here, we show that PLDα6 and PA regulate the distribution of GIBBERELLIN (GA)-INSENSITIVE DWARF1 (GID1), a soluble gibberellin receptor in rice. PLDα6-knockout (KO) plants display less sensitivity to GA than WT, and PA restores the mutant to a normal GA response. PA binds to GID1, as documented by liposome binding, fat immunoblotting, and surface plasmon resonance. Arginines 79 and 82 of GID1 are two key amino acid residues required for PA binding and also for GID1's nuclear localization. The loss of PLDα6 impedes GA-induced nuclear localization of GID1. In addition, PLDα6-KO plants attenuated GA-induced degradation of the DELLA protein SLENDER RICE1 (SLR1). These data suggest that PLDα6 and PA positively mediate GA signaling in rice via PA binding to GID1 and promotion of its nuclear translocation.
Assuntos
Giberelinas , Oryza , Regulação da Expressão Gênica de Plantas , Mutação , Oryza/genética , Ácidos Fosfatídicos , Fosfolipases , Reguladores de Crescimento de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente ModificadasRESUMO
Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non-small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53ß and p53γ, comprising exons 1 to 9ß or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53ß and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Células A549 , Animais , Humanos , Camundongos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
MOTIVATION: The concept of synergy between two agents, over a century old, is important to the fields of biology, chemistry, pharmacology and medicine. A key step in drug combination analysis is the selection of an additivity model to identify combination effects including synergy, additivity and antagonism. Existing methods for identifying and interpreting those combination effects have limitations. RESULTS: We present here a computational framework, termed response envelope analysis (REA), that makes use of 3D response surfaces formed by generalized Loewe Additivity and Bliss Independence models of interaction to evaluate drug combination effects. Because the two models imply two extreme limits of drug interaction (mutually exclusive and mutually non-exclusive), a response envelope defined by them provides a quantitatively stringent additivity model for identifying combination effects without knowing the inhibition mechanism. As a demonstration, we apply REA to representative published data from large screens of anticancer and antibiotic combinations. We show that REA is more accurate than existing methods and provides more consistent results in the context of cross-experiment evaluation. AVAILABILITY AND IMPLEMENTATION: The open-source software package associated with REA is available at: https://github.com/4dsoftware/rea. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Biologia Computacional , Software , Combinação de Medicamentos , Interações MedicamentosasRESUMO
Sequencing of clear cell renal cell carcinomas identified loss-of-function mutations of SETD2, a gene that encodes a nonredundant methytransferase responsible for histone H3 lysine 36 trimethylation (H3K36me3), and H3K36me3 is progressively deregulated in metastases. However, few data exist regarding the impact of loss of H3K36me3 on outcomes. We assessed the association of SETD2 DNA alterations and mRNA expression with overall survival using The Cancer Genome Atlas clear cell renal carcinoma data (N=411). Additionally, we assessed the association of H3K36 loss of methylation with renal cell carcinoma-specific survival and progression-free survival using an independent cohort at Mayo Clinic (N=1454). Overall survival, renal cell carcinoma-specific survival and progression-free survival were estimated using Kaplan-Meier method, and differences in survival across groups was compared using Cox regression models, adjusted for age and the Mayo SSIGN (stage, size, grade, and necrosis) score. In The Cancer Genome Atlas cohort, SETD2 DNA alterations or mRNA expression was not associated with overall survival (P>0.05). In the Mayo cohort, patients with H3K36me3-negative tumors were two times more likely to experience renal cell carcinoma-specific death than patients with H3K36me3-positive tumors (hazard ratio, 2.23; 95% confidence interval, 1.77-2.81); P<0.0001. After stratifying for the SSIGN score, H3K36me3-negative tumors in the low-risk SSIGN group had a worse renal cell carcinoma-specific survival (hazard ratio, 2.18; 95% confidence interval, 1.09-4.36); P=0.03. Although SETD2 DNA and mRNA alterations are not associated with overall survival, we provide evidence that deregulation of the H3K36me3 axis is associated with a higher risk of renal cell carcinoma-specific death. This association remains significant after stratifying for the SSIGN score, particularly among those patients with low-risk tumors.
Assuntos
Carcinoma de Células Renais/metabolismo , Metilação de DNA , Histonas/metabolismo , Neoplasias Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Intervalo Livre de Doença , Humanos , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Mutação , Fatores de Risco , Taxa de SobrevidaRESUMO
MOTIVATION: Nonlinear dose-response models are primary tools for estimating the potency [e.g. half-maximum inhibitory concentration (IC) known as IC50] of anti-cancer drugs. We present drexplorer software, which enables biologists to evaluate replicate reproducibility, detect outlier data points, fit different models, select the best model, estimate IC values at different percentiles and assess drug-drug interactions. drexplorer serves as a computation engine within the R environment and a graphical interface for users who do not have programming backgrounds.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Software , Algoritmos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Concentração Inibidora 50 , Dinâmica não Linear , Reprodutibilidade dos TestesRESUMO
The core promoter, which immediately flanks the transcription start site (TSS), plays a critical role in transcriptional regulation of eukaryotes. Recent studies on higher eukaryotes have revealed an unprecedented complexity of core promoter structures that underscores diverse regulatory mechanisms of gene expression. For unicellular eukaryotes, however, the structures of core promoters have not been investigated in detail. As an important model organism, Schizosaccharomyces pombe still lacks the precise annotation for TSSs, thus hampering the analysis of core promoter structures and their relationship to higher eukaryotes. Here we used a deep sequencing-based approach (DeepCAGE) to generate 16 million uniquely mapped tags, corresponding to 93,736 positions in the S. pombe genome. The high-resolution TSS landscape enabled identification of over 8,000 core promoters, characterization of 4 promoter classes and observation of widespread alternative promoters. The landscape also allowed precise determination of the representative TSSs within core promoters, thus redefining the 5' UTR for 82.8% of S. pombe genes. We further identified the consensus initiator (Inr) sequence--PyPyPuN(A/C)(C/A), the TATA-enriched region (between position -25 and -37) and an Inr immediate downstream motif--CC(T/A)(T/C)(T/C/A)(A/G)CCA(A/T/C), all of which were associated with highly expressed promoters. In conclusion, the detailed analysis of core promoters not only significantly improves the genome annotation of S. pombe, but also reveals that this unicellular eukaryote shares a highly similar organization in the core promoters with higher eukaryotes. These findings lend additional evidence for the power of this model system in delineating complex regulatory processes in multicellular organisms, despite its perceived simplicity.
Assuntos
Genoma Fúngico , Regiões Promotoras Genéticas , Schizosaccharomyces/genética , Software , Regiões 5' não Traduzidas/genética , Sequência de Bases , Sequência Consenso , DNA Intergênico/genética , Genes Fúngicos , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Fases de Leitura Aberta/genética , RNA não Traduzido/genética , TATA Box/genética , Sítio de Iniciação de TranscriçãoRESUMO
MOTIVATION: Identification of bimodally expressed genes is an important task, as genes with bimodal expression play important roles in cell differentiation, signalling and disease progression. Several useful algorithms have been developed to identify bimodal genes from microarray data. Currently, no method can deal with data from next-generation sequencing, which is emerging as a replacement technology for microarrays. RESULTS: We present SIBER (systematic identification of bimodally expressed genes using RNAseq data) for effectively identifying bimodally expressed genes from next-generation RNAseq data. We evaluate several candidate methods for modelling RNAseq count data and compare their performance in identifying bimodal genes through both simulation and real data analysis. We show that the lognormal mixture model performs best in terms of power and robustness under various scenarios. We also compare our method with alternative approaches, including profile analysis using clustering and kurtosis (PACK) and cancer outlier profile analysis (COPA). Our method is robust, powerful, invariant to shifting and scaling, has no blind spots and has a sample-size-free interpretation. AVAILABILITY: The R package SIBER is available at the website http://bioinformatics.mdanderson.org/main/OOMPA:Overview.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Algoritmos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Análise por Conglomerados , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , SoftwareRESUMO
Neuroblastoma is the most common and deadly solid tumor in children, and there is currently no effective treatment available for neuroblastoma patients. The repressor element-1 silencing transcription (REST) factor has been found to play important roles in the regulation of neural differentiation and tumorigenesis. Recently, a REST signature consisting of downstream targets of REST has been reported to have clinical relevance in both breast cancer and glioblastoma. However it remains unclear how the REST signature works in neuroblastoma. Publicly available datasets were mined and bioinformatic approaches were used to investigate the utility of the REST signature in neuroblastoma with both preclinical and real patient data. The REST signature was found to be associated with drug sensitivity in neuroblastoma cell lines. Further, neuroblastoma patients with enhanced REST activity are significantly associated with higher clinical stages. Loss of heterozygosity on chromosome 11q23, which occurs in a large subset of high-risk neuroblastomas, tends to be correlated with high REST activity, with marginal significance. In conclusion, the REST signature has important implications for targeted therapy, and it is a prognostic factor in neuroblastoma patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neuroblastoma/metabolismo , Proteínas Repressoras/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Cromossomos Humanos Par 11/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Estadiamento de Neoplasias , Neuroblastoma/diagnóstico , Neuroblastoma/tratamento farmacológico , Proteínas Repressoras/genéticaRESUMO
MOTIVATION: Identifying genes altered in cancer plays a crucial role in both understanding the mechanism of carcinogenesis and developing novel therapeutics. It is known that there are various mechanisms of regulation that can lead to gene dysfunction, including copy number change, methylation, abnormal expression, mutation and so on. Nowadays, all these types of alterations can be simultaneously interrogated by different types of assays. Although many methods have been proposed to identify altered genes from a single assay, there is no method that can deal with multiple assays accounting for different alteration types systematically. RESULTS: In this article, we propose a novel method, integration using item response theory (integIRTy), to identify altered genes by using item response theory that allows integrated analysis of multiple high-throughput assays. When applied to a single assay, the proposed method is more robust and reliable than conventional methods such as Student's t-test or the Wilcoxon rank-sum test. When used to integrate multiple assays, integIRTy can identify novel-altered genes that cannot be found by looking at individual assay separately. We applied integIRTy to three public cancer datasets (ovarian carcinoma, breast cancer, glioblastoma) for cross-assay type integration which all show encouraging results. AVAILABILITY AND IMPLEMENTATION: The R package integIRTy is available at the web site http://bioinformatics.mdanderson.org/main/OOMPA:Overview. CONTACT: kcoombes@mdanderson.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Neoplasias da Mama/genética , Perfilação da Expressão Gênica/métodos , Glioblastoma/genética , Modelos Genéticos , Neoplasias Ovarianas/genética , Simulação por Computador , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MutaçãoRESUMO
Loss-of-function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for patients with LKB1-deficient non-small cell lung cancer (NSCLC) remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analog. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress, and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the antitumor efficacy of 8-Cl-Ado. IMPLICATIONS: Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.
Assuntos
2-Cloroadenosina/análogos & derivados , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , 2-Cloroadenosina/farmacologia , 2-Cloroadenosina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Homeostase , Humanos , Neoplasias Pulmonares/patologia , Mutação , Oxirredução , Transdução de Sinais , TransfecçãoRESUMO
BACKGROUND: Inhibitors of apoptosis proteins (IAPs) are a family of antiapoptotic proteins modulating cell cycle, signal transduction and apoptosis. Dysregulated IAPs have been reported to contribute to tumor progression and chemoresistance in various cancers. However, existing studies were sporadic and only focus on one specific cancer with one particular gene in the IAPs family. A systematic investigation on the co-expression pattern, regulation frameworks on various pathways, prognostic utility on patient outcomes, and predictive value on drug sensitivity among all the IAPs across multiple tumor types was lacking. METHODS: Leveraging The Cancer Genome Atlas data with comprehensive genomic characterizations on 9714 patients across 32 tumor types and the Genomics of Drug Sensitivity in Cancer data with both genomic characterizations and drug sensitivity data on > 1000 cell lines, we investigated the co-expression pattern of IAPs, their regulations of apoptosis as well as other pathways and clinical relevance of IAPs for therapeutics development. RESULTS: We discovered diverse expression pattern among IAPs, varied spectrum of apoptosis regulations through IAPs and extensive regulations beyond apoptosis involving immune response, cell cycle, gene expression and DNA damage repair. Importantly, IAPs were strong prognostic factors for patient survival and tumor stage in several tumor types including brain, liver, kidney, breast and lung cancer. Further, several IAPs were found to be predictive of sensitivity to BCL-2 inhibitors (BIRC3, BIRC5, BIRC6, and BIRC7) as well as RIPK1 inhibitors (BIRC3 and BIRC6). CONCLUSION: Together, our work revealed the landscape of regulations, prognostic utilities and therapeutic relevance of IAPs across multiple tumor types.
Assuntos
Bases de Dados de Ácidos Nucleicos , Sistemas de Liberação de Medicamentos , Proteínas Inibidoras de Apoptose , Neoplasias , Proteínas Proto-Oncogênicas c-bcl-2 , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
The case-control study design is one of the main tools for detecting associations between genetic markers and diseases. It is well known that population substructure can lead to spurious association between disease status and a genetic marker if the prevalence of disease and the marker allele frequency vary across subpopulations. In this paper, we propose a novel statistical method to estimate the association in case-control studies with unmeasured population substructure. The proposed method takes two steps. First, the information on genomic markers and disease status is used to infer the population substructure; second, the association between the disease and the test marker adjusting for the population substructure is modeled and estimated parametrically through polytomous logistic regression. The performance of the proposed method, relative to the existing methods, on bias, coverage probability and computational time, is assessed through simulations. The method is applied to an end-stage renal disease study in African Americans population.
Assuntos
Estudos de Casos e Controles , Marcadores Genéticos , Viés , Frequência do Gene , Humanos , ProbabilidadeRESUMO
Renal cell carcinoma (RCC) is not sensitive to conventional radiotherapy and chemotherapy, and the effectiveness rate of molecular targeted therapy is low. Therefore, it is urgent to identify new treatment methods. Recently, adoptive Tcell therapy has provided a new option for cancer treatment. Furthermore, lowdose chemotherapy not only has no evident side effects and inhibitory effects on the human immune system, but can also enhance the immune activity of some effector cells. Therefore, it is surmised that the combination of different mechanisms of chemotherapy and immunotherapy could be a new treatment concept. In the present study, the effects of lowdose chemotherapy combined with T cells in the treatment of renal cell carcinoma were explored using cytotoxicity assays, enzymelinked immunosorbent assay (ELISA), western blot analysis and flow cytometric analysis. The results revealed that lowdose chemotherapy and T cells had synergistic effects on tumor cell elimination in vitro. The transforming growth factor (TGF)ß signaling pathway may be involved in the inhibition of Tcell functions. The targeted inhibition of TGFß signals may be a promising therapeutic strategy for the treatment of renal cancer. The present results provided a novel strategy for the combination of lowdose chemotherapy and T cells to enhance the therapeutic efficacy of RCC treatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Renais/terapia , Imunoterapia Adotiva/métodos , Neoplasias Renais/terapia , Linfócitos T Citotóxicos/transplante , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Terapia Combinada/métodos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Camundongos , Mitomicina/farmacologia , Mitomicina/uso terapêutico , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismoRESUMO
Several photorespiratory bypasses have been introduced into plants and shown to improve photosynthesis by increasing chloroplastic CO2 concentrations or optimizing energy balance. We recently reported that an engineered GOC bypass could increase photosynthesis and productivity in rice. However, the grain yield of GOC plants was unstable, fluctuating in different cultivation seasons because of varying seed setting rates. In this study, we designed a synthetic photorespiratory shortcut (the GCGT bypass) consisting of genes encoding Oryza sativa glycolate oxidase and Escherichia coli catalase, glyoxylate carboligase, and tartronic semialdehyde reductase. The GCGT bypass was guided by an optimized chloroplast transit peptide that targeted rice chloroplasts and redirected 75% of carbon from glycolate metabolism to the Calvin cycle, identical to the native photorespiration pathway. GCGT transgenic plants exhibited significantly increased biomass production and grain yield, which were mainly attributed to enhanced photosynthesis due to increased chloroplastic CO2 concentrations. Despite the increases in biomass production and grain yield, GCGT transgenic plants showed a reduced seed setting rate, a phenotype previously reported for the GOC plants. Integrative transcriptomic, physiological, and biochemical assays revealed that photosynthetic carbohydrates were not transported to grains in an efficient manner, thereby reducing the seed setting rate. Taken together, our results demonstrate that the GCGT photorespiratory shortcut confers higher yield by promoting photosynthesis in rice, mainly through increasing chloroplastic CO2 concentrations.
Assuntos
Biomassa , Luz , Oryza/crescimento & desenvolvimento , Oryza/efeitos da radiação , Fotossíntese/efeitos da radiação , Sementes/crescimento & desenvolvimento , Transporte Biológico/efeitos da radiação , Metabolismo dos Carboidratos/efeitos da radiação , Dióxido de Carbono/metabolismo , Respiração Celular/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Cloroplastos/ultraestrutura , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Metaboloma/efeitos da radiação , Oryza/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Folhas de Planta/ultraestrutura , Plantas Geneticamente Modificadas , Sementes/efeitos da radiação , Transcriptoma/genéticaRESUMO
Transketolase (TKT), which is a metabolic enzyme in the nonoxidative phase of the pentose phosphate pathway (PPP), plays an important role in providing cancer cells with raw materials for macromolecular biosynthesis. The ectopic expression of TKT in hepatocellular carcinoma (HCC) was reported previously. However, the role of TKT in the initiation of liver cancer is still obscure. In our previous study, we found that TKT deficiency protects the liver from DNA damage by increasing levels of ribose 5-phosphate and nucleotides. What's more interesting is that we found TKT deficiency reduced bile acids and loss of TKT promoted the farnesoid receptor (FXR) expression. We further showed that TKT translocated into the nucleus of HCC cell lines through interacting with the signal transducer and activator of transcription 1 (STAT1), and then the complex inhibited FXR expression by promoting the binding of histone deacetylase 3 (HDAC3) to FXR promoter.
Assuntos
Carcinoma Hepatocelular/genética , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Neoplasias Hepáticas/genética , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Transcetolase/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Fator de Transcrição STAT1/metabolismo , Transcetolase/deficiênciaRESUMO
Acquired resistance to tyrosine kinase inhibitors (TKIs) of epidermal growth factor receptor (EGFR) remains a clinical challenge. Especially challenging are cases in which resistance emerges through EGFR-independent mechanisms, such as through pathways that promote epithelial-to-mesenchymal transition (EMT). Through an integrated transcriptomic, proteomic, and drug screening approach, we identified activation of the yes-associated protein (YAP) and forkhead box protein M1 (FOXM1) axis as a driver of EMT-associated EGFR TKI resistance. EGFR inhibitor resistance was associated with broad multidrug resistance that extended across multiple chemotherapeutic and targeted agents, consistent with the difficulty of effectively treating resistant disease. EGFR TKI-resistant cells displayed increased abundance of spindle assembly checkpoint (SAC) proteins, including polo-like kinase 1 (PLK1), Aurora kinases, survivin, and kinesin spindle protein (KSP). Moreover, EGFR TKI-resistant cells exhibited vulnerability to SAC inhibitors. Increased activation of the YAP/FOXM1 axis mediated an increase in the abundance of SAC components in resistant cells. The clinical relevance of these finding was indicated by evaluation of specimens from patients with EGFR mutant lung cancer, which showed that high FOXM1 expression correlated with expression of genes encoding SAC proteins and was associated with a worse clinical outcome. These data revealed the YAP/FOXM1 axis as a central regulator of EMT-associated EGFR TKI resistance and that this pathway, along with SAC components, are therapeutic vulnerabilities for targeting this multidrug-resistant phenotype.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Receptores ErbB/genética , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/farmacologia , Proteína Forkhead Box M1/uso terapêutico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Pontos de Checagem da Fase M do Ciclo Celular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , ProteômicaRESUMO
PURPOSE: Despite a growing arsenal of approved drugs, therapeutic resistance remains a formidable and, often, insurmountable challenge in cancer treatment. The mechanisms underlying therapeutic resistance remain largely unresolved and, thus, examples of effective combinatorial or sequential strategies to combat resistance are rare. Here, we present Differential Sensitivity Analysis for Resistant Malignancies (DISARM), a novel, integrated drug screen analysis tool designed to address this dilemma. EXPERIMENTAL DESIGN: DISARM, a software package and web-based application, analyzes drug response data to prioritize candidate therapies for models with resistance to a reference drug and to assess whether response to a reference drug can be utilized to predict future response to other agents. Using cisplatin as our reference drug, we applied DISARM to models from nine cancers commonly treated with first-line platinum chemotherapy including recalcitrant malignancies such as small cell lung cancer (SCLC) and pancreatic adenocarcinoma (PAAD). RESULTS: In cisplatin-resistant models, DISARM identified novel candidates including multiple inhibitors of PI3K, MEK, and BCL-2, among other classes, across unrelated malignancies. Additionally, DISARM facilitated the selection of predictive biomarkers of response and identification of unique molecular subtypes, such as contrasting ASCL1-low/cMYC-high SCLC targetable by AURKA inhibitors and ASCL1-high/cMYC-low SCLC targetable by BCL-2 inhibitors. Utilizing these predictions, we assessed several of DISARM's top candidates, including inhibitors of AURKA, BCL-2, and HSP90, to confirm their activity in cisplatin-resistant SCLC models. CONCLUSIONS: DISARM represents the first validated tool to analyze large-scale in vitro drug response data to statistically optimize candidate drug and biomarker selection aimed at overcoming candidate drug resistance.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Antineoplásicos/uso terapêutico , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Cisplatino/efeitos adversos , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Platina/efeitos adversos , Platina/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , SoftwareRESUMO
PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is driven largely by the loss of tumor suppressor genes, including NOTCH1, but lacks a biomarker-driven targeted therapy. Although the PI3K/mTOR pathway is frequently altered in HNSCC, the disease has modest clinical response rates to PI3K/mTOR inhibitors and lacks validated biomarkers of response. We tested the hypothesis that an unbiased pharmacogenomics approach to PI3K/mTOR pathway inhibitors would identify novel, clinically relevant molecular vulnerabilities in HNSCC with loss of tumor suppressor function.Experimental Design: We assessed the degree to which responses to PI3K/mTOR inhibitors are associated with gene mutations in 59 HNSCC cell lines. Apoptosis in drug-sensitive cell lines was confirmed in vitro and in vivo. NOTCH1 pathway components and PDK1 were manipulated with drugs, gene editing, knockdown, and overexpression. RESULTS: PI3K/mTOR inhibition caused apoptosis and decreased colony numbers in HNSCC cell lines harboring NOTCH1 loss-of-function mutations (NOTCH1 MUT) and reduced tumor size in subcutaneous and orthotopic xenograft models. In all cell lines, NOTCH1 MUT was strongly associated with sensitivity to six PI3K/mTOR inhibitors. NOTCH1 inhibition or knockout increased NOTCH1 WT HNSCC sensitivity to PI3K/mTOR inhibition. PDK1 levels dropped following PI3K/mTOR inhibition in NOTCH1 MUT but not NOTCH1 WT HNSCC, and PDK1 overexpression rescued apoptosis in NOTCH1 MUT cells. PDK1 and AKT inhibitors together caused apoptosis in NOTCH1 WT HNSCC but had little effect as single agents. CONCLUSIONS: Our findings suggest that NOTCH1 MUT predicts response to PI3K/mTOR inhibitors, which may lead to the first biomarker-driven targeted therapy for HNSCC, and that targeting PDK1 sensitizes NOTCH1 WT HNSCC to PI3K/mTOR pathway inhibitors.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Receptor Notch1/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/etiologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Edição de Genes , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mutação com Perda de Função , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Glutathione (GSH)/GSH reductase (GSR) and thioredoxin/thioredoxin reductase (TXNRD) are two major compensating thiol-dependent antioxidant pathways that maintain protein dithiol/disulfide balance. We hypothesized that functional deficiency in one of these systems would render cells dependent on compensation by the other system for survival, providing a mechanism-based synthetic lethality approach for treatment of cancers. The human GSR gene is located on chromosome 8p12, a region frequently lost in human cancers. GSR deletion was detected in about 6% of lung adenocarcinomas in The Cancer Genome Atlas database. To test whether loss of GSR sensitizes cancer cells to TXNRD inhibition, we knocked out or knocked down the GSR gene in human lung cancer cells and evaluated their response to the TXNRD inhibitor auranofin. GSR deficiency sensitized lung cancer cells to this agent. Analysis of a panel of 129 non-small cell lung cancer (NSCLC) cell lines revealed that auranofin sensitivity correlated with the expression levels of the GSR, glutamate-cysteine ligase catalytic subunit (GCLC), and NAD(P)H quinone dehydrogenase 1 (NQO1) genes. In NSCLC patient-derived xenografts with reduced expression of GSR and/or GCLC, growth was significantly suppressed by treatment with auranofin. Together, these results provide a proof of concept that cancers with compromised expression of enzymes required for GSH homeostasis or with chromosome 8p deletions that include the GSR gene may be targeted by a synthetic lethality strategy with inhibitors of TXNRD. SIGNIFICANCE: These findings demonstrate that lung cancers with compromised expression of enzymes required for glutathione homeostasis, including reduced GSR gene expression, may be targeted by thioredoxin/thioredoxin reductase inhibitors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Glutamato-Cisteína Ligase/metabolismo , Glutationa Redutase/metabolismo , Glutationa/metabolismo , Mutações Sintéticas Letais , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Animais , Antirreumáticos/farmacologia , Auranofina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutationa Redutase/genética , Homeostase , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitors have failed to show clinical benefit in Kirsten rat sarcoma (KRAS) mutant lung cancer due to various resistance mechanisms. To identify differential therapeutic sensitivities between epithelial and mesenchymal lung tumors, we performed in vivo small hairpin RNA screens, proteomic profiling, and analysis of patient tumor datasets, which revealed an inverse correlation between mitogen-activated protein kinase (MAPK) signaling dependency and a zinc finger E-box binding homeobox 1 (ZEB1)-regulated epithelial-to-mesenchymal transition. Mechanistic studies determined that MAPK signaling dependency in epithelial lung cancer cells is due to the scaffold protein interleukin-17 receptor D (IL17RD), which is directly repressed by ZEB1. Lung tumors in multiple Kras mutant murine models with increased ZEB1 displayed low IL17RD expression, accompanied by MAPK-independent tumor growth and therapeutic resistance to MEK inhibition. Suppression of ZEB1 function with miR-200 expression or the histone deacetylase inhibitor mocetinostat sensitized resistant cancer cells to MEK inhibition and markedly reduced in vivo tumor growth, showing a promising combinatorial treatment strategy for KRAS mutant cancers. In human lung tumor samples, high ZEB1 and low IL17RD expression correlated with low MAPK signaling, presenting potential markers that predict patient response to MEK inhibitors.