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The cascade of immune responses involves activation of diverse immune cells and release of a large amount of cytokines, which leads to either normal, balanced inflammation or hyperinflammatory responses and even organ damage by sepsis. Conventional diagnosis of immunological disorders based on multiple cytokines in the blood serum has varied accuracy, and it is difficult to distinguish normal inflammation from sepsis. Herein, we present an approach to detect immunological disorders through rapid, ultrahigh-multiplex analysis of T cells using single-cell multiplex in situ tagging (scMIST) technology. scMIST permits simultaneous detection of 46 markers and cytokines from single cells without the assistance of special instruments. A cecal ligation and puncture sepsis model was built to supply T cells from two groups of mice that survived the surgery or died after 1 day. The scMIST assays have captured the T cell features and the dynamics over the course of recovery. Compared with cytokines in the peripheral blood, T cell markers show different dynamics and cytokine levels. We have applied a random forest machine learning model to single T cells from two groups of mice. Through training, the model has been able to predict the group of mice through T cell classification and majority rule with 94% accuracy. Our approach pioneers the direction of single-cell omics and could be widely applicable to human diseases.
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Enfermedades del Sistema Inmune , Sepsis , Humanos , Ratones , Animales , Citocinas , Inflamación , Linfocitos T , Sepsis/diagnóstico , Modelos Animales de EnfermedadRESUMEN
BackgroundImmune checkpoint inhibitors (ICIs) have modest activity in ovarian cancer (OC). To augment their activity, we used priming with the hypomethylating agent guadecitabine in a phase II study.MethodsEligible patients had platinum-resistant OC, normal organ function, measurable disease, and received up to 5 prior regimens. The treatment included guadecitabine (30 mg/m2) on days 1-4, and pembrolizumab (200 mg i.v.) on day 5, every 21 days. The primary endpoint was the response rate. Tumor biopsies, plasma, and PBMCs were obtained at baseline and after treatment.ResultsAmong 35 evaluable patients, 3 patients had partial responses (8.6%), and 8 (22.9%) patients had stable disease, resulting in a clinical benefit rate of 31.4% (95% CI: 16.9%-49.3%). The median duration of clinical benefit was 6.8 months. Long-interspersed element 1 (LINE1) was hypomethylated in post-treatment PBMCs, and methylomic and transcriptomic analyses showed activation of antitumor immunity in post-treatment biopsies. High-dimensional immune profiling of PBMCs showed a higher frequency of naive and/or central memory CD4+ T cells and of classical monocytes in patients with a durable clinical benefit or response (CBR). A higher baseline density of CD8+ T cells and CD20+ B cells and the presence of tertiary lymphoid structures in tumors were associated with a durable CBR.ConclusionEpigenetic priming using a hypomethylating agent with an ICI was feasible and resulted in a durable clinical benefit associated with immune responses in selected patients with recurrent OC.Trial registrationClinicalTrials.gov NCT02901899.FundingUS Army Medical Research and Material Command/Congressionally Directed Medical Research Programs (USAMRMC/CDMRP) grant W81XWH-17-0141; the Diana Princess of Wales Endowed Professorship and LCCTRAC funds from the Robert H. Lurie Comprehensive Cancer Center; Walter S. and Lucienne Driskill Immunotherapy Research funds; Astex Pharmaceuticals; Merck & Co.; National Cancer Institute (NCI), NIH grants CCSG P30 CA060553, CCSG P30 CA060553, and CA060553.
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Recurrencia Local de Neoplasia , Neoplasias Ováricas , Protocolos de Quimioterapia Combinada Antineoplásica , Epigénesis Genética , Epigenómica , Femenino , Humanos , Recurrencia Local de Neoplasia/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patologíaRESUMEN
How mammalian neuronal identity is progressively acquired and reinforced during development is not understood. We have previously shown that loss of RP58 (ZNF238 or ZBTB18), a BTB/POZ-zinc finger-containing transcription factor, in the mouse brain leads to microcephaly, corpus callosum agenesis, and cerebellum hypoplasia and that it is required for normal neuronal differentiation. The transcriptional programs regulated by RP58 during this process are not known. Here, we report for the first time that in embryonic mouse neocortical neurons a complex set of genes normally expressed in other cell types, such as those from mesoderm derivatives, must be actively repressed in vivo and that RP58 is a critical regulator of these repressed transcriptional programs. Importantly, gene set enrichment analysis (GSEA) analyses of these transcriptional programs indicate that repressed genes include distinct sets of genes significantly associated with glioma progression and/or pluripotency. We also demonstrate that reintroducing RP58 in glioma stem cells leads not only to aspects of neuronal differentiation but also to loss of stem cell characteristics, including loss of stem cell markers and decrease in stem cell self-renewal capacities. Thus, RP58 acts as an in vivo master guardian of the neuronal identity transcriptome, and its function may be required to prevent brain disease development, including glioma progression.
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Regulación del Desarrollo de la Expresión Génica/fisiología , Glioblastoma/metabolismo , Neuronas/metabolismo , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular/genética , Movimiento Celular/genética , Ratones , Neurogénesis/fisiología , Neuroglía/metabolismo , Proteínas Represoras/genéticaRESUMEN
Glioblastoma (GBM) is one of the most difficult cancers to effectively treat, in part because of the lack of precision therapies and limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain and blood-tumor barriers. We have developed a precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference-based spherical nucleic acids (SNAs), which consist of gold nanoparticle cores covalently conjugated with radially oriented and densely packed small interfering RNA (siRNA) oligonucleotides. On the basis of previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in nonhuman primates and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event level, followed by tumor resection. Safety assessment revealed no grade 4 or 5 treatment-related toxicities. Inductively coupled plasma mass spectrometry, x-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold enrichment observed in the tumor-associated endothelium, macrophages, and tumor cells. NU-0129 uptake into glioma cells correlated with a reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of matched primary tumor and NU-0129-treated recurrent tumor. Our results establish SNA nanoconjugates as a potential brain-penetrant precision medicine approach for the systemic treatment of GBM.
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Neoplasias Encefálicas , Glioblastoma , Nanopartículas del Metal , Ácidos Nucleicos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioblastoma/genética , Glioblastoma/terapia , Oro , Humanos , Proteínas Musculares/metabolismo , Recurrencia Local de Neoplasia , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARNRESUMEN
The tumor suppressor p53 integrates stress response pathways by selectively engaging one of several potential transcriptomes, thereby triggering cell fate decisions (e.g., cell cycle arrest, apoptosis). Foundational to this process is the binding of tetrameric p53 to 20-bp response elements (REs) in the genome (RRRCWWGYYYN0-13RRRCWWGYYY). In general, REs at cell cycle arrest targets (e.g. p21) are of higher affinity than those at apoptosis targets (e.g., BAX). However, the RE sequence code underlying selectivity remains undeciphered. Here, we identify molecular mechanisms mediating p53 binding to high- and low-affinity REs by showing that key determinants of the code are embedded in the DNA shape. We further demonstrate that differences in minor/major groove widths, encoded by G/C or A/T bp content at positions 3, 8, 13, and 18 in the RE, determine distinct p53 DNA-binding modes by inducing different Arg248 and Lys120 conformations and interactions. The predictive capacity of this code was confirmed in vivo using genome editing at the BAX RE to interconvert the DNA-binding modes, transcription pattern, and cell fate outcome.
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Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/genética , Ciclo Celular , Puntos de Control del Ciclo Celular , Línea Celular , ADN/química , Proteínas de Unión al ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica/genética , Elementos de RespuestaRESUMEN
Defining traits of platinum-tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum-tolerant cells and tumors were identified using in vitro and in vivo ovarian cancer models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were enriched in ALDH+ cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared with parental cells. Additionally, platinum-tolerant cells and tumors exhibited expression of the Wnt receptor Frizzled-7 (FZD7). Knockdown of FZD7 improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7+ from FZD7- cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation-enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protected cells from chemotherapy-induced oxidative stress. FZD7+ platinum-tolerant ovarian cancer cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63, and glutathione metabolism gene sets were strongly correlated in the ovarian cancer Tumor Cancer Genome Atlas (TCGA) database and in residual human ovarian cancer specimens after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial cancer stem cell features, characterized by FZD7 expression and dependent on the FZD7-ß-catenin-Tp63-GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum-tolerant cancer cells and provide new insight into a potential "persister cancer cell" phenotype. SIGNIFICANCE: Frizzled-7 marks platinum-tolerant cancer cells harboring stemness features and altered glutathione metabolism that depend on GPX4 for survival and are highly susceptible to ferroptosis.
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Biomarcadores de Tumor/metabolismo , Cisplatino/farmacología , Resistencia a Antineoplásicos , Ferroptosis , Receptores Frizzled/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Femenino , Receptores Frizzled/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
N 6-Methyladenosine (m6A) is the most abundant modification of mammalian mRNAs. RNA methylation fine tunes RNA stability and translation, altering cell fate. The fat mass- and obesity-associated protein (FTO) is an m6A demethylase with oncogenic properties in leukemia. Here, we show that FTO expression is suppressed in ovarian tumors and cancer stem cells (CSC). FTO inhibited the self-renewal of ovarian CSC and suppressed tumorigenesis in vivo, both of which required FTO demethylase activity. Integrative RNA sequencing and m6A mapping analysis revealed significant transcriptomic changes associated with FTO overexpression and m6A loss involving stem cell signaling, RNA transcription, and mRNA splicing pathways. By reducing m6A levels at the 3'UTR and the mRNA stability of two phosphodiesterase genes (PDE1C and PDE4B), FTO augmented second messenger 3', 5'-cyclic adenosine monophosphate (cAMP) signaling and suppressed stemness features of ovarian cancer cells. Our results reveal a previously unappreciated tumor suppressor function of FTO in ovarian CSC mediated through inhibition of cAMP signaling. SIGNIFICANCE: A new tumor suppressor function of the RNA demethylase FTO implicates m6A RNA modifications in the regulation of cyclic AMP signaling involved in stemness and tumor initiation.
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Adenosina/análogos & derivados , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Células Madre Neoplásicas/metabolismo , Neoplasias Ováricas/metabolismo , Sistemas de Mensajero Secundario , Proteínas Supresoras de Tumor/metabolismo , Regiones no Traducidas 3'/genética , Adenosina/genética , Adenosina/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Empalme Alternativo , Animales , Ascitis/metabolismo , Carcinogénesis/metabolismo , Línea Celular Tumoral , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Regulación hacia Abajo , Trompas Uterinas/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Ováricas/patología , Ovario/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Análisis de Secuencia de ARN , Esferoides Celulares , Análisis de Matrices Tisulares , Transcriptoma , Proteínas Supresoras de Tumor/genéticaRESUMEN
Identifying and evaluating the right target are the most important factors in early drug discovery phase. Most studies focus on one protein ignoring the multiple splice-variant or protein-isoforms, which might contribute to unexpected therapeutic activity or adverse side effects. Here, we present computational analysis of cancer drug-target interactions affected by alternative splicing. By integrating information from publicly available databases, we curated 883 FDA approved or investigational stage small molecule cancer drugs that target 1,434 different genes, with an average of 5.22 protein isoforms per gene. Of these, 618 genes have ≥5 annotated protein-isoforms. By analyzing the interactions with binding pocket information, we found that 76% of drugs either miss a potential target isoform or target other isoforms with varied expression in multiple normal tissues. We present sequence and structure level alignments at isoform-level and make this information publicly available for all the curated drugs. Structure-level analysis showed ligand binding pocket architectures differences in size, shape and electrostatic parameters between isoforms. Our results emphasize how potentially important isoform-level interactions could be missed by solely focusing on the canonical isoform, and suggest that on- and off-target effects at isoform-level should be investigated to enhance the productivity of drug-discovery research.
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Empalme Alternativo , Antineoplásicos/metabolismo , Biología Computacional , Simulación por Computador , Terapia Molecular Dirigida , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Perfilación de la Expresión Génica , Modelos Moleculares , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alineación de SecuenciaRESUMEN
Recent work has highlighted the tumor microenvironment as a central player in cancer. In particular, interactions between tumor and immune cells may help drive the development of brain tumors such as glioblastoma multiforme (GBM). Despite significant research into the molecular classification of glioblastoma, few studies have characterized in a comprehensive manner the immune infiltrate in situ and within different GBM subtypes.In this study, we use an unbiased, automated immunohistochemistry-based approach to determine the immune phenotype of the four GBM subtypes (classical, mesenchymal, neural and proneural) in a cohort of 98 patients. Tissue Micro Arrays (TMA) were stained for CD20 (B lymphocytes), CD5, CD3, CD4, CD8 (T lymphocytes), CD68 (microglia), and CD163 (bone marrow derived macrophages) antibodies. Using automated image analysis, the percentage of each immune population was calculated with respect to the total tumor cells. Mesenchymal GBMs displayed the highest percentage of microglia, macrophage, and lymphocyte infiltration. CD68+ and CD163+ cells were the most abundant cell populations in all four GBM subtypes, and a higher percentage of CD163+ cells was associated with a worse prognosis. We also compared our results to the relative composition of immune cell type infiltration (using RNA-seq data) across TCGA GBM tumors and validated our results obtained with immunohistochemistry with an external cohort and a different method. The results of this study offer a comprehensive analysis of the distribution and the infiltration of the immune components across the four commonly described GBM subgroups, setting the basis for a more detailed patient classification and new insights that may be used to better apply or design immunotherapies for GBM.
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Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Inmunidad Celular/inmunología , Microambiente Tumoral/inmunología , Antígenos CD20/análisis , Antígenos CD20/inmunología , Neoplasias Encefálicas/patología , Glioblastoma/patología , HumanosRESUMEN
BACKGROUND: In cystic fibrosis (CF), impaired immune cell responses, driven by the dysfunctional CF transmembrane conductance regulator (CFTR) gene, may determine the disease severity but clinical heterogeneity remains a major therapeutic challenge. The characterization of molecular mechanisms underlying impaired immune responses in CF may reveal novel targets with therapeutic potential. Therefore, we utilized simultaneous RNA sequencing targeted at identifying differentially expressed genes, transcripts, and miRNAs that characterize impaired immune responses triggered by CF and its phenotypes. METHODS: Peripheral blood mononuclear cells (PBMCs) extracted from a healthy donor were stimulated with plasma from CF patients (n = 9) and healthy controls (n = 3). The PBMCs were cultured (1 × 105 cells/well) for 9 h at 37 ° C in 5% CO2. After culture, total RNA was extracted from each sample and used for simultaneous total RNA and miRNA sequencing. RESULTS: Analysis of expression signatures from peripheral blood mononuclear cells induced by plasma of CF patients and healthy controls identified 151 genes, 154 individual transcripts, and 41 miRNAs differentially expressed in CF compared to HC while the expression signatures of 285 genes, 241 individual transcripts, and seven miRNAs differed due to CF phenotypes. Top immune pathways influenced by CF included agranulocyte adhesion, diapedesis signaling, and IL17 signaling, while those influenced by CF phenotypes included natural killer cell signaling and PI3K signaling in B lymphocytes. Upstream regulator analysis indicated dysregulation of CCL5, NF-κB and IL1A due to CF while dysregulation of TREM1 and TP53 regulators were associated with CF phenotype. Five miRNAs showed inverse expression patterns with three target genes relevant in CF-associated impaired immune pathways while two miRNAs showed inverse expression patterns with two target genes relevant to a dysregulated immune pathway associated with CF phenotypes. CONCLUSIONS: Our results indicate that miRNAs and individual transcript variants are relevant molecular targets contributing to impaired immune cell responses in CF.
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Fibrosis Quística/genética , Fibrosis Quística/inmunología , Análisis de Secuencia de ARN , Transcripción Genética/inmunología , Adolescente , Estudios de Casos y Controles , Niño , Fibrosis Quística/sangre , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , FenotipoRESUMEN
PURPOSE: Molecular cancer subtyping is an important tool in predicting prognosis and developing novel precision medicine approaches. We developed a novel platform-independent gene expression-based classification system for molecular subtyping of patients with high-grade serous ovarian carcinoma (HGSOC). METHODS: Unprocessed exon array (569 tumor and nine normal) and RNA sequencing (RNA-seq; 376 tumor) HGSOC data sets, with clinical annotations, were downloaded from the Genomic Data Commons portal. Sample clustering was performed by non-negative matrix factorization by using isoform-level expression estimates. The association between the subtypes and overall survival was evaluated by Cox proportional hazards regression model after adjusting for the covariates. A novel classification system was developed for HGSOC molecular subtyping. Robustness and generalizability of the gene signatures were validated using independent microarray and RNA-seq data sets. RESULTS: Sample clustering recaptured the four known The Cancer Genome Atlas molecular subtypes but switched the subtype for 22% of the cases, which resulted in significant (P = .006) survival differences among the refined subgroups. After adjusting for covariate effects, the mesenchymal subgroup was found to be at an increased hazard for death compared with the immunoreactive subgroup. Both gene- and isoform-level signatures achieved more than 92% prediction accuracy when tested on independent samples profiled on the exon array platform. When the classifier was applied to RNA-seq data, the subtyping calls agreed with the predictions made from exon array data for 95% of the 279 samples profiled by both platforms. CONCLUSION: Isoform-level expression analysis successfully stratifies patients with HGSOC into groups with differing prognosis and has led to the development of robust, platform-independent gene signatures for HGSOC molecular subtyping. The association of the refined The Cancer Genome Atlas HGSOC subtypes with overall survival, independent of covariates, enhances the clinical annotation of the HGSOC cohort.
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Biomarcadores de Tumor , Biología Computacional/métodos , Perfilación de la Expresión Génica , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/genética , Algoritmos , Análisis por Conglomerados , Cistadenocarcinoma Seroso/diagnóstico , Cistadenocarcinoma Seroso/etiología , Cistadenocarcinoma Seroso/mortalidad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Clasificación del Tumor , Neoplasias Ováricas/mortalidad , Pronóstico , Modelos de Riesgos ProporcionalesRESUMEN
Mutation or transcriptional up-regulation of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) promotes cancer progression through metabolic reprogramming and epigenetic deregulation of gene expression. Here, we demonstrate that IDH3α, a subunit of the IDH3 heterotetramer, is elevated in glioblastoma (GBM) patient samples compared to normal brain tissue and promotes GBM progression in orthotopic glioma mouse models. IDH3α loss of function reduces tricarboxylic acid (TCA) cycle turnover and inhibits oxidative phosphorylation. In addition to its impact on mitochondrial energy metabolism, IDH3α binds to cytosolic serine hydroxymethyltransferase (cSHMT). This interaction enhances nucleotide availability during DNA replication, while the absence of IDH3α promotes methionine cycle activity, S-adenosyl methionine generation, and DNA methylation. Thus, the regulation of one-carbon metabolism via an IDH3α-cSHMT signaling axis represents a novel mechanism of metabolic adaptation in GBM.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glicina Hidroximetiltransferasa/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Ciclo del Ácido Cítrico/genética , Citosol/metabolismo , Metilación de ADN/genética , Femenino , Glioblastoma/genética , Células HEK293 , Xenoinjertos , Humanos , Isocitrato Deshidrogenasa/genética , Ratones , Ratones SCID , Fosforilación Oxidativa , Puntos de Control de la Fase S del Ciclo Celular , TransfecciónRESUMEN
Glioblastoma, the most frequent primary malignant brain neoplasm, is genetically diverse and classified into four transcriptomic subtypes, i. e., classical, mesenchymal, proneural, and neural. Currently, detection of transcriptomic subtype is based on ex vivo analysis of tissue that does not capture the spatial tumor heterogeneity. In view of accumulative evidence of in vivo imaging signatures summarizing molecular features of cancer, this study seeks robust non-invasive radiographic markers of transcriptomic classification of glioblastoma, based solely on routine clinically-acquired imaging sequences. A pre-operative retrospective cohort of 112 pathology-proven de novo glioblastoma patients, having multi-parametric MRI (T1, T1-Gd, T2, T2-FLAIR), collected from the Hospital of the University of Pennsylvania were included. Following tumor segmentation into distinct radiographic sub-regions, diverse imaging features were extracted and support vector machines were employed to multivariately integrate these features and derive an imaging signature of transcriptomic subtype. Extracted features included intensity distributions, volume, morphology, statistics, tumors' anatomical location, and texture descriptors for each tumor sub-region. The derived signature was evaluated against the transcriptomic subtype of surgically-resected tissue specimens, using a 5-fold cross-validation method and a receiver-operating-characteristics analysis. The proposed model was 71% accurate in distinguishing among the four transcriptomic subtypes. The accuracy (sensitivity/specificity) for distinguishing each subtype (classical, mesenchymal, proneural, neural) from the rest was equal to 88.4% (71.4/92.3), 75.9% (83.9/72.8), 82.1% (73.1/84.9), and 75.9% (79.4/74.4), respectively. The findings were also replicated in The Cancer Genomic Atlas glioblastoma dataset. The obtained imaging signature for the classical subtype was dominated by associations with features related to edge sharpness, whereas for the mesenchymal subtype had more pronounced presence of higher T2 and T2-FLAIR signal in edema, and higher volume of enhancing tumor and edema. The proneural and neural subtypes were characterized by the lower T1-Gd signal in enhancing tumor and higher T2-FLAIR signal in edema, respectively. Our results indicate that quantitative multivariate analysis of features extracted from clinically-acquired MRI may provide a radiographic biomarker of the transcriptomic profile of glioblastoma. Importantly our findings can be influential in surgical decision-making, treatment planning, and assessment of inoperable tumors.
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Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-ß cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.
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Caquexia/metabolismo , Caquexia/patología , Proteínas de Transporte de Catión/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Neoplasias/metabolismo , Neoplasias/patología , Regulación hacia Arriba , Animales , Diferenciación Celular , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones Endogámicos C57BL , Cadenas Pesadas de Miosina/metabolismo , Metástasis de la Neoplasia , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Zinc/metabolismoRESUMEN
The remarkable heterogeneity of glioblastoma, across patients and over time, is one of the main challenges in precision diagnostics and treatment planning. Non-invasive in vivo characterization of this heterogeneity using imaging could assist in understanding disease subtypes, as well as in risk-stratification and treatment planning of glioblastoma. The current study leveraged advanced imaging analytics and radiomic approaches applied to multi-parametric MRI of de novo glioblastoma patients (n = 208 discovery, n = 53 replication), and discovered three distinct and reproducible imaging subtypes of glioblastoma, with differential clinical outcome and underlying molecular characteristics, including isocitrate dehydrogenase-1 (IDH1), O6-methylguanine-DNA methyltransferase, epidermal growth factor receptor variant III (EGFRvIII), and transcriptomic subtype composition. The subtypes provided risk-stratification substantially beyond that provided by WHO classifications. Within IDH1-wildtype tumors, our subtypes revealed different survival (p < 0.001), thereby highlighting the synergistic consideration of molecular and imaging measures for prognostication. Moreover, the imaging characteristics suggest that subtype-specific treatment of peritumoral infiltrated brain tissue might be more effective than current uniform standard-of-care. Finally, our analysis found subtype-specific radiogenomic signatures of EGFRvIII-mutated tumors. The identified subtypes and their clinical and molecular correlates provide an in vivo portrait of phenotypic heterogeneity in glioblastoma, which points to the need for precision diagnostics and personalized treatment.
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Neoplasias Encefálicas/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Glioblastoma/diagnóstico por imagen , Isocitrato Deshidrogenasa/análisis , Imagen por Resonancia Magnética/métodos , Receptores ErbB/análisis , Femenino , Humanos , Masculino , O(6)-Metilguanina-ADN Metiltransferasa/análisis , Pronóstico , Análisis de SupervivenciaRESUMEN
CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC's orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.
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In the originally published version of this Article, the positions of the final two authors in the author list were inadvertently inverted during the production process. This error has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
The transcribed ultraconserved regions (T-UCRs) encode long non-coding RNAs implicated in human carcinogenesis. Their mechanisms of action and the factors regulating their expression in cancers are poorly understood. Here we show that high expression of uc.339 correlates with lower survival in 210 non-small cell lung cancer (NSCLC) patients. We provide evidence from cell lines and primary samples that TP53 directly regulates uc.339. We find that transcribed uc.339 is upregulated in archival NSCLC samples, functioning as a decoy RNA for miR-339-3p, -663b-3p, and -95-5p. As a result, Cyclin E2, a direct target of all these microRNAs is upregulated, promoting cancer growth and migration. Finally, we find that modulation of uc.339 affects microRNA expression. However, overexpression or downregulation of these microRNAs causes no significant variations in uc.339 levels, suggesting a type of interaction for uc.339 that we call "entrapping". Our results support a key role for uc.339 in lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Secuencia Conservada/genética , Neoplasias Pulmonares/genética , MicroARNs/genética , ARN Largo no Codificante/metabolismo , Animales , Secuencia de Bases/genética , Carcinogénesis/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Ciclinas/genética , Ciclinas/metabolismo , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenic mutations in two isocitrate dehydrogenase (IDH)-encoding genes (IDH1 and IDH2) have been identified in acute myelogenous leukemia, low-grade glioma, and secondary glioblastoma (GBM). Our in silico and wet-bench analyses indicate that non-mutated IDH1 mRNA and protein are commonly overexpressed in primary GBMs. We show that genetic and pharmacologic inactivation of IDH1 decreases GBM cell growth, promotes a more differentiated tumor cell state, increases apoptosis in response to targeted therapies, and prolongs the survival of animal subjects bearing patient-derived xenografts (PDXs). On a molecular level, diminished IDH1 activity results in reduced α-ketoglutarate (αKG) and NADPH production, paralleled by deficient carbon flux from glucose or acetate into lipids, exhaustion of reduced glutathione, increased levels of reactive oxygen species (ROS), and enhanced histone methylation and differentiation marker expression. These findings suggest that IDH1 upregulation represents a common metabolic adaptation by GBMs to support macromolecular synthesis, aggressive growth, and therapy resistance.
Asunto(s)
Resistencia a Antineoplásicos , Glioblastoma/enzimología , Glioblastoma/patología , Isocitrato Deshidrogenasa/genética , Terapia Molecular Dirigida , Mutación/genética , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Clorhidrato de Erlotinib/farmacología , Clorhidrato de Erlotinib/uso terapéutico , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Histonas/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Lípidos/biosíntesis , Metilación , Ratones , Ratones SCID , NADP/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice's brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients' PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.