RESUMEN
The C9ORF72-linked diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by the nuclear depletion and cytoplasmic accumulation of TAR DNA-binding protein 43 (TDP-43). Recent studies have shown that the loss of TDP-43 function leads to the inclusion of cryptic exons (CE) in several RNA transcript targets of TDP-43. Here, we show for the first time the detection of CEs in a single-nuclei RNA sequencing (snRNA-seq) dataset obtained from frontal and occipital cortices of C9ORF72 patients that phenotypically span the ALS-FTD disease spectrum. We assessed each cellular cluster for detection of recently described TDP-43-induced CEs. Transcripts containing CEs in the genes STMN2 and KALRN were detected in the frontal cortex of all C9ORF72 disease groups with the highest frequency in excitatory neurons in the C9ORF72-FTD group. Within the excitatory neurons, the cluster with the highest proportion of cells containing a CE had transcriptomic similarities to von Economo neurons, which are known to be vulnerable to TDP-43 pathology and selectively lost in C9ORF72-FTD. Differential gene expression and pathway analysis of CE-containing neurons revealed multiple dysregulated metabolic processes. Our findings reveal novel insights into the transcriptomic changes of neurons vulnerable to TDP-43 pathology.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Enfermedad de Pick , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Transcriptoma , Enfermedad de Pick/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exones , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Adrenocortical carcinoma (ACC) is a rare tumor with variable prognosis even within the same tumor stage. Cancer-related sex hormones and their sulfated metabolites in body fluids can be used as tumor markers. The role of steroid sulfation in ACC has not yet been studied. MALDI mass spectrometry imaging (MALDI-MSI) is a novel tool for tissue-based chemical phenotyping. METHODS: We performed phenotyping of formalin-fixed, paraffin-embedded tissue samples from 72 ACC by MALDI-MSI at a metabolomics level. RESULTS: Tumoral steroid hormone metabolites-estradiol sulfate [hazard ratio (HR) 0.26; 95% CI, 0.10-0.69; P = 0.005] and estrone 3-sulfate (HR 0.22; 95% CI, 0.07-0.63; P = 0.003)-were significantly associated with prognosis in Kaplan-Meier analyses and after multivariable adjustment for age, tumor stage, and sex (HR 0.29; 95% CI, 0.11-0.79; P = 0.015 and HR 0.30; 95% CI, 0.10-0.91; P = 0.033, respectively). Expression of sulfotransferase SULT2A1 was associated with prognosis to a similar extent and was validated to be a prognostic factor in two published data sets. We discovered the presence of estradiol-17ß 3,17-disulfate (E2S2) in a subset of tumors with particularly poor overall survival. Electron microscopy revealed novel membrane-delimited organelles in only these tumors. By applying cluster analyses of metabolomic data, 3 sulfation-related phenotypes exhibited specific metabolic features unrelated to steroid metabolism. CONCLUSIONS: MALDI-MSI provides novel insights into the pathophysiology of ACC. Steroid hormone sulfation may be used for prognostication and treatment stratification. Sulfation-related metabolic reprogramming may be of relevance also in conditions beyond the rare ACC and can be directly investigated by the use of MALDI-MSI.
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Neoplasias de la Corteza Suprarrenal/mortalidad , Carcinoma Corticosuprarrenal/mortalidad , Biomarcadores de Tumor/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Esteroides/análisis , Adolescente , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/patología , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Niño , Estradiol/análogos & derivados , Estradiol/análisis , Estradiol/metabolismo , Estrona/análogos & derivados , Estrona/análisis , Estrona/metabolismo , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Esteroides/metabolismo , Sulfotransferasas/metabolismo , Adulto JovenRESUMEN
BACKGROUND: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a poor prognosis and few therapeutic options. Stathmin1 (STMN1) is a cytosolic protein involved in microtubule dynamics through inhibition of tubulin polymerization and promotion of microtubule depolymerization, which has been implicated in carcinogenesis and aggressive behavior in multiple epithelial malignancies. We aimed to evaluate expression of STMN1 in ACC and to elucidate how this may contribute to its malignant phenotype. METHODS: STMN1 was identified by RNA sequencing as a highly differentially expressed gene in human ACC samples compared with benign adrenal tumors. Expression was confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical (IHC) staining of a tissue microarray (TMA) from two independent cohorts. The biologic relevance of STMN1 was investigated in NCI-H295R cells by lentivirus-mediated silencing. RESULTS: Differential gene expression demonstrated an eightfold increase in STMN1 messenger RNA (mRNA) in malignant compared with benign adrenal tissue. IHC showed significantly higher expression of STMN1 protein in ACC compared with normal and benign tissues. STMN1 knockdown in an ACC cell line resulted in decreased cell viability, cell-cycle arrest at G0/G1, and increased apoptosis in serum-starved conditions compared with scramble short hairpin RNA (shRNA) controls. STMN1 knockdown also decreased migration, invasion, and anchorage-independent growth compared with controls. CONCLUSIONS: STMN1 is overexpressed in human ACC samples, and knockdown of this target in vitro resulted in a less aggressive phenotype of ACC, particularly under serum-starved conditions. Further study is needed to investigate the feasibility of interfering with STMN1 as a potential therapeutic target.
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Neoplasias de la Corteza Suprarrenal/patología , Carcinoma Corticosuprarrenal/patología , Biomarcadores de Tumor/metabolismo , Estatmina/metabolismo , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/cirugía , Adrenalectomía , Carcinoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/cirugía , Apoptosis , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Técnicas In Vitro , Persona de Mediana Edad , Pronóstico , Estatmina/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Adrenocortical carcinoma (ACC) has a poor prognosis and there is an unmet clinical need for biomarkers to improve both diagnostic and prognostic assessment. Pituitary-tumor transforming gene (PTTG1) has been shown to modulate cancer invasiveness and response to therapy. The potential role of PTTG1 protein levels in ACC has not been previously addressed. We assessed whether increased nuclear protein expression of PTTG1 distinguished ACCs from adrenocortical adenomas (ACAs). METHODS: Patients with ACC or ACA were identified from prospective tissue banks at two independent institutions. Two tissue microarrays (TMAs) consisting of adrenal specimens from 131 patients were constructed and clinically annotated. Immunohistochemical analysis for PTTG1 and Ki-67 was performed on each TMA. RESULTS: TMA-1 (n = 80) contained 20 normal adrenals, 20 ACAs, and 40 ACCs, and the validation, TMA-2 (n = 51), consisted of 10 normal adrenals, 14 ACAs, and 27 ACCs. On TMA-1, nuclear staining of PTTG1 was detected in 12 (31%) ACC specimens, while all ACAs and normal adrenal glands were negative for PTTG1. On TMA-2, 20 (74%) of the ACC tumors demonstrated PTTG1 nuclear staining of PTTG1, and 13 (93%) ACA and 4 (44%) normal adrenal glands were negative for PTTG1. ACC tumors with increased PTTG1 protein staining had a significantly higher Ki-67 index (p < 0.001) than those with lower levels of PTTG1. CONCLUSIONS: Increased nuclear protein expression of PTTG1 was observed in malignant adrenal tumors. PTTG1 correlated with Ki-67 in two independent TMAs. PTTG1 is a promising biologic marker in the evaluation of adrenal tumors.
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Neoplasias de la Corteza Suprarrenal/diagnóstico , Glándulas Suprarrenales/patología , Adenoma Corticosuprarrenal/diagnóstico , Carcinoma Corticosuprarrenal/diagnóstico , Biomarcadores de Tumor/metabolismo , Securina/metabolismo , Neoplasias de la Corteza Suprarrenal/metabolismo , Glándulas Suprarrenales/metabolismo , Adenoma Corticosuprarrenal/metabolismo , Carcinoma Corticosuprarrenal/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia , Adulto JovenRESUMEN
DLC1 encodes a RhoA GTPase-activating protein and tumor suppressor lost in cancer by genomic deletion or epigenetic silencing and loss of DLC1 gene transcription. We unexpectedly identified non-small cell lung cancer (NSCLC) cell lines and tumor tissue that expressed DLC1 mRNA yet lacked DLC1 protein expression. We determined that DLC1 was ubiquitinated and degraded by cullin 4A-RING ubiquitin ligase (CRL4A) complex interaction with DDB1 and the FBXW5 substrate receptor. siRNA-mediated suppression of cullin 4A, DDB1, or FBXW5 expression restored DLC1 protein expression in NSCLC cell lines. FBXW5 suppression-induced DLC1 reexpression was associated with a reduction in the levels of activated RhoA-GTP and in RhoA effector signaling. Finally, FBXW5 suppression caused a DLC1-dependent decrease in NSCLC anchorage-dependent and -independent proliferation. In summary, we identify a posttranslational mechanism for loss of DLC1 and a linkage between CRL4A-FBXW5-associated oncogenesis and regulation of RhoA signaling.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Cullin/metabolismo , Proteínas F-Box/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Neoplasias Pulmonares/metabolismo , Proteolisis , Proteínas Supresoras de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proteínas Cullin/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/genética , Proteínas Activadoras de GTPasa/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitinación/genéticaRESUMEN
The long-term survival of patients with glioblastoma is compromised by the proclivity for local invasion into the surrounding normal brain, escaping surgical resection and contributing to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, can stimulate glioma cell invasion via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the Rho guanosine triphosphatase family member Rac1. Here, we demonstrate that TWEAK acts as a chemotactic factor for glioma cells, a potential process for driving cell invasion into the surrounding brain tissue. TWEAK exposure induced the activation of Src family kinases (SFKs), and pharmacologic suppression of SFK activity inhibited TWEAK-induced chemotactic migration. We employed a multiplexed Luminex assay and identified Lyn as a candidate SFK activated by TWEAK. Depletion of Lyn suppressed TWEAK-induced chemotaxis and Rac1 activity. Furthermore, Lyn gene expression levels increase with primary glioma tumor grade and inversely correlate with patient survival. These results show that TWEAK-induced glioma cell chemotaxis is dependent upon Lyn kinase function and, thus, provides opportunities for therapeutic targeting of this deadly disease.
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Neoplasias Encefálicas/patología , Quimiotaxis/fisiología , Glioblastoma/patología , Factores de Necrosis Tumoral/metabolismo , Familia-src Quinasas/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Movimiento Celular , Citocina TWEAK , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Ratas Wistar , Factores de Necrosis Tumoral/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/genéticaRESUMEN
Alzheimer's disease (AD), due to its multifactorial nature and complex etiology, poses challenges for research, diagnosis, and treatment, and impacts millions worldwide. To address the need for minimally invasive, repeatable measures that aid in AD diagnosis and progression monitoring, studies leveraging RNAs associated with extracellular vesicles (EVs) in human biofluids have revealed AD-associated changes. However, the validation of AD biomarkers has suffered from the collection of samples from differing points in the disease time course or a lack of confirmed AD diagnoses. Here, we integrate clinical diagnosis and postmortem pathology data to form more accurate experimental groups and use small RNA sequencing to show that EVs from plasma can serve as a potential source of RNAs that reflect disease-related changes. Importantly, we demonstrated that these changes are identifiable in the EVs of preclinical patients, years before symptom manifestation, and that machine learning models based on differentially expressed RNAs can help predict disease conversion or progression. This research offers critical insight into early disease biomarkers and underscores the significance of accounting for disease progression and pathology in human AD studies.
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Enfermedad de Alzheimer , Vesículas Extracelulares , Humanos , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Encéfalo/patología , Vesículas Extracelulares/patología , Diagnóstico Precoz , BiomarcadoresRESUMEN
Lung cancer is the leading cause of cancer deaths worldwide; approximately 85% of these cancers are non-small cell lung cancer (NSCLC). Patients with NSCLC frequently have tumors harboring somatic mutations in the epidermal growth factor receptor (EGFR) gene that cause constitutive receptor activation. These patients have the best clinical response to EGFR tyrosine kinase inhibitors (TKIs). Herein, we show that fibroblast growth factor-inducible 14 (Fn14; TNFRSF12A) is frequently overexpressed in NSCLC tumors, and Fn14 levels correlate with p-EGFR expression. We also report that NSCLC cell lines that contain EGFR-activating mutations show high levels of Fn14 protein expression. EGFR TKI treatment of EGFR-mutant HCC827 cells decreased Fn14 protein levels, whereas EGF stimulation of EGFR wild-type A549 cells transiently increased Fn14 expression. Furthermore, Fn14 is highly expressed in EGFR-mutant H1975 cells that also contain an EGFR TKI-resistance mutation, and high TKI doses are necessary to reduce Fn14 levels. Constructs encoding EGFRs with activating mutations induced Fn14 expression when expressed in rat lung epithelial cells. We also report that short hairpin RNA-mediated Fn14 knockdown reduced NSCLC cell migration and invasion in vitro. Finally, Fn14 overexpression enhanced NSCLC cell migration and invasion in vitro and increased experimental lung metastases in vivo. Thus, Fn14 may be a novel therapeutic target for patients with NSCLC, in particular for those with EGFR-driven tumors who have either primary or acquired resistance to EGFR TKIs.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Receptores del Factor de Necrosis Tumoral/fisiología , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/secundario , Movimiento Celular/fisiología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Clorhidrato de Erlotinib , Técnicas de Silenciamiento del Gen , Genes ras/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones SCID , Mutación , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Trasplante de Neoplasias , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Ratas , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Transducción de Señal/fisiología , Receptor de TWEAK , Células Tumorales CultivadasRESUMEN
A detailed understanding of the molecular and immunological changes that occur longitudinally across tumors exposed to immune checkpoint inhibitors is a significant knowledge gap in oncology. To address this unmet need, we created a statewide biospecimen collection and clinical informatics system to enable longitudinal tumor and immune profiling and to enhance translational research. The Texas Immuno-Oncology Biorepository (TIOB) consents patients to collect, process, store, and analyze serial biospecimens of tissue, blood, urine, and stool from a diverse population of over 100,000 cancer patients treated each year across the Baylor Scott & White Health system. Here we sought to demonstrate that these samples were fit for purpose with regard to downstream multi-omic assays. Plasma, urine, peripheral blood mononuclear cells, and stool samples from 11 enrolled patients were collected from various cancer types. RNA isolated from extracellular vesicles derived from plasma and urine was sufficient for transcriptomics. Peripheral blood mononuclear cells demonstrated excellent yield and viability. Ten of 11 stool samples produced RNA quality to enable microbiome characterization. Sample acquisition and processing methods are known to impact sample quality and performance. We demonstrate that consistent acquisition methodology, sample preparation, and sample storage employed by the TIOB can produce high-quality specimens, suited for employment in a wide array of multi-omic platforms, enabling comprehensive immune and molecular profiling.
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One promising goal for utilizing the molecular information circulating in biofluids is the discovery of clinically useful biomarkers. Extracellular RNAs (exRNAs) are one of the most diverse classes of molecular cargo, easily assayed by sequencing and with expressions that rapidly change in response to subject status. Despite diverse exRNA cargo, most evaluations from biofluids have focused on small RNA sequencing and analysis, specifically on microRNAs (miRNAs). Another goal of characterizing circulating molecular information, is to correlate expression to injuries associated with specific tissues of origin. Biomarker candidates are often described as being specific, enriched in a particular tissue or associated with a disease process. Likewise, miRNA data is often reported to be specific, enriched for a tissue, without rigorous testing to support the claim. Here we provide a tissue atlas of small RNAs from 30 different tissues and three different blood cell types. We analyzed the tissues for enrichment of small RNA sequences and assessed their expression in biofluids: plasma, cerebrospinal fluid, urine, and saliva. We employed published data sets representing physiological (resting vs. acute exercise) and pathologic states (early- vs. late-stage liver fibrosis, and differential subtypes of stroke) to determine differential tissue-enriched small RNAs. We also developed an online tool that provides information about exRNA sequences found in different biofluids and tissues. The data can be used to better understand the various types of small RNA sequences in different tissues as well as their potential release into biofluids, which should help in the validation or design of biomarker studies.
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Circular RNA (circRNA) are a recently discovered class of RNA characterized by a covalently-bonded back-splice junction. As circRNAs are inherently more stable than other RNA species, they may be detected extracellularly in peripheral biofluids and provide novel biomarkers. While circRNA have been identified previously in peripheral biofluids, there are few datasets for circRNA junctions from healthy controls. We collected 134 plasma and 114 urine samples from 54 healthy, male college athlete volunteers, and used RNASeq to determine circRNA content. The intersection of six bioinformatic tools identified 965 high-confidence, characteristic circRNA junctions in plasma and 72 in urine. Highly-expressed circRNA junctions were validated by qRT-PCR. Longitudinal samples were collected from a subset, demonstrating circRNA expression was stable over time. Lastly, the ratio of circular to linear transcripts was higher in plasma than urine. This study provides a valuable resource for characterization of circRNA in plasma and urine from healthy volunteers, one that can be developed and reassessed as researchers probe the circRNA contents of biofluids across physiological changes and disease states.
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Atletas , ARN Circular/sangre , ARN Circular/orina , Adolescente , Voluntarios Sanos , Humanos , Masculino , RNA-Seq , Adulto JovenRESUMEN
Changes in the blood-based RNA transcriptome have the potential to inform biomarkers of Parkinson's disease (PD) progression. Here we sequenced a discovery set of whole-blood RNA species in 4,871 longitudinally collected samples from 1,570 clinically phenotyped individuals from the Parkinson's Progression Marker Initiative (PPMI) cohort. Samples were sequenced to an average of 100 million read pairs to create a high-quality transcriptome. Participants with PD in the PPMI had significantly altered RNA expression (>2,000 differentially expressed genes), including an early and persistent increase in neutrophil gene expression, with a concomitant decrease in lymphocyte cell counts. This was validated in a cohort from the Parkinson's Disease Biomarkers Program (PDBP) consisting of 1,599 participants and by alterations in immune cell subtypes. This publicly available transcriptomic dataset, coupled with available detailed clinical data, provides new insights into PD biological processes impacting whole blood and new paths for developing diagnostic and prognostic PD biomarkers.
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Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/genética , Progresión de la Enfermedad , Biomarcadores , Transcriptoma/genética , Análisis de Secuencia de ARN , ARNRESUMEN
BACKGROUND: Antibody-drug conjugates are an exceptional and useful therapeutic tool for multiple diseases, particularly for cancer treatment. We previously showed that the fusion of the serine protease granzyme B (GrB), the effector molecule or T and B cells, to a binding domain allows the controlled and effective delivery of the cytotoxic payload into the target cell. The production of these constructs induced the formation of high molecular aggregates with a potential impact on the efficacy and safety of the protein. METHODS: Our laboratory designed a new Fn14 targeted fusion construct designated GrB(C210A)-Fc-IT4 which contains a modified GrB payload for improved protein production and preserved biological activity. We assessed the construct's enzymatic activity, as well as in vitro cytotoxicity and internalization into target cells. We also assessed pharmacokinetics, efficacy and toxicology parameters in vivo. RESULTS: GrB(C210A)-Fc-IT4 protein exhibited high affinity and selective cytotoxicity within the nanomolar range when tested against a panel of Fn14-positive human cancer cell lines. The construct rapidly internalized into target cells, activating the caspase cascade and causing mitochondrial membrane depolarization. Pharmacokinetic studies in mice revealed that GrB(C210A)-Fc-IT4 displayed a bi-exponential clearance from plasma with a fast initial clearance (t1/2α=0.36 hour) followed by a prolonged terminal-phase plasma half-life (t1/2ß=35 hours). Mice bearing MDA-MB-231 orthotopic tumor xenografts treated with vehicle or GrB(C210A)-Fc-IT4 construct (QODx5) demonstrated tumor regression and long-term (>80 days) suppression of tumor growth. Treatment of mice bearing established, subcutaneous A549 lung tumors showed impressive, long-term tumor suppression compared with a control group treated with vehicle alone. Administration of GrB(C210A)-Fc-IT4 (100 mg/kg total dose) was well-tolerated by mice and resulted in significant reduction of tumor burden in a lung cancer patient-derived xenograft model. Toxicity studies revealed no statistically significant changes in aspartate transferase, alanine transferase or lactate dehydrogenase in treated mice. Histopathological analysis of tissues from treated mice did not demonstrate any specific drug-related changes. CONCLUSION: GrB(C210A)-Fc-IT4 demonstrated excellent, specific cytotoxicity in vitro and impressive in vivo efficacy with no significant toxicity in normal murine models. These studies show GrB(C210A)-Fc-IT4 is an excellent candidate for further preclinical development.
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Sistemas de Liberación de Medicamentos/métodos , Granzimas/metabolismo , Receptor de TWEAK/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones DesnudosRESUMEN
Rapid identification of patients suffering from cerebral ischaemia, while excluding intracerebral haemorrhage, can assist with patient triage and expand patient access to chemical and mechanical revascularization. We sought to identify blood-based, extracellular microRNAs 15 (ex-miRNAs) derived from extracellular vesicles associated with major stroke subtypes using clinical samples from subjects with spontaneous intraparenchymal haemorrhage (IPH), aneurysmal subarachnoid haemorrhage (SAH) and ischaemic stroke due to cerebral vessel occlusion. We collected blood from patients presenting with IPH (n = 19), SAH (n = 17) and ischaemic stroke (n = 21). We isolated extracellular vesicles from plasma, extracted RNA cargo, 20 sequenced the small RNAs and performed bioinformatic analyses to identify ex-miRNA biomarkers predictive of the stroke subtypes. Sixty-seven miRNAs were significantly variant across the stroke subtypes. A subset of exmiRNAs differed between haemorrhagic and ischaemic strokes, and LASSO analysis could distinguish SAH from the other subtypes with an accuracy of 0.972 ± 0.002. Further analyses predicted 25 miRNA classifiers that stratify IPH from ischaemic stroke with an accuracy of 0.811 ± 0.004 and distinguish haemorrhagic from ischaemic stroke with an accuracy of 0.813 ± 0.003. Blood-based, ex-miRNAs have predictive value, and could be capable of distinguishing between major stroke subtypes with refinement and validation. Such a biomarker could one day aid in the triage of patients to expand the pool eligible for effective treatment.
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The recent discovery of extracellular RNAs in blood, including RNAs in extracellular vesicles (EVs), combined with low-input RNA-sequencing advances have enabled scientists to investigate their role in human disease. To date, most studies have been focusing on small RNAs, and methodologies to optimize long RNAs measurement are lacking. We used plasma RNA to assess the performance of six long RNA sequencing methods, at two different sites, and we report their differences in reads (%) mapped to the genome/transcriptome, number of genes detected, long RNA transcript diversity, and reproducibility. Using the best performing method, we further compare the profile of long RNAs in the EV- and no-EV-enriched RNA plasma compartments. These results provide insights on the performance and reproducibility of commercially available kits in assessing the landscape of long RNAs in human plasma and different extracellular RNA carriers that may be exploited for biomarker discovery.
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PURPOSE: Naturally occurring primary canine lung cancers share clinicopathologic features with human lung cancers in never-smokers, but the genetic underpinnings of canine lung cancer are unknown. We have charted the genomic landscape of canine lung cancer and performed functional characterization of novel, recurrent HER2 (ERBB2) mutations occurring in canine pulmonary adenocarcinoma (cPAC). EXPERIMENTAL DESIGN: We performed multiplatform genomic sequencing of 88 primary canine lung tumors or cell lines. Additionally, in cPAC cell lines, we performed functional characterization of HER2 signaling and evaluated mutation-dependent HER2 inhibitor drug dose-response. RESULTS: We discovered somatic, coding HER2 point mutations in 38% of cPACs (28/74), but none in adenosquamous (cPASC, 0/11) or squamous cell (cPSCC, 0/3) carcinomas. The majority (93%) of HER2 mutations were hotspot V659E transmembrane domain (TMD) mutations comparable to activating mutations at this same site in human cancer. Other HER2 mutations were located in the extracellular domain and TMD. HER2 V659E was detected in the plasma of 33% (2/6) of dogs with localized HER2 V659E tumors. HER2 V659E cPAC cell lines displayed constitutive phosphorylation of AKT and significantly higher sensitivity to the HER2 inhibitors lapatinib and neratinib relative to HER2-wild-type cell lines (IC50 < 200 nmol/L in HER2 V659E vs. IC50 > 2,500 nmol/L in HER2 WT). CONCLUSIONS: This study creates a foundation for molecular understanding of and drug development for canine lung cancer. These data also establish molecular contexts for comparative studies in dogs and humans of low mutation burden, never-smoker lung cancer, and mutant HER2 function and inhibition.
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Adenocarcinoma del Pulmón/veterinaria , Enfermedades de los Perros/genética , Neoplasias Pulmonares/veterinaria , Mutación , Receptor ErbB-2/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Supervivencia Celular/efectos de los fármacos , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/patología , Perros , Femenino , Lapatinib/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Transducción de Señal , Células Tumorales CultivadasRESUMEN
BACKGROUND: The incidence of esophageal adenocarcinoma (EAC) has increased over the last several decades. Apart from mutations in TP53 gene, there are little data on genetic drivers of EAC. Liver kinase B1 (LKB1) has emerged as a multifunctional tumor suppressor regulating cell growth, differentiation, and metabolism. Somatic inactivation of LKB1 has been described in several tumor types; however, whether LKB1 inactivation has a role in EAC is unknown. Here we analyzed patient tumors to assess the prevalence of LKB1 loss in EAC. METHODS: Chromosomal deletion and expression of LKB1 in EAC were investigated using publicly available genomic data. Protein expression was assessed by immunohistochemistry (IHC) analysis for LKB1 in a tissue microarray (TMA) containing esophageal tumor specimens, including EAC. LKB1 was suppressed in EAC cells to determine the effects on cell growth in vitro. RESULTS: Analysis of EAC data in The Cancer Genome Atlas dataset revealed significant deletion of chromosome 19p13.3, containing the LKB1 gene locus. Single copy loss (shallow deletion) of LKB1 was present in 58% of EAC samples. Expression of LKB1 was significantly lower in EAC tumors compared with normal esophagus. IHC analysis showed reduced LKB1 protein expression in EAC. Suppression of LKB1 was sufficient to enhance EAC cell growth in vitro. CONCLUSIONS: Our data suggest that inactivation of LKB1 frequently occurs in EAC. Based on the reported oncogenic effects of LKB1 inactivation, our data indicate that LKB1 loss may play a significant role in EAC tumorigenesis, and point to the need for future studies.
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Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Proliferación Celular , Neoplasias Esofágicas/genética , Silenciador del Gen , Proteínas Serina-Treonina Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular/genética , Bases de Datos Genéticas , Neoplasias Esofágicas/enzimología , Neoplasias Esofágicas/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Análisis de Matrices TisularesRESUMEN
G1-S checkpoint loss contributes to carcinogenesis and increases reliance upon the G2-M checkpoint for adaptation to stress and DNA repair, making G2-M checkpoint inhibition a target for novel therapeutic development. AZD1775, an inhibitor against the critical G2-M checkpoint protein WEE1, is currently in clinical trials across a number of tumor types. AZD1775 and DNA-damaging agents have displayed favorable activity in several preclinical tumor models, often in the molecular context of TP53 loss. Whether AZD1775 efficacy is modulated by other molecular contexts remains poorly understood. The tumor suppressor serine/threonine kinase 11 (LKB1/STK11) is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC) and is commonly comutated with oncogenic KRAS mutations. We investigated the preclinical effects of AZD1775 in the context of KRAS/LKB1 in NSCLC. Using NSCLC cell lines, we found that AZD1775 alone and in combination with DNA-damaging agents (e.g., cisplatin and radiation) decreased tumor cell viability in LKB1-deficient NSCLC cells. In vitro, LKB1 deficiency enhanced DNA damage and apoptosis in response to AZD1775 exposure compared with wild-type LKB1 cells. In a genetically engineered mouse model of mutant Kras with concomitant loss of Lkb1, combined AZD1775 and cisplatin extended overall survival compared with cisplatin alone. Our data suggest that lack of phosphorylation of LKB1 by ATM was involved in AZD1775-mediated cytotoxicity. Collectively, these findings provide a clinical application for AZD1775 with DNA-damaging agents in KRAS/LKB1 NSCLC. Cancer Res; 77(17); 4663-72. ©2017 AACR.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirazoles/farmacología , Pirimidinas/farmacología , Proteínas Quinasas Activadas por AMP , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Transgénicos , Mutación/genética , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Pirimidinonas , Células Tumorales CultivadasRESUMEN
BACKGROUND: Small cell lung cancer (SCLC) that has progressed after first-line therapy is an aggressive disease with few effective therapeutic strategies. In this prospective study, we employed next-generation sequencing (NGS) to identify therapeutically actionable alterations to guide treatment for advanced SCLC patients. METHODS: Twelve patients with SCLC were enrolled after failing platinum-based chemotherapy. Following informed consent, genome-wide exome and RNA-sequencing was performed in a CLIA-certified, CAP-accredited environment. Actionable targets were identified and therapeutic recommendations made from a pharmacopeia of FDA-approved drugs. Clinical response to genomically-guided treatment was evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. RESULTS: The study completed its accrual goal of 12 evaluable patients. The minimum tumor content for successful NGS was 20%, with a median turnaround time from sample collection to genomics-based treatment recommendation of 27 days. At least two clinically actionable targets were identified in each patient, and six patients (50%) received treatment identified by NGS. Two had partial responses by RECIST 1.1 on a clinical trial involving a PD-1 inhibitor + irinotecan (indicated by MLH1 alteration). The remaining patients had clinical deterioration before NGS recommended therapy could be initiated. CONCLUSIONS: Comprehensive genomic profiling using NGS identified clinically-actionable alterations in SCLC patients who progressed on initial therapy. Recommended PD-1 therapy generated partial responses in two patients. Earlier access to NGS guided therapy, along with improved understanding of those SCLC patients likely to respond to immune-based therapies, should help to extend survival in these cases with poor outcomes.
Asunto(s)
Proteínas de Neoplasias/biosíntesis , Análisis de Secuencia de ARN/métodos , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Transcriptoma/genética , Adulto , Anciano , Biopsia , Exoma/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proyectos Piloto , Platino (Metal)/administración & dosificación , Alineación de Secuencia , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Resultado del TratamientoRESUMEN
CONTEXT: Adrenocortical carcinomas (ACC) are a rare tumor type with a poor five-year survival rate and limited treatment options. OBJECTIVE: Understanding of the molecular pathogenesis of this disease has been aided by genomic analyses highlighting alterations in TP53, WNT, and IGF signaling pathways. Further elucidation is needed to reveal therapeutically actionable targets in ACC. DESIGN: In this study, global DNA methylation levels were assessed by the Infinium HumanMethylation450 BeadChip Array on 18 ACC tumors and 6 normal adrenal tissues. A new, non-linear correlation approach, the discretization method, assessed the relationship between DNA methylation/gene expression across ACC tumors. RESULTS: This correlation analysis revealed epigenetic regulation of genes known to modulate TP53, WNT, and IGF signaling, as well as silencing of the tumor suppressor MARCKS, previously unreported in ACC. CONCLUSIONS: DNA methylation may regulate genes known to play a role in ACC pathogenesis as well as known tumor suppressors.