RESUMEN
Genome-scale technologies have enabled mapping of the complex molecular networks that govern cellular behavior. An emerging theme in the analyses of these networks is that cells use many layers of regulatory feedback to constantly assess and precisely react to their environment. The importance of complex feedback in controlling the real-time response to external stimuli has led to a need for the next generation of cell-based technologies that enable both the collection and analysis of high-throughput temporal data. Toward this end, we have developed a microfluidic platform capable of monitoring temporal gene expression from over 2,000 promoters. By coupling the "Dynomics" platform with deep neural network (DNN) and associated explainable artificial intelligence (XAI) algorithms, we show how machine learning can be harnessed to assess patterns in transcriptional data on a genome scale and identify which genes contribute to these patterns. Furthermore, we demonstrate the utility of the Dynomics platform as a field-deployable real-time biosensor through prediction of the presence of heavy metals in urban water and mine spill samples, based on the the dynamic transcription profiles of 1,807 unique Escherichia coli promoters.
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Técnicas Biosensibles/instrumentación , Monitoreo del Ambiente , Perfilación de la Expresión Génica , Aprendizaje Automático , Regiones Promotoras Genéticas/genética , Bases de Datos Genéticas , Monitoreo del Ambiente/instrumentación , Monitoreo del Ambiente/métodos , Diseño de Equipo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilación de la Expresión Génica/instrumentación , Perfilación de la Expresión Génica/métodos , Genes Bacterianos/genética , Genómica/instrumentación , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento , Metales Pesados/toxicidad , Técnicas Analíticas Microfluídicas/instrumentación , Transcriptoma/genéticaRESUMEN
Transcription factors critical for the transition of normal breast epithelium to ductal carcinoma in situ (DCIS) and invasive breast cancer are not clearly defined. Here, we report that the expression of a subset of YAP-activated and YAP-repressed genes in normal mammary and early-stage breast cancer cells is dependent on the nuclear co-activator AIB1. Gene expression, sequential ChIP, and ChIP-seq analyses show that AIB1 and YAP converge upon TEAD for transcriptional activation and repression. We find that AIB1-YAP repression of genes at the 1q21.3 locus is mediated by AIB1-dependent recruitment of ANCO1, a tumor suppressor whose expression is progressively lost during breast cancer progression. Reducing ANCO1 reverts AIB1-YAP-dependent repression, increases cell size, and enhances YAP-driven aberrant 3D growth. Loss of endogenous ANCO1 occurs during DCIS xenograft progression, a pattern associated with poor prognosis in human breast cancer. We conclude that increased expression of AIB1-YAP co-activated targets coupled with a loss of normal ANCO1 repression is critical to patterns of gene expression that mediate malignant progression of early-stage breast cancer.
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Neoplasias de la Mama , Coactivador 3 de Receptor Nuclear/genética , Proteínas Represoras/genética , Mama , Neoplasias de la Mama/genética , Humanos , Coactivador 3 de Receptor Nuclear/metabolismoRESUMEN
Connections between epigenetic reprogramming and transcription or splicing create novel mechanistic networks that can be targeted with tailored therapies. Multiple subunits of the chromatin remodeling BAF complex, including ARID1A, play a role in oncogenesis, either as tumor suppressors or oncogenes. Recent work demonstrated that EWS-FLI1, the oncogenic driver of Ewing sarcoma (ES), plays a role in chromatin regulation through interactions with the BAF complex. However, the specific BAF subunits that interact with EWS-FLI1 and the precise role of the BAF complex in ES oncogenesis remain unknown. In addition to regulating transcription, EWS-FLI1 also alters the splicing of many mRNA isoforms, but the role of splicing modulation in ES oncogenesis is not well understood. We have identified a direct connection between the EWS-FLI1 protein and ARID1A isoform protein variant ARID1A-L. We demonstrate here that ARID1A-L is critical for ES maintenance and supports oncogenic transformation. We further report a novel feed-forward cycle in which EWS-FLI1 leads to preferential splicing of ARID1A-L, promoting ES growth, and ARID1A-L reciprocally promotes EWS-FLI1 protein stability. Dissecting this interaction may lead to improved cancer-specific drug targeting.
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Carcinogénesis/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Factores de Transcripción/genética , Empalme Alternativo/genética , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Nucleares/química , Proteínas de Fusión Oncogénica/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estabilidad Proteica , Proteína Proto-Oncogénica c-fli-1/química , Proteína EWS de Unión a ARN/química , Sarcoma de Ewing/patología , Factores de Transcripción/químicaRESUMEN
The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNA-seq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.
Asunto(s)
Empalme Alternativo/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Transducción de Señal/genética , Empalme Alternativo/efectos de los fármacos , Secuencia de Bases , Línea Celular Tumoral , Exones/genética , Humanos , Indoles , Intrones/genética , Proteínas de Fusión Oncogénica/genética , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Transducción de Señal/efectos de los fármacos , Empalmosomas/efectos de los fármacos , Empalmosomas/metabolismo , Telomerasa/metabolismoRESUMEN
Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.
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Antineoplásicos/farmacología , Proteínas del Citoesqueleto/antagonistas & inhibidores , Estrés Fisiológico , Transcriptoma , Adamantano/análogos & derivados , Adamantano/farmacocinética , Adamantano/farmacología , Animales , Antineoplásicos/farmacocinética , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Línea Celular Tumoral , Perros , Femenino , Semivida , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/secundario , Fenoles/farmacocinética , Fenoles/farmacología , Quinolinas/farmacocinética , Quinolinas/farmacología , Quinolonas/farmacocinética , Quinolonas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , Indoles/farmacología , Modelos Moleculares , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , ARN/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
CD133, a cancer stem cell (CSC) marker in tumors, including melanoma, is associated with tumor recurrence, chemoresistance, and metastasis. Patient-derived melanoma cell lines were transduced with a Tet-on vector expressing CD133, generating doxycycline (Dox)-inducible cell lines. Cells were exposed to Dox for 24 h to induce CD133 expression, followed by RNA-seq and bioinformatic analyses, revealing genes and pathways that are significantly up- or downregulated by CD133. The most significantly upregulated gene after CD133 was amphiregulin (AREG), validated by qRT-PCR and immunoblot analyses. Induced CD133 expression significantly increased cell growth, percentage of cells in S-phase, BrdU incorporation into nascent DNA, and PCNA levels, indicating that CD133 stimulates cell proliferation. CD133 induction also activated EGFR and the MAPK pathway. Potential mechanisms highlighting the role(s) of CD133 and AREG in melanoma CSC were further delineated using AREG/EGFR inhibitors or siRNA knockdown of AREG mRNA. Treatment with the EGFR inhibitor gefitinib blocked CD133-induced cell growth increase and MAPK pathway activation. Importantly, siRNA knockdown of AREG reversed the stimulatory effects of CD133 on cell growth, indicating that AREG mediates the effects of CD133 on cell proliferation, thus serving as an attractive target for novel combinatorial therapeutics in melanoma and cancers with overexpression of both CD133 and AREG.
Asunto(s)
Antígeno AC133 , Anfirregulina , Proliferación Celular , Melanoma , Humanos , Antígeno AC133/metabolismo , Antígeno AC133/genética , Anfirregulina/metabolismo , Anfirregulina/genética , Línea Celular Tumoral , Proliferación Celular/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Melanoma/patología , Melanoma/metabolismo , Melanoma/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Endometriosis (ENDO) is a chronic estrogen-dependent gynecological condition that affects reproductive-age women, causing pelvic pain, infertility, and increased risk for ovarian cancer. Diabetes mellitus (DM) is a metabolic disease with significant morbidity and mortality and rising incidence worldwide. The occurrence of DM among ENDO patients remains understudied, despite commonalities in these conditions' immune, inflammatory, and metabolic dysfunctions. This pilot study evaluated whether a subset of women with ENDO manifests DM co-morbidity and if so, whether DM promotes ENDO status. Archived ectopic lesions obtained at ENDO surgery from non-diabetic (ENDO-N; n = 11) and diabetic (ENDO-DM; n = 15) patients were identified by a search of an electronic health database. Retrieved samples were analyzed by immunohistochemistry for markers of proliferation (Ki67, PTEN), steroid receptor signaling (ESR, PGR) and macrophage infiltration (CD68). Immunostaining data were expressed as percentages of immune-positive cells in lesion stroma and epithelium. In lesion stroma, the percentages of nuclear immune-positive cells were higher for ESR2 and lower for PGR-T, in ENDO-DM than ENDO-N patients. The percentages of nuclear immune-positive cells for ESR1 and PTEN tended to be higher and lower, respectively, in ENDO-DM than ENDO-N groups. In lesion glandular epithelium, the percentages of nuclear immune-positive cells were higher for ESR1 and ESR2, in ENDO-DM than ENDO-N groups. ENDO-N lesions had lower percentages of stromal CD68 immune-positive cells than ENDO-DM Type 1 lesions. Findings demonstrate DM in a subset of women with ENDO, which was associated with significant changes in lesion stromal and epithelial nuclear steroid hormone receptor levels, suggestive of disease progression.
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Diabetes Mellitus , Endometriosis , Humanos , Femenino , Endometriosis/metabolismo , Proyectos Piloto , Regulación de la Expresión Génica , Estrógenos/metabolismo , Transducción de Señal , Receptores Citoplasmáticos y Nucleares , Endometrio/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
A growing body of evidence links gut microbiota changes with inflammatory bowel disease (IBD), raising the potential benefit of exploiting metagenomics data for non-invasive IBD diagnostics. The sbv IMPROVER metagenomics diagnosis for inflammatory bowel disease challenge investigated computational metagenomics methods for discriminating IBD and nonIBD subjects. Participants in this challenge were given independent training and test metagenomics data from IBD and nonIBD subjects, which could be wither either raw read data (sub-challenge 1, SC1) or processed Taxonomy- and Function-based profiles (sub-challenge 2, SC2). A total of 81 anonymized submissions were received between September 2019 and March 2020. Most participants' predictions performed better than random predictions in classifying IBD versus nonIBD, Ulcerative Colitis (UC) versus nonIBD, and Crohn's Disease (CD) versus nonIBD. However, discrimination between UC and CD remains challenging, with the classification quality similar to the set of random predictions. We analyzed the class prediction accuracy, the metagenomics features by the teams, and computational methods used. These results will be openly shared with the scientific community to help advance IBD research and illustrate the application of a range of computational methodologies for effective metagenomic classification.
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Colitis Ulcerosa , Enfermedad de Crohn , Microbioma Gastrointestinal , Enfermedades Inflamatorias del Intestino , Humanos , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/genética , Colitis Ulcerosa/diagnóstico , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/genética , Metagenómica , Microbioma Gastrointestinal/genéticaRESUMEN
Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify cis-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing events that discriminate EwS tumor samples from the assumed cell of origin, human mesenchymal stem cells derived from bone marrow (hMSC-BM). Finally, we identify specific splicing factors PCBP2, RBMX, and SRSF9 by motif enrichment and confirm findings from tumor samples in EwS cell lines.
RESUMEN
Cell signaling pathways consist of multiple connections of different types of gene, mRNA and protein networks. It is not a trivial task to follow the signals flowing through these networks. The difficulty comes from considering the entire biological structure as a single network without breaking it into connected modules. The study of these networks simplifies if the complex system is reduced to a hierarchy of interconnected modules. Out of many potential modules, a specific one, namely the Goldbeter-Koshland switch, was encountered by the authors during their study of the Mammalian Heat Shock Response Network (MHSRN) where the switch acts as a stress sensor. Usually, only the steady state behavior of the switch is studied, in which the phosphorylated protein is given as a function of the enzyme concentration. Experimental results show that the heat shock response is still present 20 h after the temperature stress had ended. Thus, it is useful to analyze the transient behavior of the switch that couples the environment to the MHSRN. A stochastic model for the switch is proposed using the Master Equation which is subsequently transformed into an equation for the factorial cumulant generating function. This generating function can be easily read from a graphical representation of the stochastic switch. The second order approximation of the equation for the factorial cumulant generating function is solved and the time dependence of the transient regime of the mean and standard deviation is readily obtained. Using the mean and standard deviation of the switch's output as a function of the stochastic input signals that represent the environment, we classify the switches according to different criteria. The switches differ by the numerical values of the parameters that characterize the switch's chemical reactions. The classifying criteria will distinguish the switches by the levels of the response for a given transition time and by the sensitivity of the response to the enzyme levels. It is also found that the environment can drastically change the response of the switch, which has important biological consequences.
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INTRODUCTION: Few US studies have examined the usefulness of participatory surveillance during the coronavirus disease 2019 (COVID-19) pandemic for enhancing local health response efforts, particularly in rural settings. We report on the development and implementation of an internet-based COVID-19 participatory surveillance tool in rural Appalachia. METHODS: A regional collaboration among public health partners culminated in the design and implementation of the COVID-19 Self-Checker, a local online symptom tracker. The tool collected data on participant demographic characteristics and health history. County residents were then invited to take part in an automated daily electronic follow-up to monitor symptom progression, assess barriers to care and testing, and collect data on COVID-19 test results and symptom resolution. RESULTS: Nearly 6500 county residents visited and 1755 residents completed the COVID-19 Self-Checker from April 30 through June 9, 2020. Of the 579 residents who reported severe or mild COVID-19 symptoms, COVID-19 symptoms were primarily reported among women (n = 408, 70.5%), adults with preexisting health conditions (n = 246, 70.5%), adults aged 18-44 (n = 301, 52.0%), and users who reported not having a health care provider (n = 131, 22.6%). Initial findings showed underrepresentation of some racial/ethnic and non-English-speaking groups. PRACTICAL IMPLICATIONS: This low-cost internet-based platform provided a flexible means to collect participatory surveillance data on local changes in COVID-19 symptoms and adapt to guidance. Data from this tool can be used to monitor the efficacy of public health response measures at the local level in rural Appalachia.
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COVID-19/epidemiología , Recolección de Datos/métodos , Intervención basada en la Internet , Vigilancia en Salud Pública/métodos , Autoinforme , Evaluación de Síntomas , Adolescente , Adulto , Anciano , Región de los Apalaches/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Participación del Paciente , SARS-CoV-2 , Adulto JovenRESUMEN
AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an "enabler" phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population. SIGNIFICANCE: A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.
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Coactivador 3 de Receptor Nuclear/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Sistemas CRISPR-Cas , Técnicas de Cultivo Tridimensional de Células , Línea Celular Tumoral , Dexametasona/química , Progresión de la Enfermedad , Impedancia Eléctrica , Elementos de Facilitación Genéticos , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Coactivador 3 de Receptor Nuclear/química , Fenotipo , Isoformas de Proteínas , Empalme del ARN , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Tiazolidinedionas/farmacología , Pez CebraRESUMEN
Clofarabine, an FDA approved purine analog, is used in the treatment of relapsed or refractory acute lymphoblastic leukemia. Clofarabine acts by inhibiting DNA synthesis. We demonstrated that clofarabine may have a novel function though inhibiting CD99, a transmembrane protein highly expressed on Ewing Sarcoma (ES) cells. CD99 is a validated target in ES whose inhibition may lead to a high therapeutic index for patients. Here we present additional data to support the hypothesis that clofarabine acts on CD99 and regulates key signaling pathways in ES. Cellular thermal shift assay indicated a direct interaction between clofarabine and CD99 in ES cell lysates. Clofarabine induced ES cell death does not require clofarabine's conversion to its active form by deoxycytidine kinase. A phosphokinase array screen with clofarabine and a CD99 blocking antibody identified alterations in signaling pathways. CD99 inhibition with clofarabine in ES cells caused rapid and sustained phosphorylation of ERK, MSK, and CREB. However, activation of this pathway did not correlate with clofarabine induced ES cell death. In summary, we demonstrated that clofarabine may activate ERK, MSK, and CREB phosphorylation through CD99 within minutes, however this paradoxical activation and subsequent ES cell death requires additional investigation.
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Antígeno 12E7/antagonistas & inhibidores , Antimetabolitos Antineoplásicos/farmacología , Proteína de Unión a CREB/metabolismo , Clofarabina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Sarcoma de Ewing/metabolismo , Transducción de Señal/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Humanos , Fosforilación , Sarcoma de Ewing/tratamiento farmacológicoRESUMEN
Despite Ewing sarcoma (ES) being the second most common pediatric malignancy of bone and soft tissue, few novel therapeutic approaches have been introduced over the past few decades. ES contains a pathognomonic chromosomal translocation that leads to a fusion protein between EWSR1 and an ets family member, most often FLI1. EWSFLI1 is the most common type of fusion protein and is a wellvetted therapeutic target. A small molecule inhibitor of EWSFLI1, YK4279 (YK) was developed with the intention to serve as a targeted therapy option for patients with ES. The present study investigated resistance mechanisms by developing an ES cell line specifically resistant to YK. The ES cell line A4573 was treated with YK to create resistant cells by long term continuous exposure. The results revealed that resistance in A4573 was robust and sustainable, with a >27fold increase in IC50 lasting up to 16 weeks in the absence of the compound. Resistant ES cells were still sensitive to standard of care drugs, including doxorubicin, vincristine and etoposide, which may be valuable in future combination treatments in the clinic. Resistant ES cells revealed an increased expression of CD99. RNA sequencing and qPCR validation of resistant ES cells confirmed an increased expression of ANO1, BRSK2 and IGSF21, and a reduced expression of COL24A1, PRSS23 and RAB38 genes. A functional association between these genes and mechanism of resistance remains to be investigated. The present study created a cell line to investigate YK resistance.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Indoles/farmacología , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteína Proto-Oncogénica c-fli-1/antagonistas & inhibidores , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/antagonistas & inhibidores , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Antígeno 12E7/genética , Antígeno 12E7/metabolismo , Biomarcadores de Tumor , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Expresión Génica , HumanosRESUMEN
Nonalcoholic fatty liver disease (NAFLD) and its evolution to inflammatory steatohepatitis (NASH) are the most common causes of chronic liver damage and transplantation that are reaching epidemic proportions due to the upraising incidence of metabolic syndrome, obesity, and diabetes. Currently, there is no approved treatment for NASH. The mitochondrial citrate carrier, Slc25a1, has been proposed to play an important role in lipid metabolism, suggesting a potential role for this protein in the pathogenesis of this disease. Here, we show that Slc25a1 inhibition with a specific inhibitor compound, CTPI-2, halts salient alterations of NASH reverting steatosis, preventing the evolution to steatohepatitis, reducing inflammatory macrophage infiltration in the liver and adipose tissue, while starkly mitigating obesity induced by a high-fat diet. These effects are differentially recapitulated by a global ablation of one copy of the Slc25a1 gene or by a liver-targeted Slc25a1 knockout, which unravel dose-dependent and tissue-specific functions of this protein. Mechanistically, through citrate-dependent activities, Slc25a1 inhibition rewires the lipogenic program, blunts signaling from peroxisome proliferator-activated receptor gamma, a key regulator of glucose and lipid metabolism, and inhibits the expression of gluconeogenic genes. The combination of these activities leads not only to inhibition of lipid anabolic processes, but also to a normalization of hyperglycemia and glucose intolerance as well. In summary, our data show for the first time that Slc25a1 serves as an important player in the pathogenesis of fatty liver disease and thus, provides a potentially exploitable and novel therapeutic target.
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Proteínas Portadoras/antagonistas & inhibidores , Intolerancia a la Glucosa/complicaciones , Inflamación/complicaciones , Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Acetilcoenzima A/metabolismo , Animales , Glucemia/metabolismo , Proteínas Portadoras/metabolismo , Polaridad Celular , Ácido Cítrico/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Regulación hacia Abajo , Ayuno/sangre , Gluconeogénesis , Intolerancia a la Glucosa/sangre , Hepatomegalia/sangre , Hepatomegalia/complicaciones , Hepatomegalia/diagnóstico por imagen , Humanos , Hiperglucemia/sangre , Hiperglucemia/complicaciones , Inflamación/sangre , Resistencia a la Insulina , Interleucina-6/biosíntesis , Lipogénesis , Hígado/diagnóstico por imagen , Hígado/metabolismo , Hígado/patología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/diagnóstico por imagen , Obesidad/sangre , Obesidad/complicaciones , Fenotipo , Factores de Tiempo , Triglicéridos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
To explore how airborne microbial patterns change with height above the Earth's surface, we flew NASA's C-20A aircraft on two consecutive days in June 2018 along identical flight paths over the US Sierra Nevada mountain range at four different altitudes ranging from 10,000 ft to 40,000 ft. Bioaerosols were analyzed by metagenomic DNA sequencing and traditional culturing methods to characterize the composition and diversity of atmospheric samples compared to experimental controls. The relative abundance of taxa changed significantly at each altitude sampled, and the diversity profile shifted across the two sampling days, revealing a regional atmospheric microbiome that is dynamically changing. The most proportionally abundant microbial genera were Mycobacterium and Achromobacter at 10,000 ft; Stenotrophomonas and Achromobacter at 20,000 ft; Delftia and Pseudoperonospora at 30,000 ft; and Alcaligenes and Penicillium at 40,000 ft. Culture-based detections also identified viable Bacillus zhangzhouensis, Bacillus pumilus, and Bacillus spp. in the upper troposphere. To estimate bioaerosol dispersal, we developed a human exposure likelihood model (7-day forecast) using general aerosol characteristics and measured meteorological conditions. By coupling metagenomics to a predictive atmospheric model, we aim to set the stage for field campaigns that monitor global bioaerosol emissions and impacts.
RESUMEN
Liver cancer is associated with genetic mutations caused by environmental exposures, including occupational exposure to alpha radiation emitted by plutonium. We used whole exome sequencing (WES) to characterize somatic mutations in 3 histologically distinct primary liver tumors (angiosarcoma of the liver (ASL), cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC)) from Mayak worker subjects occupationally exposed to ionizing radiation (IR) to investigate the contribution of IR to the mutational landscape of liver cancer. DNA sequence analysis revealed these tumors harbor an excess of deletions, with a deletions:substitutions ratio similar to that previously reported in radiation-associated tumors. These tumors were also enriched for clustered mutations, a signature of radiation exposure. Multiple tumors displayed similarities in abrogated gene pathways including actin cytoskeletal signaling and DNA double-strand break (DSB) repair. WES identified novel candidate driver genes in ASL involved in angiogenesis and PIK3CA/AKT/mTOR signaling. We confirmed known driver genes of CCA, and identified candidate driver genes involved in chromatin remodeling. In HCC tumors we validated known driver genes, and identified novel putative driver genes involved in Wnt/ß-catenin signaling, chromatin remodeling, PIK3CA/AKT/mTOR signaling, and angiogenesis. This pilot study identifies several novel candidate driver mutations that are likely to be caused by IR exposure, and provides the first data on the mutational landscape of liver cancer after IR exposure.
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Carcinoma Hepatocelular/genética , Colangiocarcinoma/genética , Hemangiosarcoma/genética , Neoplasias Hepáticas/genética , Neoplasias Inducidas por Radiación/genética , Enfermedades Profesionales/genética , Anciano , Carcinoma Hepatocelular/patología , Colangiocarcinoma/patología , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Hemangiosarcoma/patología , Humanos , Hígado/patología , Hígado/efectos de la radiación , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Mutación/efectos de la radiación , Neoplasias Inducidas por Radiación/patología , Enfermedades Profesionales/patología , Exposición Profesional/efectos adversos , Proyectos Piloto , Residuos Radiactivos/efectos adversos , Federación de Rusia , Instalaciones de Eliminación de Residuos , Secuenciación del ExomaRESUMEN
CDK4/6 inhibitors are used in the treatment of advanced estrogen receptor (ER)(+) breast cancer. Their efficacy in ER(-) and early-stage breast cancer is currently under investigation. Here, we show that palbociclib, a CDK4/6 inhibitor, can inhibit both progression of ductal carcinoma in situ (DCIS) and growth of invasive disease in both an ER(-) basal breast cancer model (MCFDCIS) and an ER(+) luminal model (MCF7 intraductal injection). In MCFDCIS cells, palbociclib repressed cell-cycle gene expression, inhibited proliferation, induced senescence, and normalized tumorspheres formed in Matrigel while the formation of acini by normal mammary epithelial cells (MCF10A) was not affected. Palbociclib treatment of mice with MCFDCIS tumors inhibited their malignant progression and reduced proliferation of invasive lesions. Transcriptomic analysis of the tumor and stromal cell compartments showed that cell cycle and senescence genes, and MUC16, an ovarian cancer biomarker gene, were repressed during treatment. Knockdown of MUC16 in MCFDCIS cells inhibited proliferation of invasive lesions but not progression of DCIS. After cessation of palbociclib treatment genes associated with differentiation, for example, P63, inflammation, IFNγ response, and antigen processing and presentation remained suppressed in the tumor and surrounding stroma. We conclude that palbociclib can prevent progression of DCIS and is antiproliferative in ER(-) invasive disease mediated in part via MUC16. Lasting effects of CDK4/6 inhibition after drug withdrawal on differentiation and the immune response could impact the approach to treatment of early-stage ER(-) breast cancer.
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Neoplasias de la Mama/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/uso terapéutico , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/uso terapéutico , Animales , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Desnudos , Estadificación de NeoplasiasRESUMEN
Targeted monoclonal antibody therapy is a promising therapeutic strategy for cancer, and antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial mechanism underlying these approaches. The majority of patients have limited responses to monoclonal antibody therapy due to the development of resistance. Models of ADCC provide a system for uncovering immune-resistance mechanisms. We continuously exposed epidermal growth factor receptor (EGFR+) A431 cells to KIR-deficient NK92-CD16V effector cells and the anti-EGFR cetuximab. Persistent ADCC exposure yielded ADCC-resistant cells (ADCCR1) that, compared with control ADCC-sensitive cells (ADCCS1), exhibited reduced EGFR expression, overexpression of histone- and interferon-related genes, and a failure to activate NK cells, without evidence of epithelial-to-mesenchymal transition. These properties gradually reversed following withdrawal of ADCC selection pressure. The development of resistance was associated with lower expression of multiple cell-surface molecules that contribute to cell-cell interactions and immune synapse formation. Classic immune checkpoints did not modulate ADCC in this unique model system of immune resistance. We showed that the induction of ADCC resistance involves genetic and epigenetic changes that lead to a general loss of target cell adhesion properties that are required for the establishment of an immune synapse, killer cell activation, and target cell cytotoxicity.