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Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3ß, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.
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MicroARNs , MicroARNs/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN , Quinasa Tipo Polo 1RESUMEN
OBJECTIVE: Our aim is to emphasize the varied presentation of acute disseminated encephalomyelitis (ADEM) to help health care professionals improve recognition of the disease in a timely manner, thereby allowing for the selection of an appropriate treatment regimen. Therefore, this may avoid neurocognitive consequences and the ultimate fatality of the patient. PATIENTS AND METHODS: This is a retrospective case series involving 7 cases of children presenting to the Pediatric Emergency Department of Hackensack University Medical Center who were ultimately diagnosed with ADEM. RESULTS: In many of the cases, a preceding viral-like illness with nonspecific symptomatology made it difficult to accurately establish an initial diagnosis. Ultimately, the neurologic symptoms spontaneously resolved or improved with administration of high-dose steroids. CONCLUSIONS: Children presenting to the emergency department with nonspecific symptoms associated with any neurological deficits should undergo further investigation using magnetic resonance imaging and lumbar puncture to rule out rare yet possibly fatal diseases such as ADEM.
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INTRODUCTION: Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, with the hope of targeting unique aspects of metastatic tumor outgrowth, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle. METHODS: An in vivo reporter system for epithelial cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of transforming growth factor ß1 (TGF-ß1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-ß1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor type 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor tissues versus matched normal tissues. FGFR1 gene expression was manipulated using short hairpin RNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398. RESULTS: Metastatic BC cells undergo spontaneous downregulation of E-cad during primary tumor growth, and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-ß1 was sufficient to drive the metastasis of an otherwise in situ model of BC and was similarly associated with a depletion and return of E-cad expression during metastatic progression. BC cells treated and withdrawn from TGF-ß stably upregulate a truncated FGFR1-ß splice variant that lacks the outermost extracellular immunoglobulin domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-ß was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants and recombinant expression of full-length FGFR1-α also blocked pulmonary tumor outgrowth. CONCLUSION: The results of our study strongly suggest that FGFR1-ß is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitors.
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Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Factor de Crecimiento Transformador beta1/farmacología , Empalme Alternativo , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Análisis de Secuencia por Matrices de Oligonucleótidos , Compuestos de Fenilurea/farmacología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Pirimidinas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Nonconsensual sterilization is usually seen as the by-product of a classist and racist society; disability is ignored. This article examines the 1973 sterilization of two young black girls from Alabama and other precedent-setting court cases involving the sterilization of "mentally retarded" white women to make disability more central to the historical analysis of sterilization. It analyzes the concept of mental retardation and the appeal of a surgical solution to birth control, assesses judicial deliberations over the "right to choose" contraceptive sterilization when the capacity to consent is in doubt, and reflects on the shadow of eugenics that hung over the sterilization debate in the 1970s and 1980s.
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Eugenesia/historia , Esterilización Reproductiva/historia , Eugenesia/legislación & jurisprudencia , Femenino , Historia del Siglo XX , Humanos , Personas con Discapacidades Mentales , Esterilización Reproductiva/legislación & jurisprudencia , Estados UnidosRESUMEN
Breast cancer is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90 % of these patients, yet the mechanisms underlying this event remain poorly defined. WAVE3 belongs to the WASP/WAVE family of actin-binding proteins that play essential roles in regulating cell morphology, actin polymerization, cytoskeleton remodeling, cell motility, and invasion. Accordingly, we demonstrated previously that WAVE3 promotes the acquisition of invasive and metastatic phenotypes by human breast cancers. Herein, we show that transforming growth factor-ß (TGF-ß) selectively and robustly induced the expression of WAVE3 in metastatic breast cancer cells, but not in their nonmetastatic counterparts. Moreover, the induction of WAVE3 expression in human and mouse triple-negative breast cancer cells (TNBCs) by TGF-ß likely reflects its coupling to microRNA expression via a Smad2- and ß3 integrin-dependent mechanism. We further demonstrate the requirement for WAVE3 expression in mediating the initiation of epithelial-mesenchymal transition (EMT) programs stimulated by TGF-ß. Indeed, stable depletion of WAVE3 expression in human TNBC cells prevented TGF-ß from inducing EMT programs and from stimulating the proliferation, migration, and the formation of lamellipodia in metastatic TNBC cells. Lastly, we observed WAVE3 deficiency to abrogate the outgrowth of TNBC cell organoids in 3-dimensional organotypic cultures as well as to decrease the growth and metastasis of 4T1 tumors produced in syngeneic Balb/C mice. Indeed, WAVE3 deficiency significantly reduced the presence of sarcomatoid morphologies indicative of EMT phenotypes in pulmonary TNBC tumors as compared to those detected in their parental counterparts. Collectively, these findings indicate the necessity for WAVE3 expression and activity during EMT programs stimulated by TGF-ß; they also suggest that measures capable of inactivating WAVE3 may play a role in alleviating metastasis stimulated by TGF-ß.
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Factor de Crecimiento Transformador beta/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Animales , Movimiento Celular/genética , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Integrina beta3/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteína Smad2/metabolismo , Regulación hacia Arriba , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Breast cancer is a heterogeneous disease comprised of at least five major tumor subtypes that coalesce as the second leading cause of cancer death in women in the United States. Although metastasis clearly represents the most lethal characteristic of breast cancer, our understanding of the molecular mechanisms that govern this event remains inadequate. Clinically, ~30% of breast cancer patients diagnosed with early-stage disease undergo metastatic progression, an event that (a) severely limits treatment options, (b) typically results in chemoresistance and low response rates, and (c) greatly contributes to aggressive relapses and dismal survival rates. Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that regulates all phases of postnatal mammary gland development, including branching morphogenesis, lactation, and involution. TGF-ß also plays a prominent role in suppressing mammary tumorigenesis by preventing mammary epithelial cell (MEC) proliferation, or by inducing MEC apoptosis. Genetic and epigenetic events that transpire during mammary tumorigenesis conspire to circumvent the tumor suppressing activities of TGF-ß, thereby permitting late-stage breast cancer cells to acquire invasive and metastatic phenotypes in response to TGF-ß. Metastatic progression stimulated by TGF-ß also relies on its ability to induce epithelial-mesenchymal transition (EMT) and the expansion of chemoresistant breast cancer stem cells. Precisely how this metamorphosis in TGF-ß function comes about remains incompletely understood; however, recent findings indicate that the initiation of oncogenic TGF-ß activity is contingent upon imbalances between its canonical and noncanonical signaling systems. Here we review the molecular and cellular contributions of noncanonical TGF-ß effectors to mammary tumorigenesis and metastatic progression.
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Neoplasias de la Mama/metabolismo , Transformación Celular Neoplásica/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Humanas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Humanos , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/crecimiento & desarrollo , Glándulas Mamarias Humanas/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/genéticaRESUMEN
We report the development of a large scale process for heat inactivation of clinical COVID-19 samples prior to laboratory processing for detection of SARS-CoV-2 by RT-qPCR. With more than 266 million confirmed cases, over 5.26 million deaths already recorded at the time of writing, COVID-19 continues to spread in many parts of the world. Consequently, mass testing for SARS-CoV-2 will remain at the forefront of the COVID-19 response and prevention for the near future. Due to biosafety considerations the standard testing process requires a significant amount of manual handling of patient samples within calibrated microbiological safety cabinets. This makes the process expensive, effects operator ergonomics and restricts testing to higher containment level laboratories. We have successfully modified the process by using industrial catering ovens for bulk heat inactivation of oropharyngeal/nasopharyngeal swab samples within their secondary containment packaging before processing in the lab to enable all subsequent activities to be performed in the open laboratory. As part of a validation process, we tested greater than 1200 clinical COVID-19 samples and showed less than 1 Cq loss in RT-qPCR test sensitivity. We also demonstrate the bulk heat inactivation protocol inactivates a murine surrogate of human SARS-CoV-2. Using bulk heat inactivation, the assay is no longer reliant on containment level 2 facilities and practices, which reduces cost, improves operator safety and ergonomics and makes the process scalable. In addition, heating as the sole method of virus inactivation is ideally suited to streamlined and more rapid workflows such as 'direct to PCR' assays that do not involve RNA extraction or chemical neutralisation methods.
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Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Contención de Riesgos Biológicos/métodos , Calor , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/genética , Manejo de Especímenes/métodos , Inactivación de Virus , Animales , COVID-19/virología , Línea Celular , Humanos , Ratones , Virus de la Hepatitis Murina/genética , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS: We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS: AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS: Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).
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Neoplasias , Oligonucleótidos Antisentido , Animales , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Terapia de Inmunosupresión , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Linfocitos T Reguladores , Microambiente TumoralRESUMEN
On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.
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SARS-CoV-2RESUMEN
Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that functions to inhibit mammary tumorigenesis by directly inducing mammary epithelial cells (MECs) to undergo cell cycle arrest or apoptosis, and to secrete a variety of cytokines, growth factors, and extracellular matrix proteins that maintain cell and tissue homeostasis. Genetic and epigenetic events that transpire during mammary tumorigenesis typically inactivate the tumor suppressing activities of TGF-beta and ultimately confer this cytokine with tumor promoting activities, including the ability to stimulate breast cancer invasion, metastasis, angiogenesis, and evasion from the immune system. This dramatic conversion in TGF-beta function is known as the "TGF-beta paradox" and reflects a variety of dynamic alterations that occur not only within the developing mammary carcinoma, but also within the cellular and structural composition of its accompanying tumor microenvironment. Recent studies have begun to elucidate the critical importance of mammary tumor microenvironments in manifesting the TGF-beta paradox and influencing the response of developing mammary carcinomas to TGF-beta. Here we highlight recent findings demonstrating the essential function of tumor microenvironments in regulating the oncogenic activities of TGF-beta and its stimulation of metastatic progression during mammary tumorigenesis.
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Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Transformación Celular Neoplásica , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral , Animales , Células Endoteliales/patología , Células Endoteliales/fisiología , Células Epiteliales/patología , Células Epiteliales/fisiología , Femenino , Fibroblastos/patología , Fibroblastos/fisiología , Humanos , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/fisiopatología , Metástasis de la Neoplasia/patología , Transducción de SeñalRESUMEN
Epithelial-mesenchymal transition (EMT) is an essential process that drives polarized, immotile mammary epithelial cells (MECs) to acquire apolar, highly migratory fibroblastoid-like features. EMT is an indispensable process that is associated with normal tissue development and organogenesis, as well as with tissue remodeling and wound healing. In stark contrast, inappropriate reactivation of EMT readily contributes to the development of a variety of human pathologies, particularly those associated with tissue fibrosis and cancer cell invasion and metastasis, including that by breast cancer cells. Although metastasis is unequivocally the most lethal aspect of breast cancer and the most prominent feature associated with disease recurrence, the molecular mechanisms whereby EMT mediates the initiation and resolution of breast cancer metastasis remains poorly understood. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that is intimately involved in regulating numerous physiological processes, including cellular differentiation, homeostasis, and EMT. In addition, TGF-beta also functions as a powerful tumor suppressor in MECs, whose neoplastic development ultimately converts TGF-beta into an oncogenic cytokine in aggressive late-stage mammary tumors. Recent findings have implicated the process of EMT in mediating the functional conversion of TGF-beta during breast cancer progression, suggesting that the chemotherapeutic targeting of EMT induced by TGF-beta may offer new inroads in ameliorating metastatic disease in breast cancer patients. Here we review the molecular, cellular, and microenvironmental factors that contribute to the pathophysiological activities of TGF-beta during its regulation of EMT in normal and malignant MECs.
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Neoplasias de la Mama/fisiopatología , Transdiferenciación Celular , Células Epiteliales/metabolismo , Glándulas Mamarias Humanas/fisiopatología , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Desdiferenciación Celular , Diferenciación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/fisiopatología , Glándulas Mamarias Humanas/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/fisiopatología , Metástasis de la Neoplasia , Transducción de SeñalRESUMEN
Poly ADP-ribose polymerase inhibitors (PARPi) have transformed ovarian cancer (OC) treatment, primarily for tumours deficient in homologous recombination repair. Combining VEGF-signalling inhibitors with PARPi has enhanced clinical benefit in OC. To study drivers of efficacy when combining PARP inhibition and VEGF-signalling, a cohort of patient-derived ovarian cancer xenografts (OC-PDXs), representative of the molecular characteristics and drug sensitivity of patient tumours, were treated with the PARPi olaparib and the VEGFR inhibitor cediranib at clinically relevant doses. The combination showed broad anti-tumour activity, reducing growth of all OC-PDXs, regardless of the homologous recombination repair (HRR) mutational status, with greater additive combination benefit in tumours poorly sensitive to platinum and olaparib. In orthotopic models, the combined treatment reduced tumour dissemination in the peritoneal cavity and prolonged survival. Enhanced combination benefit was independent of tumour cell expression of receptor tyrosine kinases targeted by cediranib, and not associated with change in expression of genes associated with DNA repair machinery. However, the combination of cediranib with olaparib was effective in reducing tumour vasculature in all the OC-PDXs. Collectively our data suggest that olaparib and cediranib act through complementary mechanisms affecting tumour cells and tumour microenvironment, respectively. This detailed analysis of the combined effect of VEGF-signalling and PARP inhibitors in OC-PDXs suggest that despite broad activity, there is no dominant common mechanistic inter-dependency driving therapeutic benefit.
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Antineoplásicos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Quinazolinas/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Genes BRCA1/efectos de los fármacos , Genes BRCA2/efectos de los fármacos , Humanos , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.
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Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Macrófagos/efectos de los fármacos , RatonesRESUMEN
BACKGROUND: A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the in vivo composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts. RESULTS: All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, in vitro transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1. CONCLUSIONS: Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed.
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Antígenos de Superficie/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Animales , Línea Celular , Cricetinae , Proteínas ELAV , Proteína 1 Similar a ELAV , Transferencia Resonante de Energía de Fluorescencia/métodos , Ribonucleoproteína Nuclear Heterogénea D0 , Unión Proteica/genética , Multimerización de Proteína , Transporte de Proteínas/genética , ARN Mensajero/genéticaRESUMEN
Direct measurements of the lifetimes of He(79)Br(2) and Ne(79)Br(2) B-state vibrational levels 10 < or = nu' < or = 20 have been performed using time-resolved optical pump-probe spectroscopy. The values do not obey the energy gap law for direct vibrational predissociation. For both molecules, the dissociation rate for nu'=11 is much faster than for nu'=12, and the nu'=13 rate is also faster than is consistent with the energy gap law. We attribute this unexpected behavior to an electronic predissociation channel. Based on Franck-Condon factors between the Br(2) B-state vibrational wave functions and the possible Br-Br product wave functions, we surmise that either the Br(2) (3)Pi(g)(1(g)) or (2(g)) state is responsible for the electronic predissociation. To our knowledge, this is the first time electronic predissociation and direct Deltanu=-1 vibrational predissociation have been observed to be in competition for a wide range of vibrational levels. As such, this problem deserves a detailed theoretical analysis.
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We have characterized the vibrational predissociation (VP) of the Ne(2)Br(2) van der Waals complex using time- and frequency-resolved pump-probe spectroscopy. After exciting Br(2) within the complex to a vibrational level 16
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Recently, the predissociation lifetimes of the NeBr(2)(B) complex for different initial vibrational excitation (10
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We present our experience in the implementation of an antibiotic stewardship quality improvement initiative directed toward infants born at ≥35 weeks using as a primary tool the Kaiser Permanente early onset sepsis calculator (KP-EOS-C) at the Baylor Scott & White Medical Center - Frisco. After the approval and support of the medical staff and administration, we proceeded to launch an extensive educational program for all women's services nursing staff on how to utilize this calculator to communicate results to the pediatricians on staff. After implementation, we saw a 54% reduction in the number of infants undergoing sepsis workup evaluations and a 51% reduction in the number of infants receiving antibiotics (P < 0.001). We conclude that the implementation of this type of initiative may be feasible and worthwhile in other similar community hospitals, provided there is buy-in by physicians and administration as well as an extensive educational program to the hospital medical and nursing staff.
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BACKGROUND: The ability to modulate immune-inhibitory pathways using checkpoint blockade antibodies such as αPD-1, αPD-L1, and αCTLA-4 represents a significant breakthrough in cancer therapy in recent years. This has driven interest in identifying small-molecule-immunotherapy combinations to increase the proportion of responses. Murine syngeneic models, which have a functional immune system, represent an essential tool for pre-clinical evaluation of new immunotherapies. However, immune response varies widely between models and the translational relevance of each model is not fully understood, making selection of an appropriate pre-clinical model for drug target validation challenging. METHODS: Using flow cytometry, O-link protein analysis, RT-PCR, and RNAseq we have characterized kinetic changes in immune-cell populations over the course of tumor development in commonly used syngeneic models. RESULTS: This longitudinal profiling of syngeneic models enables pharmacodynamic time point selection within each model, dependent on the immune population of interest. Additionally, we have characterized the changes in immune populations in each of these models after treatment with the combination of α-PD-L1 and α-CTLA-4 antibodies, enabling benchmarking to known immune modulating treatments within each model. CONCLUSIONS: Taken together, this dataset will provide a framework for characterization and enable the selection of the optimal models for immunotherapy combinations and generate potential biomarkers for clinical evaluation in identifying responders and non-responders to immunotherapy combinations.
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Antineoplásicos Inmunológicos/farmacología , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Inmunomodulación/efectos de los fármacos , Animales , Biomarcadores de Tumor , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Microambiente TumoralRESUMEN
mTOR inhibition can promote or inhibit immune responses in a context dependent manner, but whether this will represent a net benefit or be contraindicated in the context of immunooncology therapies is less understood. Here, we report that the mTORC1/2 dual kinase inhibitor vistusertib (AZD2014) potentiates anti-tumour immunity in combination with anti-CTLA-4 (αCTLA-4), αPD-1 or αPD-L1 immune checkpoint blockade. Combination of vistusertib and immune checkpoint blocking antibodies led to tumour growth inhibition and improved survival of MC-38 or CT-26 pre-clinical syngeneic tumour models, whereas monotherapies were less effective. Underlying these combinatorial effects, vistusertib/immune checkpoint combinations reduced the occurrence of exhausted phenotype tumour infiltrating lymphocytes (TILs), whilst increasing frequencies of activated Th1 polarized T-cells in tumours. Vistusertib alone was shown to promote a Th1 polarizing proinflammatory cytokine profile by innate primary immune cells. Moreover, vistusertib directly enhanced activation of effector T-cell and survival, an effect that was critically dependent on inhibitor dose. Therefore, these data highlight direct, tumour-relevant immune potentiating benefits of mTOR inhibition that complement immune checkpoint blockade. Together, these data provide a clear rationale to investigate such combinations in the clinic.