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PTEN germline mutations account for ~0.2-1% of all autism spectrum disorder (ASD) cases, as well as ~17% of ASD patients with macrocephaly, making it one of the top ASD-associated risk genes. Individuals with germline PTEN mutations receive the molecular diagnosis of PTEN Hamartoma Tumor Syndrome (PHTS), an inherited cancer predisposition syndrome, about 20-23% of whom are diagnosed with ASD. We generated forebrain organoid cultures from gene-edited isogenic human induced pluripotent stem cells (hiPSCs) harboring a PTENG132D (ASD) or PTENM134R (cancer) mutant allele to model how these mutations interrupt neurodevelopmental processes. Here, we show that the PTENG132D allele disrupts early neuroectoderm formation during the first several days of organoid generation, and results in deficient electrophysiology. While organoids generated from PTENM134R hiPSCs remained morphologically similar to wild-type organoids during this early stage in development, we observed disrupted neuronal differentiation, radial glia positioning, and cortical layering in both PTEN-mutant organoids at the later stage of 72+ days of development. Perifosine, an AKT inhibitor, reduced over-activated AKT and partially corrected the abnormalities in cellular organization observed in PTENG132D organoids. Single cell RNAseq analyses on early-stage organoids revealed that genes related to neural cell fate were decreased in PTENG132D mutant organoids, and AKT inhibition was capable of upregulating gene signatures related to neuronal cell fate and CNS maturation pathways. These findings demonstrate that different PTEN missense mutations can have a profound impact on neurodevelopment at diverse stages which in turn may predispose PHTS individuals to ASD. Further study will shed light on ways to mitigate pathological impact of PTEN mutants on neurodevelopment by stage-specific manipulation of downstream PTEN signaling components.
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Colorectal cancer (CRC) remains a leading killer in the U.S. with resistance to treatment as the largest hurdle to cure. Colorectal cancer-initiating cells (CICs) are a self-renewing tumor population that contribute to tumor relapse. Here, we report that patient-derived CICs display relative chemoresistance compared with differentiated progeny. In contrast, conventional cell lines failed model therapeutic resistance. CICs preferentially repaired chemotherapy-induced DNA breaks, prompting us to interrogate DNA damage pathways against which pharmacologic inhibitors have been developed. We found that CICs critically depended on the key single-strand break repair mediator, poly(ADP-ribose) polymerase (PARP), to survive treatment with standard-of-care chemotherapy. Small molecule PARP inhibitors (PARPi) sensitized CICs to chemotherapy and reduced chemotherapy-treated CIC viability, self-renewal, and DNA damage repair. Although PARPi monotherapy failed to kill CICs, combined PARPi therapy with chemotherapy attenuated tumor growth in vivo. Clinical significance of PARPi for CRC patients was supported by elevated PARP levels in colorectal tumors compared with normal colon, with further increases in metastases. Collectively, our results suggest that PARP inhibition serves as a point of fragility for CICs by augmenting therapeutic efficacy of chemotherapy. Stem Cells 2019;37:42-53.
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Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Reparo do DNA/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Microambiente Tumoral/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
Growth factor-mediated proliferation and self-renewal maintain tissue-specific stem cells and are frequently dysregulated in cancers. Platelet-derived growth factor (PDGF) ligands and receptors (PDGFRs) are commonly overexpressed in gliomas and initiate tumors, as proven in genetically engineered models. While PDGFRα alterations inform intertumoral heterogeneity toward a proneural glioblastoma (GBM) subtype, we interrogated the role of PDGFRs in intratumoral GBM heterogeneity. We found that PDGFRα is expressed only in a subset of GBMs, while PDGFRß is more commonly expressed in tumors but is preferentially expressed by self-renewing tumorigenic GBM stem cells (GSCs). Genetic or pharmacological targeting of PDGFRß (but not PDGFRα) attenuated GSC self-renewal, survival, tumor growth, and invasion. PDGFRß inhibition decreased activation of the cancer stem cell signaling node STAT3, while constitutively active STAT3 rescued the loss of GSC self-renewal caused by PDGFRß targeting. In silico survival analysis demonstrated that PDGFRB informed poor prognosis, while PDGFRA was a positive prognostic factor. Our results may explain mixed clinical responses of anti-PDGFR-based approaches and suggest the need for integration of models of cancer as an organ system into development of cancer therapies.
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Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Humanos , Camundongos , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Fator de Transcrição STAT3/metabolismo , Transplante HeterólogoRESUMO
Cancer stem cells (CSCs) are a heterogeneous and dynamic self-renewing population that stands at the top of tumor cellular hierarchy and contribute to tumor recurrence and therapeutic resistance. As methods of CSC isolation and functional interrogation advance, there is a need for a reliable and accessible quantitative approach to assess heterogeneity and state transition dynamics in CSCs. We developed a high-throughput automated single cell imaging analysis (HASCIA) approach for the quantitative assessment of protein expression with single-cell resolution and applied the method to investigate spatiotemporal factors that influence CSC state transition using glioblastoma (GBM) CSCs (GSCs) as a model system. We were able to validate the quantitative nature of this approach through comparison of the protein expression levels determined by HASCIA to those determined by immunoblotting. A virtue of HASCIA was exemplified by detection of a subpopulation of SOX2-low cells, which expanded in fraction size during state transition. HASCIA also revealed that GSCs were committed to loose stem cell state at an earlier time point than the average SOX2 level decreased. Functional assessment of stem cell frequency in combination with the quantification of SOX2 expression by HASCIA defined a stable cutoff of SOX2 expression level for stem cell state. We also developed an approach to assess local cell density and found that denser monolayer areas possess higher average levels of SOX2, higher cell diversity, and a presence of a sub-population of slowly proliferating SOX2-low GSCs. HASCIA is an open source software that facilitates understanding the dynamics of heterogeneous cell population such as that of GSCs and their progeny. It is a powerful and easy-to-use image analysis and statistical analysis tool available at https://hascia.lerner.ccf.org. © 2019 International Society for Advancement of Cytometry.
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Processamento de Imagem Assistida por Computador/métodos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Análise de Célula Única/métodos , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/genética , Células Cultivadas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Neoplásicas/ultraestrutura , Fatores de Transcrição SOXB1/análise , Fatores de Transcrição SOXB1/genética , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , SoftwareRESUMO
Advanced cancers display cellular heterogeneity driven by self-renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self-renewal and tumor initiation. To overcome these barriers in triple-negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well-established pluripotency gene NANOG. NANOG-GFP+ cells gave rise to both GFP+ and GFP(-) cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self-renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well-established CSC markers CD24(-) /CD44(+) , CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG-GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule-A (JAM-A). JAM-A was highly expressed in GFP+ cells and patient-derived xenograft ALDH+ CSCs compared with the GFP(-) and ALDH(-) cells, respectively. Depletion of JAM-A compromised self-renewal, whereas JAM-A overexpression induced self-renewal in GFP(-) cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non-CSCs in TNBC that can be used to identify CSC-specific targets for the development of future therapeutic strategies.
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Genes Reporter/genética , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Glioblastoma (GBM) contains a self-renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self-renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient-derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self-renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self-renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non-CSCs, increased with exposure to oxidized low-density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages.
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Neoplasias Encefálicas/metabolismo , Antígenos CD36/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Antígenos CD36/genética , Proliferação de Células , Progressão da Doença , Feminino , Expressão Gênica , Glioblastoma/mortalidade , Glioblastoma/patologia , Estimativa de Kaplan-Meier , Lipoproteínas LDL/fisiologia , Camundongos Nus , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
Individuals with a PTEN germline mutation receive the molecular diagnosis of PTEN hamartoma tumor syndrome (PHTS). PHTS displays a complex spectrum of clinical phenotypes including harmartomas, predisposition to cancers, and autism spectrum disorder (ASD). Clear-cut genotype-phenotype correlations are yet to be established due to insufficient information on the PTEN function being impacted by mutations. To fill this knowledge gap, we compared functional impacts of two selected missense PTEN mutant alleles, G132D and M134R, each respectively being associated with distinct clinical phenotype, ASD or thyroid cancer without ASD using gene-edited human induced pluripotent stem cells (hiPSCs). In homozygous hiPSCs, PTEN expression was severely reduced by M134R mutation due to shortened protein half-life. G132D suppressed PTEN expression to a lesser extent than Μ134R mutation without altering protein half-life. When challenged with γ-irradiation, G132D heterozygous cells exited radiation-induced G2 arrest earlier than wildtype and M134R heterozygous hiPSCs despite the similar DNA damage levels as the latter two. Immunoblotting analyses suggested that γ-irradiation induced apoptosis in G132D heterozygous cells to lesser degrees than in the hiPSCs of other genotypes. These data suggest that ASD-associated G132D allele promotes genome instability by premature cell cycle reentry with incomplete DNA repair.
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Transtorno do Espectro Autista , Síndrome do Hamartoma Múltiplo , Células-Tronco Pluripotentes Induzidas , Neoplasias da Glândula Tireoide , Humanos , Transtorno do Espectro Autista/genética , Mutação em Linhagem Germinativa , Células-Tronco Pluripotentes Induzidas/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fenótipo , Pontos de Checagem do Ciclo CelularRESUMO
Alamar Blue (AB) has become an increasingly popular reagent of choice for cell viability assays. We chose AB over other reagents such as MTT and Cell-Titer Glo due to its cost effectiveness and its ability to be a nondestructive assay. While analyzing the effect of osimertinib, an EGFR inhibitor on the non-small cell lung cancer cell line, PC-9, we noticed unexpected right-shifts of dose response curves as compared to the curve obtained by Cell Titer Glo assay. Here, we describe our modified AB assay method to avoid right shift right shift in dose response curve. Unlike some of the redox drugs that were reported to directly affected AB reading, osimertinib itself did not directly increase AB reading. Yet, the removal of the drug containing medium prior to AB addition eliminated falsely increased reading giving comparable dose response curve as the one determined by Cell Titer Glo assay. When a panel of 11 drugs were assessed, we found that this modified AB assay eliminated unexpected similar right shifts detected in other epidermal growth factor receptor (EGFR) inhibitors. We also found that plate-to-plate variability can be minimized by adding an appropriate concentration of rhodamine B solution to the assay plates to calibrate fluorimeter sensitivity. This calibration method also enables a continuous longitudinal assay to monitor cell growth or recovery from drug toxicity over time. Our new modified AB assay is expected to provide accurate in vitro measurement of EGFR targeted therapies.
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In this study, we experimentally measure the frequency-dependent interactions between a gefitinib-resistant non-small cell lung cancer population and its sensitive ancestor via the evolutionary game assay. We show that cost of resistance is insufficient to accurately predict competitive exclusion and that frequency-dependent growth rate measurements are required. Using frequency-dependent growth rate data, we then show that gefitinib treatment results in competitive exclusion of the ancestor, while the absence of treatment results in a likely, but not guaranteed, exclusion of the resistant strain. Then, using simulations, we demonstrate that incorporating ecological growth effects can influence the predicted extinction time. In addition, we show that higher drug concentrations may not lead to the optimal reduction in tumor burden. Together, these results highlight the potential importance of frequency-dependent growth rate data for understanding competing populations, both in the laboratory and as we translate adaptive therapy regimens to the clinic.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Evolução Biológica , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Asymmetric cell division (ACD) enables the maintenance of a stem cell population while simultaneously generating differentiated progeny. Cancer stem cells (CSCs) undergo multiple modes of cell division during tumor expansion and in response to therapy, yet the functional consequences of these division modes remain to be determined. Using a fluorescent reporter for cell surface receptor distribution during mitosis, we found that ACD generated a daughter cell with enhanced therapeutic resistance and increased coenrichment of EGFR and neurotrophin receptor (p75NTR) from a glioblastoma CSC. Stimulation of both receptors antagonized differentiation induction and promoted self-renewal capacity. p75NTR knockdown enhanced the therapeutic efficacy of EGFR inhibition, indicating that coinheritance of p75NTR and EGFR promotes resistance to EGFR inhibition through a redundant mechanism. These data demonstrate that ACD produces progeny with coenriched growth factor receptors, which contributes to the generation of a more therapeutically resistant CSC population.
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Divisão Celular Assimétrica , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Antígeno AC133/metabolismo , Neoplasias Encefálicas/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Autorrenovação Celular , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Glioblastoma/metabolismo , Humanos , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/antagonistas & inibidores , Receptores de Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/metabolismoRESUMO
BACKGROUND: Filtering facepiece respirators (FFR) are critical for protecting essential personnel and limiting the spread of disease. Due to the current COVID-19 pandemic, FFR supplies are dwindling in many health systems, necessitating re-use of potentially contaminated FFR. Multiple decontamination solutions have been developed to meet this pressing need, including systems designed for bulk decontamination of FFR using vaporous hydrogen peroxide or ultraviolet-C (UV-C) radiation. However, the large scale on which these devices operate may not be logistically practical for small or rural health care settings or for ad hoc use at points-of-care. METHODS: Here, we present the Synchronous UV Decontamination System, a novel device for rapidly deployable, point-of-care decontamination using UV-C germicidal irradiation. We designed a compact, easy-to-use device capable of delivering over 2 J cm2 of UV-C radiation in one minute. RESULTS: We experimentally tested Synchronous UV Decontamination System' microbicidal capacity and found that it eliminates near all virus from the surface of tested FFRs, with less efficacy against pathogens embedded in the inner layers of the masks. CONCLUSIONS: This short decontamination time should enable care-providers to incorporate decontamination of FFR into a normal donning and doffing routine following patient encounters.
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COVID-19/prevenção & controle , Descontaminação/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Dispositivos de Proteção Respiratória/virologia , SARS-CoV-2 , Raios Ultravioleta , COVID-19/virologia , Descontaminação/métodos , Reutilização de Equipamento , HumanosRESUMO
Personal protective equipment (PPE) is crucially important to the safety of both patients and medical personnel, particularly in the event of an infectious pandemic. As the incidence of Coronavirus Disease 2019 (COVID-19) increases exponentially in the United States and many parts of the world, healthcare provider demand for these necessities is currently outpacing supply. In the midst of the current pandemic, there has been a concerted effort to identify viable ways to conserve PPE, including decontamination after use. In this study, we outline a procedure by which PPE may be decontaminated using ultraviolet (UV) radiation in biosafety cabinets (BSCs), a common element of many academic, public health, and hospital laboratories. According to the literature, effective decontamination of N95 respirator masks or surgical masks requires UV-C doses of greater than 1 Jcm-2, which was achieved after 4.3 hours per side when placing the N95 at the bottom of the BSCs tested in this study. We then demonstrated complete inactivation of the human coronavirus NL63 on N95 mask material after 15 minutes of UV-C exposure at 61 cm (232 µWcm-2). Our results provide support to healthcare organizations looking for methods to extend their reserves of PPE.
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COVID-19/prevenção & controle , Contenção de Riscos Biológicos/métodos , Descontaminação/métodos , Pandemias , SARS-CoV-2/efeitos da radiação , Raios Ultravioleta , COVID-19/transmissão , COVID-19/virologia , Relação Dose-Resposta à Radiação , Reutilização de Equipamento , Pessoal de Saúde/educação , Humanos , Laboratórios/organização & administração , Máscaras/virologia , Respiradores N95/virologia , Radiometria/estatística & dados numéricos , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologiaRESUMO
Quantitative cytometric studies show that cyclin D1 levels must decline during S phase for proper cell cycle progression, and that cyclin D1 decline follows phosphorylation induced by the checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). ATM is mutated in ataxia telangiectasia (AT), a disease characterized by progressive neurodegeneration. Importantly, neurodegeneration in many cases has been linked to the increased expression of cyclin D1 in neurons leading to inappropriate cell cycle entry. These facts prompted us to test the possibility that ATM normally protects against neural degeneration by suppressing cyclin D1 levels, particularly following genotoxic stress. For this purpose, neural stem cells were induced to differentiate into mature neural cells, including neurons. ATM activity in these cultures was inhibited with a specific chemical inhibitor in the presence or absence of hydrogen peroxide treatment, and the effect on cyclin D1 expression was determined by quantitative, single cell cytometric analyses. As predicted, inhibition of ATM did promote elevation of cyclin D1 in differentiated neurons, particularly under conditions of oxidative stress. The survival of differentiated neurons and of neural stem cells was reduced by such treatments. These data support our suggestion that ATM functions to maintain low levels of cyclin D1 expression in differentiated neurons; and may provide important clues in understanding neural degeneration in general.
Assuntos
Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Ciclina D1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Ciclina D1/genética , Regulação para Baixo , Citometria de Fluxo , Humanos , Neurônios/citologiaRESUMO
Advances in the treatment of Ewing's sarcoma (EWS) are desperately needed, particularly in the case of metastatic disease. A deeper understanding of collateral sensitivity, where the evolution of therapeutic resistance to one drug aligns with sensitivity to another drug, may improve our ability to effectively target this disease. For the first time in a solid tumor, we produced a temporal collateral sensitivity map that demonstrates the evolution of collateral sensitivity and resistance in EWS. We found that the evolution of collateral resistance was predictable with some drugs but had significant variation in response to other drugs. Using this map of temporal collateral sensitivity in EWS, we can see that the path toward collateral sensitivity is not always repeatable, nor is there always a clear trajectory toward resistance or sensitivity. Identifying transcriptomic changes that accompany these states of transient collateral sensitivity could improve treatment planning for patients with EWS.
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BACKGROUND: PTEN, a syndromic autism spectrum disorder (ASD) risk gene, is mutated in approximately 10% of macrocephalic ASD cases. Despite the described genetic association between PTEN and ASD and ensuing studies, we continue to have a limited understanding of how PTEN disruption drives ASD pathogenesis and maintenance. METHODS: We derived neural stem cells (NSCs) from the dentate gyrus (DG) of Ptenm3m4 mice, a model that recapitulates PTEN-ASD phenotypes. We subsequently characterized the expression of stemness factors, proliferation, and differentiation of neurons and glia in Ptenm3m4 NSCs using immunofluorescent and immunoblotting approaches. We also measured Creb phosphorylation by Western blot analysis and expression of Creb-regulated genes with qRT-PCR. RESULTS: The m3m4 mutation decreases Pten localization to the nucleus and its global expression over time. Ptenm3m4 NSCs exhibit persistent stemness characteristics associated with increased proliferation and a resistance to neuronal maturation during differentiation. Given the increased proliferation of Ptenm3m4 NSCs, a significant increase in the population of immature neurons relative to mature neurons occurs, an approximately tenfold decrease in the ratio between the homozygous mutant and wildtype. There is an opposite pattern of differentiation in some Ptenm3m4 glia, specifically an increase in astrocytes. These aberrant differentiation patterns associate with changes in Creb activation in Ptenm3m4/m3m4 NSCs. We specifically observed loss of Creb phosphorylation at S133 in Ptenm3m4/m3m4 NSCs and a subsequent decrease in expression of Creb-regulated genes important to neuronal function (i.e., Bdnf). Interestingly, Bdnf treatment is able to partially rescue the stunted neuronal maturation phenotype in Ptenm3m4/m3m4 NSCs. CONCLUSIONS: Constitutional disruption of Pten nuclear localization with subsequent global decrease in Pten expression generates abnormal patterns of differentiation, a stunting of neuronal maturation. The propensity of Pten disruption to restrain neurons to a more progenitor-like state may be an important feature contributing to PTEN-ASD pathogenesis.
Assuntos
Diferenciação Celular/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , PTEN Fosfo-Hidrolase/genética , Animais , Transtorno do Espectro Autista/etiologia , Transtorno do Espectro Autista/metabolismo , Biomarcadores , Núcleo Celular , Proliferação de Células , Autorrenovação Celular , Células Cultivadas , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Mutação , Neuroglia/citologia , Neuroglia/metabolismo , PTEN Fosfo-Hidrolase/metabolismoRESUMO
Gap-junction-mediated cell-cell communication enables tumor cells to synchronize complex processes. We previously found that glioblastoma cancer stem cells (CSCs) express higher levels of the gap junction protein Cx46 compared to non-stem tumor cells (non-CSCs) and that this was necessary and sufficient for CSC maintenance. To understand the mechanism underlying this requirement, we use point mutants to disrupt specific functions of Cx46 and find that Cx46-mediated gap-junction coupling is critical for CSCs. To develop a Cx46 targeting strategy, we screen a clinically relevant small molecule library and identify clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuates proliferation, self-renewal, and tumor growth and synergizes with temozolomide to induce apoptosis. Although clofazimine does not cross the blood-brain barrier, the combination of clofazimine derivatives optimized for brain penetrance with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy.
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Conexina 43/fisiologia , Glioblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Animais , Comunicação Celular/efeitos dos fármacos , Clofazimina/farmacologia , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Análise Mutacional de DNA , Junções Comunicantes/fisiologia , Glioblastoma/metabolismo , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
When cell cycle studies are performed following cell cycle synchronization, it is possible that critical properties of an actively cycling cell will be overlooked. For this reason past studies have not revealed critical aspects of cell cycle control; such as how a cell determines when to exit the cell cycle, or how rapidly it should cycle. To address these challenging questions we have developed a procedure to quantitate fluorescent stains in a monolayer culture, where nuclear fluorescence and cell cycle history can be assessed with accuracy on a cell by cell basis. The cell cycle position of each cell can be determined by analyzing DNA and BrdU levels. The behavior of cells in a given cell cycle position can then be studied by quantitating up to two other stained markers. When the microinjection of siRNA, neutralizing antibodies, and expression plasmids are coupled with quantitative image analysis, these cell cycle studies can be conducted following alterations in the expression levels of selected cellular targets. With these techniques we have discovered critical aspects of cell cycle control; including how cyclin D1 levels vary through the cell cycle, the molecular mechanisms governing these changes, and the biological implications of changes in cyclin D1 concentration in various cell cycle stages. Our studies with cyclin D1, coupled with similar studies of p27Kip1, form the basis of an entirely new model of cell cycle control proposed here. This model explains how cell cycle progression is terminated, and how the length of the cell cycle is regulated.
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Ciclo Celular , Ciclina D1/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Citometria de Fluxo/métodos , Bromodesoxiuridina/farmacologia , Proliferação de Células , DNA/metabolismo , Citometria de Fluxo/instrumentação , Humanos , Microscopia de Fluorescência/métodos , Fosforilação , RNA Interferente Pequeno/metabolismoRESUMO
Tumors adapt their phenotypes during growth and in response to therapies through dynamic changes in cellular processes. Connexin proteins enable such dynamic changes during development, and their dysregulation leads to disease states. The gap junction communication channels formed by connexins have been reported to exhibit tumor-suppressive functions, including in triple-negative breast cancer (TNBC). However, we find that connexin 26 (Cx26) is elevated in self-renewing cancer stem cells (CSCs) and is necessary and sufficient for their maintenance. Cx26 promotes CSC self-renewal by forming a signaling complex with the pluripotency transcription factor NANOG and focal adhesion kinase (FAK), resulting in NANOG stabilization and FAK activation. This FAK/NANOG-containing complex is not formed in mammary epithelial or luminal breast cancer cells. These findings challenge the paradigm that connexins are tumor suppressors in TNBC and reveal a unique function for Cx26 in regulating the core self-renewal signaling that controls CSC maintenance.
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Autorrenovação Celular , Conexinas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Conexina 26 , Feminino , Humanos , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos SCID , Transplante de NeoplasiasRESUMO
The second International Cancer Stem Cell Conference in Cleveland, Ohio, on September 20-23, 2016, convened 330 attendees from academic, industrial, and clinical organizations. It featured a debate on the concepts and challenges of the cancer stem cells (CSC) as well as CSC-centered scientific sessions on clinical trials, genetics and epigenetics, tumor microenvironment, immune suppression, metastasis, therapeutic resistance, and emerging novel concepts. The conference hosted 35 renowned speakers, 100 posters, 20 short talks, and a preconference workshop. The reported advances of CSC research and therapies fostered new collaborations across national and international borders, and inspired the next generation's young scientists. Cancer Res; 77(19); 5222-7. ©2017 AACR.