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1.
Cancer Discov ; 2024 Aug 07.
Artículo en Inglés | MEDLINE | ID: mdl-39109936

RESUMEN

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show non-homologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

2.
Cell Rep ; 36(9): 109632, 2021 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-34469729

RESUMEN

Tubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3-CD16-) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy.


Asunto(s)
Tolerancia Inmunológica , Células Asesinas Naturales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Quísticas, Mucinosas y Serosas/inmunología , Neoplasias Ováricas/inmunología , Escape del Tumor , Microambiente Tumoral/inmunología , Antineoplásicos/farmacología , Carboplatino/farmacología , Línea Celular Tumoral , Técnicas de Cocultivo , Citocinas/metabolismo , Citotoxicidad Inmunológica , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Quísticas, Mucinosas y Serosas/tratamiento farmacológico , Neoplasias Quísticas, Mucinosas y Serosas/metabolismo , Neoplasias Quísticas, Mucinosas y Serosas/patología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fenotipo , Receptores de Células Asesinas Naturales/metabolismo , Tetraspanina 29/metabolismo , Trogocitosis , Escape del Tumor/efectos de los fármacos
3.
FASEB J ; 35(3): e21423, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33605480

RESUMEN

The degree that maternal glycemia affects placental metabolism of trophoblast cell types [cytotrophoblast (CTB) and syncytiotrophoblast (SCT)] in pregnant persons with gestational diabetes mellitus (GDM) is unknown. We tested the hypotheses that (a) hyperglycemia suppresses the metabolic rates of CTB and SCT; and (b) low placental metabolic activity from GDM placentas is due to decreased oxygen consumption of CTB. Trophoblast cells isolated from GDM and non-GDM term placentas were cultured for 8-hour (CTB) and following syncytialization at 72-hour (SCT) in 5 mM of glucose or 25 mM of glucose. Oxygen consumption rates, glycolysis, ATP levels, and lipid droplet morphometries were determined in CTB and SCT. In CTB from GDM placentas compared to control CTB: (a) oxidative phosphorylation was decreased by 44% (41.8 vs 74.2 pmol O2 /min/100 ng DNA, P = .002); (b) ATP content was 39% lower (1.1 × 10-7 vs 1.8 × 10-7  nM/ng DNA, P = .046); and (c) lipid droplets were two times larger (31.0 vs 14.4 µm2 /cell, P < .001) and 1.7 times more numerous (13.5 vs 7.9 lipid droplets/cell, P < .001). Hyperglycemia suppressed CTB glycolysis by 55%-60% (mean difference 20.4 [GDM, P = .008] and 15.4 [non-GDM, P = .029] mpH/min/100 ng DNA). GDM SCT was not metabolically different from non-GDM SCT. However, GDM SCT had significantly decreased expression of genes associated with differentiation including hCG, GCM1, and syncytin-1. We conclude that suppressed metabolic activity by the GDM placenta is attributable to metabolic dysfunction of CTB, not SCT. Critical placental hormone expression and secretion are decreased in GDM trophoblasts.


Asunto(s)
Diabetes Gestacional/metabolismo , Hiperglucemia/metabolismo , Lípidos , Mitocondrias/metabolismo , Diferenciación Celular , Femenino , Glucosa/metabolismo , Glucólisis/fisiología , Humanos , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/fisiología , Placenta/metabolismo , Embarazo , Trofoblastos/metabolismo
4.
Cancer Discov ; 11(6): 1562-1581, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33451982

RESUMEN

Mutations in ARID1A rank among the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout (KO) in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity, and mucinous differentiation. Genetic WNT/ß-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of microsatellite instability and Epstein-Barr virus-associated subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with nonessential WNT-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells. SIGNIFICANCE: We establish the first human forward genetic modeling of a commonly mutated tumor suppressor gene, ARID1A. Our study integrates diverse modalities including CRISPR/Cas9 genome editing, organoid culture, systems biology, and small-molecule screening to derive novel insights into early transformation mechanisms of ARID1A-deficient gastric cancers.See related commentary by Zafra and Dow, p. 1327.This article is highlighted in the In This Issue feature, p. 1307.


Asunto(s)
Sistemas CRISPR-Cas , Transformación Celular Neoplásica , Proteínas de Unión al ADN/genética , Neoplasias Gástricas/genética , Factores de Transcripción/genética , Humanos , Modelos Biológicos , Mutación
5.
J Clin Neurosci ; 78: 413-415, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32631721

RESUMEN

Tenosynovial giant cell tumors (TGCTs) are benign neoplasms that arise from the synovium of tendon sheaths, bursae, and joints. We report a rare presentation of TGCT involving the suboccipital spine.


Asunto(s)
Neoplasias Encefálicas/patología , Tumores de Células Gigantes/patología , Membrana Sinovial/patología , Tumor de Células Gigantes de las Vainas Tendinosas/patología , Humanos , Lóbulo Occipital/patología , Neoplasias de la Columna Vertebral/patología
6.
Cell Stem Cell ; 26(1): 4-6, 2020 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-31951587

RESUMEN

Two recent papers in Cell Stem Cell and Nature Medicine (Yao et al. [2019] and Ganesh et al. [2019]) demonstrate the successful use of rectal cancer patient-derived organoids to predict patient responses to neoadjuvant chemoradiation therapy, paving the way toward a new paradigm for precision medicine.


Asunto(s)
Organoides , Neoplasias del Recto , Humanos , Terapia Neoadyuvante , Medicina de Precisión
7.
8.
Placenta ; 72-73: 1-9, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30501875

RESUMEN

INTRODUCTION: The placenta employs an efficient and selective fatty acid transport system to supply lipids for fetal development. Disruptions in placental fatty acid transport lead to restricted fetal growth along with cardiovascular and neurologic deficits. Nevertheless, little is known about the molecular mechanisms involved in human placental fatty acid trafficking during the initial steps of uptake, or the importance of fatty acid chain length in determining uptake rates. METHODS: We employed BODIPY fluorophore conjugated fatty acid analogues of three chain lengths, medium (BODIPY-C5), long (BODIPY-C12), and very-long (BODIPY-C16), to study fatty acid uptake in isolated human trophoblast and explants using confocal microscopy. The three BODIPY-labeled fatty acids were added to freshly isolated explants and tracked for up to 30 min. Fatty acid uptake kinetics were quantified in trophoblast (cytotrophoblast and syncytiotrophoblast together) and the fetal capillary lumen. RESULTS: Long- (BODIPY-C12) and Very long-chain (BODIPY-C16) fatty acids accumulated more rapidly in the trophoblast layer than did medium-chain (BODIPY-C5) whereas BODIPY-C5 accumulated more rapidly in the fetal capillary than did the longer chain length fatty acids. The long-chain fatty acids, BODIPY-C12 and BODIPY-C16, are esterified and stored in lipid droplets in the cytotrophoblast layer, but medium-chain fatty acid, BODIPY-C5, is not. DISCUSSION: Fatty acids accumulate in trophoblast and fetal capillaries inversely according to their chain length. BODIPY-C5 accumulates in the fetal capillary in concentrations far greater than in the trophoblast, suggesting that medium-chain length BODIPY-labeled fatty acids are capable of being transported against a concentration gradient.


Asunto(s)
Ácidos Grasos/metabolismo , Microscopía Confocal/métodos , Placenta/metabolismo , Trofoblastos/metabolismo , Adulto , Transporte Biológico , Compuestos de Boro , Capilares/metabolismo , Células Cultivadas , Ácidos Grasos/química , Femenino , Feto/irrigación sanguínea , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Humanos , Cinética , Embarazo
9.
Sci Rep ; 7: 42941, 2017 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-28230167

RESUMEN

The syncytiotrophoblast (SCT) at the maternal-fetal interface has been presumed to be the primary driver of placental metabolism, and the underlying progenitor cytotrophoblast cells (CTB) an insignificant contributor to placental metabolic activity. However, we now show that the metabolic rate of CTB is much greater than the SCT. The oxygen consumption and extracellular acidification rate, a measure of glycolysis, are both greater in CTB than in SCT in vitro (CTB: 96 ± 16 vs SCT: 46 ± 14 pmol O2 × min-1 × 100 ng DNA-1, p < 0.001) and (CTB: 43 ± 6.7 vs SCT 1.4 ± 1.0 ∆mpH × min-1 × 100 ng DNA-1, p < 0.0001). Mitochondrial activity, as determined by using the mitochondrial activity-dependent dye Mitotracker CM-H2TMRosa, is higher in CTB than in SCT in culture and living explants. These data cast doubt on the previous supposition that the metabolic rate of the placenta is dominated by the SCT contribution. Moreover, differentiation into SCT leads to metabolic suppression. The normal suppression of metabolic activity during CTB differentiation to SCT is prevented with a p38 MAPK signaling inhibitor and epidermal growth factor co-treatment. We conclude that the undifferentiated CTB, in contrast to the SCT, is highly metabolically active, has a high level of fuel flexibility, and contributes substantially to global metabolism in the late gestation human placenta.


Asunto(s)
Glucólisis , Fosforilación Oxidativa , Placenta/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Ácidos Grasos no Esterificados/metabolismo , Femenino , Glucólisis/efectos de los fármacos , Humanos , Imidazoles/farmacología , Análisis de Flujos Metabólicos , Microscopía Fluorescente , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Embarazo , Piridinas/farmacología , Nacimiento a Término , Trofoblastos/citología , Trofoblastos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
10.
PLoS One ; 7(7): e41717, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22860009

RESUMEN

It is becoming increasingly clear that cells are remarkably sensitive to the biophysical cues of their microenvironment and that these cues play a significant role in influencing their behaviors. In this study, we investigated whether the early pre-implantation embryo is sensitive to mechanical cues, i.e. the elasticity of the culture environment. To test this, we have developed a new embryo culture system where the mechanical properties of the embryonic environment can be precisely defined. The contemporary standard environment for embryo culture is the polystyrene petri dish (PD), which has a stiffness (1 GPa) that is six orders of magnitude greater than the uterine epithelium (1 kPa). To approximate more closely the mechanical aspects of the in vivo uterine environment we used polydimethyl-siloxane (PDMS) or fabricated 3D type I collagen gels (1 kPa stiffness, Col-1k group). Mouse embryo development on alternate substrates was compared to that seen on the petri dish; percent development, hatching frequency, and cell number were observed. Our results indicated that embryos are sensitive to the mechanical environment on which they are cultured. Embryos cultured on Col-1k showed a significantly greater frequency of development to 2-cell (68 ± 15% vs. 59 ± 18%), blastocyst (64 ± 9.1% vs. 50 ± 18%) and hatching blastocyst stages (54 ± 25% vs. 21 ± 16%) and an increase in the number of trophectodermal cell (TE,65 ± 13 vs. 49 ± 12 cells) compared to control embryos cultured in PD (mean ± S.D.; p<.01). Embryos cultured on Col-1k and PD were transferred to recipient females and observed on embryonic day 12.5. Both groups had the same number of fetuses, however the placentas of the Col-1k fetuses were larger than controls, suggesting a continued effect of the preimplantation environment. In summary, characteristics of the preimplantation microenvironment affect pre- and post-implantation growth.


Asunto(s)
Medios de Cultivo/química , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Animales , Blastocisto/citología , Blastómeros/fisiología , Colágeno Tipo I/química , Dimetilpolisiloxanos/química , Ectodermo/citología , Elasticidad , Transferencia de Embrión , Embrión de Mamíferos/citología , Embrión de Mamíferos/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Placenta/citología , Poliestirenos/química , Embarazo , Propiedades de Superficie , Trofoblastos/citología , Zona Pelúcida/fisiología
11.
Endocrinology ; 153(7): 3457-67, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22562173

RESUMEN

More than 4.5 million children have been conceived by in vitro fertilization (IVF). Interestingly, singleton IVF offspring born at term have an increased incidence of low birth weight. The mechanism responsible for the lower birth weight is unknown, but alterations in placental function are possible. Hence, the goal of our study was to examine placental growth and function in mice generated in vivo or in vitro. To assess placental function, blastocysts were generated by IVF or produced by natural mating (control group); both IVF and control blastocysts were transferred to pseudopregnant recipients. Placental weights did not differ at embryonic d 15.5 (E15.5) but were increased at E18.5 in the IVF group (25.4%, P < 0.001) compared with control. Proliferation was increased in IVF placentae, whereas overall placental gross morphology and apoptosis were not affected. Both fetal weights (16.4% lower at E15.5 and 8.8% lower at E18.5, P < 0.05) and fetal to placental ratios were lower (P < 0.001) in the IVF compared with the control group at both time points, whereas birth weights did not differ. At E18.5, the mRNA for selected glucose, system A amino acid transporters, and imprinted genes were down-regulated in IVF placentae. GLUT3 protein level was decreased in the IVF group (P < 0.05). Importantly, intrajugular injections of (14)C-methyl-D-glucose or (14)C-MeAIB tracers (n = 6 litters per group) showed that placental transport of glucose and amino acids were 24.8% (not significant) and 58.1% (P < 0.05) lower in the IVF group. Fetal accumulation of glucose was not different, but amino acid accumulation was significantly (36 %) lower in IVF fetuses (P < 0.05). We conclude that IVF alters both fetal and placental growth and, importantly, decreases placental transport efficiency in mice conceived by IVF.


Asunto(s)
Fertilización In Vitro/métodos , Placenta/fisiología , Aminoácidos/metabolismo , Animales , Apoptosis , Transporte Biológico , Blastocisto/metabolismo , Transferencia de Embrión , Femenino , Peso Fetal , Glucosa/metabolismo , Ratones , Modelos Animales , Tamaño de los Órganos , Placenta/metabolismo , Embarazo , Preñez , Factores de Tiempo
12.
Artículo en Inglés | MEDLINE | ID: mdl-20890961

RESUMEN

In nearly all aspects of biology, forces are a relevant regulator of life's form and function. More recently, science has established that cells are exquisitely sensitive to forces of varying magnitudes and time scales, and they convert mechanical stimuli into a chemical response. This phenomenon, termed mechanotransduction, is an integral part of cellular physiology and has a profound impact on the development of the organism. Furthermore, malfunctioning mechanical properties or mechanotransduction often leads to pathology of the organism. In this review, we describe mechanotransduction and the theories underlying how forces may be sensed, from the molecular to organism scale. The influence of mechanotransduction on normal and abnormal development, such as stem cell differentiation and cancer, is also reviewed. Studies illustrate the diversity of mechanotransduction, and the major role it has on organism homeostasis. Cells employ a variety of mechanisms, which differ depending upon cell type and environment, to sense and respond to forces.


Asunto(s)
Mecanotransducción Celular/fisiología , Animales , Fenómenos Fisiológicos Celulares , Epitelio/crecimiento & desarrollo , Epitelio/fisiología , Adhesiones Focales/fisiología , Homeostasis , Canales Iónicos/fisiología , Transducción de Señal , Células Madre/citología , Células Madre/fisiología , Estrés Mecánico
13.
Biophys J ; 97(12): 3095-104, 2009 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-20006946

RESUMEN

Filamins are actin binding proteins that contribute to cytoskeletal integrity and biochemical scaffolds during mechanochemical signal transductions. Structurally, human filamins are dimers composed of an actin-binding domain with 24 immunoglobulin (Ig)-like repeats. In this study, we focus on the recently solved high-resolution crystal structure of Ig-like repeats 19-21 of filamin-A (IgFLNa-R19-R21). IgFLNa-R19-21 is of marked importance because it contains the binding site for integrins and facilitates the dynamic ability of filamin-A to communicate with the extracellular environment. However, the structure of filamin-A shows an interesting domain arrangement where the integrin binding site on IgFLNa-R21 is hindered sterically by IgFLNa-R20. Thus, a number of hypotheses on the regulation of filamin-A exist. Using molecular dynamics simulations we evaluated the effects of two primary regulators of filamin-A, force and phosphorylation. We find that a tensile force of 40 pN is sufficient to initiate the partial removal of the autoinhibition on the integrin binding site of IgFLNa-R21. Force coupled to phosphorylation at Ser(2152), however, affords complete dissociation of autoinhibition with a decreased force requirement. Phosphorylation seems to decrease the threshold for removing the IgFLNa-R20 beta-strand inhibitor within 300 ps with 40 pN tensile force. Furthermore, the molecular dynamic trajectories illustrate phosphorylation of Ser(2152) without force is insufficient to remove autoinhibition. We believe the results of this study implicate filamin-A as a tunable mechanosensor, where its sensitivity can be modulated by the degree of phosphorylation.


Asunto(s)
Proteínas Contráctiles/metabolismo , Integrinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Fenómenos Biomecánicos , Proteínas Contráctiles/antagonistas & inhibidores , Proteínas Contráctiles/química , Filaminas , Humanos , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/química , Simulación de Dinámica Molecular , Fosforilación , Unión Proteica , Desnaturalización Proteica , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Serina , Solventes/química
14.
Biophys J ; 94(3): 1075-83, 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17921200

RESUMEN

Rearrangement of the actin cytoskeleton is integral to cell shape and function. Actin-binding proteins, e.g., filamin, can naturally contribute to the mechanics and function of the actin cytoskeleton. The molecular mechanical bases for filamin's function in actin cytoskeletal reorganization are examined here using molecular dynamics simulations. Simulations are performed by applying forces ranging from 25 pN to 125 pN for 2.5 ns to the rod domain of filamin. Applying small loads ( approximately 25 pN) to filamin's rod domain supplies sufficient energy to alter the conformation of the N-terminal regions of the rod. These forces break local hydrogen bond coordination often enough to allow side chains to find new coordination partners, in turn leading to drastic changes in the conformation of filamin, for example, increasing the hydrophobic character of the N-terminal rod region and, alternatively, activating the C-terminal region to become increasingly stiff. These changes in conformation can lead to changes in the affinity of filamin for its binding partners. Therefore, filamin can function to transduce mechanical signals as well as preserve topology of the actin cytoskeleton throughout the rod domain.


Asunto(s)
Proteínas Contráctiles/química , Proteínas Contráctiles/ultraestructura , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/ultraestructura , Modelos Químicos , Modelos Moleculares , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/ultraestructura , Simulación por Computador , Elasticidad , Filaminas , Mecánica , Conformación Proteica , Estructura Terciaria de Proteína , Estrés Mecánico
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