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CD14 is an innate immune receptor that senses pathogen-associated molecular patterns, such as lipopolysaccharide, to activate the innate immune response. Although CD14 is known to be glycosylated, detailed understanding about the structural and functional significance of this modification is still missing. Herein, an NMR and MS-based study, assisted by MD simulations, has provided a 3D-structural model of glycosylated CD14. Our results reveal the existence of a key N-glycosylation site at Asn282 that exclusively contains unprocessed oligomannnose N-glycans that perfectly fit the concave cavity of the bent-solenoid shaped protein. This site is not accessible to glycosidases and is fundamental for protein folding and secretion. A second N-site at Asn151 displays mostly complex N-glycans, with the typical terminal epitopes of the host cell-line expression system (i.e. ßGal, α2,3 and α2,6 sialylated ßGal, here), but also particularities, such as the lack of core fucosylation. The glycan at this site points outside the protein surface, resulting in N-glycoforms fully exposed and available for interactions with lectins. In fact, NMR experiments show that galectin-4, proposed as a binder of CD14 on monocytes to induce their differentiation into macrophages-like cells, interacts in vitro with CD14 through the recognition of the terminal glycoepitopes on Asn151. This work provides key information about CD14 glycosylation, which helps to better understand its functional roles and significance. Although protein glycosylation is known to be dynamic and influenced by many factors, some of the features found herein (presence of unprocessed N-glycans and lack of core Fuc) are likely to be protein specific.
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Lectinas , Polisacáridos , Glicosilación , Polisacáridos/química , Lectinas/metabolismo , Línea Celular , Lipopolisacáridos/metabolismoRESUMEN
A significant number of breast cancers develop resistance to hormone therapy. This progression, while posing a major clinical challenge, is difficult to predict. Despite important contributions made by cell models and clinical studies to tackle this problem, both present limitations when taken individually. Experiments with cell models are highly reproducible but do not reflect the indubitable heterogenous landscape of breast cancer. On the other hand, clinical studies account for this complexity but introduce uncontrolled noise due to external factors. Here, we propose a new approach for biomarker discovery that is based on a combined analysis of sequencing data from controlled MCF7 cell experiments and heterogenous clinical samples that include clinical and sequencing information from The Cancer Genome Atlas. Using data from differential gene expression analysis and a Bayesian logistic regression model coupled with an original simulated annealing-type algorithm, we discovered a novel 6-gene signature for stratifying patient response to hormone therapy. The experimental observations and computational analysis built on independent cohorts indicated the superior predictive performance of this gene set over previously known signatures of similar scope. Together, these findings revealed a new gene signature to identify patients with breast cancer with an increased risk of developing resistance to endocrine therapy.
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Neoplasias de la Mama , Perfilación de la Expresión Génica , Humanos , Femenino , Teorema de Bayes , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Hormonas/uso terapéutico , PronósticoRESUMEN
On 8 December 2022 the organizing committee of the European Network for Breast Development and Cancer labs (ENBDC) held its fifth annual Think Tank meeting in Amsterdam, the Netherlands. Here, we embraced the opportunity to look back to identify the most prominent breakthroughs of the past ten years and to reflect on the main challenges that lie ahead for our field in the years to come. The outcomes of these discussions are presented in this position paper, in the hope that it will serve as a summary of the current state of affairs in mammary gland biology and breast cancer research for early career researchers and other newcomers in the field, and as inspiration for scientists and clinicians to move the field forward.
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Neoplasias de la Mama , Glándulas Mamarias Humanas , Humanos , Femenino , Mama , BiologíaRESUMEN
In healthy tissues, cells are in mechanical homeostasis. During cancer progression, this equilibrium is disrupted. Cancer cells alter their mechanical phenotype to a softer and more fluid-like one than that of healthy cells. This is connected to cytoskeletal remodeling, changed adhesion properties, faster cell proliferation and increased cell motility. In this work, we investigated the mechanical properties of breast cancer cells representative of different breast cancer subtypes, using MCF-7, tamoxifen-resistant MCF-7, MCF10A and MDA-MB-231 cells. We derived viscoelastic properties from atomic force microscopy force spectroscopy measurements and showed that the mechanical properties of the cells are associated with cancer cell malignancy. MCF10A are the stiffest and least fluid-like cells, while tamoxifen-resistant MCF-7 cells are the softest ones. MCF-7 and MDA-MB-231 show an intermediate mechanical phenotype. Confocal fluorescence microscopy on cytoskeletal elements shows differences in actin network organization, as well as changes in focal adhesion localization. These findings provide further evidence of distinct changes in the mechanical properties of cancer cells compared to healthy cells and add to the present understanding of the complex alterations involved in tumorigenesis.
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Neoplasias de la Mama , Citoesqueleto , Humanos , Femenino , Línea Celular Tumoral , Citoesqueleto/metabolismo , Células MCF-7 , Actinas/metabolismo , Tamoxifeno/farmacología , Tamoxifeno/metabolismo , Neoplasias de la Mama/metabolismo , Microscopía de Fuerza Atómica/métodosRESUMEN
Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that act as biological sensors and use a combination of mechanisms to modulate positively and negatively gene expression in a spatial and temporal manner. The highly orchestrated biological actions of several NRs influence the proliferation, differentiation, and apoptosis of many different cell types. Synthetic ligands for several NRs have been the focus of extensive drug discovery efforts for cancer intervention. This review summarizes the roles in tumour growth and metastasis of several relevant NR family members, namely androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor (TR), retinoic acid receptors (RARs), retinoid X receptors (RXRs), peroxisome proliferator-activated receptors (PPARs), and liver X receptors (LXRs). These studies are key to develop improved therapeutic agents based on novel modes of action with reduced side effects and overcoming resistance.
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Hormonas , Lípidos , Neoplasias , Receptores Citoplasmáticos y Nucleares , Animales , HumanosRESUMEN
The hybrid compound [Cu(cyclam)(H2O)2]0.5[{Cu(cyclam)}1.5{B-H2As2Mo6O26(H2O)}]·9H2O (1) (cyclam = 1,4,8,11-tetraazacyclotetradecane) was synthesized in aqueous solution by reacting the {Cu(cyclam)}2+ complex with a mixture of heptamolybdate and an arsenate(V) source. Crystal packing of 1 exhibits a supramolecular open-framework built of discrete covalent molybdoarsenate/metalorganic units and additional [Cu(cyclam)(H2O)2]2+ cations, the stacking of which generates squarelike channels parallel to the z axis with an approximate cross section of 10 × 11 Å2 where all the hydration water molecules are hosted. Thermal evacuation of solvent molecules yields a new anhydrous crystalline phase, but compound 1 does not preserve its single-crystalline nature upon heating. However, when crystals are dehydrated under vacuum, they undergo a structural transformation that proceeds via a single-crystal-to-single-crystal pathway, leading to the anhydrous phase [{Cu(cyclam)}2(A-H2As2Mo6O26)] (2). Total dehydration results in important modifications within the inorganic cluster skeleton which reveals an unprecedented solid-state B to A isomerization of the polyoxoanion. This transition also involves changes in the CuII bonding scheme that lead to covalent cluster/metalorganic layers by retaining the open-framework nature of 1. Compound 2 adsorbs ambient moisture upon air exposure, but it does not revert back to 1, and the hydrated phase [{Cu(cyclam)}2(A-H2As2Mo6O26)]·6H2O (2h) is obtained instead. Structural variations between 1 and 2 are reflected in electron paramagnetic resonance spectroscopy measurements, and the permanent microporosity of 2 provides interesting functionalities to the system such as the selective adsorption of gaseous CO2 over N2.
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Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.
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Adenosina Trifosfato/metabolismo , Epilepsia del Lóbulo Temporal/fisiopatología , Microglía/patología , Neuronas/metabolismo , Fagocitosis/fisiología , Adulto , Animales , Apoptosis/fisiología , Receptor 1 de Quimiocinas CX3C , Humanos , Ácido Kaínico/toxicidad , Antígenos Comunes de Leucocito/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Monocitos/patología , Neuronas/patología , Receptores CCR2/genética , Receptores CCR2/metabolismo , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Convulsiones/inducido químicamente , Convulsiones/fisiopatologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pbio.1002466.].
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[This corrects the article DOI: 10.1371/journal.pbio.1002466.].
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Atomic force microscopy (AFM) combined with fluorescence microscopy has been used to quantify cytomechanical modifications induced by resveratrol (at a fixed concentration of 50 µM) in a breast cancer cell line (MCF-7) upon temporal variation. Cell indentation methodology has been utilized to determine simultaneous variations of Young's modulus, the maximum adhesion force, and tether formation, thereby determining cell motility and adhesiveness. Effects of treatment were measured at several time-points (0-6 h, 24 h, and 48 h); longer exposures resulted in cell death. Our results demonstrated that AFM can be efficiently used as a diagnostic tool to monitor irreversible morpho/nano-mechanical changes in cancer cells during the early steps of drug treatment.
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Neoplasias de la Mama/fisiopatología , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Módulo de Elasticidad/efectos de los fármacos , Microscopía de Fuerza Atómica/métodos , Resveratrol/farmacología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Humanos , Células MCF-7 , Fenómenos Mecánicos/efectos de los fármacos , Resveratrol/uso terapéuticoRESUMEN
Mammary gland biologists gathered for the ninth annual workshop of the European Network for Breast Development and Cancer (ENBDC) at Weggis on the shores of Lake Lucerne in March 2017. The main themes were oestrogen receptor alpha signalling, new techniques for mammary cell culture, CRISPR screening and proteogenomics.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Transducción de Señal , Animales , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula , Femenino , Pruebas Genéticas , Humanos , Organoides , Proteómica/métodos , Técnicas de Cultivo de TejidosRESUMEN
The seventh annual meeting of the European Network of Breast Development and Cancer Laboratories, held in Weggis, Switzerland, in April 2015, was focused on techniques for the study of normal and cancer stem cells, cell fate decisions, cancer initiation and progression.
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Neoplasias de la Mama/patología , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/patología , Animales , Diferenciación Celular/fisiología , Progresión de la Enfermedad , Femenino , Humanos , Células Madre Neoplásicas/patologíaRESUMEN
The reaction between neutral bis(picolinate)copper(II) complexes and copper(II)-monosubstituted Keggin-type phosphotungstate anions formed in situ leads to the formation of the hybrid [C(NH2)3]10[{PW11O39Cu(H2O)}2{Cu(pic)2}]·10H2O compound (1, pic = picolinate) in the presence of structure-directing guanidinium cations. Single-crystal X-ray diffraction studies demonstrate that 1 contains dimeric {PW11O39Cu(H2O)}2{Cu(pic)2} molecular species constituted by two Keggin-type anions linked by one {Cu(pic)2} octahedral complex through axial coordination to their terminal oxygen atoms. The extensive hydrogen-bonding network established by guanidium cations and Keggin clusters plays a key role in retaining the crystallinity of the system throughout dehydration to allow a single-crystal-to-single-crystal (SCSC) transformation into the anhydrous [C(NH2)3]10[{PW11O39Cu}2{Cu(pic)2}] (2a) at 170 °C. Structural modifications involve the re-orientation, shifting in ca. 1.5 Å and condensation of all the {PW11O39Cu} units to result in {PW11O39Cu}n chains in an unprecedented solid-state polymerisation. This phase transition also implies the cleavage of Cu-O bonds induced by the rotation and translation of Keggin-type anions, in such a way that hybrid dimeric units in 1 are dismantled and {Cu(pic)2} complexes become square planar. The irreversibility of the phase transition has been confirmed by combined thermal and diffractometric analyses, which evidence that the anhydrous phase adsorbs only one water molecule per cluster to become the [C(NH2)3]10[{PW11O39Cu}2{Cu(pic)2}]·2H2O (2h) hydrated derivative without any significant alteration in its cell parameters, nor in its crystalline structure. Phase transformations have been monitored by electron paramagnetic resonance spectroscopy.
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Cell mechanical properties have been proposed as label free markers for diagnostic purposes in diseases such as cancer. Cancer cells show altered mechanical phenotypes compared to their healthy counterparts. Atomic Force Microscopy (AFM) is a widely utilized tool to study cell mechanics. These measurements often need skilful users, physical modelling of mechanical properties and expertise in data interpretation. Together with the need to perform many measurements for statistical significance and to probe wide enough areas in tissue structures, the application of machine learning and artificial neural network techniques to automatically classify AFM datasets has received interest recently. We propose the use of self-organizing maps (SOMs) as unsupervised artificial neural network applied to mechanical measurements performed via AFM on epithelial breast cancer cells treated with different substances that affect estrogen receptor signalling. We show changes in mechanical properties due to treatments, as estrogen softened the cells, while resveratrol led to an increase in cell stiffness and viscosity. These data were then used as input for SOMs. Our approach was able to distinguish between estrogen treated, control and resveratrol treated cells in an unsupervised manner. In addition, the maps enabled investigation of the relationship of the input variables.
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Algoritmos , Neoplasias , Microscopía de Fuerza Atómica/métodos , Resveratrol , Viscosidad , Redes Neurales de la ComputaciónRESUMEN
Cells generate traction forces to probe the mechanical properties of the surroundings and maintain a basal equilibrium state of stress. Traction forces are also implicated in cell migration, adhesion and ECM remodeling, and alteration of these forces is often observed in pathologies such as cancer. Thus, analyzing the traction forces is important for studies of cell mechanics in cancer and metastasis. In this primer, the methodology for conducting two-dimensional traction force microscopy (2D-TFM) experiments is reported. As a practical example, we analyzed the traction forces generated by three human breast cancer cell lines of different metastatic potential: MCF10-A, MCF-7 and MDA-MB-231 cells, and studied the effects of actin cytoskeleton disruption on those traction forces. Contrary to what is often reported in literature, lower traction forces were observed in cells with higher metastatic potential (MDA-MB-231). Implications of substrate stiffness and concentration of extracellular matrix proteins in such findings are discussed in the text. RESEARCH HIGHLIGHTS: Traction force microscopy (TFM) is suitable for studying and quantifying cell-substrate and cell-cell forces. TFM is suitable for investigating the relationship between chemical to mechanical signal transduction and vice versa. TFM can be combined with classical indentation studies providing a compact picture of cell mechanics. TFM still needs new physico-chemical (sample preparation) and computational approaches for more accurate data evaluation.
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Fenómenos Mecánicos , Tracción , Humanos , Microscopía de Fuerza Atómica/métodos , Adhesión Celular/fisiología , Movimiento Celular/fisiologíaRESUMEN
Retinoic acid and Wnt/ß-catenin signals play important roles during neuronal differentiation but less is known about noncanonical Wnt signals in this context. We examined retinoic acid and Wnt signaling in two human embryonal carcinoma cell lines, NTERA-2 (clone D1), which undergoes neuronal differentiation in response to retinoic acid, and 2102Ep, which does not. Retinoic acid treatment inhibited ß-catenin/Tcf activity in NTERA-2 cells but not in 2102Ep cells. Inhibition occurred downstream of ß-catenin but did not involve competition between retinoic acid receptors and ß-catenin for binding to p300 or Tcf-4. Ectopic expression of FZD1 partially restored inhibition in 2102Ep cells, suggesting the involvement of Wnt ligands. Retinoic acid treatment of NTERA-2 cells induced the expression of Wnt-4 and Wnt-11, both of which were able to inhibit ß-catenin/Tcf activity. Wnt-4 and Wnt-11 were found at cell borders in islands of cells that expressed OCT4 and GFAP and were predominantly negative for Nestin, PAX6, and GATA6. Gene silencing of Wnt-4, but not Wnt-11, reduced retinoic acid downregulation of OCT4 and Nanog and upregulation of PAX6, ASCL1, HOXC5, and NEUROD1, suggesting that Wnt-4 promotes early neuronal differentiation. Gene expression analysis of NTERA-2 cells stably overexpressing Wnt-11 suggested that Wnt-11 potentiates retinoic acid induction of early neurogenesis. Consistent with this, overexpression of Wnt-11 maintained a population of proliferating progenitor cells in cultures treated with retinoic acid for several weeks. These observations highlight the distinct roles of two noncanonical Wnts during the early stages of retinoic acid-induced neuronal differentiation.
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Células Madre Embrionarias/fisiología , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Neuronas/citología , Tretinoina/fisiología , Proteínas Wnt/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular Tumoral , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Receptores Frizzled/metabolismo , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neurogénesis/genética , Transducción de Señal , Transfección , Tretinoina/farmacología , Regulación hacia Arriba , Proteínas Wnt/genética , Proteína Wnt4 , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
Cells sense stiffness of surrounding tissues and adapt their activity, proliferation, motility and mechanical properties based on such interactions. Cells probe the stiffness of the substrate by anchoring and pulling to their surroundings, transmitting force to the extracellular matrix and other cells, and respond to the resistance they sense, mainly through changes in their cytoskeleton. Cancer and other diseases alter stiffness of tissues, and the response of cancer cells to this stiffness can also be affected. In the present study we show that MCF-7 breast cancer cells seeded on polyacrylamide gels have the ability to detect the stiffness of the substrate and alter their mechanical properties in response. MCF-7 cells plated on soft substrates display lower stiffness and viscosity when compared to those seeded on stiffer gels or glass. These differences can be associated with differences in the morphology and cytoskeleton organisation, since cells seeded on soft substrates have a round morphology, while cells seeded on stiffer substrates acquire a flat and spread morphology with formation of actin filaments, similar to that observed when seeded on glass. These findings show that MCF-7 cells can detect the stiffness of the surrounding microenvironment and thus, modify their mechanical properties.
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Células MCF-7 , HumanosRESUMEN
The identification and characterization of normal and breast cancer stem cells have provided a new vision of breast tumorigenesis. Cancer stem cells may be responsible for breast tumor initiation, progression and development of resistance to therapy. Most breast cancers express the estrogen receptor, and several studies have linked long-term estrogen exposure to enhanced breast cancer risk; however, estrogen receptor-positive tumors usually present a better prognosis than estrogen receptor-negative ones. The finding that estrogen reduces the pool of human breast stem cells may explain the more differentiated phenotype observed in estrogen receptor-positive tumors. In this article, our current understanding of the complex role of estrogen in human breast stem cells is discussed in the context of breast malignancy.
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Diferenciación Celular , Estrógenos/metabolismo , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Receptores de Estrógenos/metabolismo , Células Madre/metabolismoRESUMEN
In this work we present a unified method to study the mechanical properties of cells using the atomic force microscope. Stress relaxation and creep compliance measurements permitted us to determine, the relaxation times, the Young moduli and the viscosity of breast cancer cells (MCF-7). The results show that the mechanical behaviour of MCF-7 cells responds to a two-layered model of similar elasticity but differing viscosity. Treatment of MCF-7 cells with an actin-depolymerising agent results in an overall decrease in both cell elasticity and viscosity, however to a different extent for each layer. The layer that undergoes the smaller decrease (36-38%) is assigned to the cell membrane/cortex while the layer that experiences the larger decrease (70-80%) is attributed to the cell cytoplasm. The combination of the method presented in this work, together with the approach based on stress relaxation microscopy (Moreno-Flores et al 2010 J. Biomech. 43 349-54), constitutes a unique AFM-based experimental framework to study cell mechanics. This methodology can also be extended to study the mechanical properties of biomaterials in general.