RESUMEN
Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs. The combinations were derived from a pool of unique-sequence scFv Ab fragments retrieved from two SARS-CoV-2-naïve human phage display libraries. Following recombinant expression to full-length human IgG1 candidates, a biolayer interferometric analysis mapped epitopes to bins and confirmed that up to four Abs from across the bins can exist simultaneously on the spike glycoprotein trimer. Not all the bins of Abs interfered with the spike protein binding to angiotensin converting enzyme 2 (ACE2) in competitive binding assays, nor neutralized the pseudovirus or authentic virus in vitro, but when combined in vivo, their inclusion resulted in a much stronger viral clearance in the lungs of intranasally challenged hamsters, compared to that of those treated with mono ACE2 blockers. In addition, the Ab mixtures activated in vitro reporter cells expressing Fc-gamma receptors (FcγRs) involved in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The best four-Ab combination neutralized seventeen variants of concern from Wuhan-Hu1 to Omicron BA.4/BA.5 in vitro.
RESUMEN
Mechanically induced signaling pathways are important drivers of tumor progression. However, if and how mechanical signals affect metastasis or therapy response remains poorly understood. We previously found that the channel-kinase TRPM7, a regulator of cellular tension implicated in mechano-sensory processes, is required for breast cancer metastasis in vitro and in vivo. Here, we show that TRPM7 contributes to maintaining a mesenchymal phenotype in breast cancer cells by tensional regulation of the EMT transcription factor SOX4. The functional consequences of SOX4 knockdown closely mirror those produced by TRPM7 knockdown. By traction force measurements, we demonstrate that TRPM7 reduces cytoskeletal tension through inhibition of myosin II activity. Moreover, we show that SOX4 expression and downstream mesenchymal markers are inversely regulated by cytoskeletal tension and matrix rigidity. Overall, our results identify SOX4 as a transcription factor that is uniquely sensitive to cellular tension and indicate that TRPM7 may contribute to breast cancer progression by tensional regulation of SOX4.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción SOXC/metabolismo , Canales Catiónicos TRPM/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Citoesqueleto/genética , Citoesqueleto/metabolismo , Citoesqueleto/patología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Factores de Transcripción SOXC/genética , Canales Catiónicos TRPM/genética , Resistencia a la TracciónRESUMEN
Dendritic cell (DC) migration is essential for efficient host defense against pathogens and cancer, as well as for the efficacy of DC-based immunotherapies. However, the molecules that induce the migratory phenotype of DCs are poorly defined. Based on a large-scale proteome analysis of maturing DCs, we identified the GPI-anchored protein semaphorin 7A (Sema7A) as being highly expressed on activated primary myeloid and plasmacytoid DCs in human and mouse. We demonstrate that Sema7A deficiency results in impaired chemokine CCL21-driven DC migration in vivo. Impaired formation of actin-based protrusions, resulting in slower three-dimensional migration, was identified as the mechanism underlying the DC migration defect. Furthermore, we show, by atomic force microscopy, that Sema7A decreases adhesion strength to extracellular matrix while increasing the connectivity of adhesion receptors to the actin cytoskeleton. This study demonstrates that Sema7A controls the assembly of actin-based protrusions that drive DC migration in response to CCL21.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Antígenos CD/fisiología , Movimiento Celular/fisiología , Quimiocina CCL21/metabolismo , Células Dendríticas/fisiología , Matriz Extracelular/metabolismo , Semaforinas/fisiología , Animales , Antígenos CD/genética , Adhesión Celular , Movimiento Celular/genética , Células Cultivadas , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/fisiología , Humanos , Ratones , Ratones Noqueados , Microscopía de Fuerza Atómica , Interferencia de ARN , ARN Interferente Pequeño , Semaforinas/genéticaRESUMEN
Neuroblastoma is an embryonal tumor derived from poorly differentiated neural crest cells. Current research is aimed at identifying the molecular mechanisms that maintain the progenitor state of neuroblastoma cells and to develop novel therapeutic strategies that induce neuroblastoma cell differentiation. Mechanisms controlling neural crest development are typically dysregulated during neuroblastoma progression, and provide an appealing starting point for drug target discovery. Transcriptional programs involved in neural crest development act as a context dependent gene regulatory network. In addition to BMP, Wnt and Notch signaling, activation of developmental gene expression programs depends on the physical characteristics of the tissue microenvironment. TRPM7, a mechanically regulated TRP channel with kinase activity, was previously found essential for embryogenesis and the maintenance of undifferentiated neural crest progenitors. Hence, we hypothesized that TRPM7 may preserve progenitor-like, metastatic features of neuroblastoma cells. Using multiple neuroblastoma cell models, we demonstrate that TRPM7 expression closely associates with the migratory and metastatic properties of neuroblastoma cells in vitro and in vivo. Moreover, microarray-based expression profiling on control and TRPM7 shRNA transduced neuroblastoma cells indicates that TRPM7 controls a developmental transcriptional program involving the transcription factor SNAI2. Overall, our data indicate that TRPM7 contributes to neuroblastoma progression by maintaining progenitor-like features.
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Metástasis de la Neoplasia/genética , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/citología , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/fisiología , Canales Catiónicos TRPM/fisiología , Animales , Neoplasias de la Médula Ósea/secundario , División Celular , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Neoplasias Hepáticas/secundario , Ratones , Cresta Neural/citología , Neuroblastoma/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Factores de Transcripción de la Familia Snail , Factores de Transcripción/fisiología , Transcripción Genética , Microambiente TumoralRESUMEN
Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1(+/-) mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1(+/-) mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1(+/-) mice. Lastly, we found that Ehmt1(+/-) mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1(+/-) mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1(+/-) mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1(+/-) mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation.
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Huesos/metabolismo , Anomalías Craneofaciales/enzimología , Anomalías Craneofaciales/patología , Regulación del Desarrollo de la Expresión Génica/fisiología , Cardiopatías Congénitas/enzimología , Cardiopatías Congénitas/patología , N-Metiltransferasa de Histona-Lisina/deficiencia , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/patología , Cráneo/anomalías , Análisis de Varianza , Animales , Inmunoprecipitación de Cromatina , Deleción Cromosómica , Cromosomas Humanos Par 9/enzimología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Masculino , Ratones , Ratones Noqueados , Hipotonía Muscular/genética , Hipotonía Muscular/patología , Osteopontina , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
TRPM7 encodes a Ca2+-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Canales Catiónicos TRPM/biosíntesis , Animales , Neoplasias de la Mama/genética , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/patología , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proteínas Serina-Treonina Quinasas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Estrógenos/biosíntesis , Receptores de Estrógenos/metabolismo , Canales Catiónicos TRPM/deficiencia , Canales Catiónicos TRPM/genéticaRESUMEN
The ability of cells to respond to mechanical stimulation is crucial to a variety of biological processes, including cell migration, axonal outgrowth, perception of pain, cardiovascular responses and kidney physiology. The translation of mechanical cues into cellular responses, a process known as mechanotransduction, typically takes place in specialized multiprotein structures such as cilia, cell-cell or cell-matrix adhesions. Within these structures, mechanical forces such as shear stress and membrane stretch activate mechanosensitive proteins, which set off a series of events that lead to altered cell behavior. Members of the transient receptor potential (TRP) family of cation channels are emerging as important players in mechanotransductory pathways. Localized within mechanosensory structures, they are activated by mechanical stimuli and trigger fast as well as sustained cytoskeletal responses. In this review, we will provide an overview of how TRP channels affect cytoskeletal dynamics in various mechano-regulated processes.
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Citoesqueleto/fisiología , Mecanotransducción Celular , Canales de Potencial de Receptor Transitorio/fisiología , Animales , Adhesión Celular , Movimiento Celular , Citoesqueleto/metabolismo , Humanos , Canales de Potencial de Receptor Transitorio/clasificación , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
The 9q34.3 subtelomeric deletion syndrome is a newly defined mental retardation syndrome, caused by haplo-insufficiency of the euchromatin histone methyltransferase 1 (EHMT1) gene. Patients also have childhood hypotonia, facial dysmorphisms, delay in reaching developmental milestones, and behavioral problems like aggressive outbursts, hypoactivity, or autistic-like features. Male and female heterozygous Ehmt1 knockout mice (Ehmt1(+/-), aged 1-20 months, kept on a C57BL/6J background), were used to investigate whether they mimic the patients behavioral characteristics by comparing their behavior to wildtype littermates. The Ehmt1(+/-) mice revealed reduced activity and exploration, with increased anxiety compared to wildtype mice when exposed to novel environments in the open field, object exploration, marble burying, light-dark box, mirrored chamber and T-maze tests. They also demonstrated diminished social play when encountering a mouse from a different litter, and a delayed or absent response to social novelty when exposed to a stranger mouse. However, no differences in phenotyper home cage locomotor activity or rotarod motor function were observed between Ehmt1(+/-) and wildtype mice. Together, these results indicate that the hypoactivity and the autistic-like features of 9q34.3 subtelomeric deletion syndrome patients are recapitulated in this Ehmt1(+/-) mouse model, and that the hypoactivity is apparently not caused by any motor dysfunction. Together, these observations make it plausible that the Ehmt1(+/-) mouse is a faithful mammalian model for the autistic-like behavioral features of patients with the 9q34.3 subtelomeric deletion syndrome.