Chromatin regulation by Histone H4 acetylation at Lysine 16 during cell death and differentiation in the myeloid compartment

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death

Blockage of Squamous Cancer Cell Collective Invasion by FAK Inhibition Is Released by CAFs and MMP-2

Metastasis remains a clinically unsolved issue in cancer that is initiated by the acquisition of collective migratory properties of cancer cells. Phenotypic and functional heterogeneity that arise among cancer cells within the same tumor increase cellular plasticity and promote metastasis, however, their impact on collective cell migration is incompletely understood. Here, we show that in vitro collective cancer cell migration depends on FAK and MMP-2 and on the presence of cancer-associated fibroblasts (CAFs). The absence of functional FAK rendered cancer cells incapable of invading the surrounding stroma. However, CAFs and cancer cells over-expressing MMP-2 released FAK-deficient cells from this constraint by taking the leader positions in the invasive tracks, pushing FAK-deficient squamous cell carcinoma (SCC) cells towards the stroma and leading to the transformation of non-invasive cells into invasive cells. Our cell-based studies and the RNAseq data from the TCGA cohort of patients with head and neck squamous cell carcinomas reveal that, although both FAK and MMP-2 over-expression are associated with epithelial–mesenchymal transition, it is only MMP-2, not FAK, that functions as an independent prognostic factor. Given the significant role of MMP-2 in cancer dissemination, targeting of this molecule, better than FAK, presents a more promising opportunity to block metastasis

Interacciones célula-microambiente tisular en carcinomas epidermoides de cabeza y cuello: Identificación de mecanismos moleculares

La principal causa de las muertes asociadas al cáncer es debida al desarrollo de metástasis. En este proceso tiene gran importancia la reprogramación metabólica, comunicación intercelular e invasión de las células tumorales. El objetivo de la tesis doctoral fue profundizar en el conocimiento de los mecanismos moleculares implicados en esos procesos para identificar aquellos que puedan servir como dianas para fármacos antitumorales. Los resultados obtenidos indicaron que: (1) la hipoxia intratumoral induce reprogramación metabólica mediada por HIF-1α que incluye sobre-expresión de enzimas glucolíticos y de la vía miR-210/ISCU lo que se asocia a menor supervivencia global de la enfermedad; (2) la interacción de las células con la matriz extracelular, a través de las adhesiones focales y la proteína Focal Adhesion Kinase (FAK), ejerce un papel importante en la invasión celular a través, al menos parcialmente, de la secreción de la metaloproteasa MMP-2; (3) la proteína FAK participa también en la comunicación intercelular a través de tunneling nanotubes, unas estructuras claves para la transferencia de autofagosomas y mitocondiras entre células tumorales. Colectivamente, nuestros resultados proporcionan nuevas vulnerabilidades que pueden ser explotadas para erradicar eficazmente las células tumorales

Identification and Characterization of Tunneling Nanotubes for Intercellular Trafficking

International audienceAbstract Tunneling nanotubes (TNTs) are thin membranous channels providing a direct cytoplasmic connection between remote cells. They are commonly observed in different cell cultures and increasing evidence supports their role in intercellular communication, and pathogen and amyloid protein transfer. However, the study of TNTs presents several pitfalls (e.g., difficulty in preserving such delicate structures, possible confusion with other protrusions, structural and functional heterogeneity, etc.) and therefore requires thoroughly designed approaches. The methods described in this protocol represent a guideline for the characterization of TNTs (or TNT‐like structures) in cell culture. Specifically, optimized protocols to (1) identify TNTs and the cytoskeletal elements present inside them; (2) evaluate TNT frequency in cell culture; (3) unambiguously distinguish them from other cellular connections or protrusions; (4) monitor their formation in living cells; (5) characterize TNTs by a micropatterning approach; and (6) investigate TNT ultrastructure by cryo‐EM are provided. Finally, this article describes how to assess TNT‐mediated cell‐to‐cell transfer of cellular components, which is a fundamental criterion for identifying functional TNTs. © 2023 Wiley Periodicals LLC. Basic Protocol 1 : Identification of tunneling nanotubes Alternate Protocol 1 : Identifying the cytoskeletal elements present in tunneling nanotubes Alternate Protocol 2 : Distinguishing tunneling nanotubes from intercellular bridges formed during cell division Basic Protocol 2 : Deciphering tunneling nanotube formation and lifetime by live fluorescent microscopy Alternate Protocol 3 : Deciphering tunneling nanotube formation using a live‐compatible dye Basic Protocol 3 : Assessing tunneling nanotubes functionality in intercellular transfer Alternate Protocol 4 : Flow cytometry approach to quantify the rate of vesicle or mitochondria transfer Support Protocol : Controls to support TNT‐mediated transfer Basic Protocol 4 : Studies of tunneling nanotubes by cell micropatterning Basic Protocol 5 : Characterization of the ultrastructure of tunneling nanotubes by cryo‐E

Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes

Tunneling nanotubes (TnTs) are thin channels that temporally connect nearby cells allowing the cell-to-cell trafficking of biomolecules and organelles. The presence or absence of TnTs in human neoplasms and the mechanisms of TnT assembly remains largely unexplored. In this study, we have identified TnTs in tumor cells derived from squamous cell carcinomas (SCC) cultured under bi-dimensional and tri-dimensional conditions and also in human SCC tissues. Our study demonstrates that TnTs are not specific of epithelial or mesenchymal phenotypes and allow the trafficking of endosomal/ lysosomal vesicles, mitochondria, and autophagosomes between both types of cells. We have identified focal adhesion kinase (FAK) as a key molecule required for TnT assembly via a mechanism involving the MMP-2 metalloprotease. We have also found that the FAK inhibitor PF-562271, which is currently in clinical development for cancer treatment, impairs TnT formation. Finally, FAK-deficient cells transfer lysosomes/autophagosomes to FAK-proficient cells via TnTs which may represent a novel mechanism to adapt to the stress elicited by impaired FAK signaling. Collectively, our results strongly suggest a link between FAK, MMP-2, and TnT, and unveil new vulnerabilities that can be exploited to efficiently eradicate cancer cells.This work was supported by the Instituto de Salud Carlos III-Fondo de Investigación Sanitaria (FIS PI11/929); Red Temática de Investigación Cooperativa en Cáncer, CIBERONC, Instituto de Salud Carlos III (ISCIII) Spanish Ministerio de Economia y Competitividad & European Regional Development Fund (ERDF) (RD12/0036/0015); Spanish Ministerio de Economía y Competitividad (MAT2014-51937-C3-1-P and Acciones de Dinamización “Red de Excelencia” MAT2015-68837-REDT); and Fundación Bancaria Caja de Ahorros de Asturias-Instituto Universitario de Oncología del Principado de AsturiasPeer Reviewe

Compuestos y sus usos como sondas fluorescentes

Compuestos y sus usos como sondas fluorescentes. Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o marcaje fluorescente especifico de mitocondrias en células vivas.Peer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)A1 Solicitud de patente con informe sobre el estado de la técnic

Compuestos y sus usos como sondas fluorescentes

Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o marcaje fluorescente especifico de mitocondrias en células vivas.Peer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)B2 Patente con examen previ

Compounds and their uses as fluorescent probes

Organic dyes based on F-BODIPY derivatives and carnitine, method of producing same and their use for specific fluorescent labeling or tagging of mitochondria in live cellsPeer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)A1 Solicitud de patente con informe sobre el estado de la técnic

Compuestos y sus usos como sondas fluorescentes

[EN] Organic dyes based on F -BODIPY derivatives and camitine, method for producing same and their use for the specific fluorescent labelling or tagging of mitochondria in living cells.[ES] Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o mareaje fluorescente específico de mitocondrias en células vivas.Peer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)A1 Solicitud de patente con informe sobre el estado de la técnic