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The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.
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How cancer cells determine their shape in response to three-dimensional (3D) geometric and mechanical cues is unclear. We develop an approach to quantify the 3D cell shape of over 60,000 melanoma cells in collagen hydrogels using high-throughput stage-scanning oblique plane microscopy (ssOPM). We identify stereotypic and environmentally dependent changes in shape and protrusivity depending on whether a cell is proximal to a flat and rigid surface or is embedded in a soft environment. Environmental sensitivity metrics calculated for small molecules and gene knockdowns identify interactions between the environment and cellular factors that are important for morphogenesis. We show that the Rho guanine nucleotide exchange factor (RhoGEF) TIAM2 contributes to shape determination in environmentally independent ways but that non-muscle myosin II, microtubules, and the RhoGEF FARP1 regulate shape in ways dependent on the microenvironment. Thus, changes in cancer cell shape in response to 3D geometric and mechanical cues are modulated in both an environmentally dependent and independent fashion.
Asunto(s)
Forma de la Célula , Factores de Intercambio de Guanina Nucleótido , Humanos , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Línea Celular Tumoral , Microtúbulos/metabolismo , Miosina Tipo II/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Melanoma/patología , Melanoma/metabolismoRESUMEN
BACKGROUND: From January 2017 to June 2018 more than 7000 measles cases were reported in Italy, of which more than 400 among unvaccinated healthcare workers. We described a measles outbreak occurred in Western Liguria, Italy, characterized by a high involvement of healthcare workers and hospital visitors. METHODS: Suspected measles cases and data regarding vaccination status and clinical management of the patients were collected by reviewing 3 different surveillance systems: the routine mandatory notification system, the National Integrated Surveillance System for Measles and Rubella and the regional reference laboratory for measles diagnosis. RESULTS: Thirty-six cases were reported, with a median age of 31 years and >95% in unvaccinated subjects. One death occurred, 15 cases were hospitalized. Hospital transmission was confirmed or suspected in 12 cases; amongst this cases, 5 were healthcare workers (a gynaecologist, an obstetric nurse, a radiologist, a physiotherapist and a nurse working in an infectious disease ward), all certified unvaccinated. Phylogenetic analysis revealed the circulation of a single B3 genotype variant. CONCLUSIONS: Our experience highlighted the key role of nosocomial transmission and the need for targeted strategies, in particular (i) to implement a measles catch-up immunization campaign in susceptible groups, especially in healthcare workers, (ii) to intensify the check of immunisation status of healthcare workers and to offer vaccination for those who need it, (iii) to improve timeliness and completeness of surveillance systems. Efforts are needed to guarantee the safety of the hospital and the reliability of the healthcare workers. Only high vaccination coverage among HCWs can prevent the diffusion of measles in the hospital setting.
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Brotes de Enfermedades/estadística & datos numéricos , Transmisión de Enfermedad Infecciosa de Paciente a Profesional/estadística & datos numéricos , Sarampión/epidemiología , Adolescente , Adulto , Niño , Preescolar , Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Infección Hospitalaria/transmisión , Infección Hospitalaria/virología , Femenino , Humanos , Lactante , Italia/epidemiología , Masculino , Sarampión/prevención & control , Vacuna Antisarampión/uso terapéutico , Virus del Sarampión/genética , Personal de Hospital/estadística & datos numéricos , Filogenia , Adulto JovenRESUMEN
Spiradenoma and cylindroma are distinctive skin adnexal tumors with sweat gland differentiation and potential for malignant transformation and aggressive behaviour. We present the genomic analysis of 75 samples from 57 representative patients including 15 cylindromas, 17 spiradenomas, 2 cylindroma-spiradenoma hybrid tumors, and 24 low- and high-grade spiradenocarcinoma cases, together with morphologically benign precursor regions of these cancers. We reveal somatic or germline alterations of the CYLD gene in 15/15 cylindromas and 5/17 spiradenomas, yet only 2/24 spiradenocarcinomas. Notably, we find a recurrent missense mutation in the kinase domain of the ALPK1 gene in spiradenomas and spiradenocarcinomas, which is mutually exclusive from mutation of CYLD and can activate the NF-κB pathway in reporter assays. In addition, we show that high-grade spiradenocarcinomas carry loss-of-function TP53 mutations, while cylindromas may have disruptive mutations in DNMT3A. Thus, we reveal the genomic landscape of adnexal tumors and therapeutic targets.
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Carcinoma Adenoide Quístico/genética , Enzima Desubiquitinante CYLD/genética , Proteínas Quinasas/genética , Neoplasias de las Glándulas Sudoríparas/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Adenoide Quístico/patología , Estudios de Cohortes , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Análisis Mutacional de ADN , Femenino , Humanos , Mutación con Pérdida de Función , Masculino , Persona de Mediana Edad , Mutación Missense , Dominios Proteicos/genética , Neoplasias de las Glándulas Sudoríparas/patología , Glándulas Sudoríparas/patología , Proteína p53 Supresora de Tumor/genética , Secuenciación del ExomaRESUMEN
Following DNA damage caused by exogenous sources, such as ionizing radiation, the tumour suppressor p53 mediates cell cycle arrest via expression of the CDK inhibitor, p21. However, the role of p21 in maintaining genomic stability in the absence of exogenous DNA-damaging agents is unclear. Here, using live single-cell measurements of p21 protein in proliferating cultures, we show that naturally occurring DNA damage incurred over S-phase causes p53-dependent accumulation of p21 during mother G2- and daughter G1-phases. High p21 levels mediate G1 arrest via CDK inhibition, yet lower levels have no impact on G1 progression, and the ubiquitin ligases CRL4Cdt2 and SCFSkp2 couple to degrade p21 prior to the G1/S transition. Mathematical modelling reveals that a bistable switch, created by CRL4Cdt2, promotes irreversible S-phase entry by keeping p21 levels low, preventing premature S-phase exit upon DNA damage. Thus, we characterize how p21 regulates the proliferation-quiescence decision to maintain genomic stability.