Anti‐cancer drugs often increase reactive oxygen species (ROS) and cause DNA damage. Here, we highlight a new cross talk between chronic oxidative stress and the histone variant H2AX, a key player in ...DNA repair. We observe that persistent accumulation of ROS, due to a deficient JunD‐/Nrf2‐antioxidant response, reduces H2AX protein levels. This effect is mediated by an enhanced interaction of H2AX with the E3 ubiquitin ligase RNF168, which is associated with H2AX poly‐ubiquitination and promotes its degradation by the proteasome. ROS‐mediated H2AX decrease plays a crucial role in chemosensitivity. Indeed, cycles of chemotherapy that sustainably increase ROS reduce H2AX protein levels in Triple‐Negative breast cancer (TNBC) patients. H2AX decrease by such treatment is associated with an impaired NRF2‐antioxidant response and is indicative of the therapeutic efficiency and survival of TNBC patients. Thus, our data describe a novel ROS‐mediated regulation of H2AX turnover, which provides new insights into genetic instability and treatment efficacy in TNBC patients.
Synopsis
This work gives new insights into the regulation of H2AX protein turnover under chronic oxidative stress that affects DNA damage response. H2AX degradation upon chronic stress sensitizes tumour cells to chemotherapy and is indicative of better survival in triple‐negative breast cancer patients.
Physiological conditions of chronic oxidative stress, mediated by the loss of JunD or Nrf2 transcription factors, are associated with a reduced protein level of the histone variant H2AX.
Under conditions of chronic stress due to junD or Nrf2 deficiency, H2AX protein is targeted for degradation by the proteasome.
ROS‐dependent H2AX degradation is mediated by enhanced interaction of H2AX protein with the E3 ubiquitin ligase RNF168.
H2AX decrease by chronic oxidative stress increases tumour cell genomic instability and death.
Chemosensitivity and survival of triple‐negative breast cancer patients are improved by stress‐mediated H2AX degradation following successive cycles of chemotherapy.
This work gives new insights into the regulation of H2AX protein turnover under chronic oxidative stress that affects DNA damage response. H2AX degradation upon chronic stress sensitizes tumour cells to chemotherapy and is indicative of better survival in triple‐negative breast cancer patients.
Dilute gas‐particle mixtures in which the particles are carried by the turbulent fluid are found in various geophysical contexts, from cold snow avalanches to hot pyroclastic density currents. Though ...previous studies suggest that such mixtures have maximum particle concentrations of a few volume percent, the dependence of this maximum concentration on the Reynolds number is unclear. We addressed this issue through laboratory experiments in a vertical pipe, where dilute gas‐particle mixtures were created by injecting a turbulent air flow from below. Nearly monodisperse mixtures of glass beads of different grain sizes (77 to 1,550 μm) were used with varying bulk concentrations from 0.025 to 8 vol. %. To create quasi‐static mixtures, the mean air velocity matched the terminal settling velocity for the grain sizes investigated. The maximum Reynolds numbers of the mixtures were ~104–106. The air pressure indicated full support of the particle weight at concentrations down to 0.025 vol. %. Above a critical particle concentration, at which all the particles were suspended, subsequent additional particles were not maintained in the mixture and led to the formation of clusters that settled downward in the pipe to form a dense fluidized bed. Maximum mean particle concentrations of the dilute mixtures increased from ~1 to ~2.8 vol. % and reached a plateau at increasing mixture Reynolds number. These results give insights into the maximum particle concentrations of geophysical turbulent gas‐particle mixtures and may serve to constrain observations as well as the input and output data of models.
Key Points
We investigated experimentally dilute turbulent gas‐particle mixtures with Reynolds numbers up to Remix ~ 106
The mixtures had maximum particle concentrations controlled by the formation of particle clusters that segregated from the mixtures
Maximum mean particle concentrations increased with Remix and were ~1‐2.8 vol. %
Numerical simulations, performed with a new two-phase bi-projection scheme, of the collapse of columns of glass beads with aspect ratios equal to 0.7 and 2 over a horizontal plane are reported and ...comparison with experiments are presented. A level-set formulation for the Navier–Stokes equations is used, so that the interface between the granular material and the ambient air is tracked. The granular flow is modeled with a viscoplastic rheology, derived from the μ(I)–rheology, resulting in a Drucker–Prager plasticity criterion combined with a spatio-temporal variable viscosity depending on the pressure and on the shear rate. The computational effort is reduced by using instead a constant viscosity. The dependence of the results upon the value of the constant viscosity, which has been reduced by two orders of magnitude, is very weak suggesting that these granular flows are mainly governed by the Drucker–Prager plasticity criterion. The rheology is formulated as a projection, allowing for an efficient computation of the plastic part of the stress tensor. Coulomb friction conditions are applied on the walls. The dynamics of the collapse and the morphology of the final deposit are accurately reproduced. Sensitivity of the results, with respect to the resolution and the basal friction coefficient, is also studied. During the collapse, the granular material consists of a basal deposit overlain by a flowing layer, which are separated by an interface that migrates upwards until the flowing layer is consumed. The time evolution of this static-mobile interface is quantified and a good agreement is found with experiments. To the best of our knowledge this is the first simulation of internal flow dynamics validated by experiments. We also report results obtained with a viscoplastic model where the dynamic pressure in the yield is replaced by the hydrostatic pressure, depending on the granular flow height. This model produces non-physically relevant solutions. Nevertheless, from a numerical point of view, it provides interesting and challenging test cases for two-phases viscoplastic simulations as the interface rolls up before the head of the granular mass falls on the bottom wall.
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•A level-set bi-projection scheme is used to simulate dambreak granular flows.•Results are compared with experimental data for columns of glass beads.•A viscoplastic rheology with a Drucker–Prager criterion is used.•The dynamics of the collapse and the morphology of the deposit are recovered.•The granular flow separates into a basal deposit overlain by a flowing layer.
A new numerical scheme for solving incompressible Bingham flows with variable density, plastic viscosity, and yield stress is proposed. The mathematical and computational difficulties due to the ...nondifferentiable definition of the stress tensor in the plug regions, i.e., where the strain-rate tensor vanishes, is overcome by using a projection formulation as in the Uzawa-like method for viscoplastic flows. This projection definition of the plastic tensor is coupled with a fractional time-stepping scheme designed for Newtonian incompressible flows with variable density. The plastic tensor is treated implicitly in the first substep of the fractional time-stepping scheme and a fixed-point iterative procedure is used for its computation. A pseudotime relaxation term is added into the Bingham projection whose effect is to ensure a geometric convergence of the fixed-point algorithm. This is a key feature of the bi-projection scheme which provides a fast and accurate computation of the plastic tensor. Stability and error analyses of the bi-projection scheme are provided. The use of the discrete divergence-free velocity to convect the density in the mass conservation equation allows us to derive lower and upper bounds for the discrete density. The error induced by the pseudotime relaxation term is controlled by a prescribed numerical parameter so that a first-order estimate of the time error is derived for the velocity field and the density, as well as the dependent parameters that are the plastic viscosity and the yield stress.
Gas production is expected in radioactive-waste storage structures. This will induce a slow increase in gas pressure, which necessitates the study of gas transfer at a low pressure. In this special ...case, calculations of the flow through storing materials while solely using permeability and Darcy’s law are likely to be inadequate, as diffusion may play a crucial role in the process. The gas permeability and gas diffusion coefficient of industrial concrete have then been measured on the dry material. Diffusion tests were performed with a new device, specially designed for this study. The diffusion coefficient was directly measured with the use of the first Fick’s law, as the test was analyzed under a steady state. Using some simplified hypotheses, it was then possible to compare the proportion of flow occurring due to diffusion with the one occurring due to permeation. The tendency is very clear and unambiguously shows that diffusion is predominant at a very low injection pressure but becomes negligible as soon as the gas pressure exceeds a moderate value.
Breast cancers are composed of molecularly distinct subtypes with different clinical outcomes and responses to therapy. To discover potential therapeutic targets for the poor prognosis-associated ...triple-negative breast cancer (TNBC), gene expression profiling was carried out on a cohort of 130 breast cancer samples. Polo-like kinase 1 (PLK1) was found to be significantly overexpressed in TNBC compared with the other breast cancer subtypes. High PLK1 expression was confirmed by reverse phase protein and tissue microarrays. In triple-negative cell lines, RNAi-mediated PLK1 depletion or inhibition of PLK1 activity with a small molecule (BI-2536) induced an increase in phosphorylated H2AX, G(2)-M arrest, and apoptosis. A soft-agar colony assay showed that PLK1 silencing impaired clonogenic potential of TNBC cell lines. When cells were grown in extracellular matrix gels (Matrigel), and exposed to BI-2536, apoptosis was observed specifically in TNBC cancerous cells, and not in a normal cell line. When administrated as a single agent, the PLK1 inhibitor significantly impaired tumor growth in vivo in two xenografts models established from biopsies of patients with TNBC. Most importantly, the administration of BI-2536, in combination with doxorubicin + cyclophosphamide chemotherapy, led to a faster complete response compared with the chemotherapy treatment alone and prevented relapse, which is the major risk associated with TNBC. Altogether, our observations suggest PLK1 inhibition as an attractive therapeutic approach, in association with conventional chemotherapy, for the management of patients with TNBC.
Triple-negative breast cancer (TNBC) patients commonly exhibit poor prognosis and high relapse after treatment, but there remains a lack of biomarkers and effective targeted therapies for this ...disease. Here, we report evidence highlighting the cell-cycle-related kinase CDK7 as a driver and candidate therapeutic target in TNBC. Using publicly available transcriptomic data from a collated set of TNBC patients (
= 383) and the METABRIC TNBC dataset (
= 217), we found
mRNA levels to be correlated with patient prognosis. High CDK7 protein expression was associated with poor prognosis within the RATHER TNBC cohort (
= 109) and the METABRIC TNBC cohort (
= 203). The highly specific CDK7 kinase inhibitors, BS-181 and THZ1, each downregulated CDK7-mediated phosphorylation of RNA polymerase II, indicative of transcriptional inhibition, with THZ1 exhibiting 500-fold greater potency than BS-181. Mechanistic investigations revealed that the survival of MDA-MB-231 TNBC cells relied heavily on the BCL-2/BCL-XL signaling axes in cells. Accordingly, we found that combining the BCL-2/BCL-XL inhibitors ABT-263/ABT199 with the CDK7 inhibitor THZ1 synergized in producing growth inhibition and apoptosis of human TNBC cells. Collectively, our results highlight elevated CDK7 expression as a candidate biomarker of poor prognosis in TNBC, and they offer a preclinical proof of concept for combining CDK7 and BCL-2/BCL-XL inhibitors as a mechanism-based therapeutic strategy to improve TNBC treatment.
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DNA damage triggers a complex signaling cascade involving a multitude of phosphorylation events. We found that the threonine 7 (Thr-7) residue of heat shock protein 90α (Hsp90α) was phosphorylated ...immediately after DNA damage. The phosphorylated Hsp90α then accumulated at sites of DNA double strand breaks and formed repair foci with slow kinetics, matching the repair kinetics of complex DNA damage. The phosphorylation of Hsp90α was dependent on phosphatidylinositol 3-kinase-like kinases, including the DNA-dependent protein kinase (DNA-PK) in particular. DNA-PK plays an essential role in the repair of DNA double strand breaks by nonhomologous end-joining and in the signaling of DNA damage. It is also present in the cytoplasm of the cell and has been suggested to play a role in cytoplasmic signaling pathways. Using stabilized double-stranded DNA molecules to activate DNA-PK, we showed that an active DNA-PK complex could be assembled in the cytoplasm, resulting in phosphorylation of the cytoplasmic pool of Hsp90α. In vivo, reverse phase protein array data for tumors revealed that basal levels of Thr-7-phosphorylated Hsp90α were correlated with phosphorylated histone H2AX levels. The Thr-7 phosphorylation of the ubiquitously produced and secreted Hsp90α may therefore serve as a surrogate biomarker of DNA damage. These findings shed light on the interplay between central DNA repair enzymes and an essential molecular chaperone.
DNA damage triggers a complex signaling cascade that remains incompletely understood.
The essential chaperone Hsp90α is phosphorylated by DNA damage signaling kinases and accumulates at DNA damage sites.
Hsp90α is directly involved in DNA repair.
Our results provide an explanation for the radiosensitizing effect of Hsp90 inhibitors and identify phosphorylated Hsp90α as a potential biomarker for genetic instability.
Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The ...molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.
Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 ...complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.
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