O-GlcNAcylation-Inducing Treatments Inhibit Estrogen Receptor α Expression and Confer Resistance to 4-OH-Tamoxifen in Human Breast Cancer-Derived MCF-7 Cells.

PLoS OneInternational audienceO-GlcNAcylation (addition of N-acetyl-glucosamine on serine or threonine residues) is a post-translational modification that regulates stability, activity or localization of cytosolic and nuclear proteins. O-linked N-acetylgluocosmaine transferase (OGT) uses UDP-GlcNAc, produced in the hexosamine biosynthetic pathway to O-GlcNacylate proteins. Removal of O-GlcNAc from proteins is catalyzed by the β-N-Acetylglucosaminidase (OGA). Recent evidences suggest that O-GlcNAcylation may affect the growth of cancer cells. However, the consequences of O-GlcNAcylation on anti-cancer therapy have not been evaluated. In this work, we studied the effects of O-GlcNAcylation on tamoxifen-induced cell death in the breast cancer-derived MCF-7 cells. Treatments that increase O-GlcNAcylation (PUGNAc and/or glucosoamine) protected MCF-7 cells from death induced by tamoxifen. In contrast, inhibition of OGT expression by siRNA potentiated the effect of tamoxifen on cell death. Since the PI-3 kinase/Akt pathway is a major regulator of cell survival, we used BRET to evaluate the effect of PUGNAc+glucosamine on PIP3 production. We observed that these treatments stimulated PIP3 production in MCF-7 cells. This effect was associated with an increase in Akt phosphorylation. However, the PI-3 kinase inhibitor LY294002, which abolished the effect of PUGNAc+glucosamine on Akt phosphorylation, did not impair the protective effects of PUGNAc+glucosamine against tamoxifen-induced cell death. These results suggest that the protective effects of O-GlcNAcylation are independent of the PI-3 kinase/Akt pathway. As tamoxifen sensitivity depends on the estrogen receptor (ERα) expression level, we evaluated the effect of PUGNAc+glucosamine on the expression of this receptor. We observed that O-GlcNAcylation-inducing treatment significantly reduced the expression of ERα mRNA and protein, suggesting a potential mechanism for the decreased tamoxifen sensitivity induced by these treatments. Therefore, our results suggest that inhibition of O-GlcNAcylation may constitute an interesting approach to improve the sensitivity of breast cancer to anti-estrogen therapy

Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma

BACKGROUND High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models

PURPOSE ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma

Development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (BRET)-based phosphatidyl inositol-3 phosphate (PIP3) biosensor.

International audienceStimulation of tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived breast cancer cell line, that stably co-expresses the PH domain of Akt fused to Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different ligands, including insulin, the insulin analogue glargine, IGF1, IGF2 and EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of ligand-induced BRET. Cetuximab, a humanized anti-EGF receptor monoclonal antibody used for the treatment of cancer, completely inhibited EGF-induced BRET, and the tyrosine kinase inhibitor tyrphostine AG1024 inhibited insulin effect on PIP3 production. Moreover, the effects of insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and cancer

Influence of rumen protozoa on methane emissions in ruminants: A meta-analysis approach

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The PI3K stimulatory activity present in human serum can be evaluated using the MCF-7/B2 cells.

<p>(A, B) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with PBS containing 0%, 1%, 2%, 5% or 10% human serum that had been previously heated or not at 95°C during 1 h, and light emission acquisition started immediately. (A) A typical real-time BRET experiment is shown. (B) Means ± SEM of BRET values at the plateau of 3 to 7 independent experiments are shown. (C) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with 5% human serum previously submitted or not to centrifugation on a centrifugal filter device with a molecular weight cut-off of 3 kDa. A typical real-time BRET experiment (left panel) and means ± SEM of BRET values at the plateau (right panel) are shown (n = 3). (D) MCF-7/B2 cells were starved overnight in culture medium containing only 0.1% FBS. Cells were then stimulated with 5% human serum that had been pre-incubated for 1 h in presence of 50 μM IGFBP1. A typical real-time BRET experiment (left panel) and means ± SEM of BRET values at the plateau (right panel) are shown (n = 3). Statistical analysis was performed using ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.01; ***, P<0.001; NS, Non significant.</p

Effect of inhibitors of the PI3K/Akt signaling pathway on BRET signal in MCF-7/B2 cells.

<p>(A) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 20 ng/μl of the humanized anti-EGFR antibody Cetuximab. Cells were then stimulated with EGF (32 nM), and light emission acquisition started immediately. A typical real-time BRET experiment (left panel) and the mean ± SEM of BRET values at the peak (right panel) are shown (n = 3). (B) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 25 μM of the tyrphostin AG1024. Cells were then stimulated with 100 nM insulin, and light emission acquisition started immediately. A typical real-time BRET experiment (left panel) and the mean ± SEM of BRET values at the plateau (right panel) are shown (n = 3). (C) MCF-7/B2 cells were preincubated for 1 h in absence or presence of 25 μM of the PI3K inhibitor LY294002. Cells were then stimulated with 10 nM IGF1 and light emission acquisition started immediately. A typical real-time BRET experiments (left panel) and the mean ± SEM of BRET values at the plateau (right panel) of 3 independent experiments are shown. (D, E) MCF-7/B2 cells were preincubated for 4 h in absence or presence of 10 μM of the inhibitors of Akt-PH/PIP₃ interaction PIT-1 (D) and DMPIT-1 (E). Cells were then stimulated with 10 nM IGF1, and light emission acquisition started immediately. Typical real-time BRET experiments (left panels) and the mean ± SEM of BRET values at the plateau (right panels) of 3 to 4 independent experiments are shown. Statistical analysis was performed using ANOVA followed by Tukey’s test. *, P<0.05; **, P<0.01; ***, P<0.001; NS, Non significant.</p

Effect of IGF1, IGF2, EGF and glargine on PIP₃ production in MCF-7/B2 cells.

<p>(A) Typical experiments showing real-time effects of IGF1, IGF2, EGF and glargine on PIP₃ production in MCF-7/B2 cells. (B) Dose-dependent effect of IGF1, IGF2, EGF, glargine and insulin on PIP₃ production in MCF-7/B2 cells. Ligand-induced BRET (BRET above basal) at the plateau (IGF1, IGF2, Insulin, Glargine) or at the peak (EGF) was determined for each ligand concentration and was used to establish dose-response curves. Results are means ± S.E.M. of 3 to 8 independent experiments. EC50 for insulin, IGF1, IGF2, EGF and glargine are given in the result section.</p

Development of a human breast cancer-derived clone stably expressing a BRET-based PIP₃ biosensor.

<p>(A) Principle of the BRET-based assay used to monitor PIP₃ production in living cells. Activation of the PI-3 kinase by tyrosine kinase receptors induces phosphorylation of PIP₂ into PIP₃ and recruitment of Akt to the plasma membrane through its PH domain. To monitor PIP₃ production, MCF-7 cells were stably transfected with the PH domain of Akt fused to luciferase (Luc-Akt-PH) and YFP fused to a membrane localization sequence (YFP-membrane). Recruitment of Luc-Akt-PH to the plasma membrane by PIP₃ results in energy transfer between Luc-Akt-PH and YFP-membrane. This permits to study the pharmacological properties of ligands that activate this pathway, and to evaluate the effects of inhibitory molecules acting on (I) the extracellular part of receptors, (II) the tyrosine kinase activity of the receptors, (III) the catalytic activity of the PI3K, and (IV) the interaction between PIP₃ and the PH domain of Akt. (B) Transfection of a MCF-7 clone stably expressing Luc-Akt-PH with YFP-membrane cDNA gave rise to 4 sub-clones stably expressing both constructs. Ligand-induced BRET could be detected in only 3 sub-clones (B2, D2, C2). Preliminary BRET experiments indicated that insulin-induced BRET was higher with clone B2. (C) Surface expression of pEYFP-Mem in MCF-7/B2 cells was studied by fluorescence microscopy. YFP fluorescence was detected using a FITC filter and nuclei were visualized using a DAPI filter. The image was obtained by deconvolution analysis. (D) Western-blotting experiment showed that expression of Luc-Akt-PH alone or together with YFP-membrane does not affect insulin-induced phosphorylation of endogenous Akt. (E) Insulin dose-dependently stimulated PIP₃ production in MCF-7/B2 cells. Left panel: typical real-time experiment showing basal and insulin-stimulated BRET. Right panel: insulin-induced BRET (BRET above basal at the plateau) was determined for each insulin concentration to establish dose-response curves. Results are means ± S.E.M. (standard error of the mean) of 6 to 11 independent experiments. The freeze-thaw cycle did not affect the sensitivity of the cells to insulin stimulation.</p