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1.
Front Endocrinol (Lausanne) ; 13: 888802, 2022.
Article in English | MEDLINE | ID: mdl-36034422

ABSTRACT

Background: Breast cancer cells enter into the cell cycle following progestin exposure by the activation of signalling cascades involving a plethora of enzymes, transcription factors and co-factors that transmit the external signal from the cell membrane to chromatin, ultimately leading to a change of the gene expression program. Although many of the events within the signalling network have been described in isolation, how they globally team up to generate the final cell response is unclear. Methods: In this study we used antibody microarrays and phosphoproteomics to reveal a dynamic global signalling map that reveals new key regulated proteins and phosphor-sites and links between previously known and novel pathways. T47D breast cancer cells were used, and phospho-sites and pathways highlighted were validated using specific antibodies and phenotypic assays. Bioinformatic analysis revealed an enrichment in novel signalling pathways, a coordinated response between cellular compartments and protein complexes. Results: Detailed analysis of the data revealed intriguing changes in protein complexes involved in nuclear structure, epithelial to mesenchyme transition (EMT), cell adhesion, as well as transcription factors previously not associated with breast cancer cell proliferation. Pathway analysis confirmed the key role of the MAPK signalling cascade following progesterone and additional hormone regulated phospho-sites were identified. Full network analysis shows the activation of new signalling pathways previously not associated with progesterone signalling in T47D breast cancer cells such as ERBB and TRK. As different post-translational modifications can mediate complex crosstalk mechanisms and massive PARylation is also rapidly induced by progestins, we provide details of important chromatin regulatory complexes containing both phosphorylated and PARylated proteins. Conclusions: This study contributes an important resource for the scientific community, as it identifies novel players and connections meaningful for breast cancer cell biology and potentially relevant for cancer management.


Subject(s)
Breast Neoplasms , Progesterone , Chromatin , Female , Humans , Phosphorylation , Progestins , Receptors, Progesterone , Transcription Factors
2.
Genes Dev ; 33(13-14): 782-798, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31171699

ABSTRACT

Mouse embryonic stem cell (ESC) cultures contain a rare cell population of "2C-like" cells resembling two-cell embryos, the key stage of zygotic genome activation (ZGA). Little is known about positive regulators of the 2C-like state and two-cell stage embryos. Here we show that GADD45 (growth arrest and DNA damage 45) proteins, regulators of TET (TET methylcytosine dioxygenase)-mediated DNA demethylation, promote both states. Methylome analysis of Gadd45a,b,g triple-knockout (TKO) ESCs reveal locus-specific DNA hypermethylation of ∼7000 sites, which are enriched for enhancers and loci undergoing TET-TDG (thymine DNA glycosylase)-mediated demethylation. Gene expression is misregulated in TKOs, notably upon differentiation, and displays signatures of DNMT (DNA methyltransferase) and TET targets. TKOs manifest impaired transition into the 2C-like state and exhibit DNA hypermethylation and down-regulation of 2C-like state-specific genes. Gadd45a,b double-mutant mouse embryos display embryonic sublethality, deregulated ZGA gene expression, and developmental arrest. Our study reveals an unexpected role of GADD45 proteins in embryonic two-cell stage regulation.


Subject(s)
Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Demethylation , Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Animals , Cells, Cultured , Gene Knockout Techniques , Mice
3.
Genes Dev ; 32(11-12): 742-762, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29884649

ABSTRACT

Changes in DNA methylation are among the best-documented epigenetic alterations accompanying organismal aging. However, whether and how altered DNA methylation is causally involved in aging have remained elusive. GADD45α (growth arrest and DNA damage protein 45A) and ING1 (inhibitor of growth family member 1) are adapter proteins for site-specific demethylation by TET (ten-eleven translocation) methylcytosine dioxygenases. Here we show that Gadd45a/Ing1 double-knockout mice display segmental progeria and phenocopy impaired energy homeostasis and lipodystrophy characteristic of Cebp (CCAAT/enhancer-binding protein) mutants. Correspondingly, GADD45α occupies C/EBPß/δ-dependent superenhancers and, cooperatively with ING1, promotes local DNA demethylation via long-range chromatin loops to permit C/EBPß recruitment. The results indicate that enhancer methylation can affect aging and imply that C/EBP proteins play an unexpected role in this process. Our study suggests a causal nexus between DNA demethylation, metabolism, and organismal aging.


Subject(s)
Aging, Premature/genetics , Aging/genetics , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Cycle Proteins/metabolism , DNA Demethylation , Inhibitor of Growth Protein 1/metabolism , Nuclear Proteins/metabolism , Animals , Cell Cycle Proteins/genetics , Cells, Cultured , Homeostasis/genetics , Inhibitor of Growth Protein 1/genetics , Lipodystrophy/genetics , Mice , Mice, Knockout , Nuclear Proteins/genetics
4.
Metabolism ; 87: 70-79, 2018 10.
Article in English | MEDLINE | ID: mdl-29928894

ABSTRACT

BACKGROUND: The impact of nutrition on the evolution towards type 2 diabetes has recently received increasing attention because of the effect on chromatin structure and gene expression. PURPOSE: Evaluate the effect of high-fat diet on chromatin remodelling and expression of Ped/Pea-15, a gene commonly overexpressed in individuals at risk of type 2 diabetes. METHODS: We used mouse and cell models to investigate Ped/Pea-15 transcriptional regulation by high-fat diet and glucose, respectively. Chromatin structure and histone modification marks were assessed by Micrococcal Nuclease Protection and Chromatin Immunoprecipitation assays. RESULTS: Sixteen-week exposure of C57BL/6J mice to a high-fat diet impaired glucose tolerance and enhanced Ped/Pea-15 expression in their skeletal muscle tissue. This effect was associated with increased chromatin accessibility at specific regulatory sites at the Ped/Pea-15 gene. In particular, the region at -1900 to -1300 bp from Ped/Pea-15 transcription start site was revealed to feature enhancer activity as demonstrated by its function in the luciferase assay, increased p300 recruitment and H3K4me1 and H3K27Ac levels, all marks of functionally active enhancers. Returning mice to a standard chow diet was accompanied by rapid loss of acetylation of K27 on histone H3 and p300 recruitment at Ped/Pea-15. In contrast, the increased H3K4me1, which accompanied the high-fat diet exposure, remained stable. Incubation of muscle cells in culture medium supplemented with 25 mM glucose (HG) increased Ped/Pea-15 mRNA expression and H3K4me1 at the enhancer region. These effects became measurable upon 72 h of exposure to the HG medium and were not rescued upon returning the cells to the 5 mM glucose-containing medium. Interestingly, after 25 mM and sequential 5 mM glucose treatments, re-exposure of the same cells to HG medium further enhanced Ped/Pea-15 expression and increased H3K4me1 above the levels induced by the initial HG challenge already upon 24 h. CONCLUSION: Transient exposure to HFD or HG unveiled the presence of an enhancer element at the Ped/Pea-15 gene. Epigenetic changes imposed at this region by diets, which impair glucose tolerance generate metabolic memory of the nutritional injury and leave Ped/Pea-15 induction in a poised state.


Subject(s)
Diet, High-Fat , Enhancer Elements, Genetic/genetics , Epigenesis, Genetic/drug effects , Histocompatibility Antigens Class I/genetics , Muscle, Skeletal/metabolism , Phosphoproteins/genetics , Animals , Apoptosis Regulatory Proteins , Chromatin/drug effects , Chromatin/genetics , Chromatin Immunoprecipitation , Diet , Gene Expression Regulation/drug effects , Glucose/pharmacology , Glucose Intolerance/metabolism , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/drug effects
5.
J Immunol ; 198(9): 3426-3435, 2017 05 01.
Article in English | MEDLINE | ID: mdl-28341671

ABSTRACT

Thymic stromal lymphopoietin (TSLP) is a cytokine produced mainly by epithelial cells in response to inflammatory or microbial stimuli and binds to the TSLP receptor (TSLPR) complex, a heterodimer composed of TSLPR and IL-7 receptor α (CD127). TSLP activates multiple immune cell subsets expressing the TSLPR complex and plays a role in several models of disease. Although human monocytes express TSLPR and CD127 mRNAs in response to the TLR4 agonist LPS, their responsiveness to TSLP is poorly defined. We demonstrate that TSLP enhances human CD14+ monocyte CCL17 production in response to LPS and IL-4. Surprisingly, only a subset of CD14+ CD16- monocytes, TSLPR+ monocytes (TSLPR+ mono), expresses TSLPR complex upon LPS stimulation in an NF-κB- and p38-dependent manner. Phenotypic, functional, and transcriptomic analysis revealed specific features of TSLPR+ mono, including higher CCL17 and IL-10 production and increased expression of genes with important immune functions (i.e., GAS6, ALOX15B, FCGR2B, LAIR1). Strikingly, TSLPR+ mono express higher levels of the dendritic cell marker CD1c. This evidence led us to identify a subset of peripheral blood CD14+ CD1c+ cells that expresses the highest levels of TSLPR upon LPS stimulation. The translational relevance of these findings is highlighted by the higher expression of TSLPR and CD127 mRNAs in monocytes isolated from patients with Gram-negative sepsis compared with healthy control subjects. Our results emphasize a phenotypic and functional heterogeneity in an apparently homogeneous population of human CD14+ CD16- monocytes and prompt further ontogenetic and functional analysis of CD14+ CD1c+ and LPS-activated CD14+ CD1c+ TSLPR+ mono.


Subject(s)
Cell Differentiation , Cytokines/metabolism , Monocytes/immunology , Receptors, Cytokine/metabolism , Sepsis/immunology , Antigens, CD1/metabolism , Arachidonate 15-Lipoxygenase/genetics , Cells, Cultured , Chemokine CCL17/metabolism , Gene Expression Regulation , Humans , Immunophenotyping , Intercellular Signaling Peptides and Proteins/genetics , Interleukin-4/immunology , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/immunology , Receptors, Cytokine/genetics , Receptors, IgG/genetics , Receptors, Immunologic/genetics , Thymic Stromal Lymphopoietin
6.
Crit Rev Food Sci Nutr ; 57(12): 2589-2599, 2017 Aug 13.
Article in English | MEDLINE | ID: mdl-26357880

ABSTRACT

Polyphenols are the most abundant phytochemicals in fruits, vegetables, and plant-derived beverages. Recent findings suggest that polyphenols display the ability to reverse adverse epigenetic regulation involved in pathological conditions, such as obesity, metabolic disorder, cardiovascular and neurodegenerative diseases, and various forms of cancer. Epigenetics, defined as heritable changes to the transcriptome, independent from those occurring in the genome, includes DNA methylation, histone modifications, and posttranscriptional gene regulation by noncoding RNAs. Sinergistically and cooperatively, these processes regulate gene expression by changing chromatin organization and DNA accessibility. Such induced epigenetic changes can be inherited during cell division, resulting in permanent maintenance of the acquired phenotype, but they may also occur throughout an individual life-course and may ultimately influence phenotypic outcomes (health and disease risk). In the last decade, a number of studies have shown that nutrients can affect metabolic traits by altering the structure of chromatin and directly regulate both transcription and translational processes. In this context, dietary polyphenol-targeted epigenetics becomes an attractive approach for disease prevention and intervention. Here, we will review how polyphenols, including flavonoids, curcuminoids, and stilbenes, modulate the establishment and maintenance of key epigenetic marks, thereby influencing gene expression and, hence, disease risk and health.


Subject(s)
Chromatin Assembly and Disassembly , Epigenesis, Genetic/drug effects , Polyphenols/pharmacology , Chromatin , DNA Methylation , Epigenesis, Genetic/genetics , Histones , Humans , Molecular Targeted Therapy
7.
Oncotarget ; 7(2): 1500-15, 2016 Jan 12.
Article in English | MEDLINE | ID: mdl-26625205

ABSTRACT

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human solid tumor and current treatments are ineffective in increasing patients' survival. Thus, the development of new therapeutic approaches for ATC is needed. We have previously shown that the oncolytic adenovirus dl922-947 induces ATC cell death in vitro and tumor regression in vivo. However, the impact of dl922-947 on the pro-tumorigenic ATC microenvironment is still unknown. Since viruses are able to regulate cytokine and chemokine production from infected cells, we sought to investigate whether dl922-947 virotherapy has such effect on ATC cells, thereby modulating ATC microenvironment. dl922-947 decreased IL-8/CXCL8 and MCP-1/CCL2 production by the ATC cell lines 8505-c and BHT101-5. These results correlated with dl922-947-mediated reduction of NF-κB p65 binding to IL8 promoter in 8505-c and BHT101-5 cells and CCL2 promoter in 8505-c cells. IL-8 stimulates cancer cell proliferation, survival and invasion, and also angiogenesis. dl922-947-mediated reduction of IL-8 impaired ATC cell motility in vitro and ATC-induced angiogenesis in vitro and in vivo. We also show that dl922-947-mediated reduction of the monocyte-attracting chemokine CCL2 decreased monocyte chemotaxis in vitro and tumor macrophage density in vivo. Interestingly, dl922-947 treatment induced the switch of tumor macrophages toward a pro-inflammatory M1 phenotype, likely by increasing the expression of the pro-inflammatory cytokine interferon-γ. Altogether, we demonstrate that dl922-947 treatment re-shape the pro-tumorigenic ATC microenvironment by modulating cancer-cell intrinsic factors and the immune response. An in-depth knowledge of dl922-947-mediated effects on ATC microenvironment may help to refine ATC virotherapy in the context of cancer immunotherapy.


Subject(s)
Adenoviridae/pathogenicity , Chemokine CCL2/metabolism , Interleukin-8/metabolism , Macrophages/metabolism , Neovascularization, Pathologic , Oncolytic Virotherapy , Oncolytic Viruses/pathogenicity , Thyroid Carcinoma, Anaplastic/therapy , Thyroid Neoplasms/therapy , Animals , Binding Sites , Cell Line, Tumor , Cell Plasticity , Chemokine CCL2/genetics , Chemotaxis , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Host-Pathogen Interactions , Humans , Interleukin-8/genetics , Macrophages/virology , Mice, Nude , Phenotype , Promoter Regions, Genetic , Thyroid Carcinoma, Anaplastic/metabolism , Thyroid Carcinoma, Anaplastic/pathology , Thyroid Carcinoma, Anaplastic/virology , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology , Thyroid Neoplasms/virology , Time Factors , Transcription Factor RelA/metabolism , Transfection , Tumor Microenvironment , Xenograft Model Antitumor Assays
8.
Diabetologia ; 59(1): 176-186, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26453063

ABSTRACT

AIMS/HYPOTHESIS: Chronic hyperglycaemia worsens insulin resistance in individuals with type 2 diabetes. Whether this effect is contributed by epigenetic dysregulation and which genes are involved remain unclear. Prep1 (also known as Pknox1) is a gene exerting major effects on the sensitivity of the glucose transport machinery to insulin. Here, we show that dysregulation of Prep1 expression by high glucose levels is associated with histone modifications at its 5' regulatory region. METHODS: We used mouse and cell models to investigate Prep1 transcriptional regulation by glucose. RESULTS: Differentiated L6 skeletal muscle cells were grown in the presence of either 5.5 or 25 mmol/l glucose (normal [NG] and high glucose [HG], respectively). The HG exposure increased nuclear factor κ light chain enhancer of activated B cells (NF-κB) p65 binding and recruitment of the su(var)3-9, enhancer-of-zeste, trithorax domain-containing lysine methyltransferase 7 (SET7) histone methyltransferase and p300 acetyltransferase to the 5' region of Prep1, leading to enhanced transcription. In addition, chromatin immunoprecipitation assays revealed concomitantly increased histone H3 mono- and dimethylation and acetylation at Lys4 and Lys9/14, respectively. Skeletal muscle tissue from streptozotocin-treated diabetic mice also showed Prep1 overexpression accompanied by similarly increased recruitment of NF-κB p65 and histone modifications at the 5' region of Prep1. In these same mice, as well as in Prep1-overexpressing L6 cells, Prep1-induced recruitment of the repressor complex myocyte enhancer factor 2 (MEF2)/histone deacetylase 5 (HDAC5) at the Glut4 promoter was also increased, leading to reduced Glut4 expression. CONCLUSIONS/INTERPRETATION: These studies indicate that HG exposure induces NF-κB recruitment and histone modification at the Prep1 5' region, thereby enhancing the transcription of Prep1 and repressing that of Glut4. Histone changes at the Prep1 gene may contribute to insulin resistance in individuals with type 2 diabetes.


Subject(s)
Diabetes Mellitus, Type 2/blood , Gene Expression Regulation , Glucose/metabolism , Histones/metabolism , Homeodomain Proteins/metabolism , Protein Processing, Post-Translational , Animals , Blood Glucose/analysis , Cell Line , Diabetes Mellitus, Type 2/metabolism , Epigenesis, Genetic , Inflammation , Insulin Resistance , MEF2 Transcription Factors/metabolism , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , NF-kappa B/metabolism
9.
J Biol Chem ; 287(51): 42951-61, 2012 Dec 14.
Article in English | MEDLINE | ID: mdl-23105093

ABSTRACT

The gene network responsible for inflammation-induced insulin resistance remains enigmatic. In this study, we show that, in L6 cells, rosiglitazone- as well as pioglitazone-dependent activation of peroxisome proliferator-activated receptor-γ (PPARγ) represses transcription of the ped/pea-15 gene, whose increased activity impairs glucose tolerance in mice and humans. Rosiglitazone enhanced insulin-induced glucose uptake in L6 cells expressing the endogenous ped/pea-15 gene but not in cells expressing ped/pea-15 under the control of an exogenous promoter. The ability of PPARγ to affect ped/pea-15 expression was also lost in cells and in C57BL/6J transgenic mice expressing ped/pea-15 under the control of an exogenous promoter, suggesting that ped/pea-15 repression may contribute to rosiglitazone action on glucose disposal. Indeed, high fat diet mice showed insulin resistance and increased ped/pea-15 levels, although these effects were reduced by rosiglitazone treatment. Both supershift and ChIP assays revealed the presence of the AP-1 component c-JUN at the PED/PEA-15 promoter upon 12-O-tetradecanoylphorbol-13-acetate stimulation of the cells. In these experiments, rosiglitazone treatment reduced c-JUN presence at the PED/PEA-15 promoter. This effect was not associated with a decrease in c-JUN expression. In addition, c-jun silencing in L6 cells lowered ped/pea-15 expression and caused nonresponsiveness to rosiglitazone, although c-jun overexpression enhanced the binding to the ped/pea-15 promoter and blocked the rosiglitazone effect. These results indicate that PPARγ regulates ped/pea-15 transcription by inhibiting c-JUN binding at the ped/pea-15 promoter. Thus, ped/pea-15 is downstream of a major PPARγ-regulated inflammatory network. Repression of ped/pea-15 transcription might contribute to the PPARγ regulation of muscle sensitivity to insulin.


Subject(s)
Gene Expression Regulation , Glucose/metabolism , Insulin/metabolism , Muscle Cells/metabolism , PPAR gamma/metabolism , Phosphoproteins/genetics , Transcription Factor AP-1/metabolism , Animals , Apoptosis Regulatory Proteins , Diet, High-Fat , Feeding Behavior/drug effects , Gene Expression Regulation/drug effects , Gene Silencing/drug effects , HeLa Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle Cells/drug effects , Muscle, Skeletal/cytology , Phosphoproteins/metabolism , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Binding/genetics , Rats , Rosiglitazone , Signal Transduction/drug effects , Signal Transduction/genetics , Tetradecanoylphorbol Acetate/pharmacology , Thiazolidinediones/pharmacology , Transcription, Genetic/drug effects
10.
Clin Exp Metastasis ; 29(6): 585-601, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22484917

ABSTRACT

Prostate and breast cancer are major causes of death worldwide, mainly due to patient relapse upon disease recurrence through formation of metastases. Chemokines are small proteins with crucial roles in the immune system, and their regulation is finely tuned in early inflammatory responses. They are key molecules during inflammatory processes, and many studies are focusing on their regulatory functions in tumor growth and angiogenesis during metastatic cell seeding and spreading. Bindarit is an anti-inflammatory indazolic derivative that can inhibit the synthesis of MCP-1/CCL2, with a potential inhibitory function in tumor progression and metastasis formation. We show here that in vitro, bindarit can modulate cancer-cell proliferation and migration, mainly through negative regulation of TGF-ß and AKT signaling, and it can impair the NF-κB signaling pathway through enhancing the expression of the NF-κB inhibitor IkB-α. In vivo administration of bindarit results in impaired metastatic disease in prostate cancer xenograft mice (PC-3M-Luc2 cells injected intra-cardially) and impairment of local tumorigenesis in syngeneic Balb/c mice injected under the mammary gland with murine breast cancer cells (4T1-Luc cells). In addition, bindarit treatment significantly decreases the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in 4T1-Luc primary tumors. Overall, our data indicate that bindarit is a good candidate for new therapies against prostate and breast tumorigenesis, with an action through impairment of inflammatory cell responses during formation of the tumor-stroma niche microenvironment.


Subject(s)
Breast Neoplasms/pathology , Chemokine CCL2/biosynthesis , Indazoles/pharmacology , Propionates/pharmacology , Prostatic Neoplasms/pathology , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Chemokine CCL2/chemistry , Chemokine CCL2/metabolism , Female , Humans , Macrophages/metabolism , Male , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Neoplasm Metastasis , Neoplasm Transplantation , Signal Transduction
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