Publications by authors named "Véronique Bourdeau"

30 Publications

  • Page 1 of 1

A hydride transfer complex reprograms NAD metabolism and bypasses senescence.

Mol Cell 2021 Sep;81(18):3848-3865.e19

CRCHUM, 900 Saint-Denis St, Montréal, QC H2X 0A9, Canada; Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada. Electronic address:

Metabolic rewiring and redox balance play pivotal roles in cancer. Cellular senescence is a barrier for tumorigenesis circumvented in cancer cells by poorly understood mechanisms. We report a multi-enzymatic complex that reprograms NAD metabolism by transferring reducing equivalents from NADH to NADP. This hydride transfer complex (HTC) is assembled by malate dehydrogenase 1, malic enzyme 1, and cytosolic pyruvate carboxylase. HTC is found in phase-separated bodies in the cytosol of cancer or hypoxic cells and can be assembled in vitro with recombinant proteins. HTC is repressed in senescent cells but induced by p53 inactivation. HTC enzymes are highly expressed in mouse and human prostate cancer models, and their inactivation triggers senescence. Exogenous expression of HTC is sufficient to bypass senescence, rescue cells from complex I inhibitors, and cooperate with oncogenic RAS to transform primary cells. Altogether, we provide evidence for a new multi-enzymatic complex that reprograms metabolism and overcomes cellular senescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2021.08.028DOI Listing
September 2021

Phosphorylation of SOCS1 Inhibits the SOCS1-p53 Tumor Suppressor Axis.

Cancer Res 2019 07 17;79(13):3306-3319. Epub 2019 May 17.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec, Canada.

Expression of the suppressor of cytokine signaling-1 (SOCS1) is inactivated in hematopoietic and solid cancers by promoter methylation, miRNA-mediated silencing, and mutations. Paradoxically, SOCS1 is also overexpressed in many human cancers. We report here that the ability of SOCS1 to interact with p53 and regulate cellular senescence depends on a structural motif that includes tyrosine (Y)80 in the SH2 domain of SOCS1. Mutations in this motif are found at low frequency in some human cancers, and substitution of Y80 by a phosphomimetic residue inhibits p53-SOCS1 interaction and its functional consequences, including stimulation of p53 transcriptional activity, growth arrest, and cellular senescence. Mass spectrometry confirmed SOCS1 Y80 phosphorylation in cells, and a new mAb was generated to detect its presence in tissues by IHC. A tyrosine kinase library screen identified the SRC family as Y80-SOCS1 kinases. SRC family kinase inhibitors potentiated the SOCS1-p53 pathway and reinforced SOCS1-induced senescence. Samples from human lymphomas that often overexpress SOCS1 also displayed SRC family kinase activation, constitutive phosphorylation of SOCS1 on Y80, and SOCS1 cytoplasmic localization. Collectively, these results reveal a mechanism that inactivates the SOCS1-p53 senescence pathway and suggest that inhibition of SRC family kinases as personalized treatment in patients with lymphomas may be successful. SIGNIFICANCE: These findings show that SOCS1 phosphorylation by the SRC family inhibits its tumor-suppressive activity, indicating that patients with increased SOCS1 phosphorylation may benefit from SRC family kinase inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-18-1503DOI Listing
July 2019

Ribosomal protein RPL22/eL22 regulates the cell cycle by acting as an inhibitor of the CDK4-cyclin D complex.

Cell Cycle 2019 Mar - Apr;18(6-7):759-770. Epub 2019 Mar 28.

a Department of Biochemistry and Molecular Medicine , Université de Montréal , Montréal , Québec , Canada.

Senescence is a tumor suppressor program characterized by a stable growth arrest while maintaining cell viability. Senescence-associated ribogenesis defects (SARD) have been shown to regulate senescence through the ability of the ribosomal protein S14 (RPS14 or uS11) to bind and inhibit the cyclin-dependent kinase 4 (CDK4). Here we report another ribosomal protein that binds and inhibits CDK4 in senescent cells: L22 (RPL22 or eL22). Enforcing the expression of RPL22/eL22 is sufficient to induce an RB and p53-dependent cellular senescent phenotype in human fibroblasts. Mechanistically, RPL22/eL22 can interact with and inhibit CDK4-Cyclin D1 to decrease RB phosphorylation both in vitro and in cells. Briefly, we show that ribosome-free RPL22/eL22 causes a cell cycle arrest which could be relevant during situations of nucleolar stress such as cellular senescence or the response to cancer chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15384101.2019.1593708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6464582PMC
April 2020

Circumventing senescence is associated with stem cell properties and metformin sensitivity.

Aging Cell 2019 04 6;18(2):e12889. Epub 2019 Jan 6.

Department of Biochemistry and Molecular Medicine and CR-CHUM, Université de Montréal, Montréal, Québec, Canada.

Most cancers arise in old individuals, which also accumulate senescent cells. Cellular senescence can be experimentally induced by expression of oncogenes or telomere shortening during serial passage in culture. In vivo, precursor lesions of several cancer types accumulate senescent cells, which are thought to represent a barrier to malignant progression and a response to the aberrant activation of growth signaling pathways by oncogenes (oncogene toxicity). Here, we sought to define gene expression changes associated with cells that bypass senescence induced by oncogenic RAS. In the context of pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS induces benign pancreatic intraepithelial neoplasias (PanINs), which exhibit features of oncogene-induced senescence. We found that the bypass of senescence in PanINs leads to malignant PDAC cells characterized by gene signatures of epithelial-mesenchymal transition, stem cells, and mitochondria. Stem cell properties were similarly acquired in PanIN cells treated with LPS, and in primary fibroblasts and mammary epithelial cells that bypassed Ras-induced senescence after reduction of ERK signaling. Intriguingly, maintenance of cells that circumvented senescence and acquired stem cell properties was blocked by metformin, an inhibitor of complex I of the electron transport chain or depletion of STAT3, a protein required for mitochondrial functions and stemness. Thus, our studies link bypass of senescence in premalignant lesions to loss of differentiation, acquisition of stemness features, and increased reliance on mitochondrial functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/acel.12889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413657PMC
April 2019

Senescence-associated ribosome biogenesis defects contributes to cell cycle arrest through the Rb pathway.

Nat Cell Biol 2018 07 25;20(7):789-799. Epub 2018 Jun 25.

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, Quebec, Canada.

Cellular senescence is a tumour suppressor programme characterized by a stable cell cycle arrest. Here we report that cellular senescence triggered by a variety of stimuli leads to diminished ribosome biogenesis and the accumulation of both rRNA precursors and ribosomal proteins. These defects were associated with reduced expression of several ribosome biogenesis factors, the knockdown of which was also sufficient to induce senescence. Genetic analysis revealed that Rb but not p53 was required for the senescence response to altered ribosome biogenesis. Mechanistically, the ribosomal protein S14 (RPS14 or uS11) accumulates in the soluble non-ribosomal fraction of senescent cells, where it binds and inhibits CDK4 (cyclin-dependent kinase 4). Overexpression of RPS14 is sufficient to inhibit Rb phosphorylation, inducing cell cycle arrest and senescence. Here we describe a mechanism for maintaining the senescent cell cycle arrest that may be relevant for cancer therapy, as well as biomarkers to identify senescent cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41556-018-0127-yDOI Listing
July 2018

Quantitative SUMO proteomics reveals the modulation of several PML nuclear body associated proteins and an anti-senescence function of UBC9.

Sci Rep 2018 05 17;8(1):7754. Epub 2018 May 17.

Institute of Research in Immunology and Cancer, Université de Montréal, Montréal, QC H3C 3J7, Canada.

Several regulators of SUMOylation have been previously linked to senescence but most targets of this modification in senescent cells remain unidentified. Using a two-step purification of a modified SUMO3, we profiled the SUMO proteome of senescent cells in a site-specific manner. We identified 25 SUMO sites on 23 proteins that were significantly regulated during senescence. Of note, most of these proteins were PML nuclear body (PML-NB) associated, which correlates with the increased number and size of PML-NBs observed in senescent cells. Interestingly, the sole SUMO E2 enzyme, UBC9, was more SUMOylated during senescence on its Lys-49. Functional studies of a UBC9 mutant at Lys-49 showed a decreased association to PML-NBs and the loss of UBC9's ability to delay senescence. We thus propose both pro- and anti-senescence functions of protein SUMOylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-25150-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5958138PMC
May 2018

CDK4-CDK6 inhibitors induce autophagy-mediated degradation of DNMT1 and facilitate the senescence antitumor response.

Autophagy 2016 10 17;12(10):1965-1966. Epub 2016 Aug 17.

a Department of Biochemistry and Molecular Medicine , Université de Montréal , Montréal , Québec , Canada.

Senescence is a natural anticancer defense program disabled in tumor cells. We discovered that deregulated CDK4 (cyclin dependant kinase 4) and CDK6 activities contribute to senescence bypass during tumorigenesis and that their inhibition restores the senescence response in tumor cells. CDK4 and CDK6 phosphorylate RB1/RB, preventing its inhibitory interaction with the E2Fs, the cell cycle transcription factors. However, we also found that CDK4 interacts and phosphorylates the DNMT1 (DNA methyltransferase 1) protein protecting it from macroautophagy/autophagy-mediated protein degradation. This discovery highlights a new epigenetic component of CDK4-CDK6 signaling that could be exploited in cancer treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15548627.2016.1214779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5079664PMC
October 2016

A CDK4/6-Dependent Epigenetic Mechanism Protects Cancer Cells from PML-induced Senescence.

Cancer Res 2016 06 29;76(11):3252-64. Epub 2016 Mar 29.

Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada.

Promyelocytic leukemia (PML) plays a tumor suppressive role by inducing cellular senescence in response to oncogenic stress. However, tumor cell lines fail to engage in complete senescence upon PML activation. In this study, we investigated the mechanisms underlying resistance to PML-induced senescence. Here, we report that activation of the cyclin-dependent kinases CDK4 and CDK6 are essential and sufficient to impair senescence induced by PML expression. Disrupting CDK function by RNA interference or pharmacological inhibition restored senescence in tumor cells and diminished their tumorigenic potential in mouse xenograft models. Complete senescence correlated with an increase in autophagy, repression of E2F target genes, and an gene expression signature of blocked DNA methylation. Accordingly, treatment of tumor cells with inhibitors of DNA methylation reversed resistance to PML-induced senescence. Further, CDK inhibition with palbociclib promoted autophagy-dependent degradation of the DNA methyltransferase DNMT1. Lastly, we found that CDK4 interacted with and phosphorylated DNMT1 in vitro, suggesting that CDK activity is required for its stabilization. Taken together, our findings highlight a potentially valuable feature of CDK4/6 inhibitors as epigenetic modulators to facilitate activation of senescence programs in tumor cells. Cancer Res; 76(11); 3252-64. ©2016 AACR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-15-2347DOI Listing
June 2016

Dataset of STAT5A status in breast cancer.

Data Brief 2016 Jun 4;7:490-2. Epub 2016 Mar 4.

Université de Montréal, Département de Biochimie, Montréal, Québec, Canada H3C 3J7.

We analysed STAT5A gene expression in breast cancer using the Oncomine database. We exemplify four representative studies showing that STAT5A is generally downregulated in breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dib.2016.02.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792860PMC
June 2016

STAT5A is regulated by DNA damage via the tumor suppressor p53.

Cytokine 2016 06 11;82:70-9. Epub 2016 Feb 11.

Université de Montréal, Département de biochimie, Montréal, Québec H3C 3J7, Canada. Electronic address:

Here we report that the STAT5A transcription factor is a direct p53 transcriptional target gene. STAT5A is well expressed in p53 wild type cells but not in p53-null cells. Inhibition of p53 reduces STAT5A expression. DNA damaging agents such as doxorubicin also induced STAT5A expression in a p53 dependent manner. Two p53 binding sites were mapped in the STAT5A gene and named PBS1 and PBS2; these sites were sufficient to confer p53 responsiveness in a luciferase reporter gene. Chromatin immunoprecipitation experiments revealed that PBS2 has constitutive p53 bound to it, while p53 binding to PBS1 required DNA damage. In normal human breast lobules, weak p53 staining correlated with regions of intense STAT5A staining. Interestingly, in a cohort of triple negative breast tumor tissues there was little correlation between regions of p53 and STAT5A staining, likely reflecting a high frequency of p53 mutations that stabilize the protein in these tumors. We thus reveal an unexpected connection between cytokine signaling and p53.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cyto.2016.01.013DOI Listing
June 2016

Sponges against miR-19 and miR-155 reactivate the p53-Socs1 axis in hematopoietic cancers.

Cytokine 2016 06 1;82:80-6. Epub 2016 Feb 1.

Département de biochimie et médecine moléculaire, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec H3C 3J7, Canada. Electronic address:

Normal cell proliferation is controlled by a balance between signals that promote or halt cell proliferation. Micro RNAs are emerging as key elements in providing fine signal balance in different physiological situations. Here we report that STAT5 signaling induces the miRNAs miR-19 and miR-155, which potentially antagonize the tumor suppressor axis composed by the STAT5 target gene SOCS1 (suppressor of cytokine signaling-1) and its downstream effector p53. MiRNA sponges against miR-19 or miR-155 inhibit the functions of these miRNAs and potentiate the induction of SOCS1 and p53 in mouse leukemia cells and in human myeloma cells. Adding a catalytic RNA motif of the hammerhead type within miRNA sponges against miR-155 leads to decreased miR-155 levels and increased their ability of inhibiting cell growth and cell migration in myeloma cells. The results indicate that antagonizing miRNA activity can reactivate tumor suppressor pathways downstream cytokine stimulation in tumor cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cyto.2016.01.015DOI Listing
June 2016

Structural and functional characterization of the phosphorylation-dependent interaction between PML and SUMO1.

Structure 2015 Jan 11;23(1):126-138. Epub 2014 Dec 11.

Département de Biochimie et Médicine Moléculaire, Université de Montréal, C.P. 6128 Succursale Centre-Ville, Montréal, QC H3C 3J7, Canada. Electronic address:

PML and several other proteins localizing in PML-nuclear bodies (PML-NB) contain phosphoSIMs (SUMO-interacting motifs), and phosphorylation of this motif plays a key role in their interaction with SUMO family proteins. We examined the role that phosphorylation plays in the binding of the phosphoSIMs of PML and Daxx to SUMO1 at the atomic level. The crystal structures of SUMO1 bound to unphosphorylated and tetraphosphorylated PML-SIM peptides indicate that three phosphoserines directly contact specific positively charged residues of SUMO1. Surprisingly, the crystal structure of SUMO1 bound to a diphosphorylated Daxx-SIM peptide indicate that the hydrophobic residues of the phosphoSIM bind in a manner similar to that seen with PML, but important differences are observed when comparing the phosphorylated residues. Together, the results provide an atomic level description of how specific acetylation patterns within different SUMO family proteins can work together with phosphorylation of phosphoSIM's regions of target proteins to regulate binding specificity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.str.2014.10.015DOI Listing
January 2015

Engaging a senescent response to cure leukemia.

Nat Med 2014 Feb;20(2):123-4

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Québec, Canada.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm.3469DOI Listing
February 2014

CHES1/FOXN3 regulates cell proliferation by repressing PIM2 and protein biosynthesis.

Mol Biol Cell 2014 Mar 8;25(5):554-65. Epub 2014 Jan 8.

Département de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, QC H3C 3J7, Canada.

The expression of the forkhead transcription factor checkpoint suppressor 1 (CHES1), also known as FOXN3, is reduced in many types of cancers. We show here that CHES1 decreases protein synthesis and cell proliferation in tumor cell lines but not in normal fibroblasts. Conversely, short hairpin RNA-mediated depletion of CHES1 increases tumor cell proliferation. Growth suppression depends on the CHES1 forkhead DNA-binding domain and correlates with the nuclear localization of CHES1. CHES1 represses the expression of multiple genes, including the kinases PIM2 and DYRK3, which regulate protein biosynthesis, and a number of genes in cilium biogenesis. CHES1 binds directly to the promoter of PIM2, and in cells expressing CHES1 the levels of PIM2 are reduced, as well as the phosphorylation of the PIM2 target 4EBP1. Overexpression of PIM2 or eIF4E partially reverses the antiproliferative effect of CHES1, indicating that PIM2 and protein biosynthesis are important targets of the antiproliferative effect of CHES1. In several human hematopoietic cancers, CHES1 and PIM2 expressions are inversely correlated, suggesting that repression of PIM2 by CHES1 is clinically relevant.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E13-02-0110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937083PMC
March 2014

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation.

Genes Dev 2013 Apr 18;27(8):900-15. Epub 2013 Apr 18.

Département de Biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.203984.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650227PMC
April 2013

Metformin inhibits the senescence-associated secretory phenotype by interfering with IKK/NF-κB activation.

Aging Cell 2013 Jun 23;12(3):489-98. Epub 2013 Apr 23.

Département de Biochimie, Université de Montréal, Pavillon Roger-Gaudry 2900 boul. Édouard Montpetit, Montréal, QC, H3C 3J7, Canada.

We show that the antidiabetic drug metformin inhibits the expression of genes coding for multiple inflammatory cytokines seen during cellular senescence. Conditioned medium (CM) from senescent cells stimulates the growth of prostate cancer cells but treatment of senescent cells with metformin inhibited this effect. Bioinformatic analysis of genes downregulated by metformin suggests that the drug blocks the activity of the transcription factor NF-κB. In agreement, metformin prevented the translocation of NF-κB to the nucleus and inhibited the phosphorylation of IκB and IKKα/β, events required for activation of the NF-κB pathway. These effects were not dependent on AMPK activation or on the context of cellular senescence, as metformin inhibited the NF-κB pathway stimulated by lipopolysaccharide (LPS) in ampk null fibroblasts and in macrophages. Taken together, our results provide a novel mechanism for the antiaging and antineoplastic effects of metformin reported in animal models and in diabetic patients taking this drug.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/acel.12075DOI Listing
June 2013

Retinoblastoma-independent regulation of cell proliferation and senescence by the p53-p21 axis in lamin A /C-depleted cells.

Aging Cell 2011 Oct 29;10(5):789-97. Epub 2011 May 29.

Biochemistry Department, Université de Montréal, Montréal, QC, Canada.

The expression of A-type lamin is downregulated in several cancers, and lamin defects are the cause of several diseases including a form of accelerated aging. We report that depletion of lamin A/C expression in normal human cells leads to a dramatic downregulation of the Rb family of tumor suppressors and a defect in cell proliferation. Lamin A/C-depleted cells exhibited a flat morphology and accumulated markers of cellular senescence. This senescent phenotype was accompanied by engagement of the p53 tumor suppressor and induction of the p53 target gene p21 and was prevented by small hairpin RNAs against p53, p21, or by the oncoprotein Mdm2. The expression of E2F target genes, normally required for cell cycle progression, was downregulated after lamin A/C depletion but restored after the inactivation of p53. A similar senescence response was observed in myoblasts from a patient with a lamin A mutation causing muscular dystrophy. We thus reveal a previously unnoticed mechanism of controlling cell cycle genes expression, which depends on p53 but does not require the retinoblastoma family of tumor suppressors and that can be relevant to understand the pathogenesis of laminopathies and perhaps aging.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1474-9726.2011.00719.xDOI Listing
October 2011

Regulation of E2Fs and senescence by PML nuclear bodies.

Genes Dev 2011 Jan;25(1):41-50

Biochemistry Department, Université de Montréal, Montréal, Québec H3C3J7, Canada.

The tumor suppressor PML (promyelocytic leukemia protein) regulates cellular senescence and terminal differentiation, two processes that implicate a permanent exit from the cell cycle. Here, we show that the mechanism by which PML induces a permanent cell cycle exit and activates p53 and senescence involves a recruitment of E2F transcription factors bound to their promoters and the retinoblastoma (Rb) proteins to PML nuclear bodies enriched in heterochromatin proteins and protein phosphatase 1α. Blocking the functions of the Rb protein family or adding back E2Fs to PML-expressing cells can rescue their defects in E2F-dependent gene expression and cell proliferation, inhibiting the senescent phenotype. In benign prostatic hyperplasia, a neoplastic disease that displays features of senescence, PML was found to be up-regulated and forming nuclear bodies. In contrast, PML bodies were rarely visualized in prostate cancers. The newly defined PML/Rb/E2F pathway may help to distinguish benign tumors from cancers, and suggest E2F target genes as potential targets to induce senescence in human tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.1975111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012935PMC
January 2011

Ligand-dependent corepressor LCoR is an attenuator of progesterone-regulated gene expression.

J Biol Chem 2009 Oct 10;284(44):30275-87. Epub 2009 Sep 10.

Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada.

Ligand-dependent corepressor LCoR interacts with the progesterone receptor (PR) and estrogen receptor ERalpha in the presence of hormone. LCoR contains tandem N-terminal PXDLS motifs that recruit C-terminal-binding protein (CtBP) corepressors as well as a C-terminal helix-turn-helix (HTH) domain. Here, we analyzed the function of these domains in coregulation of PR- and ERalpha-regulated gene expression. LCoR and CtBP1 colocalize in nuclear bodies that also contain CtBP-interacting protein CtIP and polycomb group repressor complex marker BMI1. Coexpression of CtBP1 in MCF7 or T47D breast cancer cells augmented corepression by LCoR, whereas coexpression of CtIP did not, consistent with direct interaction of LCoR with CtBP1, but not CtIP. The N-terminal region containing the PXDLS motifs is necessary and sufficient for CTBP1 recruitment and essential for full corepression. However, LCoR function was also strongly dependent on the helix-turn-helix domain, as its deletion completely abolished corepression. LCoR, CtBP, and CtIP were recruited to endogenous PR- and ERalpha-stimulated genes in a hormone-dependent manner. Similarly, LCoR was recruited to estrogen-repressed genes, whereas hormone treatment reduced CtBP1 binding. Small interfering RNA-mediated knockdown of LCoR or CtBP1 augmented expression of progesterone- and estrogen-stimulated reporter genes as well as endogenous progesterone-stimulated target genes. In contrast, their ablation had gene-specific effects on ERalpha-regulated transcription that generally led to reduced gene expression. Taken together, these results show that multiple domains contribute to LCoR function. They also reveal a role for LCoR and CtBP1 as attenuators of progesterone-regulated transcription but suggest that LCoR and CtBP1 can act to enhance transcription of some genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.051201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781583PMC
October 2009

Function of histone deacetylase 6 as a cofactor of nuclear receptor coregulator LCoR.

J Biol Chem 2009 Oct 10;284(44):30264-74. Epub 2009 Sep 10.

Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada.

Ligand-dependent corepressor LCoR was identified as a protein that interacts with the estrogen receptor alpha (ERalpha) ligand binding domain in a hormone-dependent manner. LCoR also interacts directly with histone deacetylase 3 (HDAC3) and HDAC6. Notably, HDAC6 has emerged as a marker of breast cancer prognosis. However, although HDAC3 is nuclear, HDAC6 is cytoplasmic in many cells. We found that HDAC6 is partially nuclear in estrogen-responsive MCF7 cells, colocalizes with LCoR, represses transactivation of estrogen-inducible reporter genes, and augments corepression by LCoR. In contrast, no repression was observed upon HDAC6 expression in COS7 cells, where it is exclusively cytoplasmic. LCoR binds to HDAC6 in vitro via a central domain, and repression by LCoR mutants lacking this domain was attenuated. Kinetic chromatin immunoprecipitation assays revealed hormone-dependent recruitment of LCoR to promoters of ERalpha-induced target genes in synchrony with ERalpha. HDAC6 was also recruited to these promoters, and repeat chromatin immunoprecipitation experiments confirmed the corecruitment of LCoR with ERalpha and with HDAC6. Remarkably, however, although we find evidence for corecruitment of LCoR and ERalpha on genes repressed by the receptor, LCoR and HDAC6 failed to coimmunoprecipitate, suggesting that they are part of distinct complexes on these genes. Although small interfering RNA-mediated knockdown of LCoR or HDAC6 augmented expression of an estrogen-sensitive reporter gene in MCF7 cells, unexpectedly their ablation led to reduced expression of some endogenous estrogen target genes. Taken together, these data establish that HDAC6 can function as a cofactor of LCoR but suggest that they may act in enhance expressing some target genes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.045526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781582PMC
October 2009

Mitochondrial dysfunction contributes to oncogene-induced senescence.

Mol Cell Biol 2009 Aug 15;29(16):4495-507. Epub 2009 Jun 15.

Département de Biochimie, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec H3C3J7, Canada.

The expression of oncogenic ras in normal human cells quickly induces an aberrant proliferation response that later is curtailed by a cell cycle arrest known as cellular senescence. Here, we show that cells expressing oncogenic ras display an increase in the mitochondrial mass, the mitochondrial DNA, and the mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest. By the time the cells entered senescence, dysfunctional mitochondria accumulated around the nucleus. The mitochondrial dysfunction was accompanied by oxidative DNA damage, a drop in ATP levels, and the activation of AMPK. The increase in mitochondrial mass and ROS in response to oncogenic ras depended on intact p53 and Rb tumor suppression pathways. In addition, direct interference with mitochondrial functions by inhibiting the expression of the Rieske iron sulfur protein of complex III or the use of pharmacological inhibitors of the electron transport chain and oxidative phosphorylation was sufficient to trigger senescence. Taking these results together, this work suggests that mitochondrial dysfunction is an effector pathway of oncogene-induced senescence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.01868-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725737PMC
August 2009

PML links aberrant cytokine signaling and oncogenic stress to cellular senescence.

Front Biosci (Landmark Ed) 2009 Jan 1;14:475-85. Epub 2009 Jan 1.

Departement de Biochimie, Universite de Montreal, C.P. 6128, Succ. Centre-Ville, Montreal, Quebec, Canada H3C 3J7.

Senescence is a tumor suppressor mechanism triggered by oncogenic stimuli and characterized by a permanent cell cycle arrest mediated by tumor suppressors such as p53, Rb and PML. PML itself is critical for the formation of nuclear bodies (PML bodies) that accumulate in senescent cells rendering them suitable markers for the senescence phenotype. The mechanism of PML-induction during senescence is complex and includes increased PML gene transcription by p53 or transcription factors of the interferon/Jak/Stat pathway. In turn, PML engages both p53 and Rb, although the precise molecular processes are unknown. PML interacts with the DNA-binding domain of p53 facilitating p53 modifications. PML can also interact with Rb and may play a role in Rb-dependent gene silencing during senescence. Recent studies suggest an additional connection between PML and the senescence program. Senescence involves a constitutive activation of the DNA damage response. Intriguingly, proteins that signal DNA damage or help repairing it localize to PML bodies, suggesting that PML may play a role in the DNA damage response during senescence. We think that the discovery of factors acting upstream or downstream PML may help to understand how cells bypass senescence on their way to tumorigenesis. More importantly the PML pathway may eventually lead to novel anti-cancer therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2741/3256DOI Listing
January 2009

Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells.

Nucleic Acids Res 2008 Jan 5;36(1):76-93. Epub 2007 Nov 5.

Institute for Research in Immunology and Cancer and Biochemistry Department, Université de Montréal, C.P. 6128 Succursale Centre Ville, Montréal, QC H3C 3J7, Canada.

Estrogen receptors (ERs), which mediate the proliferative action of estrogens in breast cancer cells, are ligand-dependent transcription factors that regulate expression of their primary target genes through several mechanisms. In addition to direct binding to cognate DNA sequences, ERs can be recruited to DNA through other transcription factors (tethering), or affect gene transcription through modulation of signaling cascades by non-genomic mechanisms of action. To better characterize the mechanisms of gene regulation by estrogens, we have identified more than 700 putative primary and about 1300 putative secondary target genes of estradiol in MCF-7 cells through microarray analysis performed in the presence or absence of the translation inhibitor cycloheximide. Although siRNA-mediated inhibition of ERalpha expression antagonized the effects of estradiol on up- and down-regulated primary target genes, estrogen response elements (EREs) were enriched only in the vicinity of up-regulated genes. Binding sites for several other transcription factors, including proteins known to tether ERalpha, were enriched in up- and/or down-regulated primary targets. Secondary estrogen targets were particularly enriched in sites for E2F family members, several of which were transcriptionally regulated by estradiol, consistent with a major role of these factors in mediating the effects of estrogens on gene expression and cellular growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkm945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2248750PMC
January 2008

Regulation of GREB1 transcription by estrogen receptor alpha through a multipartite enhancer spread over 20 kb of upstream flanking sequences.

J Biol Chem 2007 Jun 26;282(24):17335-9. Epub 2007 Apr 26.

Institute for Research in Immunology and Cancer, Department of Biochemistry, Université de Montréal, Montréal, Québec H3C 3J7, Canada.

Estrogen receptors activate transcription in part through direct interactions with specific DNA motifs, called estrogen response elements (EREs). Here we show that the strong and sustained induction of the gene regulated in breast cancer 1 (GREB1), a gene of unknown function that has been previously suggested to play a role in the effects of estradiol on breast cancer cell proliferation (Rae, J. M., Johnson, M. D., Scheys, J. O., Cordero, K. E., Larios, J. M., and Lippman, M. E. (2005) Breast Cancer Res. Treat 92, 141-149), is mediated by binding of estrogen receptor alpha (ERalpha) to three consensus EREs spread over approximately 20 kb of upstream flanking sequences. In addition to ERalpha, coactivator SRC-3, acetylated histones and phosphorylated RNA polymerase II (P-polII) were detected on all three EREs in the presence of estrogen, while basal recruitment of ERalpha and P-polII was observed only on the proximal element. Chromatin loops were formed between each ERE and the GREB1 transcriptional start site in the presence of estrogen but not of a total antiestrogen. Furthermore, estradiol induced physical association between EREs, suggesting that these elements function as a potent multipartite enhancer to regulate GREB1 transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.C700030200DOI Listing
June 2007

An E2F/miR-20a autoregulatory feedback loop.

J Biol Chem 2007 Jan 29;282(4):2135-43. Epub 2006 Nov 29.

Département de Biochimie, Université de Montréal, Montréal, Quebec H3C 3J7, Canada.

The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1-3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17-92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17-92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3'-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17-92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17-92 cluster. Our data also point toward an anti-apoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17-92 cluster. Altogether, these results suggest that the autoregulation between E2F1-3 and miR-20a is important for preventing an abnormal accumulation of E2F1-3 and may play a role in the regulation of cellular proliferation and apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M608939200DOI Listing
January 2007

Convergence of vitamin D and retinoic acid signalling at a common hormone response element.

EMBO Rep 2006 Feb;7(2):180-5

Department of Medicine, McGill University, 3655 Drummond St, Montreal, Quebec, Canada H3G 1Y6.

Although 1,25-dihydroxyvitamin D3 (1,25D3) and retinoic acid (RA) have distinct developmental and physiological roles, both regulate the cell cycle. We provide molecular and genomic evidence that their cognate nuclear receptors regulate common genes through everted repeat TGA(C/T)TPyN8PuG(G/T)TCA (ER8) response elements. ER8 motifs were found in the promoters of several target genes of 1,25D3 and/or RA. Notably, an element was characterized in the cyclin-dependent kinase (CDK) inhibitor p19ink4d gene, and 1,25D3- or RA-induced p19INK4D) expression. P19ink4d knockdown together with depletion of p27kip1, another CDK inhibitor regulated by 1,25D3 and RA, rendered cells resistant to ligand-induced growth arrest. Remarkably, p19INK4D-deficient cells showed increased autophagic cell death, which was markedly enhanced by 1,25D3, but not RA, and attenuated by loss of p27KIP1. These results show a limited crosstalk between 1,25D3 and RA signalling by means of overlapping nuclear receptor DNA binding specificities, and uncover a role for p19INK4D in control of cell survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.embor.7400594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1369248PMC
February 2006

Large-scale in silico and microarray-based identification of direct 1,25-dihydroxyvitamin D3 target genes.

Mol Endocrinol 2005 Nov 7;19(11):2685-95. Epub 2005 Jul 7.

Department of Physiology, McIntyre Building, Room 1128, McGill University, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada.

1alpha,25-Dihydroxyvitamin D3 [1,25(OH)2D3] regulates calcium homeostasis and controls cellular differentiation and proliferation. The vitamin D receptor (VDR) is a ligand-regulated transcription factor that recognizes cognate vitamin D response elements (VDREs) formed by direct or everted repeats of PuG(G/T)TCA motifs separated by 3 or 6 bp (DR3 or ER6). Here, we have identified direct 1,25(OH)2D3 target genes by combining 35,000+ gene microarrays and genome-wide screens for consensus DR3 and ER6 elements, and DR3 elements containing single nucleotide substitutions. We find that the effect of a nucleotide substitution on VDR binding in vitro does not predict VDRE function in vivo, because substitutions that disrupted binding in vitro were found in several functional elements. Hu133A microarray analyses, performed with RNA from human SCC25 cells treated with 1,25(OH)2D3 and protein synthesis inhibitor cycloheximide, identified more than 900 regulated genes. VDREs lying within -10 to +5 kb of 5'-ends were assigned to 65% of these genes, and VDR binding was confirmed to several elements in vivo. A screen of the mouse genome identified more than 3000 conserved VDREs, and 158 human genes containing conserved elements were 1,25(OH2)D3-regulated on Hu133A microarrays. These experiments also revealed 16 VDREs in 11 of 12 genes induced more than 10-fold in our previous microarray study, five elements in the human gene encoding the epithelial calcium channel TRPV6, as well as novel 1,25(OH2)D3 target genes implicated in regulation of cell cycle progression. The combined approaches used here thus provide numerous insights into the direct target genes underlying the broad physiological actions of 1,25(OH)2D3.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2005-0106DOI Listing
November 2005

Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.

J Immunol 2004 Sep;173(5):2909-12

Department of Physiology, McGill University, Montreal, Quebec, Canada.

The hormonal form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is an immune system modulator and induces expression of the TLR coreceptor CD14. 1,25(OH)(2)D(3) signals through the vitamin D receptor, a ligand-stimulated transcription factor that recognizes specific DNA sequences called vitamin D response elements. In this study, we show that 1,25(OH)(2)D(3) is a direct regulator of antimicrobial innate immune responses. The promoters of the human cathelicidin antimicrobial peptide (camp) and defensin beta2 (defB2) genes contain consensus vitamin D response elements that mediate 1,25(OH)(2)D(3)-dependent gene expression. 1,25(OH)(2)D(3) induces antimicrobial peptide gene expression in isolated human keratinocytes, monocytes and neutrophils, and human cell lines, and 1,25(OH)(2)D(3) along with LPS synergistically induce camp expression in neutrophils. Moreover, 1,25(OH)(2)D(3) induces corresponding increases in antimicrobial proteins and secretion of antimicrobial activity against pathogens including Pseudomonas aeruginosa. 1,25(OH)(2)D(3) thus directly regulates antimicrobial peptide gene expression, revealing the potential of its analogues in treatment of opportunistic infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.173.5.2909DOI Listing
September 2004

Genome-wide identification of high-affinity estrogen response elements in human and mouse.

Mol Endocrinol 2004 Jun 4;18(6):1411-27. Epub 2004 Mar 4.

Département de Biochimie, Université de Montréal, Montréal, Québec, Canada H3C 3J7.

Although estrogen receptors (ERs) recognize 15-bp palindromic estrogen response elements (EREs) with maximal affinity in vitro, few near-consensus sequences have been characterized in estrogen target genes. Here we report the design of a genome-wide screen for high-affinity EREs and the identification of approximately 70000 motifs in the human and mouse genomes. EREs are enriched in regions proximal to the transcriptional start sites, and approximately 1% of elements appear conserved in the flanking regions (-10 kb to +5 kb) of orthologous human and mouse genes. Conserved and nonconserved elements were also found, often in multiple occurrences, in more than 230 estrogen-stimulated human genes previously identified from expression studies. In genes containing known EREs, we also identified additional distal elements, sometimes with higher in vitro binding affinity and/or better conservation between the species considered. Chromatin immunoprecipitation experiments in breast cancer cell lines indicate that most novel elements present in responsive genes bind ERalpha in vivo, including some EREs located up to approximately 10 kb from transcriptional start sites. Our results demonstrate that near-consensus EREs occur frequently in both genomes and that whereas chromatin structure likely modulates access to binding sites, far upstream elements can be evolutionarily conserved and bind ERs in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2003-0441DOI Listing
June 2004
-->