Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia.

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

FONCTIONS UBIQUITINE-DEPENDANTES DE LA DEACETYLASE HDAC6

At the start of my Ph.D., the lab had discovered and characterized HDAC6, an unusual deacetylase that possesses two deacetylase domains and directly binds to ubiquitin. Moreover, the lab had found that HDAC6 interacts with UFD3/PLAP, a regulator of ubiquitin turnover, and VCP, a mouse homologue of the chaperone Cdc48. However, nothing was known about HDAC6 biological function, especially its role in the ubiquitination pathway. We first observed that HDAC6 over-expression slows down the degradation of polyubiquitinated protein, via ZnF-UBP, its ubiquitin binding domain. Through a series of experiments, we have shown that, actually, HDAC6-VCP complex directly regulates the level of poly-ubiquitinated proteins. We then discovered that HDAC6 controls the cellular response triggered by the accumulation of poly-ubiquitinated proteins, and have unraveled mechanisms involved in this control. The accumulation of poly-ubiquitinated proteins could be toxic for cells if no cellular response is engaged. Moreover, it has been well-known for about ten years that such accumulation activates the transcription factor Heat Shock Factor 1 (HSF1) to promote cellular survival. We found that, in the absence of stress, HDAC6 and HSF1 are in a complex with VCP and HSP90. However, when the pool of poly-ubiquitinated proteins increases, such as during heat shock, HDAC6 is released from the complex in a ubiquitin and ZnF-UBP dependent manner. Such a release then enables VCP to activate HSF1. In conclusion, we propose that HDAC6-p97/VCP complexe appears as a master regulator during both poly-ubiquitinated protein management and cellular stress response engagement.Avant le début de ma thèse, le laboratoire avait découvert et caractérisé HDAC6, une Histone Déacétylase atypique qui possède deux domaines déacétylases et peut interagir directement avec l'ubiquitine, grâce à son domaine ZnF-UBP. De plus, le laboratoire avait montré que HDAC6 interagit avec UFD3/PLAP, un régulateur du recyclage de l'ubiquitine, et p97/VCP, un orthologue murin de la chaperonne de levure Cdc48p. Cependant, aucune fonction biologique dans la voie d'ubiquitination des protéines n'était connue pour HDAC6. Nous avons tout d'abord observé que la surexpression de HDAC6 ralenti la dégradation des protéines poly-ubiquitinées, via son ZnF-UBP, son domaine de liaison à l'ubiquitine. Grâce à une série d'expériences, nous avons pu montrer que les complexes HDAC6-p97/VCP régulent directement la stabilité des protéines poly-ubiquitinées. L'accumulation intracellulaire de protéines poly-ubiquitinées peut être toxique pour les cellules si aucune réponse cellulaire n'est engagée. En réalité, une telle accumulation active le facteur de transcription Heat Shock Factor 1 (HSF1) afin de promouvoir la survie de la cellule. Grâce à ces considérations, nous avons découvert que HDAC6 contrôle la réponse cellulaire à l'accumulation de protéines poly-ubiquitinées et avons disséqué les mécanismes impliqués dans ce contrôle. Nous avons trouvé qu'en l'absence de stress, HDAC6 et HSF1 sont en complexes avec p97/VCP et HSP90. Cependant, lorsque la concentration intracellulaire en protéines poly-ubiquitinées augmente, comme lors d'une inhibition du protéasome, HDAC6 est re-larguée du complexe de manière ubiquitine et ZnF-UBP dépendante. Un tel re-largage permet ensuite à p97/VCP d'activer HSF1 et d'engager la cellule dans la réponse au stress

Fonctions ubiquitine-dépendantes de la déacétylase HDAC6

Avant le début de ma thèse, le laboratoire avait découvert et caractérisé HDAC6, une histone déacétylase atypique qui possède deux domaines déacétylase et peut interagir directement avec l'ubiquitine, grâce à son domaine ZnF-UBP. De plus, le laboratoire avait montré que HDAC6 interagit avec UFD3/PLAP, un régulateur du turnover de l'ubiquitine, et p97/VCP, un homologue murin de la chaperonne de levure Cdc48p. Cependant, aucune fonction biologique n'était connue pour HDAC6, notamment dans la voie d'ubiquitination des protéines. Nous avons tout d'abord observé que la surexpression de HDAC6 ralenti la dégradation des protéines polyubiquitinées, via son ZnF-UBP, son domaine de liaison à l'ubiquitine. Grâce a une série d'expériences, nous avons pu montrer que les complexes HDAC6-p97/VCP régulent directement la stabilité des protéines poly-ubiquitinées. L'accumulation intracellulaire de protéines poly-ubiquitinées peut être toxique pour les cellules si aucune réponse cellulaire n'est engagée. En réalité, une telle accumulation active le facteur de transcription Heat Shock Factor 1 (HSFI) afin de promouvoir la survie de la cellule. Grâce à ces considérations, nous avons découvert que HDAC6 et p97/VCP contrôlent la réponse cellulaire à l'accumulation de protéines poly-ubiquitinées.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Regulation of protein turnover by acetyltransferases and deacetylases.

International audienceLysine acetylation was first discovered as a post-translational modification of histones and has long been considered as a direct regulator of chromatin structure and function. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are the enzymes involved in this modification and they were thought to act as critical gene silencers or activators. Further investigations indicated that lysine acetylation can also occur in non-histone proteins and pointed to HATs and HDACs as multifunctional factors, acting not only on transcription but also on a variety of other cellular processes. One of these processes is the regulation of protein stability. Indeed, at least four independent HATs, namely CBP, p300, PCAF and TAF1, and one HDAC, HDAC6, possess intrinsic ubiquitin-linked functions in addition to their regular HAT/HDAC activities. Furthermore HATs and HDACs can be found in multi-subunit complexes with enzymes of the ubiquitination machinery. Moreover, lysine acetylation itself was found to directly or indirectly affect protein stability. These observations reveal therefore a tight link between protein lysine acetylation and ubiquitination and designate the acetylation machinery as a determinant element in the control of cellular proteolytic activities

Heat-shock factor 1 controls genome-wide acetylation in heat-shocked cells.

International audienceA major regulatory function has been evidenced here for HSF1, the key transcription factor of the heat-shock response, in a large-scale remodeling of the cell epigenome. Indeed, upon heat shock, HSF1, in addition to its well-known transactivating activities, mediates a genome-wide and massive histone deacetylation. Investigating the underlying mechanisms, we show that HSF1 specifically associates with and uses HDAC1 and HDAC2 to trigger this heat-shock-dependent histone deacetylation. This work therefore identifies HSF1 as a master regulator of global chromatin acetylation and reveals a cross-talk between HSF1 and histone deacetylases in the general control of genome organization in response to heat shock

Nuclear envelope deformation controls cell cycle progression in response to mechanical force

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HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates.

International audienceA cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation