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.

Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying signaling pathway usage.

Upon activation by different transmembrane receptors, the same signaling protein can induce distinct cellular responses. A way to decipher the mechanisms of such pleiotropic signaling activity is to directly manipulate the decision-making activity that supports the selection between distinct cellular responses. We developed an optogenetic probe (optoSRC) to control SRC signaling, an example of a pleiotropic signaling node, and we demonstrated its ability to generate different acto-adhesive structures (lamellipodia or invadosomes) upon distinct spatio-temporal control of SRC kinase activity. The occurrence of each acto-adhesive structure was simply dictated by the dynamics of optoSRC nanoclusters in adhesive sites, which were dependent on the SH3 and Unique domains of the protein. The different decision-making events regulated by optoSRC dynamics induced distinct downstream signaling pathways, which we characterized using time-resolved proteomic and network analyses. Collectively, by manipulating the molecular mobility of SRC kinase activity, these experiments reveal the pleiotropy-encoding mechanism of SRC signaling.

Cross-talk between the calcium channel TRPV4 and reactive oxygen species interlocks adhesive and degradative functions of invadosomes.

Invadosomes support cell invasion by coupling both acto-adhesive and extracellular matrix degradative functions, which are apparently antagonistic. ß1-integrin dynamics regulate this coupling, but the actual sensing mechanism and effectors involved have not yet been elucidated. Using genetic and reverse genetic approaches combined with biochemical and imaging techniques, we now show that the calcium channel TRPV4 colocalizes with ß1-integrins at the invadosome periphery and regulates its activation and the coupling of acto-adhesive and degradative functions. TRPV4-mediated regulation of podosome function depends on its ability to sense reactive oxygen species (ROS) in invadosomes' microenvironment and involves activation of the ROS/calcium-sensitive kinase Ask1 and binding of the motor MYO1C. Furthermore, disease-associated TRPV4 gain-of-function mutations that modulate ECM degradation are also implicated in the ROS response, which provides new perspectives in our understanding of the pathophysiology of TRPV4 channelopathies.

ERRα Expression in Bone Metastases Leads to an Exacerbated Antitumor Immune Response.

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFß3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFß signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.

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

The shape of the cell nucleus can vary considerably during developmental and pathological processes; however, the impact of nuclear morphology on cell behavior is not known. Here, we observed that the nuclear envelope flattens as cells transit from G1 to S phase and inhibition of myosin II prevents nuclear flattening and impedes progression to S phase. Strikingly, we show that applying compressive force on the nucleus in the absence of myosin II-mediated tension is sufficient to restore G1 to S transition. Using a combination of tools to manipulate nuclear morphology, we observed that nuclear flattening activates a subset of transcription factors, including TEAD and AP1, leading to transcriptional induction of target genes that promote G1 to S transition. In addition, we found that nuclear flattening mediates TEAD and AP1 activation in response to ROCK-generated contractility or cell spreading. Our results reveal that the nuclear envelope can operate as a mechanical sensor whose deformation controls cell growth in response to tension.

The CCM1-CCM2 complex controls complementary functions of ROCK1 and ROCK2 that are required for endothelial integrity.

Endothelial integrity relies on a mechanical crosstalk between intercellular and cell-matrix interactions. This crosstalk is compromised in hemorrhagic vascular lesions of patients carrying loss-of-function mutations in cerebral cavernous malformation (CCM) genes. RhoA/ROCK-dependent cytoskeletal remodeling is central to the disease, as it causes unbalanced cell adhesion towards increased cell-extracellular matrix adhesions and destabilized cell-cell junctions. This study reveals that CCM proteins directly orchestrate ROCK1 and ROCK2 complementary roles on the mechanics of the endothelium. CCM proteins act as a scaffold, promoting ROCK2 interactions with VE-cadherin and limiting ROCK1 kinase activity. Loss of CCM1 (also known as KRIT1) produces excessive ROCK1-dependent actin stress fibers and destabilizes intercellular junctions. Silencing of ROCK1 but not ROCK2 restores the adhesive and mechanical homeostasis of CCM1 and CCM2-depleted endothelial monolayers, and rescues the cardiovascular defects of ccm1 mutant zebrafish embryos. Conversely, knocking down Rock2 but not Rock1 in wild-type zebrafish embryos generates defects reminiscent of the ccm1 mutant phenotypes. Our study uncovers the role of the CCM1-CCM2 complex in controlling ROCK1 and ROCK2 to preserve endothelial integrity and drive heart morphogenesis. Moreover, it solely identifies the ROCK1 isoform as a potential therapeutic target for the CCM disease.

Roles of paxillin family members in adhesion and ECM degradation coupling at invadosomes.

Invadosomes are acto-adhesive structures able to both bind the extracellular matrix (ECM) and digest it. Paxillin family members-paxillin, Hic-5, and leupaxin-are implicated in mechanosensing and turnover of adhesion sites, but the contribution of each paxillin family protein to invadosome activities is unclear. We use genetic approaches to show that paxillin and Hic-5 have both redundant and distinctive functions in invadosome formation. The essential function of paxillin-like activity is based on the coordinated activity of LD motifs and LIM domains, which support invadosome assembly and morphology, respectively. However, paxillin preferentially regulates invadosome assembly, whereas Hic-5 regulates the coupling between ECM degradation and acto-adhesive functions. Mass spectrometry analysis revealed new partners that are important for paxillin and Hic-5 specificities: paxillin regulates the acto-adhesive machinery through janus kinase 1 (JAK1), whereas Hic-5 controls ECM degradation via IQGAP1. Integrating the redundancy and specificities of paxillin and Hic-5 in a functional complex provides insights into the coupling between the acto-adhesive and ECM-degradative machineries in invadosomes.

A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery.

Circadian clocks in mammals are built on a negative feedback loop in which the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1) drives the expression of its own inhibitors, the PERIOD and CRYPTOCHROME proteins. Reactivation of CLOCK-BMAL1 occurs at a specific time several hours after PERIOD and CRYPTOCHROME protein turnover, but the mechanism underlying this process is unknown. We found that mouse BMAL1 complexes include TRAP150 (thyroid hormone receptor-associated protein-150; also known as THRAP3). TRAP150 is a selective coactivator for CLOCK-BMAL1, which oscillates under CLOCK-BMAL1 transcriptional control. TRAP150 promotes CLOCK-BMAL1 binding to target genes and links CLOCK-BMAL1 to the transcriptional machinery at target-gene promoters. Depletion of TRAP150 caused low-amplitude, long-period rhythms, identifying it as a positive clock element. The activity of TRAP150 defines a positive feedback loop within the clock and provides a potential mechanism for timing the reactivation of circadian transcription.

Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast.

RNA interference (RNAi) silences gene expression by acting both at the transcriptional and post-transcriptional levels in a broad range of eukaryotes. In the fission yeast Schizosaccharomyces pombe the RNA-Induced Transcriptional Silencing (RITS) RNAi complex mediates heterochromatin formation at non-coding and repetitive DNA. However, the targeting and role of RITS at other genomic regions, including protein-coding genes, remain unknown. Here we show that RITS localizes to specific meiotic genes and mRNAs. Remarkably, RITS is guided to these meiotic targets by the RNA-binding protein Mmi1 and its associated RNA surveillance machinery that together degrade selective meiotic mRNAs during vegetative growth. Upon sexual differentiation, RITS localization to the meiotic genes and mRNAs is lost. Large-scale identification of Mmi1 RNA targets reveals that RITS subunit Chp1 associates with the vast majority of them. In addition, loss of RNAi affects the effective repression of sexual differentiation mediated by the Mmi1 RNA surveillance machinery. These findings uncover a new mechanism for recruiting RNAi to specific meiotic genes and suggest that RNAi participates in the control of sexual differentiation in fission yeast.

Identification of RACK1 and protein kinase Calpha as integral components of the mammalian circadian clock.

At the core of the mammalian circadian clock is a negative feedback loop in which the dimeric transcription factor CLOCK-BMAL1 drives processes that in turn suppress its transcriptional activity. To gain insight into the mechanisms of circadian feedback, we analyzed mouse protein complexes containing BMAL1. Receptor for activated C kinase-1 (RACK1) and protein kinase C-alpha (PKCalpha) were recruited in a circadian manner into a nuclear BMAL1 complex during the negative feedback phase of the cycle. Overexpression of RACK1 and PKCalpha suppressed CLOCK-BMAL1 transcriptional activity, and RACK1 stimulated phosphorylation of BMAL1 by PKCalpha in vitro. Depletion of endogenous RACK1 or PKCalpha from fibroblasts shortened the circadian period, demonstrating that both molecules function in the clock oscillatory mechanism. Thus, the classical PKC signaling pathway is not limited to relaying external stimuli but is rhythmically activated by internal processes, forming an integral part of the circadian feedback loop.

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

A 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.

Regulation of protein turnover by acetyltransferases and deacetylases.

Lysine 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.

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

A 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.

HDAC6-p97/VCP controlled polyubiquitin chain turnover.

HDAC6 is a unique cytoplasmic deacetylase capable of interacting with ubiquitin. Using a combination of biophysical, biochemical and biological approaches, we have characterized the ubiquitin-binding domain of HDAC6, named ZnF-UBP, and investigated its biological functions. These studies show that the three Zn ion-containing HDAC6 ZnF-UBP domain presents the highest known affinity for ubiquitin monomers and mediates the ability of HDAC6 to negatively control the cellular polyubiquitin chain turnover. We further show that HDAC6-interacting chaperone, p97/VCP, dissociates the HDAC6-ubiquitin complexes and counteracts the ability of HDAC6 to promote the accumulation of polyubiquitinated proteins. We propose that a finely tuned balance of HDAC6 and p97/VCP concentrations determines the fate of ubiquitinated misfolded proteins: p97/VCP would promote protein degradation and ubiquitin turnover, whereas HDAC6 would favour the accumulation of ubiquitinated protein aggregates and inclusion body formation.

Regulatory cross-talk between lysine acetylation and ubiquitination: role in the control of protein stability.

It is now becoming apparent that cross-talk between two protein lysine modifications, acetylation and ubiquitination, is a critical regulatory mechanism controlling vital cellular functions. The most apparent effect is the inhibition of proteasome-mediated protein degradation by lysine acetylation. Analysis of the underlying mechanisms, however, shows that, besides a direct competition between the two lysine modifications, more complex and indirect processes also connect these two signalling pathways. These findings point to protein lysine acetylation as a potential regulator of various cellular functions involving protein ubiquitination.