C/EBPß-LIP mediated activation of the malate-aspartate shuttle sensitizes cells to glycolysis inhibition.

OBJECTIVE: Cancer cells use glycolysis for generation of metabolic intermediates and ATP needed for cell growth and proliferation. The transcription factor C/EBPß-LIP stimulates glycolysis and mitochondrial respiration in cancer cells. We initially observed that high expression of C/EBPß-LIP makes cells vulnerable to treatment with the glycolysis inhibitor 2-deoxyglucose. The aim of the study was to uncover the involved mechanisms of C/EBPß-LIP induced sensitivity to glycolysis inhibition. METHODS: We used genetically engineered cell lines to examine the effect of C/EBPß-LIP and -LAP protein isoforms on glycolysis and NADH/NAD+ metabolism in mouse embryonic fibroblasts (MEFs), and triple negative breast cancer (TNBC) cells that endogenously express high levels of C/EBPß-LIP. Analyses included assays of cell proliferation, cell survival and metabolic flux (OCR and ECAR by Seahorse XF96). Small molecule inhibitors were used to identify underlying metabolic pathways that mediate sensitivity to glycolysis inhibition induced by C/EBPß-LIP. RESULTS: The transcription factor C/EBPß-LIP stimulates both glycolysis and the malate-aspartate shuttle (MAS) and increases the sensitivity to glycolysis inhibition (2-deoxyglucose) in fibroblasts and breast cancer cells. Inhibition of glycolysis with ongoing C/EBPß-LIP-induced MAS activity results in NADH depletion and apoptosis that can be rescued by inhibiting either the MAS or other NAD+-regenerating processes. CONCLUSION: This study indicates that a low NADH/NAD+ ratio is an essential mediator of 2-deoxyglucose toxicity in cells with high cytoplasmic NAD+-regeneration capacity and that simultaneous inhibition of glycolysis and lowering of the NADH/NAD+ ratio may be considered to treat cancer.

Enhanced C/EBPß function promotes hypertrophic versus hyperplastic fat tissue growth and prevents steatosis in response to high-fat diet feeding.

Chronic obesity is correlated with severe metabolic and cardiovascular diseases as well as with an increased risk for developing cancers. Obesity is usually characterized by fat accumulation in enlarged - hypertrophic - adipocytes that are a source of inflammatory mediators, which promote the development and progression of metabolic disorders. Yet, in certain healthy obese individuals, fat is stored in metabolically more favorable hyperplastic fat tissue that contains an increased number of smaller adipocytes that are less inflamed. In a previous study, we demonstrated that loss of the inhibitory protein-isoform C/EBPß-LIP and the resulting augmented function of the transactivating isoform C/EBPß-LAP promotes fat metabolism under normal feeding conditions and expands health- and lifespan in mice. Here, we show that in mice on a high-fat diet, LIP-deficiency results in adipocyte hyperplasia associated with reduced inflammation and metabolic improvements. Furthermore, fat storage in subcutaneous depots is significantly enhanced specifically in LIP-deficient male mice. Our data identify C/EBPß as a regulator of adipocyte fate in response to increased fat intake, which has major implications for metabolic health and aging.

Oncogenic and Tumor-Suppressive Functions of the RNA Demethylase FTO.

The epitranscriptome represents the more than 140 types of chemically varying and reversable RNA modifications affecting RNA fate. Among these, the most relevant for this review are the mRNA modifications N6-methyladenosine and N6,2'-O-dimethyladenosine. Epitranscriptomic mRNA biology involves RNA methyltransferases (so-called "writers"), RNA demethylases ("erasers"), and RNA-binding proteins ("readers") that interact with methylation sites to determine the functional outcome of the modification. In this review, we discuss the role of a specific RNA demethylase encoded by the fat mass and obesity-associated gene (FTO) in cancer. FTO initially became known as the strongest genetic link for human obesity. Only in 2010, 16 years after its discovery, was its enzymatic function as a demethylase clarified, and only recently has its role in the development of cancer been revealed. FTO functions are challenging to study and interpret because of its genome-wide effects on transcript turnover and translation. We review the discovery of FTO and its enzymatic function, the tumor-promoting and suppressive roles of FTO in selected cancer types, and its potential as a therapeutic target.

C/EBPß isoform-specific regulation of migration and invasion in triple-negative breast cancer cells.

The transcription factor C/EBPß is a master regulator of mammary gland development and tissue remodelling during lactation. The CEBPB-mRNA is translated into three distinct protein isoforms named C/EBPß-LAP1, -LAP2 and -LIP that are functionally different. The smaller isoform LIP lacks the N-terminal transactivation domains and is considered to act as an inhibitor of the transactivating LAP1/2 isoforms by competitive binding for the same DNA recognition sequences. Aberrantly high expression of LIP is associated with mammary epithelial proliferation and is found in grade III, estrogen receptor (ER) and progesterone (PR) receptor-negative human breast cancer. Here, we show that reverting the high LIP/LAP ratios in triple-negative breast cancer (TNBC) cell lines into low LIP/LAP ratios by overexpression of LAP reduces migration and matrix invasion of these TNBC cells. In addition, in untransformed MCF10A human mammary epithelial cells overexpression of LIP stimulates migration. Knockout of CEBPB in TNBC cells where LIP expression prevails, resulted in strongly reduced migration that was accompanied by a downregulation of genes involved in cell migration, extracellular matrix production and cytoskeletal remodelling, many of which are epithelial to mesenchymal transition (EMT) marker genes. Together, this study suggests that the LIP/LAP ratio is involved in regulating breast cancer cell migration and invasion. This study together with studies from others shows that understanding the functions the C/EBPß-isoforms in breast cancer development may reveal new avenues of treatment.

Obesity and its impact on COVID-19.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic has proven a challenge to healthcare systems since its first appearance in late 2019. The global spread and devastating effects of coronavirus disease 2019 (COVID-19) on patients have resulted in countless studies on risk factors and disease progression. Overweight and obesity emerged as one of the major risk factors for developing severe COVID-19. Here we review the biology of coronavirus infections in relation to obesity. In particular, we review literature about the impact of adiposity-related systemic inflammation on the COVID-19 disease severity, involving cytokine, chemokine, leptin, and growth hormone signaling, and we discuss the involvement of hyperactivation of the renin-angiotensin-aldosterone system (RAAS). Due to the sheer number of publications on COVID-19, we cannot be completed, and therefore, we apologize for all the publications that we do not cite.

Leptin levels in SARS-CoV-2 infection related respiratory failure: A cross-sectional study and a pathophysiological framework on the role of fat tissue.

Obesity is a risk factor for SARS-CoV-2 infected patients to develop respiratory failure. Leptin produced in visceral fat might play a role in the deterioration to mechanical ventilation. A cross sectional study was performed. The mean BMI was 31 kg/m2 (range 24.8-48.4) for the 31 SARS-CoV-2 ventilated patients and 26 kg/m2 (range 22.4-33.5) for 8 critically ill non-infected control patients. SARS-CoV-2 infected patients with a similar BMI as control patients appear to have significantly higher levels of serum leptin. The mean leptin level was 21.2 (6.0-85.2) vs 5.6 (2.4-8.2) ug/L for SARS-CoV-2 and controls respectively (p = 0.0007). With these findings we describe a clinical and biological framework that may explain these clinical observations. The ACE2 utilization by the virus leads to local pulmonary inflammation due to ACE2-ATII disbalance. This might be enhanced by an increase in leptin production induced by SARS-CoV-2 infection of visceral fat. Leptin receptors in the lungs are now more activated to enhance local pulmonary inflammation. This adds to the pre-existent chronic inflammation in obese patients. Visceral fat, lung tissue and leptin production play an interconnecting role. This insight can lead the way to further research and treatment.

Emerging Role of C/EBPß and Epigenetic DNA Methylation in Ageing.

Changes in epigenetic DNA methylation are the most promising predictor of biological age and lifespan in humans, but whether methylation changes affect ageing is unresolved. Here, we discuss converging data, which indicate that one mode by which aberrant DNA methylation can affect ageing is via CCAAT/enhancer binding protein beta (C/EBPß). This basic leucine-zipper (bZIP) transcription factor is controlled by the lifespan regulator mechanistic/mammalian target of rapamycin complex 1 (mTORC1) and plays an important role in energy homeostasis and adipose tissue differentiation. Emerging evidence indicates that access of C/EBPß proteins to cognate binding sites is regulated by DNA demethylation via ten-eleven translocation (TET) methylcytosine dioxygenases and their adaptor proteins growth arrest and DNA damage-inducible protein 45 alpha (GADD45α) and inhibitor of growth 1 (ING1). We discuss the emerging causal nexus between C/EBPß, energy metabolism, and DNA demethylation in organismal ageing.

Latest advances in aging research and drug discovery.

An increasing aging population poses a significant challenge to societies worldwide. A better understanding of the molecular, cellular, organ, tissue, physiological, psychological, and even sociological changes that occur with aging is needed in order to treat age-associated diseases. The field of aging research is rapidly expanding with multiple advances transpiring in many previously disconnected areas. Several major pharmaceutical, biotechnology, and consumer companies made aging research a priority and are building internal expertise, integrating aging research into traditional business models and exploring new go-to-market strategies. Many of these efforts are spearheaded by the latest advances in artificial intelligence, namely deep learning, including generative and reinforcement learning. To facilitate these trends, the Center for Healthy Aging at the University of Copenhagen and Insilico Medicine are building a community of Key Opinion Leaders (KOLs) in these areas and launched the annual conference series titled "Aging Research and Drug Discovery (ARDD)" held in the capital of the pharmaceutical industry, Basel, Switzerland (www.agingpharma.org). This ARDD collection contains summaries from the 6th annual meeting that explored aging mechanisms and new interventions in age-associated diseases. The 7th annual ARDD exhibition will transpire 2nd-4th of September, 2020, in Basel.

C/EBPß-LIP induces cancer-type metabolic reprogramming by regulating the let-7/LIN28B circuit in mice.

The transcription factors LAP1, LAP2 and LIP are derived from the Cebpb-mRNA through the use of alternative start codons. High LIP expression has been associated with human cancer and increased cancer incidence in mice. However, how LIP contributes to cellular transformation is poorly understood. Here we present that LIP induces aerobic glycolysis and mitochondrial respiration reminiscent of cancer metabolism. We show that LIP-induced metabolic programming is dependent on the RNA-binding protein LIN28B, a translational regulator of glycolytic and mitochondrial enzymes with known oncogenic function. LIP activates LIN28B through repression of the let-7 microRNA family that targets the Lin28b-mRNA. Transgenic mice overexpressing LIP have reduced levels of let-7 and increased LIN28B expression, which is associated with metabolic reprogramming as shown in primary bone marrow cells, and with hyperplasia in the skin. This study establishes LIP as an inducer of cancer-type metabolic reprogramming and as a regulator of the let-7/LIN28B regulatory circuit.

Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphoma.

The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient-sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC-driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR-15a. TSC1 knockdown results in elevated mTORC1-dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC-driven cancers.

Reduced expression of C/EBPß-LIP extends health and lifespan in mice.

Ageing is associated with physical decline and the development of age-related diseases such as metabolic disorders and cancer. Few conditions are known that attenuate the adverse effects of ageing, including calorie restriction (CR) and reduced signalling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Synthesis of the metabolic transcription factor C/EBPß-LIP is stimulated by mTORC1, which critically depends on a short upstream open reading frame (uORF) in the Cebpb-mRNA. Here, we describe that reduced C/EBPß-LIP expression due to genetic ablation of the uORF delays the development of age-associated phenotypes in mice. Moreover, female C/EBPßΔuORF mice display an extended lifespan. Since LIP levels increase upon aging in wild type mice, our data reveal an important role for C/EBPß in the aging process and suggest that restriction of LIP expression sustains health and fitness. Thus, therapeutic strategies targeting C/EBPß-LIP may offer new possibilities to treat age-related diseases and to prolong healthspan.

A p300 and SIRT1 Regulated Acetylation Switch of C/EBPα Controls Mitochondrial Function.

Cellular metabolism is a tightly controlled process in which the cell adapts fluxes through metabolic pathways in response to changes in nutrient supply. Among the transcription factors that regulate gene expression and thereby cause changes in cellular metabolism is the basic leucine-zipper (bZIP) transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα). Protein lysine acetylation is a key post-translational modification (PTM) that integrates cellular metabolic cues with other physiological processes. Here, we show that C/EBPα is acetylated by the lysine acetyl transferase (KAT) p300 and deacetylated by the lysine deacetylase (KDAC) sirtuin1 (SIRT1). SIRT1 is activated in times of energy demand by high levels of nicotinamide adenine dinucleotide (NAD+) and controls mitochondrial biogenesis and function. A hypoacetylated mutant of C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. Our study identifies C/EBPα as a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply.

Roquin Suppresses the PI3K-mTOR Signaling Pathway to Inhibit T Helper Cell Differentiation and Conversion of Treg to Tfr Cells.

Roquin proteins preclude spontaneous T cell activation and aberrant differentiation of T follicular helper (Tfh) or T helper 17 (Th17) cells. Here we showed that deletion of Roquin-encoding alleles specifically in regulatory T (Treg) cells also caused the activation of conventional T cells. Roquin-deficient Treg cells downregulated CD25, acquired a follicular Treg (Tfr) cell phenotype, and suppressed germinal center reactions but could not protect from colitis. Roquin inhibited the PI3K-mTOR signaling pathway by upregulation of Pten through interfering with miR-17∼92 binding to an overlapping cis-element in the Pten 3' UTR, and downregulated the Foxo1-specific E3 ubiquitin ligase Itch. Loss of Roquin enhanced Akt-mTOR signaling and protein synthesis, whereas inhibition of PI3K or mTOR in Roquin-deficient T cells corrected enhanced Tfh and Th17 or reduced iTreg cell differentiation. Thereby, Roquin-mediated control of PI3K-mTOR signaling prevents autoimmunity by restraining activation and differentiation of conventional T cells and specialization of Treg cells.

Identification of an RNA Polymerase III Regulator Linked to Disease-Associated Protein Aggregation.

Protein aggregation is associated with age-related neurodegenerative disorders, such as Alzheimer's and polyglutamine diseases. As a causal relationship between protein aggregation and neurodegeneration remains elusive, understanding the cellular mechanisms regulating protein aggregation will help develop future treatments. To identify such mechanisms, we conducted a forward genetic screen in a C. elegans model of polyglutamine aggregation and identified the protein MOAG-2/LIR-3 as a driver of protein aggregation. In the absence of polyglutamine, MOAG-2/LIR-3 regulates the RNA polymerase III-associated transcription of small non-coding RNAs. This regulation is lost in the presence of polyglutamine, which mislocalizes MOAG-2/LIR-3 from the nucleus to the cytosol. We then show biochemically that MOAG-2/LIR-3 can also catalyze the aggregation of polyglutamine-expanded huntingtin. These results suggest that polyglutamine can induce an aggregation-promoting activity of MOAG-2/LIR-3 in the cytosol. The concept that certain aggregation-prone proteins can convert other endogenous proteins into drivers of aggregation and toxicity adds to the understanding of how cellular homeostasis can be deteriorated in protein misfolding diseases.

A screening strategy for the discovery of drugs that reduce C/EBPß-LIP translation with potential calorie restriction mimetic properties.

An important part of the beneficial effects of calorie restriction (CR) on healthspan and lifespan is mediated through regulation of protein synthesis that is under control of the mechanistic target of rapamycin complex 1 (mTORC1). As one of its activities, mTORC1 stimulates translation into the metabolic transcription factor CCAAT/Enhancer Binding Protein ß (C/EBPß) isoform Liver-specific Inhibitory Protein (LIP). Regulation of LIP expression strictly depends on a translation re-initiation event that requires a conserved cis-regulatory upstream open reading frame (uORF) in the C/EBPß-mRNA. We showed before that suppression of LIP in mice, reflecting reduced mTORC1-signaling at the C/EBPß level, results in CR-type of metabolic improvements. Hence, we aim to find possibilities to pharmacologically down-regulate LIP in order to induce CR-mimetic effects. We engineered a luciferase-based cellular reporter system that acts as a surrogate for C/EBPß-mRNA translation, emulating uORF-dependent C/EBPß-LIP expression under different translational conditions. By using the reporter system in a high-throughput screening (HTS) strategy we identified drugs that reduce LIP. The drug Adefovir Dipivoxil passed all counter assays and increases fatty acid ß-oxidation in a hepatoma cell line in a LIP-dependent manner. Therefore, these drugs that suppress translation into LIP potentially exhibit CR-mimetic properties.

Shwachman-Bodian-Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPß mRNAs.

Mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene cause Shwachman-Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -ß mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPß-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5' untranslated regions (5' UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -ß deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype.

Deficiency in mTORC1-controlled C/EBPß-mRNA translation improves metabolic health in mice.

The mammalian target of rapamycin complex 1 (mTORC1) is a central regulator of physiological adaptations in response to changes in nutrient supply. Major downstream targets of mTORC1 signalling are the mRNA translation regulators p70 ribosomal protein S6 kinase 1 (S6K1p70) and the 4E-binding proteins (4E-BPs). However, little is known about vertebrate mRNAs that are specifically controlled by mTORC1 signalling and are engaged in regulating mTORC1-associated physiology. Here, we show that translation of the CCAAT/enhancer binding protein beta (C/EBPß) mRNA into the C/EBPß-LIP isoform is suppressed in response to mTORC1 inhibition either through pharmacological treatment or through calorie restriction. Our data indicate that the function of 4E-BPs is required for suppression of LIP. Intriguingly, mice lacking the cis-regulatory upstream open reading frame (uORF) in the C/EBPß-mRNA, which is required for mTORC1-stimulated translation into C/EBPß-LIP, display an improved metabolic phenotype with features also found under calorie restriction. Thus, our data suggest that translational adjustment of C/EBPß-isoform expression is one of the key processes that direct metabolic adaptation in response to changes in mTORC1 activity.

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation.

Damaged replication forks activate poly(ADP-ribose) polymerase 1 (PARP1), which catalyses poly(ADP-ribose) (PAR) formation; however, how PARP1 or poly(ADP-ribosyl)ation is involved in the S-phase checkpoint is unknown. Here we show that PAR, supplied by PARP1, interacts with Chk1 via a novel PAR-binding regulatory (PbR) motif in Chk1, independent of ATR and its activity. iPOND studies reveal that Chk1 associates readily with the unperturbed replication fork and that PAR is required for efficient retention of Chk1 and phosphorylated Chk1 at the fork. A PbR mutation, which disrupts PAR binding, but not the interaction with its partners Claspin or BRCA1, impairs Chk1 and the S-phase checkpoint activation, and mirrors Chk1 knockdown-induced hypersensitivity to fork poisoning. We find that long chains, but not short chains, of PAR stimulate Chk1 kinase activity. Collectively, we disclose a previously unrecognized mechanism of the S-phase checkpoint by PAR metabolism that modulates Chk1 activity at the replication fork.

Transcriptional repression of ATF4 gene by CCAAT/enhancer-binding protein ß (C/EBPß) differentially regulates integrated stress response.

Different environmental stresses induce the phosphorylation of eIF2 (eIF2∼P), repressing global protein synthesis coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in metabolism and nutrient uptake, antioxidation, and regulation of apoptosis. Because ATF4 is a common downstream target that integrates signaling from different eIF2 kinases and their respective stress signals, the eIF2∼P/ATF4 pathway is collectively referred to as the integrated stress response. Although eIF2∼P elicits translational control in response to many different stresses, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. The rationale for this discordant induction of ATF4 expression and eIF2∼P in response to UV irradiation is that transcription of ATF4 is repressed, and therefore ATF4 mRNA is not available for preferential translation. In this study, we show that C/EBPß is a transcriptional repressor of ATF4 during UV stress. C/EBPß binds to critical elements in the ATF4 promoter, resulting in its transcriptional repression. Expression of C/EBPß increases in response to UV stress, and the liver-enriched inhibitory protein (LIP) isoform of C/EBPß, but not the liver-enriched activating protein (LAP) version, represses ATF4 transcription. Loss of the liver-enriched inhibitory protein isoform results in increased ATF4 mRNA levels in response to UV irradiation and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2∼P and translational control combined with transcription factors regulated by alternative signaling pathways can direct programs of gene expression that are specifically tailored to each environmental stress.

Eukaryotic initiation factor 2alpha phosphorylation is required for B-cell maturation and function in mice.

BACKGROUND: The control of translation initiation is a crucial component in the regulation of gene expression. The eukaryotic initiation factor 2α (eIF2α) mediates binding of the initiator transfer-messenger-RNA to the AUG initiation codon, and thus controls a rate-limiting step in translation initiation. Phosphorylation of eIF2α at serine 51 is linked to cellular stress response and attenuates translation initiation. The biochemistry of translation inhibition mediated by eIF2α phosphorylation is well characterized, yet the physiological importance in hematopoiesis remains only partially known. DESIGN AND METHODS: Using hematopoietic stem cells carrying a non-phosphorylatable mutant form of eIF2α (eIF2αAA), we examined the efficiency of reconstitution in wild-type and B-cell-deficient microMT C57BL/6 recipients in two independent models. RESULTS: We provide evidence that phosphorylation-deficient eIF2α mutant hematopoietic stem cells may repopulate lethally irradiated mice but have a defect in the development and maintenance of newly formed B cells in the bone marrow and of naïve follicular B cells in the periphery. The mature B-cell compartment is markedly reduced in bone marrow, spleen and peripheral blood, and B-cell receptor-mediated proliferation in vitro and serum immunoglobulin secretion in vivo are impaired. CONCLUSIONS: The data suggest that regulation of translation through eIF2α phosphorylation is dispensable in hematopoietic reconstitution but essential during late B-cell development.