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.

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.

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.

Argonaute 2 RNA Immunoprecipitation Reveals Distinct miRNA Targetomes of Primary Burkitt Lymphoma Tumors and Normal B Cells.

miRNAs are small noncoding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been linked to Burkitt lymphoma (BL) pathogenesis. To date, the number of known pathogenesis-related miRNA-target gene interactions is limited. Here, we determined for the first time the miRNA targetomes of primary BL tumors and normal B cells. AGO2-RNA immunoprecipitation of two frozen diagnostic BL tissue samples and three CD19+ B-cell samples isolated from routinely removed tonsils showed distinct miRNA targetomes of BL and normal B cells. In contrast to normal B cells, miRNA target genes in BL were enriched for targets of the oncogenic miR-17 to 92 cluster, and were involved mainly in cell cycle and cell death. Immunohistochemistry on BL and tonsil tissues confirmed altered protein levels for two of six selected miRNA targets, in line with the differential AGO2-IP enrichment between BL and normal B cells. A comparison of AGO2-IP-enriched genes in primary BL cases with BL cell lines indicated that despite a considerable overlap, the miRNA targetomes of BL cell lines show substantial differences with the targetomes of primary BL tumors. In summary, we identified distinct miRNA targetomes of BL and normal B cells, and showed both the necessity and feasibility of studying miRNA-target gene interactions in primary tumors.

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.

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.

Long Noncoding RNA Expression Profiling in Normal B-Cell Subsets and Hodgkin Lymphoma Reveals Hodgkin and Reed-Sternberg Cell-Specific Long Noncoding RNAs.

Hodgkin lymphoma (HL) is a malignancy of germinal center (GC) B-cell origin. To explore the role of long noncoding RNAs (lncRNAs) in HL, we studied lncRNA expression patterns in normal B-cell subsets, HL cell lines, and tissues. Naive and memory B cells showed a highly similar lncRNA expression pattern, distinct from GC-B cells. Significant differential expression between HL and normal GC-B cells was observed for 475 lncRNA loci. For two validated lncRNAs, an enhanced expression was observed in HL, diffuse large B-cell lymphoma, and lymphoblastoid cell lines. For a third lncRNA, increased expression levels were observed in HL and part of Burkitt lymphoma cell lines. RNA fluorescence in situ hybridization on primary HL tissues revealed a tumor cell-specific expression pattern for all three lncRNAs. A potential cis-regulatory role was observed for 107 differentially expressed lncRNA-mRNA pairs localizing within a 60-kb region. Consistent with a cis-acting role, we showed a preferential nuclear localization for two selected candidates. Thus, we showed dynamic lncRNA expression changes during the transit of normal B cells through the GC reaction and widely deregulated lncRNA expression patterns in HL. Three lncRNAs showed a tumor cell-specific expression pattern in HL tissues and might therefore be of value as a biomarker.

Inhibition of the miR-155 target NIAM phenocopies the growth promoting effect of miR-155 in B-cell lymphoma.

Several studies have indicated an important role for miR-155 in the pathogenesis of B-cell lymphoma. Highly elevated levels of miR-155 were indeed observed in most B-cell lymphomas with the exception of Burkitt lymphoma (BL). However, the molecular mechanisms that underlie the oncogenic role of miR-155 in B-cell lymphoma are not well understood. To identify the miR-155 targets relevant for B-cell lymphoma, we performed RNA immunoprecipitation of Argonaute 2 in Hodgkin lymphoma (HL) cells upon miR-155 inhibition and in BL cells upon ectopic expression of miR-155. We identified 54 miR-155-specific target genes in BL cells and confirmed miR-155 targeting of DET1, NIAM, TRIM32, HOMEZ, PSIP1 and JARID2. Five of these targets are also regulated by endogenous miR-155 in HL cells. Both overexpression of miR-155 and inhibition of expression of the novel miR-155 target gene NIAM increased proliferation of BL cells. In primary B-cell lymphoma NIAM-positive cases have significant lower levels of miR-155 as compared to NIAM-negative cases, suggesting that NIAM is also regulated by miR-155 in primary B-cell lymphoma. Thus, our data indicate an oncogenic role for miR-155 in B-cell lymphoma which involves targeting the tumor suppressor NIAM.

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.

Long noncoding RNAs as a novel component of the Myc transcriptional network.

Myc is a well-known transcription factor with important roles in cell cycle, apoptosis, and cellular transformation. Long noncoding RNAs (lncRNAs) have recently emerged as an important class of regulatory RNAs. Here, we show that lncRNAs are a main component of the Myc-regulated transcriptional program using the P493-6 tetracycline-repressible myc model. We demonstrate that both Myc-induced mRNAs and lncRNAs are significantly enriched for Myc binding sites. In contrast to Myc-repressed mRNAs, Myc-repressed lncRNAs are significantly enriched for Myc binding sites. Subcellular localization analysis revealed that compared to mRNAs, lncRNAs more often have a specific subcellular localization with a markedly higher percentage of nuclear enrichment within the Myc-repressed lncRNA set. Parallel analysis of differentially expressed lncRNAs and mRNAs identified 105 juxtaposed lncRNA-mRNA pairs, indicative for regulation in cis. To support the potential relevance of the Myc-regulated lncRNAs in cellular transformation, we analyzed their expression in primary Myc-high and Myc-low B-cell lymphomas. In total, 54% of the lncRNAs differentially expressed between the lymphoma subsets were identified as Myc-regulated in P493-6 cells. This study is the first to show that lncRNAs are an important factor within the Myc-regulated transcriptional program and indicates a marked difference between Myc-repressed lncRNAs and mRNAs.

Cellular localization and processing of primary transcripts of exonic microRNAs.

Processing of miRNAs occurs simultaneous with the transcription and splicing of their primary transcripts. For the small subset of exonic miRNAs it is unclear if the unspliced and/or spliced transcripts are used for miRNA biogenesis. We assessed endogenous levels and cellular location of primary transcripts of three exonic miRNAs. The ratio between unspliced and spliced transcripts varied markedly, i.e. >1 for BIC, <1 for pri-miR-146a and variable for pri-miR-22. Endogenous unspliced transcripts were located almost exclusively in the nucleus and thus available for miRNA processing for all three miRNAs. Endogenous spliced pri-miRNA transcripts were present both in the nucleus and in the cytoplasm and thus only partly available for miRNA processing. Overexpression of constructs containing the 5' upstream exonic or intronic sequence flanking pre-miR-155 resulted in strongly enhanced miR-155 levels, indicating that the flanking sequence does not affect processing efficiency. Exogenously overexpressed full-length spliced BIC transcripts were present both in the nucleus and in the cytoplasm, were bound by the Microprocessor complex and resulted in enhanced miR-155 levels. We conclude that both unspliced and spliced transcripts of exonic miRNAs can be used for pre-miRNA cleavage. Splicing and cytoplasmic transport of spliced transcripts may present a mechanism to regulate levels of exonic microRNAs.