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.

Hepatic glutamine synthetase controls N5-methylglutamine in homeostasis and cancer.

Glutamine synthetase (GS) activity is conserved from prokaryotes to humans, where the ATP-dependent production of glutamine from glutamate and ammonia is essential for neurotransmission and ammonia detoxification. Here, we show that mammalian GS uses glutamate and methylamine to produce a methylated glutamine analog, N5-methylglutamine. Untargeted metabolomics revealed that liver-specific GS deletion and its pharmacological inhibition in mice suppress hepatic and circulating levels of N5-methylglutamine. This alternative activity of GS was confirmed in human recombinant enzyme and cells, where a pathogenic mutation in the active site (R324C) promoted the synthesis of N5-methylglutamine over glutamine. N5-methylglutamine is detected in the circulation, and its levels are sustained by the microbiome, as demonstrated by using germ-free mice. Finally, we show that urine levels of N5-methylglutamine correlate with tumor burden and GS expression in a ß-catenin-driven model of liver cancer, highlighting the translational potential of this uncharacterized metabolite.

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.

Ultrasensitive Photonic Microsystem Enabling Sub-micrometric Monitoring of Arterial Oscillations for Advanced Cardiovascular Studies.

Cardiovascular diseases are the first cause of death globally. Their early diagnosis requires ultrasensitive tools enabling the detection of minor structural and functional alterations in small arteries. Such analyses have been traditionally performed with video imaging-based myographs, which helped to investigate the pathophysiology of the microvessels. Since new vascular questions have emerged, substantial modifications are necessary to improve the performance of imaging and tracking software, reducing the cost and minimizing the microvessel cleaning and manipulation. To address these limitations, we present a photonic microsystem fabricated in polydimethylsiloxane and integrating micro-optical elements and a lightguide-cantilever for sub-micrometric analysis of small arteries (between 125 and 400 µm of basal diameter). This technology enables simultaneous measurement of arterial distension, stiffness, vasomotion, and heartbeat and without the need for advanced imaging system. The microsystem has a limit of detection of 2 µm, five times lower than video imaging-based myographs, is two times more sensitive than them (0.5 µm/mmHg), reduces variability to half and doubles the linear range reported in these myographs. More importantly, it allows the analysis of intact arteries preserving the integrity and function of surrounding tissues. Assays can be conducted in three configurations according to the surrounding tissue: (i) isolated arteries (in vitro) where the surrounding tissue is partially removed, (ii) non-isolated arteries (in vivo) with surrounding tissue partially removed, and (iii) intact arteries in vivo preserving surrounding tissue as well as function and integrity. This technology represents a step forward in the prediction of cardiovascular risk.

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.

Cell Culture Medium Formulation and Its Implications in Cancer Metabolism.

Historic cell culture media were designed to ensure continuous cancer cell proliferation in vitro. However, their composition does not recapitulate the nutritional environment of the tumor. Recent studies show that novel media formulations alleviate the nonphysiological constraints imposed by historic media, and lead to cell culture results that are more relevant to tumor metabolism.

Microfluidic-controlled optical router for lab on a chip.

In multiplexed analysis, lab on a chip (LoC) devices are advantageous due to the low sample and reagent volumes required. Although optical detection is preferred for providing high sensitivity in a contactless configuration, multiplexed optical LoCs are limited by the technological complexity for integrating multiple light sources and detectors in a single device. To address this issue, we present a microfluidic-controlled optical router that enables measurement in four individual optical channels using a single light source and detector, and without movable parts. The optofluidic device is entirely fabricated in polydimethylsiloxane (PDMS) by soft-lithography, compatible with standard microfabrication technologies, enabling monolithic integration in LoCs. In the device, in-coupled light from an optical fiber is collimated by a polymeric micro-lens and guided through a set of four sequentially connected micro-chambers. When a micro-chamber is filled with water, light is transmitted to the next one. If it is empty of liquid, however, total internal reflection (TIR) occurs at the PDMS-air interface, re-directing the light to the output optical fiber. The router presents high performance, with low cross-talk (<2%) and high switching frequencies (up to 0.343 ± 0.006 Hz), and provides a stable signal for up to 91% of the switching time. With this miniaturized, low-cost, simple and robust design, we expect the current technology to be integrated in the new generation of multiplexed photonic LoCs for biomarker analysis, even at the point of care.

Label-Free Multi Parameter Optical Interrogation of Endothelial Activation in Single Cells using a Lab on a Disc Platform.

Cellular activation and inflammation leading to endothelial dysfunction is associated with cardiovascular disease (CVD). We investigated whether a single cell label-free multi parameter optical interrogation system can detect endothelial cell and endothelial progenitor cell (EPC) activation in vitro and ex vivo, respectively. Cultured human endothelial cells were exposed to increasing concentrations of tumour necrosis factor alpha (TNF-α) or lipopolysaccharide (LPS) before endothelial activation was validated using fluorescence-activated cell sorting (FACS) analysis of inflammatory marker expression (PECAM-1, E-selectin and ICAM-1). A centrifugal microfluidic system and V-cup array was used to capture individual cells before optical measurement of light scattering, immunocytofluorescence, auto-fluorescence (AF) and cell morphology was determined. In vitro, TNF-α promoted specific changes to the refractive index and cell morphology of individual cells concomitant with enhanced photon activity of fluorescently labelled inflammatory markers and increased auto-fluorescence (AF) intensity at three different wavelengths, an effect blocked by inhibition of downstream signalling with Iκß. Ex vivo, there was a significant increase in EPC number and AF intensity of individual EPCs from CVD patients concomitant with enhanced PECAM-1 expression when compared to normal controls. This novel label-free 'lab on a disc' (LoaD) platform can successfully detect endothelial activation in response to inflammatory stimuli in vitro and ex vivo.

Improving the metabolic fidelity of cancer models with a physiological cell culture medium.

Currently available cell culture media may not reproduce the in vivo metabolic environment of tumors. To demonstrate this, we compared the effects of a new physiological medium, Plasmax, with commercial media. We prove that the disproportionate nutrient composition of commercial media imposes metabolic artifacts on cancer cells. Their supraphysiological concentrations of pyruvate stabilize hypoxia-inducible factor 1α in normoxia, thereby inducing a pseudohypoxic transcriptional program. In addition, their arginine concentrations reverse the urea cycle reaction catalyzed by argininosuccinate lyase, an effect not observed in vivo, and prevented by Plasmax in vitro. The capacity of cancer cells to form colonies in commercial media was impaired by lipid peroxidation and ferroptosis and was rescued by selenium present in Plasmax. Last, an untargeted metabolic comparison revealed that breast cancer spheroids grown in Plasmax approximate the metabolic profile of mammary tumors better. In conclusion, a physiological medium improves the metabolic fidelity and biological relevance of in vitro cancer models.

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.

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.

Plug and measure - a chip-to-world interface for photonic lab-on-a-chip applications.

The integration of detection mechanisms with microfluidics may be one of the most promising routes towards widespread application of Lab-on-a-Chip (LoC) devices. Photonic detection methods like in the so-called Photonic Lab-on-a-Chip (PhLoC) have advantages such as being non-invasive, easy to sterilize and highly sensitive even with short integration times and thus allow in situ monitoring and quantification of biological and chemical processes. The readout of such detection methods usually requires special training of potential users, as in most cases they are confronted with the need of establishing fiber-optics connections to and from the PhLoC and/or rely on the use of complex laboratory equipment. Here, we present a low-cost and robust chip-to-world interface (CWI), fabricated by CO2-laser machining, facilitating the non-expert use of PhLoCs. Fiber-optics with standard SMA-connectors (non-pigtailed) and PhLoCs can be plugged into the CWI without the need for further adjustments. This standardization bestows great versatility on the interface, providing a direct link between PhLoCs and a wide range of light sources and photo-detectors. The ease-of-use of the proposed simple plug mechanism represents a step forward in terms of user-friendliness and may lead PhLoC devices to practical applications.

Photonic Lab-on-a-Chip: Integration of Optical Spectroscopy in Microfluidic Systems.

The integration of micro-optical elements with microfluidics leads to the highly promising photonic lab-on-a-chip analytical systems (PhLoCs). In this work, we re-examine the main principles which are underneath the on-chip spectrophotometric detection, approaching the PhLoC concept to a nonexpert audience.

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.

Microbial trench-based optofluidic system for reagentless determination of phenolic compounds.

Phenolic compounds are one of the main contaminants of soil and water due to their toxicity and persistence in the natural environment. Their presence is commonly determined with bulky and expensive instrumentation (e.g. chromatography systems), requiring sample collection and transport to the laboratory. Sample transport delays data acquisition, postponing potential actions to prevent environmental catastrophes. This article presents a portable, miniaturized, robust and low-cost microbial trench-based optofluidic system for reagentless determination of phenols in water. The optofluidic system is composed of a poly(methyl methacrylate) structure, incorporating polymeric optical elements and miniaturized discrete auxiliary components for optical transduction. An electronic circuit, adapted from a lock-in amplifier, is used for system control and interfering ambient light subtraction. In the trench, genetically modified bacteria are stably entrapped in an alginate hydrogel for quantitative determination of model phenol catechol. Alginate is also acting as a diffusion barrier for compounds present in the sample. Additionally, the superior refractive index of the gel (compared to water) confines the light in the lower level of the chip. Hence, the optical readout of the device is only altered by changes in the trench. Catechol molecules (colorless) in the sample diffuse through the alginate matrix and reach bacteria, which degrade them to a colored compound. The absorbance increase at 450 nm reports the presence of catechol simply, quickly (~10 min) and quantitatively without addition of chemical reagents. This miniaturized, portable and robust optofluidic system opens the possibility for quick and reliable determination of environmental contamination in situ, thus mitigating the effects of accidental spills.

A multiple path photonic lab on a chip for parallel protein concentration measurements.

We propose a PDMS-based photonic system for the accurate measurement of protein concentration with minute amounts of the sample. As opposed to the state of the art approach, in the multiple path photonic lab on a chip (MPHIL), analyte concentration or molar absorptivity is obtained with a single injection step, by performing simultaneous parallel optical measurements varying the optical path length. Also, as opposed to the standard calibration protocol, the MPHIL approach does not require a series of measurements at different concentrations. MPHIL has three main advantages: firstly the possibility of dynamically selecting the path length, always working in the absorbance vs. concentration linear range for each target analyte. Secondly, a dramatic reduction of the total volume of the sample required to obtain statistically reliable results. Thirdly, since only one injection is required, the measurement time is minimized, reducing both contamination and signal drifts. These characteristics are clearly advantageous when compared to commercial micro-spectrophotometers. The MPHIL concept was validated by testing three commercial proteins, lysozyme (HEWL), glucose isomerase (d-xylose-ketol-isomerase, GI) and Aspergillus sp. lipase L (BLL), as well as two proteins expressed and purified for this study, B. cereus formamidase (FASE) and dihydropyrimidinase from S. meliloti CECT41 (DHP). The use of MPHIL is also proposed for any spectrophotometric measurement in the UV-VIS range, as well as for its integration as a concentration measurement platform in more advanced photonic lab on a chip systems.

Tetrac can replace thyroid hormone during brain development in mouse mutants deficient in the thyroid hormone transporter mct8.

The monocarboxylate transporter 8 (MCT8) plays a critical role in mediating the uptake of thyroid hormones (THs) into the brain. In patients, inactivating mutations in the MCT8 gene are associated with a severe form of psychomotor retardation and abnormal serum TH levels. Here, we evaluate the therapeutic potential of the TH analog 3,5,3',5'-tetraiodothyroacetic acid (tetrac) as a replacement for T(4) in brain development. Using COS1 cells transfected with TH transporter and deiodinase constructs, we could show that tetrac, albeit not being transported by MCT8, can be metabolized to the TH receptor active compound 3,3',5-triiodothyroacetic acid (triac) by type 2 deiodinase and inactivated by type 3 deiodinase. Triac in turn is capable of replacing T(3) in primary murine cerebellar cultures where it potently stimulates Purkinje cell development. In vivo effects of tetrac were assessed in congenital hypothyroid Pax8-knockout (KO) and Mct8/Pax8 double-KO mice as well as in Mct8-KO and wild-type animals after daily injection of tetrac (400 ng/g body weight) during the first postnatal weeks. This treatment was sufficient to promote TH-dependent neuronal differentiation in the cerebellum, cerebral cortex, and striatum but was ineffective in suppressing hypothalamic TRH expression. In contrast, TSH transcript levels in the pituitary were strongly down-regulated in response to tetrac. Based on our findings we propose that tetrac administration offers the opportunity to provide neurons during the postnatal stage with a potent TH receptor agonist, thereby eventually reducing the neurological damage in patients with MCT8 mutations without deteriorating the thyrotoxic situation in peripheral tissues.