Extracellular Citrate Research Articles

Anticancer effects of high-dose extracellular citrate treatment in pancreatic cancer cells under different glucose concentrations

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive solid tumor. Recently, the uptake of extracellular citrate by the sodium-dependent citrate transporter (NaCT), encoded by SLC13A5, has been demonstrated to exert profound effects on cancer cell metabolism. However, research on the function of extracellular citrate in PDAC pathogenesis and the relationship between NaCT expression and the tumor metabolic microenvironment is limited. Therefore, we aimed to evaluate the expression of citrate transporters across a spectrum of glucose concentrations in pancreatic cancer and systematically explore the effects of sodium citrate treatment on pancreatic cancer cells at different glucose concentrations. We observed a positive correlation between glucose concentration and NaCT expression in PDAC cell lines. Extracellular sodium citrate significantly reduced cell viability partially due to reduction in intracellular Ca2+ levels and decreased the migration of human PDAC cells. Furthermore, we observed a decrease in the levels of the stem cell marker prominin I (CD133) following sodium citrate treatment. Notably, the combination treatment of gemcitabine and extracellular sodium citrate exhibited a synergistic anticancer effect in both two-dimensional (2D) and three-dimensional (3D) culture systems. Additionally, we confirmed that pH slightly increased upon administration of sodium citrate, indicating that this could potentially augment the efficacy of gemcitabine. Altogether, these findings suggest that exogenous sodium citrate treatment, particularly in combination with gemcitabine, may represent a novel therapeutic strategy for treating PDAC. This approach holds promise for disrupting PDAC cell metabolism and inhibiting tumor progression.

Expression of the plasma membrane citrate carrier (pmCiC) in human cancerous tissues-correlation with tumour aggressiveness.

We have recently shown that cancer cells of various origins take up extracellular citrate through the plasma membrane citrate carrier (pmCiC), a specific plasma membrane citrate transporter. Extracellular citrate is required to support cancer cell metabolism, in particular fatty acid synthesis, mitochondrial activity, protein synthesis and histone acetylation. In addition, cancer cells tend to acquire a metastatic phenotype in the presence of extracellular citrate. Our recent study also showed that cancer-associated stromal cells synthesise and release citrate and that this process is controlled by cancer cells. In the present study, we evaluated the expression of pmCiC, fibroblast activation protein-α (FAP) and the angiogenesis marker cluster of differentiation 31 (CD31) in human cancer tissues of different origins. In the cohort studied, we found no correlation between disease stage and the expression of FAP or CD31. However, we have identified a clear correlation between pmCiC expression in cancer cells and cancer-associated stroma with tumour stage. It can be concluded that pmCiC is increased in cancer cells and in cancer-supporting cells in the tumour microenvironment at the later stages of cancer development, particularly at the metastatic sites. Therefore, pmCiC expression has the potential to serve as a prognostic marker, although further studies are needed.

Gene Expression Reprogramming by Citrate Supplementation Reduces HepG2 Cell Migration and Invasion.

Citrate, which is obtained from oxaloacetate and acetyl-CoA by citrate synthase in mitochondria, plays a key role in both normal and cancer cell metabolism. In this work, we investigated the effect of 10 mM extracellular citrate supplementation on HepG2 cells. Gene expression reprogramming was evaluated by whole transcriptome analysis using gene set enrichment analysis (GSEA). The transcriptomic data were validated through analyzing changes in the mRNA levels of selected genes by qRT-PCR. Citrate-treated cells exhibited the statistically significant dysregulation of 3551 genes; 851 genes were upregulated and 822 genes were downregulated. GSEA identified 40 pathways affected by differentially expressed mRNAs. The most affected biological processes were related to lipid and RNA metabolism. Several genes of the cytochrome P450 family were upregulated in treated cells compared to controls, including the CYP3A5 gene, a tumor suppressor in hepatocellular carcinoma (HCC) that plays an important protective role in HCC metastasis. The citrate-induced dysregulation of cytochromes could both improve the effectiveness of chemotherapeutics used in combination and reduce the aggressiveness of tumors by diminishing cell migration and invasion.

The citrate transporter SLC13A5 as a therapeutic target for kidney disease: evidence from Mendelian randomization to inform drug development

BackgroundSolute carrier family 13 member 5 (SLC13A5) is a Na+-coupled citrate co-transporter that mediates entry of extracellular citrate into the cytosol. SLC13A5 inhibition has been proposed as a target for reducing progression of kidney disease. The aim of this study was to leverage the Mendelian randomization paradigm to gain insight into the effects of SLC13A5 inhibition in humans, towards prioritizing and informing clinical development efforts.MethodsThe primary Mendelian randomization analyses investigated the effect of SLC13A5 inhibition on measures of kidney function, including creatinine and cystatin C-based measures of estimated glomerular filtration rate (creatinine-eGFR and cystatin C-eGFR), blood urea nitrogen (BUN), urine albumin-creatinine ratio (uACR), and risk of chronic kidney disease and microalbuminuria. Secondary analyses included a paired plasma and urine metabolome-wide association study, investigation of secondary traits related to SLC13A5 biology, a phenome-wide association study (PheWAS), and a proteome-wide association study. All analyses were compared to the effect of genetically predicted plasma citrate levels using variants selected from across the genome, and statistical sensitivity analyses robust to the inclusion of pleiotropic variants were also performed. Data were obtained from large-scale genetic consortia and biobanks, with sample sizes ranging from 5023 to 1,320,016 individuals.ResultsWe found evidence of associations between genetically proxied SLC13A5 inhibition and higher creatinine-eGFR (p = 0.002), cystatin C-eGFR (p = 0.005), and lower BUN (p = 3 × 10−4). Statistical sensitivity analyses robust to the inclusion of pleiotropic variants suggested that these effects may be a consequence of higher plasma citrate levels. There was no strong evidence of associations of genetically proxied SLC13A5 inhibition with uACR or risk of CKD or microalbuminuria. Secondary analyses identified evidence of associations with higher plasma calcium levels (p = 6 × 10−13) and lower fasting glucose (p = 0.02). PheWAS did not identify any safety concerns.ConclusionsThis Mendelian randomization analysis provides human-centric insight to guide clinical development of an SLC13A5 inhibitor. We identify plasma calcium and citrate as biologically plausible biomarkers of target engagement, and plasma citrate as a potential biomarker of mechanism of action. Our human genetic evidence corroborates evidence from various animal models to support effects of SLC13A5 inhibition on improving kidney function.

Bempedoic Acid Unveils Therapeutic Potential in Non-Alcoholic Fatty Liver Disease: Suppression of the Hepatic PXR-SLC13A5/ACLY Signaling Axis.

The hepatic SLC13A5/SLC25A1-ATP-dependent citrate lyase (ACLY) signaling pathway, responsible for maintaining the citrate homeostasis, plays a crucial role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Bempedoic acid (BA), an ACLY inhibitor commonly used for managing hypercholesterolemia, has shown promising results in addressing hepatic steatosis. This study aimed to elucidate the intricate relationships in processes of hepatic lipogenesis among SLC13A5, SLC25A1, and ACLY and to examine the therapeutic potential of BA in NAFLD, providing insights into its underlying mechanism. In murine primary hepatocytes and HepG2 cells, the silencing or pharmacological inhibition of SLC25A1/ACLY resulted in significant upregulation of SLC13A5 transcription and activity. This increase in SLC13A5 activity subsequently led to enhanced lipogenesis, indicating a compensatory role of SLC13A5 when the SLC25A1/ACLY pathway was inhibited. However, BA effectively counteracted this upregulation, reduced lipid accumulation, and ameliorated various biomarkers of NAFLD. The disease-modifying effects of BA were further confirmed in NAFLD mice. Mechanistic investigations revealed that BA could reverse the elevated transcription levels of SLC13A5 and ACLY, and the subsequent lipogenesis induced by PXR activation in vitro and in vivo. Importantly, this effect was diminished when PXR was knocked down, suggesting the involvement of the hepatic PXR-SLC13A5/ACLY signaling axis in the mechanism of BA action. In conclusion, SLC13A5-mediated extracellular citrate influx emerges as an alternative pathway to SLC25A1/ACLY in the regulation of lipogenesis in hepatocytes, BA exhibits therapeutic potential in NAFLD by suppressing the hepatic PXR-SLC13A5/ACLY signaling axis, while PXR, a key regulator in drug metabolism may be involved in the pathogenesis of NAFLD. SIGNIFICANCE STATEMENT: This work describes that bempedoic acid, an ATP-dependent citrate lyase (ACLY) inhibitor, ameliorates hepatic lipid accumulation and various hallmarks of non-alcoholic fatty liver disease. Suppression of hepatic SLC25A1-ACLY pathway upregulates SLC13A5 transcription, which in turn activates extracellular citrate influx and the subsequent DNL. Whereas in hepatocytes or the liver tissue challenged with high energy intake, bempedoic acid reverses compensatory activation of SLC13A5 via modulating the hepatic PXR-SLC13A5/ACLY axis, thereby simultaneously downregulating SLC13A5 and ACLY.

Mapping the Metabolic Niche of Citrate Metabolism and SLC13A5.

The small molecule citrate is a key molecule that is synthesized de novo and involved in diverse biochemical pathways influencing cell metabolism and function. Citrate is highly abundant in the circulation, and cells take up extracellular citrate via the sodium-dependent plasma membrane transporter NaCT encoded by the SLC13A5 gene. Citrate is critical to maintaining metabolic homeostasis and impaired NaCT activity is implicated in metabolic disorders. Though citrate is one of the best known and most studied metabolites in humans, little is known about the consequences of altered citrate uptake and metabolism. Here, we review recent findings on SLC13A5, NaCT, and citrate metabolism and discuss the effects on metabolic homeostasis and SLC13A5-dependent phenotypes. We discuss the "multiple-hit theory" and how stress factors induce metabolic reprogramming that may synergize with impaired NaCT activity to alter cell fate and function. Furthermore, we underline how citrate metabolism and compartmentalization can be quantified by combining mass spectrometry and tracing approaches. We also discuss species-specific differences and potential therapeutic implications of SLC13A5 and NaCT. Understanding the synergistic impact of multiple stress factors on citrate metabolism may help to decipher the disease mechanisms associated with SLC13A5 citrate transport disorders.

Dyskeratosis Congenita Links Telomere Attrition to Age-Related Systemic Energetics.

The underlying mechanisms of plasma metabolite signatures of human aging and age-related diseases are not clear but telomere attrition and dysfunction are central to both. Dyskeratosis congenita (DC) is associated with mutations in the telomerase enzyme complex (TERT, TERC, and DKC1) and progressive telomere attrition. We analyzed the effect of telomere attrition on senescence-associated metabolites in fibroblast-conditioned media and DC patient plasma. Samples were analyzed by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. We showed extracellular citrate was repressed by canonical telomerase function in vitro and associated with DC leukocyte telomere attrition in vivo, leading to the hypothesis that altered citrate metabolism detects telomere dysfunction. However, elevated citrate and senescence factors only weakly distinguished DC patients from controls, whereas elevated levels of other tricarboxylic acid cycle (TCA) metabolites, lactate, and especially pyruvate distinguished them with high significance. The DC plasma signature most resembled that of patients with loss of function pyruvate dehydrogenase complex mutations and that of older subjects but significantly not those of type 2 diabetes, lactic acidosis, or elevated mitochondrial reactive oxygen species. Additionally, our data are consistent with further metabolism of citrate and lactate in the liver and kidneys. Citrate uptake in certain organs modulates age-related disease in mice and our data have similarities with age-related disease signatures in humans. Our results have implications for the role of telomere dysfunction in human aging in addition to its early diagnosis and the monitoring of anti-senescence therapeutics, especially those designed to improve telomere function.

The Role of Citrate Homeostasis in Merkel Cell Carcinoma Pathogenesis.

Simple SummaryMerkel cell carcinoma (MCC) is a rare but highly aggressive skin cancer. Despite important progress, overall understanding of the events that drive MCC carcinogenesis remains incomplete. We discovered that the plasma membrane citrate transporter (pmCiC) is upregulated in Merkel cell carcinoma cell lines. Cancer cells import extracellular citrate via pmCiC to support their metabolism, which is critical to support proliferation and metastatic spread. In this study, we show that inhibition of pmCiC can decrease the growth rate of Merkel cell carcinoma cell lines. Targeting pmCiC and thereby the tumor metabolism should be considered further as a potential anti-cancer therapy.Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.

Extracellular Citrate Treatment Induces HIF1α Degradation and Inhibits the Growth of Low-Glycolytic Hepatocellular Carcinoma under Hypoxia.

Simple SummaryPatients with low-glycolytic hepatocellular carcinoma (HCC) show better clinical outcomes than those with hypoxic and high-glycolytic HCC. Low-glycolytic HCCs seem to utilize carbon sources other than glucose for metabolic fuel and tumor growth. However, by increasing tumor size, its outgrowth perfusion generates hypoxic foci inside the tumor and becomes more aggressive and resistant to therapy. In this study, we found that SLC13A5/NaCT is an important solute carrier (SLC) in low-glycolytic HCCs. To adapt to hypoxic conditions, low-glycolytic cancer cells have to switch metabolism from oxidative phosphorylation to hypoxia-induced glycolysis by the upregulation of HIF1α. However, extracellular citrate treatment in HCCs with high SLC13A5/NaCT expression had reduced glucose uptake due to HIF1α degradation, inducing the failure of metabolic adaptation to hypoxia, resulting in anti-cancer effects in in vitro and in vivo animal models.HCC is well known for low glycolysis in the tumors, whereas hypoxia induces glycolytic phenotype and tumor progression. This study was conducted to evaluate the expression of SLCs in human HCCs and investigated whether extracellular nutrient administration related to SLCs in low-glycolytic HCC can prevent hypoxic tumor progression. SLCs expression was screened according to the level of glycolysis in HCCs. Then, whether extracellular nutrient treatment can affect hypoxic tumor progression, as well as the mechanisms, were evaluated in an in vitro cell line and an in vivo animal model. Low-glycolytic HCCs showed high SLC13A5/NaCT and SLC16A1/MCT1 but low SLC2A1/GLUT1 and HIF1α/HIF1α expression. Especially, high SLC13A5 expression was significantly associated with good overall survival in the Cancer Genome Atlas (TCGA) database. In HepG2 cells with the highest NaCT expression, extracellular citrate treatment upon hypoxia induced HIF1α degradation, which led to reduced glycolysis and cellular proliferation. Finally, in HepG2-animal models, the citrate-treated group showed smaller tumor with less hypoxic areas than the vehicle-treated group. In patients with HCC, SLC13A5/NaCT is an important SLC, which is associated with low glycolysis and good prognosis. Extracellular citrate treatment induced the failure of metabolic adaptation to hypoxia and tumor growth inhibition, which can be a potential therapeutic strategy in HCCs.

Carbon sources and pathways for citrate secreted by human prostate cancer cells determined by NMR tracing and metabolic modeling

Prostate epithelial cells have the unique capacity to secrete large amounts of citrate, but the carbon sources and metabolic pathways that maintain this production are not well known. We mapped potential pathways for citrate carbons in the human prostate cancer metastasis cell lines LNCaP and VCaP, for which we first established that they secrete citrate (For LNCaP 5.6 ± 0.9 nmol/h per 106 cells). Using 13C-labeled substrates, we traced the incorporation of 13C into citrate by NMR of extracellular fluid. Our results provide direct evidence that glucose is a main carbon source for secreted citrate. We also demonstrate that carbons from supplied glutamine flow via oxidative Krebs cycle and reductive carboxylation routes to positions in secreted citrate but likely do not contribute to its net synthesis. The potential anaplerotic carbon sources aspartate and asparagine did not contribute to citrate carbons. We developed a quantitative metabolic model employing the 13C distribution in extracellular citrate after 13C glucose and pyruvate application to assess intracellular pathways of carbons for secreted citrate. From this model, it was estimated that in LNCaP about 21% of pyruvate entering the Krebs cycle is converted via pyruvate carboxylase as an anaplerotic route at a rate more than sufficient to compensate carbon loss of this cycle by citrate secretion. This model provides an estimation of the fraction of molecules, including citrate, leaving the Krebs cycle at every turn. The measured ratios of 13C atoms at different positions in extracellular citrate may serve as biomarkers for (malignant) epithelial cell metabolism.

Metabolic Alterations in Cellular Senescence: The Role of Citrate in Ageing and Age-Related Disease.

Recent mouse model experiments support an instrumental role for senescent cells in age-related diseases and senescent cells may be causal to certain age-related pathologies. A strongly supported hypothesis is that extranuclear chromatin is recognized by the cyclic GMP–AMP synthase-stimulator of interferon genes pathway, which in turn leads to the induction of several inflammatory cytokines as part of the senescence-associated secretory phenotype. This sterile inflammation increases with chronological age and age-associated disease. More recently, several intracellular and extracellular metabolic changes have been described in senescent cells but it is not clear whether any of them have functional significance. In this review, we highlight the potential effect of dietary and age-related metabolites in the modulation of the senescent phenotype in addition to discussing how experimental conditions may influence senescent cell metabolism, especially that of energy regulation. Finally, as extracellular citrate accumulates following certain types of senescence, we focus on the recently reported role of extracellular citrate in aging and age-related pathologies. We propose that citrate may be an active component of the senescence-associated secretory phenotype and via its intake through the diet may even contribute to the cause of age-related disease.

Extracellular citrate serves as a DAMP to activate macrophages and promote LPS-induced lung injury in mice

Citrate has a prominent role as a substrate in cellular energy metabolism. Recently, citrate has been shown to drive inflammation. However, the role of citrate in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains unclear. Here, we aimed to clarify whether extracellular citrate aggravated the LPS-induced ALI and the potential mechanism. Our findings demonstrated that extracellular citrate aggravated the pathological lung injury induced by LPS in mice, characterized by up-regulation of pro-inflammatory factors and over-activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in the lungs. In vitro, we found that citrate treatment significantly augmented the expression of NLRP3 and pro-IL-1β and enhanced the translocation of NF-κB/p65 into the nucleus. Furthermore, extracellular citrate plus adenosine-triphosphate (ATP) significantly increased the production of reactive oxygen species (ROS) in primary murine macrophages. Inhibiting the production of ROS with a ROS scavenger N-acetyl-L-cysteine (NAC) attenuated the activation of NLRP3 inflammasome. Altogether, we conclude that extracellular citrate may serve as a damage-associated molecular pattern (DAMP) and aggravates LPS-induced ALI by activating the NLRP3 inflammasome.

Drosophila INDY and Mammalian INDY: Major Differences in Transport Mechanism and Structural Features despite Mostly Similar Biological Functions.

INDY (I’m Not Dead Yet) is a plasma membrane transporter for citrate, first identified in Drosophila. Partial deficiency of INDY extends lifespan in this organism in a manner similar to that of caloric restriction. The mammalian counterpart (NaCT/SLC13A5) also transports citrate. In mice, it is the total, not partial, absence of the transporter that leads to a metabolic phenotype similar to that caloric restriction; however, there is evidence for subtle neurological dysfunction. Loss-of-function mutations in SLC13A5 (solute carrier gene family 13, member A5) occur in humans, causing a recessive disease, with severe clinical symptoms manifested by neonatal seizures and marked disruption in neurological development. Though both Drosophila INDY and mammalian INDY transport citrate, the translocation mechanism differs, the former being a dicarboxylate exchanger for the influx of citrate2− in exchange for other dicarboxylates, and the latter being a Na+-coupled uniporter for citrate2−. Their structures also differ as evident from only ~35% identity in amino acid sequence and from theoretically modeled 3D structures. The varied biological consequences of INDY deficiency across species, with the beneficial effects predominating in lower organisms and detrimental effects overwhelming in higher organisms, are probably reflective of species-specific differences in tissue expression and also in relative contribution of extracellular citrate to metabolic pathways in different tissues

Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1.

Pain at the injection site is a common complaint of patients receiving therapeutic formulations containing citric acid. Despite the widely acknowledged role of acid-sensing ion channels (ASICs) in acid-related perception, the specific ASIC subtype mediating pain caused by subcutaneous acid injection and the mechanism by which citrate affects this process are less clear. Here, male mice subjected to intraplantar acid injection responded by executing a withdrawal reflex, and this response was abolished by ASIC1 but not ASIC2 knockout. Although intraplantar injection of neutral citrate solution did not produce this response, intraplantar injection of acidic citrate solution produced a withdrawal reflex greater than that produced by acidity alone. Consistent with the behavioral data, neutral citrate failed to produce an electrophysiological response in HEK293 cells, which express ASIC1, but acidic citrate produced a whole-cell inward current greater than that produced by acidity alone. Saturating the intracellular solution with citrate had no effect on the potentiating effect of extracellular citrate, suggesting that citrate acted extracellularly to potentiate ASIC1. Moreover, exposure to citrate immediately before acid stimulation failed to potentiate ASIC1 currents, which ruled out the involvement of a metabotropic receptor gated by a citrate metabolite. Finally, removal of calcium ions from the extracellular solution mimicked the potentiating effect of citrate and prevented citrate from further potentiating ASIC1. Our data demonstrate that ASIC1 is necessary for the nociceptive response caused by subcutaneous acid infusion and that neutral citrate, despite not inducing ASIC1 currents or nociceptive behavior on its own, potentiates acid nociception by removing the inhibitory effect of extracellular calcium ions on ASIC1.SIGNIFICANCE STATEMENT Citric acid is a common ingredient used in pharmaceutical formulations. Despite the widespread clinical use of these formulations, it remains unclear how citric acid causes pain when injected into patients. We identified ASIC1 as the key receptor used to detect injection-site pain caused by acid, and we showed that neutral citrate does not stimulate ASIC1; instead, citrate substantially potentiates ASIC1 activation when injected simultaneously with acid. In addition, we demonstrated that citrate potentiates ASIC1 by removing the inhibitory action of calcium on the extracellular side of the receptor. Given that injection-site pain is the primary complaint of patients receiving citrate-containing medical products, our data provide mechanistic insight into a common medical complaint and suggest a means of avoiding injection pain.

Cancer-associated cells release citrate to support tumour metastatic progression.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

Potential Involvement of Extracellular Citrate in Brain Tumor Progression.

Brain tissue is known to have elevated citrate levels, necessary to regulate ion chelation, neuron excitability, and are also necessary for the supply of necessary energy substrates to neurons. Importantly, citrate also acts as a central substrate in cancer metabolism. Recent studies have shown that extracellular citrate levels in the brain undergo significant changes during tumor development and may play a dual role in tumor progression, as well as cancer cell aggressiveness. In the present article, we review available literature describing changes of citrate levels in brain tissue, blood, and cerebrospinal fluid, as well as intracellular alterations during tumor development before and after metastatic progression. Based on the available literature and our recent findings, we hypothesize that changes in extracellular citrate levels may be related to the increased consumption of this metabolite by cancer cells. Interestingly, cancerassociated cells, including reactive astrocytes, might be a source of citrate. Extracellular citrate uptake mechanisms, as well as potential citrate synthesis and release by surrounding stroma, could provide novel targets for anti-cancer treatments of primary brain tumors and brain metastases.

Extracellular Citrate Fuels Cancer Cell Metabolism and Growth.

Cancer cells need excess energy and essential nutrients/metabolites not only to divide and proliferate but also to migrate and invade distant organs for metastasis. Fatty acid and cholesterol synthesis, considered a hallmark of cancer for anabolism and membrane biogenesis, requires citrate. We review here potential pathways in which citrate is synthesized and/or supplied to cancer cells and the impact of extracellular citrate on cancer cell metabolism and growth. Cancer cells employ different mechanisms to support mitochondrial activity and citrate synthesis when some of the necessary substrates are missing in the extracellular space. We also discuss the different transport mechanisms available for the entry of extracellular citrate into cancer cells and how citrate as a master metabolite enhances ATP production and fuels anabolic pathways. The available literature suggests that cancer cells show an increased metabolic flexibility with which they tackle changing environmental conditions, a phenomenon crucial for cancer cell proliferation and metastasis.

Extracellular Citrate Is a Trojan Horse for Cancer Cells.

The first intermediate in the mitochondrial tricarboxylic acid (TCA) cycle is citrate, which is essential and acts as a metabolic regulator for glycolysis, TCA cycle, gluconeogenesis, and fatty acid synthesis. Within the cytosol, citrate is cleaved by ATP citrate lyase (ACLY) into oxaloacetate (OAA) and acetyl-CoA; OAA can be used for neoglucogenesis or in the TCA cycle, while acetyl-CoA is the precursor of some biosynthetic processes, including the synthesis of fatty acids. Accumulating evidence suggests that citrate is involved in numerous physiological and pathophysiological processes such as inflammation, insulin secretion, neurological disorders, and cancer. Considering the crucial role of citrate to supply the acetyl-CoA pool for fatty acid synthesis and histone acetylation in tumors, in this study we evaluated the effect of citrate added to the growth medium on lipid deposition and histone H4 acetylation in hepatoma cells (HepG2). At low concentration, citrate increased both histone H4 acetylation and lipid deposition; at high concentration, citrate inhibited both, thus suggesting a crucial role of acetyl-CoA availability, which prompted us to investigate the effect of citrate on ACLY. In HepG2 cells, the expression of ACLY is correlated with histone acetylation, which, in turn, depends on citrate concentration. A decrease in H4 acetylation was also observed when citrate was added at a high concentration to immortalized human hepatic cells, whereas ACLY expression was unaffected, indicating a lack of control by histone acetylation. Considering the strong demand for acetyl-CoA but not for OAA in tumor cells, the exogenous citrate would behave like a trojan horse that carries OAA inside the cells and reduces ACLY expression and cellular metabolism. In addition, this study confirmed the already reported dual role of citrate both as a promoter of cell proliferation (at lower concentrations) and as an anticancer agent (at higher concentrations), providing useful tips on the use of citrate for the treatment of tumors.

The membrane protein ANKH is crucial for bone mechanical performance by mediating cellular export of citrate and ATP.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.

Potential Use of Gluconate in Cancer Therapy.

We have recently discovered that cancer cells take up extracellular citrate through plasma membrane citrate transporter (pmCiC) and advantageously use citrate for their metabolism. Citrate uptake can be blocked with gluconate and this results in decreased tumor growth and altered metabolic characteristics of tumor tissue. Interestingly, gluconate, considered to be physiologically neutral, is incidentally used in medicine as a cation carrier, but not as a therapeutically active substance. In this review we discuss the results of our recent research with available literature and suggest that gluconate may be useful in the treatment of cancer.