Ethanolamine Kinase Research Articles

Whole Blood Transcriptome Analysis in Dairy Ewes Fed a Dietary Grape Pomace Supplementation

The present study aims to evaluate the effect of a dietary supplementation with 10% grape pomace (GP) on the whole blood transcriptome of lactating ewes. By applying a log2FC higher than 0.5 or lower than −0.5 and a false discovery rate (FDR) <0.05, the down-regulation of genes coding for plexin C1, ethanolamine kinase 1, tax1-binding protein 1, transmembrane 9 superfamily member 2, and Beclin-1 was observed in animals that received the dietary supplementation. This aspect was also accompanied by a reduction in the blood activity of matrix metalloproteinase 9 (MMP-9; p < 0.05), a gelatinase commonly involved in both acute and chronic pathological events. The ELISA test on other factors involved in inflammatory processes, interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α), as well as in the antioxidant response, glutathione peroxidase (GPx), and catalase (CAT), did not reveal any significant changes (p > 0.05). Overall, the introduction of GP in the diet of ewes gave indications of greater efficacy in preserving animal welfare, with interesting cues regarding the valorization of a by-product with a high biological value.

Identification and Characterization of Entamoeba histolytica Choline Kinase.

Entamoeba histolytica is one of the death-causing parasites in the world. Study on its lipid composition revealed that it is predominated by phosphatidylcholine and phosphatidylethanolamine. Further study revealed that its phosphorylated metabolites might be produced by the Kennedy pathway. Here, we would like to report on the characterizations of enzymes from this pathway that would provide information for the design of novel inhibitors against these enzymes in future. E. histolytica HM-1:IMSS genomic DNA was isolated and two putative choline/ethanolamine kinase genes (EhCK1 and EhCK2) were cloned and expressed from Escherichia coli BL21 strain. Enzymatic characterizations were further carried out on the purified enzymes. EhCK1 and EhCK2 were identified from E. histolytica genome. The deduced amino acid sequences were more identical to its homologues in human (35-48%) than other organisms. The proteins were clustered as ethanolamine kinase in the constructed phylogeny tree. Sequence analysis showed that they possessed all the conserved motifs in choline kinase family: ATP-binding loop, Brenner's phosphotransferase motif, and choline kinase motif. Here, the open reading frames were cloned, expressed, and purified to apparent homogeneity. EhCK1 showed activity with choline but not ethanolamine. The biochemical characterization showed that it had a Vmax of 1.9 ± 0.1µmol/min/mg. Its Km for choline and ATP was 203 ± 26µM and 3.1 ± 0.4mM, respectively. In contrast, EhCK2 enzymatic activity was only detected when Mn2+ was used as the co-factor instead of Mg2+ like other choline/ethanolamine kinases. Highly sensitive and specific antibody against EhCK1 was developed and used to confirm the endogenous EhCK1 expression using immunoblotting. With the understanding of EhC/EK importance in phospholipid metabolism and their unique characteristic, EhC/EK could be a potential target for future anti-amoebiasis study.

A pan-cancer analysis of the oncogenic role of Golgi transport 1B in human tumors.

Owing to the aggressiveness and treatment-refractory nature of cancer, ideal candidates for early diagnosis and treatment are needed. Golgi transport 1B (GOLT1B) has been associated with cellular malignant behaviors and immune responses in colorectal and lung cancer, but a systematic pan-cancer analysis on GOLT1B has not been conducted. The expression status and clinical association of GOLT1B in The Cancer Genome Atlas (TCGA) were analyzed. Genetic and methylation alterations in GOLT1B were explored. The relationship between GOLT1B and immune cell infiltration was also investigated. Genes related to GOLT1B expression were selected and analyzed. GOLT1B was highly expressed in most tumors, and there was a positive correlation between GOLT1B expression and clinical pathological parameters. High expression levels of GOLT1B have been associated with poor prognosis of most cancers. Copy number amplification was the primary type of GOLT1B genetic alterations, which was related to the prognosis of pan-cancer cases. There were different levels of GOLT1B promoter methylation across cancer types. The methylation level of the probe cg07371838 and cg25816357 was closely associated with prognosis in diverse cancers. There was also a positive correlation between GOLT1B genetic alterations and CD4+ T lymphocytes, especially the Th2 subset, as well as between GOLT1B expression and the estimated infiltration value of cancer-associated fibroblasts. Serine/threonine kinase receptor-associated protein (STRAP), integrator complex subunit 13 (INTS13), and ethanolamine kinase 1 (ETNK1) were the most relevant genes for GOLT1B expression, and their interactions with GOLT1B were involved in regulating the transforming growth factor (TGF)-β receptor signaling pathway and epithelial-mesenchymal transition (EMT). This pan-cancer analysis provided a comprehensive understanding of the oncogenic role of GOLT1B, highlighting a potential mechanism whereby GOLT1B influences the tumor microenvironment, as well as cancer immunotherapy.

MiR-708–3p promotes gastric cancer progression through downregulating ETNK1

MicroRNAs (miRNAs) are small, evolutionarily conserved, non-coding RNAs playing a role in the proliferation, metastasis, apoptosis, chemo-sensitivity, and chemo-resistance of gastric cancer, as well as the stemness of gastric cancer stem cells. miR-708–3p induces gastric cancer cell chemo-resistance, but its actual role in gastric cancer progression remains unclear. This paper shows that miR-708–3p is upregulated in gastric cancer samples and that a high miR-708–3p expression in gastric cancer patients is associated with poor overall survival. Our functional study results indicate that miR-708–3p overexpression promotes gastric cancer cell proliferation and migration, inhibits cell apoptosis, and facilitates the transition from the G0/G1 to the G2/M phase. Furthermore, reducing miR-708–3p levels yielded opposite effects. Next, our in vivo experiments revealed that miR-708–3p advanced gastric cancer cell growth in nude mice. The underlying mechanism was the regulation of ethanolamine kinase 1 (ETNK1) expression by miR-708–3p, which bound to the 3′UTR of the ETNK1 gene in gastric cancer cells. Finally, the recovery assay results showed that ETNK1 overexpression could slow miR-708-3p-induced gastric cancer progression. In conclusion, we identified a new miR-708–3p/ETNK1 pathway involved in gastric cancer progression. These results may offer new targets for gastric cancer therapy and markers for gastric cancer prognosis.

B cell-derived anti-beta 2 glycoprotein I antibody mediates hyperhomocysteinemia-aggravated hypertensive glomerular lesions by triggering ferroptosis

Hyperhomocysteinemia (HHcy) is a risk factor for chronic kidney diseases (CKDs) that affects about 85% CKD patients. HHcy stimulates B cells to secrete pathological antibodies, although it is unknown whether this pathway mediates kidney injury. In HHcy-treated 2-kidney, 1-clip (2K1C) hypertensive murine model, HHcy-activated B cells secreted anti-beta 2 glycoprotein I (β2GPI) antibodies that deposited in glomerular endothelial cells (GECs), exacerbating glomerulosclerosis and reducing renal function. Mechanistically, HHcy 2K1C mice increased phosphatidylethanolamine (PE) (18:0/20:4, 18:0/22:6, 16:0/20:4) in kidney tissue, as determined by lipidomics. GECs oxidative lipidomics validated the increase of oxidized phospholipids upon Hcy-activated B cells culture medium (Hcy-B CM) treatment, including PE (18:0/20:4 + 3[O], PE (18:0a/22:4 + 1[O], PE (18:0/22:4 + 2[O] and PE (18:0/22:4 + 3[O]). PE synthases ethanolamine kinase 2 (etnk2) and ethanolamine-phosphate cytidylyltransferase 2 (pcyt2) were increased in the kidney GECs of HHcy 2K1C mice and facilitated polyunsaturated PE synthesis to act as lipid peroxidation substrates. In HHcy 2K1C mice and Hcy-B CM-treated GECs, the oxidative environment induced by iron accumulation and the insufficient clearance of lipid peroxides caused by transferrin receptor (TFR) elevation and down-regulation of SLC7A11/glutathione peroxidase 4 (GPX4) contributed to GECs ferroptosis of the kidneys. In vivo, pharmacological depletion of B cells or inhibition of ferroptosis mitigated the HHcy-aggravated hypertensive renal injury. Consequently, our findings uncovered a novel mechanism by which B cell-derived pathogenic anti-β2GPI IgG generated by HHcy exacerbated hypertensive kidney damage by inducing GECs ferroptosis. Targeting B cells or ferroptosis may be viable therapeutic strategies for ameliorating lipid peroxidative renal injury in HHcy patients with hypertensive nephropathy.

ETNK2 Low-Expression Predicts Poor Prognosis in Renal Cell Carcinoma with Immunosuppressive Tumor Microenvironment.

The ethanolamine kinase 2 (ETNK2) gene is implicated in carcinogenesis, but its expression and involvement in kidney renal clear cell carcinoma (KIRC) remain unknown. Initially, we conducted a pan-cancer study in which we searched the Gene Expression Profiling Interactive Analysis, the UALCAN, and the Human Protein Atlas databases to determine the expression level of the ETNK2 gene in KIRC. The Kaplan-Meier curve was then used to calculate the overall survival (OS) of KIRC patients. We then used the differentially expressed genes (DEGs) and enrichment analysis to explain the mechanism of the ETNK2 gene. Finally, the immune cell infiltration analysis was performed. Although the ETNK2 gene expression was lower in KIRC tissues, the findings illustrated a link between the ETNK2 gene expression and a shorter OS time for KIRC patients. DEGs and enrichment analysis revealed that the ETNK2 gene in KIRC involved multiple metabolic pathways. Finally, the ETNK2 gene expression has been linked to several immune cell infiltrations. According to the findings, the ETNK2 gene plays a crucial role in tumor growth. It can potentially serve as a negative prognostic biological marker for KIRC by modifying immune infiltrating cells.

Screening of Specific and Common Pathways in Breast Cancer Cell Lines MCF-7 and MDA-MB-231 Treated with Chlorophyllides Composites.

Our previous findings have shown that the chlorophyllides composites have anticancer activities to breast cancer cell lines (MCF-7 and MDA-MB-231). In the present study, microarray gene expression profiling was utilized to investigate the chlorophyllides anticancer mechanism on the breast cancer cells lines. Results showed that chlorophyllides composites induced upregulation of 43 and 56 differentially expressed genes (DEG) in MCF-7 and MDA-MB-231 cells, respectively. In both cell lines, chlorophyllides composites modulated the expression of annexin A4 (ANXA4), chemokine C-C motif receptor 1 (CCR1), stromal interaction molecule 2 (STIM2), ethanolamine kinase 1 (ETNK1) and member of RAS oncogene family (RAP2B). Further, the KEGG annotation revealed that chlorophyllides composites modulated DEGs that are associated with the endocrine system in MCF-7 cells and with the nervous system in MDA-MB-231 cells, respectively. The expression levels of 9 genes were validated by quantitative reverse transcription PCR (RT-qPCR). The expression of CCR1, STIM2, ETNK1, MAGl1 and TOP2A were upregulated in both chlorophyllides composites treated-MCF-7 and MDA-MB-231 cells. The different expression of NLRC5, SLC7A7 and PKN1 provided valuable information for future investigation and development of novel cancer therapy.

BUMPY STEM Is an Arabidopsis Choline/Ethanolamine Kinase Required for Normal Development and Chilling Responses.

Phospholipid biosynthesis is a core metabolic pathway that affects all aspects of plant growth and development. One of the earliest step in this pathway is mediated by choline/ethanolamine kinases (CEKs), enzymes in the Kennedy pathway that catalyze the synthesis of the polar head groups found on the most abundant plant phospholipids. The Arabidopsis genome encodes four CEKs. CEK1-3 have been well characterized using viable mutants while CEK4 encodes an essential gene, making it difficult to characterize its effects on plant development and responses to the environment. We have isolated an EMS-induced allele of CEK4 called bumpy stem (bst). bst plants are viable, allowing the effects of decreased CEK4 function to be characterized throughout the Arabidopsis life cycle. bst mutants have a range of developmental defects including ectopic stem growths at the base of their flowers, reduced fertility, and short roots and stems. They are also sensitive to cold temperatures. Supplementation with choline, phosphocholine, ethanolamine, and phosphoethanolamine rescues bst root phenotypes, highlighting the flow of metabolites between the choline and ethanolamine branches of the Kennedy pathway. The identification of bst and characterization of its phenotypes defines new roles for CEK4 that go beyond its established biochemical function as an ethanolamine kinase.

PI4-kinase and PfCDPK7 signaling regulate phospholipid biosynthesis in Plasmodium falciparum.

PfCDPK7 is an atypical member of the calcium-dependent protein kinase (CDPK) family and is crucial for the development of Plasmodium falciparum. However, the mechanisms whereby PfCDPK7 regulates parasite development remain unknown. Here, we perform quantitative phosphoproteomics and phospholipid analysis and find that PfCDPK7 promotes phosphatidylcholine (PC) synthesis by regulating two key enzymes involved in PC synthesis, phosphoethanolamine-N-methyltransferase (PMT) and ethanolamine kinase (EK). In the absence of PfCDPK7, both enzymes are hypophosphorylated and PMT is degraded. We further find that PfCDPK7 interacts with 4'-phosphorylated phosphoinositides (PIPs) generated by PI4-kinase. Inhibition of PI4K activity disrupts the vesicular localization PfCDPK7. P.falciparum PI4-kinase, PfPI4K is a prominent drug target and one of its inhibitors, MMV39048, has reached Phase I clinical trials. Using this inhibitor, we demonstrate that PfPI4K controls phospholipid biosynthesis and may act in part by regulating PfCDPK7 localization and activity. These studies not only unravel a signaling pathway involving PfPI4K/4'-PIPs and PfCDPK7 but also provide novel insights into the mechanism of action of a promising series of candidate anti-malarial drugs.

Hepatic metastasis of gastric cancer is associated with enhanced expression of ethanolamine kinase 2 via the p53\u2013Bcl-2 intrinsic apoptosis pathway

BackgroundGastric cancer (GC) with hepatic metastasis has a poor prognosis. Understanding the molecular mechanisms involved in hepatic metastasis may contribute to the development of sensitive diagnostic biomarkers and novel therapeutic strategies.MethodsWe performed transcriptome analysis of surgically resected specimens from patients with advanced GC. One of the genes identified as specifically associated with hepatic metastasis was selected for detailed analysis. GC cell lines with knockout of the candidate gene were evaluated in vitro and in vivo. Expression of the candidate gene was analysed in GC tissues from 300 patients.ResultsEthanolamine kinase 2 (ETNK2) was differentially upregulated in GC patients with hepatic metastasis. ETNK2 expression was elevated in GC cell lines derived from haematogenous metastases. ETNK2 knockout significantly suppressed proliferation, invasion, and migration; increased apoptosis; reduced Bcl-2 protein expression; and increased phosphorylated p53 expression. In mouse xenograft models, ETNK2 knockout virtually abolished hepatic metastasis. Stratification of GC patients based on ETNK2 mRNA level revealed significant associations between high ETNK2 tumour expression and both hepatic recurrence and worse prognosis.ConclusionsUpregulation of ETNK2 in GC enhances hepatic metastasis, possibly via dysregulation of p53–Bcl-2-associated apoptosis. ETNK2 expression may serve as a biomarker for predicting hepatic recurrence and a therapeutic target.

Kennedy pathway regulation by miR-16-1/15a cluster family during T cell activation

Abstract The Kennedy pathway in the endoplasmic reticulum is vital for the synthesis of phosphatidylethanolamine (PE) that is incorporated into cell membranes and lipid-based signaling molecules. The three steps of the Kennedy pathway are catalyzed by ethanolamine kinase, CTP-phosphoethanolamine Cytidyltransferase, and selenoprotein I (SELENOI). Our data suggest that two of the three enzymes (ethanolamine kinase and SELENOI) included in this pathway are upregulated during T cell activation as a mechanism for increasing PE, to support increased membrane synthesis and lipidated signaling proteins. Moreover, we have analyzed micro-RNAs (miRs) and found that miR-16-1 and miR-15a, which are linked in the genome and are co-expressed, have sequences predicted to target ethanolamine kinase and SELENOI. Thus, we hypothesized that levels of miR-16-1/15a decrease upon T cell activation to allow increased expression of ethanolamine kinase and SELENOI, thereby driving the increased synthesis of PE. Using real-time PCR and western blot analyses, we found that both miR-16-1 and miR-15a decreased, while levels of ethanolamine kinase and SELENOI increased in mouse primary T cells upon T cell receptor (TCR) activation. This coincided with increased levels of different PE species as measured by LC-MS. Overall, these data support the model of miR-16-1/15a regulation of the Kennedy pathway, in which miR-16-1/15a levels are higher in naïve T cells as a means to keep PE synthesis low. Upon TCR engagement, these miRs are reduced and all three enzymes are upregulated, thereby allowing increased PE synthesis required by activated T cells.

The Four Arabidopsis Choline/Ethanolamine Kinase Isozymes Play Distinct Roles in Metabolism and Development.

Phosphatidylcholine and phosphatidylethanolamine are two major phospholipid classes in eukaryotes. Each biosynthesis pathway starts with the phosphorylation of choline (Cho) or ethanolamine (Etn) catalyzed by either choline or ethanolamine kinase (CEK). Arabidopsis contains four CEK isoforms, but their isozyme-specific roles in metabolism and development are poorly described. Here, we showed that these four CEKs have distinct substrate specificities in vitro. While CEK1 and CEK2 showed substrate preference for Cho over Etn, CEK3 and CEK4 had clear substrate specificity for Cho and Etn, respectively. In vivo, CEK1, CEK2, and CEK3 exhibited kinase activity for Cho but not Etn, although the latter two isoforms showed rather minor contributions to total Cho kinase activity in both shoots and roots. The knockout mutants of CEK2 and CEK3 both affected root growth, and these isoforms had nonoverlapping cell-type-specific expression patterns in the root meristematic zone. In-depth phenotype analysis, as well as chemical and genetic complementation, revealed that CEK3, a Cho-specific kinase, is involved in cell elongation during root development. Phylogenetic analysis of CEK orthologs in Brassicaceae species showed evolutionary divergence between Etn kinases and Cho kinases. Collectively, our results demonstrate the distinct roles of the four CEK isoforms in Cho/Etn metabolism and plant development.

Abstract C022: Large scale phenotypic screening using PRISM, integrated with functional genomic screening and transcriptional profiling accelerates target identification of cytotoxic small molecules

Abstract A comprehensive analysis of FDA approved drugs has shown that drug discovery through phenotypic screening yields a higher percentage of first-in-class compounds, compared to canonical target-based drug discovery. This has led to a renewed interest in phenotypic screening to identify novel mechanisms of action. The PRISM technology of adding a unique DNA barcode to cancer cell lines has been able to reduce the cost and time associated with phenotypic viability screening. Using the PRISM method, we profiled the cytotoxic activity of over 4,100 bioactive compounds in 578 barcoded cancer cell lines at one dose, followed by 8-point dose of 1100 active compounds. The use of genomically characterized cell lines from the Cancer Cell Line Encyclopedia (CCLE) allows us to identify baseline features associated with sensitivity to a particular compound. One of the compounds from the screen that we selected for follow up was BRD-K01353379, which killed 28 cancer cell lines of diverse lineages and showed a correlation between high expression of Ethanolamine Kinase 1 (ETNK1) and reduction of cell viability. Interestingly, BRD-3379 showed a similar PRISM cytotoxic profile to 2,3-DCPE, that was screened in our Repurposing effort. 2,3-DCPE is structurally similar to BRD-3379 and has been reported to induce cytotoxicity through upregulation of p21 and downregulation of Bcl-XL. To elucidate the mechanism of action of this compound we performed a genome-wide CRISPR-Cas9 resistance screen to identify molecular targets that are required for sensitivity to BRD-3379. Here, we found that loss of expression of the PRISM biomarker ETNK1 conferred resistance in a BRD-3379 sensitive cell line, JHH-7. Subsequently, we profiled metabolites in a cell line sensitive to BRD-3379 and found that the compound was phosphorylated. We confirmed in a biochemical assay that BRD-3379 is in fact a substrate of recombinant ETNK1, which phosphorylates the ethanolamine moiety of BRD-3379. Next, we transcriptionally profiled the effect of BRD-3379 by bulk RNAseq in a sensitive and non-sensitive cell line, as well as in a pool of 100 cell lines using single cell RNAseq [McFarland et al., manuscript in preparation]. Both bulk RNAseq and single cell transcriptional analysis showed that upregulation of both p21 and p57 is strongly associated with sensitivity to BRD-3379. This mechanism of action is similar to that of 2,3-DCPE. Here, we have demonstrated that by combining large-scale cell line viability screening, genomic characterization of these cell lines, genome wide CRISPR-Cas9 resistance screens, transcriptional profiling, and biochemical assays, we can facilitate rapid identification of molecular targets of small molecules and their respective mechanism of action by which they induce cytotoxicity. Citation Format: Kevin Larpenteur, Lucian de Waal, Li Wang, Mustafa Kocak, Jordan Bryan, Samantha Bender, Sulyman Barkho, Matt Kukurugya, Amy Goodale, Brenton Paolella, Min Cho, James McFarland, Michael Rothberg, Cong Zhu, Colin Trepiccio, Aviad Tsherniak, David Root, Bryson Bennett, Federica Piccioni, Josh Bittker, James Rong, Christopher Mader, David Stokoe, Ari Firestone, Todd R Golub, Jennifer Roth. Large scale phenotypic screening using PRISM, integrated with functional genomic screening and transcriptional profiling accelerates target identification of cytotoxic small molecules [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C022. doi:10.1158/1535-7163.TARG-19-C022

MiR-199a-3p targets ETNK1 to promote invasion and migration in gastric cancer cells and is associated with poor prognosis

PurposeTo investigate the prognostic significance of miR-199a-3p and its role in invasion and metastasis in gastric cancer. MethodsmiR-199a-3p expression in 436 formalin-fixed and 39 frozen gastric cancer tissues was investigated by in situ hybridization and RT-PCR, respectively. The role of miR-199a-3p in the migration and invasion of gastric cancer cells was determined in overexpression and inhibitor studies using transwell assays and the SGC-7901, BGC-823 and MGC-803 gastric cancer cells lines. The effect of miR-199a-3p expression on ethanolamine kinase 1 (ETNK1) levels was determined by western botting. ResultsmiR-199a-3p was significantly up-regulated in AGS, SGC-7901, BGC-823 and MGC-803 gastric cancer cells, when compared with GES-1 non-malignant gastric epithelial cells. In situ hybridization studies revealed that human non-tumor gastric mucosa samples were negative for miR-199a-3p expression, while 162 of 436 (37.16%) cases of gastric cancer demonstrated positive expression. miR-199a-3p overexpression was associated with tumor size, Lauren classification, depth of invasion, lymph node and distant metastasis, TNM stage and prognosis. In patients with I, II and III stage tumors, high miR-199a-3p expression was associated with a significantly lower 5-year survival rate. miR-199a-3p overexpression was associated with increased cell migration and invasion. ETNK1 expression was inhibited following miR-199a-3p overexpression in BGC-823 and SGC-7901 cells, and elevated following miR-199a-3p suppression in MGC-803 cells. ConclusionmiR-199a-3p is highly expressed in gastric cancer, and correlates with invasion, metastasis and prognosis. miR-199a-3p regulates the invasion and migration of gastric cancer cells by targeting ETNK1. Consequently, miR-199a-3p may serve as a prognostic indicator in gastric cancer.

A rapid and adaptable lipidomics method for quantitative UPLC-mass spectrometric analysis of phosphatidylethanolamine and phosphatidylcholine in vitro, and in cells.

Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are highly prevalent phospholipids in mammalian membranes. There are currently no methods for detection of minute levels of these phospholipids or simultaneously with products of the utilization of these phospholipid substrates by phospholipase A2 (PLA2) enzymes. To examine the substrate utilization of PE and PC by PLA2, we developed a method to accurately detect and measure specific forms of PE and PC as low as 50 femtomoles. Validation of this method consisted of an enzymatic assay to monitor docosahexaenoic acid and arachidonic acid release from the hydrolysis of PE and PC by group IV phospholipase A2 (cPLA2α) coupled to the generation of lyso-PE (LPE) and lyso-PC (LPC). In addition, the PE and PC profiles of RAW 264.7 macrophages were monitored with zymosan/lipopolysaccharide-treatment. Finally, genetic validation for the specificity of the method consisted of the downregulation of two biosynthetic enzymes responsible for the production of PE and PC, choline kinase A (CHKA) and ethanolamine kinase 1 (ETNK1). This new UPLC ESI-MS/MS method provides accurate and highly sensitive detection of PE and PC species containing AA and DHA allowing for the specific examination of the substrate utilization of these phospholipids by PLA2 in vitro and in cells.

Molecular causes of elevated phosphoethanolamine in breast and pancreatic cancer cells.

Elevated phosphoethanolamine (PE) is frequently observed in MRS studies of human cancers and xenografts. The role of PE in cell survival and the molecular causes underlying this increase are, however, relatively underexplored. In this study, we investigated the roles of ethanolamine kinases (Etnk-1 and 2) and choline kinases (Chk-α and β) in contributing to increased PE in human breast and pancreatic cancer cells. We investigated the effect of silencing Etnk-1 and Etnk-2 on cell viability as a potential therapeutic strategy. Both breast and pancreatic cancer cells showed higher PE compared with their nonmalignant counterparts. We identified Etnk-1 as a major cause of the elevated PE levels in these cancer cells, with little or no contribution from Chk-α, Chk-β, or Etnk-2. The increase of PE observed in pancreatic cancer cells in culture was replicated in the corresponding tumor xenografts. Downregulation of Etnk-1 with siRNA resulted in cell cytotoxicity that correlated with PE levels in breast and pancreatic cancer cells. Etnk-1 may provide a potential therapeutic target in breast and pancreatic cancers.

Mutant IDH1 gliomas downregulate phosphocholine and phosphoethanolamine synthesis in a 2-hydroxyglutarate-dependent manner

BackgroundMagnetic resonance spectroscopy (MRS) studies have identified elevated levels of the phospholipid precursor phosphocholine (PC) and phosphoethanolamine (PE) as metabolic hallmarks of cancer. Unusually, however, PC and PE levels are reduced in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas that produce the oncometabolite 2-hydroxyglutarate (2-HG) relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was to determine the molecular mechanism underlying this unusual metabolic reprogramming in IDHmut gliomas.MethodsSteady-state PC and PE were quantified using 31P-MRS. To quantify de novo PC and PE synthesis, we used 13C-MRS and measured flux to 13C-PC and 13C-PE in cells incubated with [1,2-13C]-choline and [1,2-13C]-ethanolamine. The activities of choline kinase (CK) and ethanolamine kinase (EK), the enzymes responsible for PC and PE synthesis, were quantified using 31P-MR-based assays. To interrogate the role of 2-HG, we examined IDHwt cells incubated with 2-HG and, conversely, IDHmut cells treated with the IDHmut inhibitor AGI-5198. To examine the role of hypoxia-inducible factor 1-α (HIF-1α), we silenced HIF-1α using RNA interference. To confirm our findings in vivo and in the clinic, we studied IDHwt and IDHmut orthotopic tumor xenografts and glioma patient biopsies.ResultsDe novo synthesis of PC and PE was reduced in IDHmut cells relative to IDHwt. Concomitantly, CK activity and EK activity were reduced in IDHmut cells. Pharmacological manipulation of 2-HG levels established that 2-HG was responsible for reduced CK activity, EK activity, PC and PE. 2-HG has previously been reported to stabilize levels of HIF-1α, a known regulator of CK activity. Silencing HIF-1α in IDHmut cells restored CK activity, EK activity, PC and PE to IDHwt levels. Our findings were recapitulated in IDHmut orthotopic tumor xenografts and, most importantly, in IDHmut patient biopsies, validating our findings in vivo and in the clinic.ConclusionsThis study identifies, to our knowledge for the first time, a direct role for 2-HG in the downregulation of CK and EK activity, and thereby, PC and PE synthesis in IDHmut gliomas. These results highlight the unusual reprogramming of phospholipid metabolism in IDHmut gliomas and have implications for the identification of MRS-detectable metabolic biomarkers associated with 2-HG status.

AP4 Transcription Factor Binding Site is a Repressor Element in ek2 Promoter of Human Liver Carcinoma Cell Line, HepG2

Ethanolamine kinase (EK) is the first enzyme in the Kennedy pathway for the biosynthesis of phosphatidylethanolamine. Although EK has been reported to be involved in phospholipid biosynthesis, carcinogenesis, cell growth, muscle development and sex determination during embryonic development, little is known about its transcriptional regulation by endogenous or exogenous signals. Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. Compared to ek1 gene, ek2 is expressed at a higher level in liver and EK2 isoforms also accept choline as substrate besides ethanolamine, which could contribute to liver carcinogenesis. The main aim of this study was to analyze and characterize the human ek2 promoter in cultured mammalian cells. Human ek2 (2011 bp) promoter was cloned into reporter vector, pGL4.10 [luc2] and the promoter activities were studied in human liver carcinoma (HepG2 cells). Sequence analyses showed that ek2 promoter contains numerous putative transcription factor binding sites including AP4 and it is devoid of a recognizable consensus TATA box but it contains a high number of guanine (G) and cytosine (C) nucleotides. PCR mutagenesis of three nucleotides at E-box motif of AP4 transcription binding site located between -293 and -276 of ek2 promoter was successfully performed to show that AP4 transcription factor binding site acts as a repressive element in the regulation of ek2 expression. AP4 upregulation has been implicated in bad prognosis of carcinoma, therefore the regulatory role of AP4 binding site reported in this study could be a link between ek2 and carcinogenesis. Although further studies need to be carried out to understand and to determine the repression mechanism of AP4 in ek2 promoter, the characterization and analysis of ek promoter performed in this study provide important understanding of its basal transcriptional regulation which would allow us to control ek expression levels in pathologic conditions that involve this gene.

Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death.

Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite’s growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase.

Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells.

BackgroundEthanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied.Methodology/Principal FindingsIn this report, we mapped the important regulatory regions in the human ek1 promoter. 5’ deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells.Conclusion/SignificanceIn conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner.