Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Activation of phosphatidylcholine-cycle enzymes in human epithelial ovarian cancer cells

ABSTRACT: Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) can provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. Biochemical, protein and mRNA expression analyses demonstrated that the increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and non-tumoral immortalized counterparts (EONT) mainly rely upon: 1) ChoK activation, consistent with higher protein content and increased ChoK? mRNA expression levels; 2) PC-plc activation, consistent with higher, previously reported, protein expression. More limited and variable sources of PCho could derive, in some EOC cells, from activation of Phospholipase D or GPC-pd. Phospholipase A2 activity and isoforms’ expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoK? mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from EOC patients. Overall, we demonstrated that the elevated PCho pool detected in EOC cells primarily resulted from the upregulation/activation of ChoK and PC-plc involved in the biosynthetic and in a degradative pathway of the PC-cycle, respectively. 20 EOC frozen surgical specimens, 8 EOC cell lines and Ovarian Surface Epithelial (OSE) cells (4 different preparations, 3 of them pooled in one sample).

ORGANISM(S): Homo sapiens

SUBMITTER: Loris De Cecco

PROVIDER: E-GEOD-19352 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Activation of phosphatidylcholine cycle enzymes in human epithelial ovarian cancer cells.

Iorio Egidio E Ricci Alessandro A Bagnoli Marina M Pisanu Maria Elena ME Castellano Giancarlo G Di Vito Massimo M Venturini Elisa E Glunde Kristine K Bhujwalla Zaver M ZM Mezzanzanica Delia D Canevari Silvana S Podo Franca F

Cancer research 20100223 5

Altered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) could provide choline-based imaging approaches as powerful tools to improve diagnosis and identify new therapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho) in EOC compared with normal and nontumoral immortalized counterparts (EONT) may derive from (a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation, (b) increased PC-specific phospholipase C (P ...[more]

PMID: 20179205

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Project description:Previously, we have identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that GALNT3 is strongly overexpressed in both LMP and HG serous EOC tumors, thus suggesting that epigenetic mechanisms might be implicated in GALNT3 overexpression in serous epithelial ovarian cancer (EOC). Moreover, GALNT3 expression significantly correlated with shorter progression-free survival (PFS) periods in serous EOC patients with advanced disease. Knockdown of the GALNT3 expression in EOC cells led to sharp decrease of cell proliferation and induced S-phase cell cycle arrest. Additionally, GALNT3 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon GALNT3 suppression, while some tumor suppressor genes were induced. Moreover, GALNT3 downregulation was associated with reduced MUC1 protein expression in EOC cells, probably related to destabilization of the MUC1 protein due to lack of GALNT3 glycosylation activity.Taken together, our data are indicative for a strong oncogenic potential of the GALNT3 gene in advanced EOC and identify this transferase as a novel EOC biomarker and putative EOC therapeutic target. Our findings also suggest that GALNT3 overexpression might contribute to ovarian etiology through aberrant mucin O-glycosylation. To better understand the molecular mechanisms of GALNT3 gene action in ovarian cancer cells, we employed the Agilent Whole Human Genome microarrays, containing ~ 44,000 genes to identify global gene expression changes upon GALNT3 suppression in A2780s cells. We compared the gene expression of the previously selected clone shRNA- GALNT3-knockdown clones 1 & 2 (sh-cl1 & sh-cl2) against the corresponding control (ctrl) clone. The microarray experiments were performed in duplicates, as four hybridizations were carried out for the GALNT3-suppressing cell clones against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.

Project description:Detection of the abnormal phosphatidylcholine (PtdCho) metabolism in EOC by analysis of magnetic resonance profile (MRS), showed a significant increase of PCho content in EOC cells as compared with non tumoral counterparts associated with activation of choline kinase (ChoK), the enzyme responsible for PCho production following choline phosphorylation in the PtdCho biosynthetic pathway (Kennedy’s pathway). The α-isoform of ChoK has an essential role in growth control and signal transduction and it has been implicated in the carcinogenic process. Aim of the study is the evaluation of the biological relevance of ChoK expression and activity to define its possible role as a non-invasively detectable EOC biomarker and druggable target. We specifically silenced ChoKα expression by transient RNA interference in two different EOC cell lines and evaluated the biological effects related to metabolic profiles, cell proliferation, cell cycle regulation and alteration of global gene expression to possibly identify new pathways implicated in altered choline metabolism. Following ChoKα silencing we observed a 70% reduction of mRNA and protein expression with a similar reduction in PCho accumulation as assessed by HMRS analysis. The ChoKα silencing was accompanied by 20% inhibition of cell growth and similar percentage of cells blocked in the G1-phase of cell cycle. No alteration in cell morphology and modulation of the main survival signaling pathways (PI3K and MAPK-related signaling) were observed. By gene expression analysis we found 476 differentially expressed genes (p< 0.01; FDR 25%) in silenced cells. By transcriptome analysis of CHKA silenced cells as compared to controls, among the most relevant co-repressed genes we found CyclinA, related to regulation of cell cycle progression, and cytokines genes (IL-6 and IL-8) related to inflammation and EOC aggressiveness. Acyl-CoA synthetase medium-chain family member 3 (ACSM3), phosphatidic acid phosphatase type 2 (PPAP2A) and Osteoprotegerin were among the most up-regulated genes. All co-modulated genes have been validated by RTqPCR also in independent biological replicates. Ingenuity System Pathways Analysis (IPA) identified a network of molecules most significantly affected by CHKA silencing whose main functions is related to cell morphology cellular assembly and organization, cellular function and maintenance. Also, the cellular functions predicted to be mostly affected by silencing were cellular movement and cell death that are expected to be respectively decreased and increased in silenced cells. a total of 12 samples were analyzed: Two EOC cell line SKOV3 and INTOV11 were transiently silenced with unrelated or CHKA-specific siRNA pool. Three independent experiment were run for each cell line.

Dataset Information

Activation of phosphatidylcholine-cycle enzymes in human epithelial ovarian cancer cells

Publications

Activation of phosphatidylcholine cycle enzymes in human epithelial ovarian cancer cells.

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