Egidio Iorio

Alterations of Choline Phospholipid Metabolism in Ovarian Tumor Progression

Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholine-producing enzymes, and uptake of [methyl-14C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the 1H NMR total choline resonance (3.20-3.24 ppm) revealed intracellular [phosphocholine] and [total choline] of 2.3 +/- 0.9 and 5.2 +/- 2.4 nmol/10(6) cells, respectively, with a glycerophosphocholine/phosphocholine ratio of 0.95 +/- 0.93 in EONT cells; average [phosphocholine] was 3- to 8-fold higher in EOC cells (P < 0.0001), becoming the predominant phosphatidylcholine metabolite, whereas average glycerophosphocholine/phosphocholine values decreased significantly to < or =0.2. Two-dimensional (phosphocholine/total choline, [total choline]) and (glycerophosphocholine/total choline, [total choline]) maps allowed separate clustering of EOC from EONT cells (P < 0.0001, 95% confidence limits). Rates of choline kinase activity in EOC cells were 12- to 24-fold higher (P < 0.03) than those in EONT cells (basal rate, 0.5 +/- 0.1 nmol/10(6) cells/h), accounting for a consistently elevated (5- to 15-fold) [methyl-14C]choline uptake after 1-hour incubation (P < 0.0001). The overall activity of phosphatidylcholine-specific phospholipase C and phospholipase D was also higher ( approximately 5-fold) in EOC cells, suggesting that both biosynthetic and catabolic pathways of the phosphatidylcholine cycle likely contribute to phosphocholine accumulation. Evidence of abnormal phosphatidylcholine metabolism might have implications in EOC biology and might provide an avenue to the development of noninvasive clinical tools for EOC diagnosis and treatment follow-up.

Activation of Phosphatidylcholine Cycle Enzymes in Human Epithelial Ovarian Cancer Cells

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 (PC-plc) activity, and (c) increased intracellular choline production by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or by phospholipase D (pld)-mediated PC catabolism followed by choline phosphorylation. Biochemical, protein, and mRNA expression analyses showed that the most relevant changes in EOC cells were (a) 12-fold to 25-fold ChoK activation, consistent with higher protein content and increased ChoKalpha (but not ChoKbeta) mRNA expression levels; and (b) 5-fold to 17-fold PC-plc activation, consistent with higher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassium xanthate (D609) in OVCAR3 and SKOV3 cancer cells induced a 30% to 40% reduction of PCho content and blocked cell proliferation. More limited and variable sources of PCho could derive, in some EOC cells, from 2-fold to 4-fold activation of pld or GPC-pd. Phospholipase A2 activity and isoform expression levels were lower or unchanged in EOC compared with EONT cells. Increased ChoKalpha mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgical specimens isolated from patients with EOC. Overall, we showed that the elevated PCho pool detected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involved in PC biosynthesis and degradation, respectively.

Phosphatidylcholine-Specific Phospholipase C Activation in Epithelial Ovarian Cancer Cells

Elucidation of the mechanisms responsible for aberrant phosphatidylcholine (PC) metabolism in cancer cells may allow identification of novel biomarkers of tumor progression and design of new targeted anticancer therapies. We recently reported up-regulation of PC-specific phospholipases in epithelial ovarian cancer cells (EOC) compared with nontumoral (normal or immortalized) counterparts (EONT). In the present study, we focused, in the same cell systems, on levels, subcellular localization, and activity of PC-specific phospholipase C (PC-PLC), for which a key role in cell proliferation, differentiation, and apoptosis has been shown in several mammalian cells. A 66-kDa PC-PLC isoform, detected in nuclear and cytoplasmic compartments of both EOC and EONT cells, accumulated on the external plasma membrane of cancer cells only, where it colocalized with beta1 integrin, in nonraft membrane domains. PC-PLC activity was 3-fold higher in total cell lysates and 5-fold higher in membrane-enriched fractions of EOC compared with EONT cells. Serum deprivation induced in EOC, but not in EONT, cells a 3-fold decrease in PC-PLC activity, associated with a 40% drop in S-phase fraction. The recovery of both variables to their original levels in serum-restimulated (or lysophosphatidic acid-restimulated) EOC cells was strongly delayed, for at least 24 h, in the presence of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609). The S-phase of serum-restimulated EONT cells was not sensitive to D609. These findings warrant further investigations on the role of PC-PLC and on the effects of its inhibition on the pathways responsible for constitutive EOC cell stimulation and cell proliferation.

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

An altered redox balance mediates the hypersensitivity of Cockayne syndrome primary fibroblasts to oxidative stress.

Cockayne syndrome (CS) is a rare hereditary multisystem disease characterized by neurological and development impairment, and premature aging. Cockayne syndrome cells are hypersensitive to oxidative stress, but the molecular mechanisms involved remain unresolved. Here we provide the first evidence that primary fibroblasts derived from patients with CS‐A and CS‐B present an altered redox balance with increased steady‐state levels of intracellular reactive oxygen species (ROS) and basal and induced DNA oxidative damage, loss of the mitochondrial membrane potential, and a significant decrease in the rate of basal oxidative phosphorylation. The Na/K‐ATPase, a relevant target of oxidative stress, is also affected with reduced transcription in CS fibroblasts and normal protein levels restored upon complementation with wild‐type genes. High‐resolution magnetic resonance spectroscopy revealed a significantly perturbed metabolic profile in CS‐A and CS‐B primary fibroblasts compared with normal cells in agreement with increased oxidative stress and alterations in cell bioenergetics. The affected processes include oxidative metabolism, glycolysis, choline phospholipid metabolism, and osmoregulation. The alterations in intracellular ROS content, oxidative DNA damage, and metabolic profile were partially rescued by the addition of an antioxidant in the culture medium suggesting that the continuous oxidative stress that characterizes CS cells plays a causative role in the underlying pathophysiology. The changes of oxidative and energy metabolism offer a clue for the clinical features of patients with CS and provide novel tools valuable for both diagnosis and therapy.

Inhibition of phosphatidylcholine-specific phospholipase C downregulates HER2 overexpression on plasma membrane of breast cancer cells

IntroductionOverexpression on plasma membrane of human epidermal growth factor receptor 2 (HER2) is reported in 25% to 30% of breast cancers. Heterodimer formation with cognate members of the epidermal growth factor receptor (EGFR) family, such as HER3 and EGFR, activates abnormal cell-signalling cascades responsible for tumorigenesis and further transcriptional HER2 gene upregulation. Targeting the molecular mechanisms controlling HER2 overexpression and recycling may effectively deactivate this feedback-amplification loop. We recently showed that inactivation of phosphatidylcholine-specific phospholipase C (PC-PLC) may exert a pivotal role in selectively modulating the expression on the membrane of specific receptors or proteins relevant to cell function. In the present study, we investigated the capability of PC-PLC inhibition to target the molecular mechanisms controlling HER2 overexpression on the membrane of breast cancer cells by altering the rates of its endocytosis and lysosomal degradation.MethodsLocalization on the membrane and interaction of PC-PLC with HER2, EGFR, and HER3 were investigated on HER2-overexpressing and HER2-low breast cancer cell lines, by using confocal laser scanning microscopy, flow cytometry, cell-surface biotinylation, isolation of lipid rafts, and immunoprecipitation experiments. The effects of the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609) on HER2 expression on the membrane and on the levels of overall HER2, HER2-HER3, and HER2-EGFR contents were monitored in the HER2-overexpressing SKBr3 cells, after either transient or continuous receptor engagement with anti-HER2 monoclonal antibodies, including trastuzumab. Changes of HER2 expression and cell proliferation were examined in SKBr3, BT-474, and MDA-MB-453 cells continuously exposed to D609 alone or combined with trastuzumab.ResultsPC-PLC selectively accumulates on the plasma membrane of HER2-overexpressing cells, where it colocalizes and associates with HER2 in raft domains. PC-PLC inhibition resulted in enhanced HER2 internalization and lysosomal degradation, inducing downmodulation of HER2 expression on the membrane. Moreover, PC-PLC inhibition resulted in strong retardation of HER2 reexpression on the membrane and a decrease in the overall cellular contents of HER2, HER2-HER3, and HER2-EGFR heterodimers. The PC-PLC inhibitor also induced antiproliferative effects, especially in trastuzumab-resistant cells.ConclusionsThe results pointed to PC-PLC inhibition as a potential means to counteract the tumorigenic effects of HER2 amplification and complement the effectiveness of current HER2-targeting therapies.

MR evaluation of response to targeted treatment in cancer cells.

The development of molecular technologies, together with progressive sophistication of molecular imaging methods, has allowed the further elucidation of the multiple mutations and dysregulatory effects of pathways leading to oncogenesis. Acting against these pathways by specifically targeted agents represents a major challenge for current research efforts in oncology. As conventional anatomically based pharmacological endpoints may be inadequate to monitor the tumor response to these targeted treatments, the identification and use of more appropriate, noninvasive pharmacodynamic biomarkers appear to be crucial to optimize the design, dosage and schedule of these novel therapeutic approaches. An aberrant choline phospholipid metabolism and enhanced flux of glucose derivatives through glycolysis, which sustain the redirection of mitochondrial ATP to glucose phosphorylation, are two major hallmarks of cancer cells. This review focuses on the changes detected in these pathways by MRS in response to targeted treatments. The progress and limitations of our present understanding of the mechanisms underlying MRS‐detected phosphocholine accumulation in cancer cells are discussed in the light of gene and protein expression and the activation of different enzymes involved in phosphatidylcholine biosynthesis and catabolism. Examples of alterations induced in the MRS choline profile of cells exposed to different agents or to tumor environmental factors are presented. Current studies aimed at the identification in cancer cells of MRS‐detected pharmacodynamic markers of therapies targeted against specific conditional or constitutive cell receptor stimulation are then reviewed. Finally, the perspectives of present efforts addressed to identify enzymes of the phosphatidylcholine cycle as possible novel targets for anticancer therapy are summarized. Copyright

Abnormal Choline Phospholipid Metabolism in Breast and Ovary Cancer:Molecular Bases for Noninvasive Imaging Approaches

Elevated contents of choline phospholipid metabolites are typically detected by nuclear magnetic resonance spectroscopy (MRS) in human and animal tumors. An increase in the intensity of the 1 H-MRS profile of total cholinecontaining compounds (tCho, 3.2 ppm) is today considered as a common feature in different types of cancer, beyond their otherwise wide phenotypic variability. This finding fostered investigations on the molecular mechanisms underlying the observed spectral changes and on correlations between aberrant phospholipid metabolism and tumor progression. At the clinical level, efforts are addressed to evaluate effectiveness and potential use of in vivo localized MRS and choline-based positron emission tomography (Cho-PET) in cancer diagnosis. Aims of this article are: a) to overview recent advances in the identification of biochemical pathways responsible for the altered 1 H-MRS tCho profile in breast and ovary cancer cells, as a basis for interpreting in vivo MR spectra and enhanced uptake of radiolabeled choline in PET; b) to summarize recent developments of in vivo 1 H-MRS methods in breast cancer diagnosis; c) to discuss the potentialities of complementing current diagnostic modalities with noninvasive MRS and Cho-PET methods to monitor biochemical alterations associated with progression, relapse and therapy response in ovary cancer.

GLYCOLYTIC PHENOTYPE AND AMP KINASE MODIFY THE PATHOLOGIC RESPONSE OF TUMOR XENOGRAFTS TO VEGF NEUTRALIZATION

VEGF antagonists are now widely used cancer therapeutics, but predictive biomarkers of response or toxicity remain unavailable. In this study, we analyzed the effects of anti-VEGF therapy on tumor metabolism and therapeutic response by using an integrated set of imaging techniques, including bioluminescence metabolic imaging, 18-fluorodeoxyglucose positron emission tomography, and MRI imaging and spectroscopy. Our results revealed that anti-VEGF therapy caused a dramatic depletion of glucose and an exhaustion of ATP levels in tumors, although glucose uptake was maintained. These metabolic changes selectively accompanied the presence of large necrotic areas and partial tumor regression in highly glycolytic tumors. In addition, we found that the central metabolic protein kinase AMP-activated protein kinase (AMPK)-a cellular sensor of ATP levels that supports cell viability in response to energy stress-was activated by anti-VEGF therapy in experimental tumors. AMPK-α2 attenuation increased glucose consumption, tumor cell sensitivity to glucose starvation, and tumor necrosis following anti-VEGF therapy. Taken together, our findings reveal functional links between the Warburg effect and the AMPK pathway with therapeutic responses to VEGF neutralization in tumor xenograft models.

Inhibition of phosphatidylcholine-specific phospholipase C results in loss of mesenchymal traits in metastatic breast cancer cells

IntroductionAcquisition of mesenchymal characteristics confers to breast cancer (BC) cells the capability of invading tissues different from primary tumor site, allowing cell migration and metastasis. Regulators of the mesenchymal-epithelial transition (MET) may represent targets for anticancer agents. Accruing evidence supports functional implications of choline phospholipid metabolism in oncogene-activated cell signaling and differentiation. We investigated the effects of D609, a xanthate inhibiting phosphatidylcholine-specific phospholipase C (PC-PLC) and sphingomyelin synthase (SMS), as a candidate regulator of cell differentiation and MET in the highly metastatic BC cell line MDA-MB-231.MethodsPC-PLC expression and activity were investigated using confocal laser scanning microscopy (CLSM), immunoblotting and enzymatic assay on human MDA-MB-231 compared with MCF-7 and SKBr3 BC cells and a nontumoral immortalized counterpart (MCF-10A). The effects of D609 on PC-PLC and SMS activity, loss of mesenchymal markers and changes in migration and invasion potential were monitored in MDA-MB-231 cells by enzymatic assays, CLSM, immunoblotting and transwell chamber invasion combined with scanning electron microscopy examinations. Cell proliferation, formation and composition of lipid bodies and cell morphology were investigated in D609-treated BC cells by cell count, CLSM, flow-cytometry of BODIPY-stained cells, nuclear magnetic resonance and thin-layer chromatography.ResultsPC-PLC (but not phospholipase D) showed 2- to 6-fold activation in BC compared with nontumoral cells, the highest activity (up to 0.4 pmol/μg protein/min) being detected in the poorly-differentiated MDA-MB-231 cells. Exposure of the latter cells to D609 (50 μg/mL, 24-72 h) resulted into 60-80% PC-PLC inhibition, while SMS was transiently inhibited by a maximum of 21%. These features were associated with progressive decreases of mesenchymal traits such as vimentin and N-cadherin expression, reduced galectin-3 and milk fat globule EGF-factor 8 levels, β-casein formation and decreased in vitro cell migration and invasion. Moreover, proliferation arrest, changes in cell morphology and formation of cytosolic lipid bodies typical of cell differentiation were induced by D609 in all investigated BC cells.ConclusionsThese results support a critical involvement of PC-PLC in controlling molecular pathways responsible for maintaining a mesenchymal-like phenotype in metastatic BC cells and suggests PC-PLC deactivation as a means to promote BC cell differentiation and possibly enhance the effectiveness of antitumor treatments.