Activation of phosphatidylcholine-cycle enzymes in humanepithelial ovarian cancer cells

Egidio Iorio1,a, Alessandro Ricci1,a, Marina Bagnoli2, Maria Elena Pisanu1, GiancarloCastellano2, Massimo Di Vito1, Elisa Venturini3, Kristine Glunde4, Zaver M. Bhujwalla4, DeliaMezzanzanica2, Silvana Canevari2,*, and Franca Podo1,*1 Department of Cell Biology and Neurosciences, Section of Molecular and Cellular Imaging, IstitutoSuperiore di Sanità, Rome, Italy2 Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS IstitutoNazionale dei Tumori, Milan, Italy3 Cogentech-Consortium for Genomic Technologies, Milan, Italy4 JHU ICMIC Program, Russell H. Morgan Department of Radiology and Radiological Science,Johns Hopkins University School of Medicine, Baltimore, MD 21205

AbstractAltered phosphatidylcholine (PC) metabolism in epithelial ovarian cancer (EOC) can providecholine-based imaging approaches as powerful tools to improve diagnosis and identify newtherapeutic targets. The increase in the major choline-containing metabolite phosphocholine (PCho)in EOC compared with normal and non-tumoral immortalized counterparts (EONT), may derivefrom a) enhanced choline transport and choline kinase (ChoK)-mediated phosphorylation; b)increased PC-specific phospholipase C (PC-plc) activity; c) increased intracellular cholineproduction by PC deacylation plus glycerophosphocholine-phosphodiesterase (GPC-pd) or byphospholipase D (pld)-mediated PC catabolism, followed by choline phosphorylation. Biochemical,protein and mRNA expression analyses demonstrated that the most relevant changes in EOC cellswere: 1) 12- to 25-fold ChoK activation, consistent with higher protein content and increasedChoKα (but not ChoKβ) mRNA expression levels; 2) 5- to 17-fold PC-plc activation, consistent withhigher, previously reported, protein expression. PC-plc inhibition by tricyclodecan-9-yl-potassiumxanthate (D609) induced in OVCAR3 and SKOV3 cancer cells a 30-to-40% reduction of PChocontent and blocked cell proliferation. More limited and variable sources of PCho could derive, insome EOC cells, from 2- to 4-fold activation of pld or GPC-pd. Phospholipase A2 activity andisoforms’ expression levels were lower or unchanged in EOC compared with EONT cells. IncreasedChoKα mRNA, as well as ChoK and PC-plc protein expression, were also detected in surgicalspecimens isolated from EOC patients. Overall, we demonstrated that the elevated PCho pooldetected in EOC cells primarily resulted from upregulation/activation of ChoK and PC-plc involvedin the biosynthetic and in a degradative pathway of the PC-cycle, respectively.

Requests for reprints: Franca Podo, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299,00161 Rome, Italy. Phone +39-06-49902686; Fax +39-06-49387144; franca.podo@iss.it or Silvana Canevari, Department ofExperimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133 Milan, Italy.Phone +39-02-23902567; Fax +39-02-23903073; silvana.canevari@istitutotumori.mi.it.aE. Iorio and A. Ricci contributed equally to this work.Potential conflicts of interest: No conflicts of interest are disclosed.

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Published in final edited form as:Cancer Res. 2010 March 1; 70(5): 2126–2135. doi:10.1158/0008-5472.CAN-09-3833.

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Keywordsovarian cancer; phosphatidylcholine metabolism; NMR; choline kinase; phospholipase D;phospholipase C; glycerophosphocholine-phosphodiesterase

IntroductionDespite progress in clinical oncology, epithelial ovarian cancer (EOC) continues to be thegynecological malignancy with the highest death rate in industrialized countries and a 5-yearsurvival as low as 44% [1]. Suitable molecular imaging approaches could facilitate elucidationof mechanisms of EOC progression, improve clinical diagnosis and follow-up, and identifynew therapeutic targets.

Detection of an aberrant phosphatidylcholine (PC) metabolism in tumors by magneticresonance spectroscopy (MRS) [2,3] allowed for the identification of novel indicators of invivo tumor progression by using choline-based MRS and positron emission tomography (PET)[4–8]. An elevation of the 1H MRS resonance at 3.2 ppm, mainly due to headgroups of choline-containing metabolites (tCho), is a common feature in several cancers including EOC [9–17].Changes of the tCho spectral profile reflect altered contents and fluxes of phosphocholine(PCho), glycerophosphocholine (GPC) and free choline (Cho) in the PC-cycle (Fig. 1A).

Major mechanisms of PCho accumulation in tumor cells include enhanced choline transportand choline kinase (ChoK)-mediated phosphorylation and activation of PC-specificphospholipases [13,15,17–19]. These biochemical features can represent fingerprints oftumour progression and potential therapeutic targets [6,20–22].

Previous studies demonstrated that ChoK is upregulated by oncogenes, growth factors andcarcinogens [22,23] and its ChoKα isoform can be constitutively activated in human tumorcells [24], in which it may act as prognostic factor [25,26]. Furthermore, specificpharmacologic or si-RNA ChoK inhibition have antiproliferative effects on cancer cells [18,27].

A neutral-active PC-specific phospholipase C (PC-plc) is also activated in EOC compared withnon-tumoral EONT cells [15,28], inhibition of this enzyme being associated with reducedresponse of EOC cells to mitogens [28].

To further elucidate the mechanisms underlying the altered tCho profile in EOC cells, we herereport on measurements of absolute activity rates of ChoK, PC-plc, phospholipase D (pld), andglycerophosphocholine-phosphodiesterase (GPC-pd), as well as on differential metabolicfluxes through the PC deacylation pathway in EOC and EONT cells. Comparative analyses ofmRNA expression levels were also performed for: ChoKα and ChoKβ isoforms,citydylyltransferase (ct) and phosphocholine transferase (pct), pld1, pld2 and nineteenphospholipase A2 isoforms. The role of PC-plc in the intracellular accumulation of PCho inEOC cells and the effect of its inhibition on cell growth have been investigated. Analyses arealso reported on ChoKα mRNA expression and on ChoK and PC-plc protein expression in aset of surgical specimens from EOC patients.

Materials and MethodsEpithelial ovarian non-tumoral and EOC cells

Ovary surface epithelial (OSE) cells, their stably immortalized non-tumoral variants (IOSEand hTERT) and the serous EOC cell lines OVCA3, SKOV3, CABAI and IGROV1 were

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prepared and cultured as described [15]. Four additional cell lines were used for microarrayanalysis: OVCA432 [15], INT-Ov1 and INT-Ov2 [29] and OAW42 (kindly provided by Dr.A. Ullrich, Max-Planck Institute, Germany). Tumor lines, but OAW42 maintained in MEM,were maintained as described [15].

Chemicals1,2dihexanoyl-sn-glycero-3-phosphocholine (C6PC) was purchased from Avanti Polar Lipids,Inc. (Alabarter, Alabama, US); trimethylsilyl-propionic-2,2,3,3-d4 acid sodium salt (TSP)from Merck & Co, Montreal, Canada; tetramethylsilane, CDCl3 and CD3OD from CambridgeIsotope Laboratories, Inc. (Andover, MA, US); tricyclodecan-9-yl-potassium xanthate (D609),sn-glycero-3-phosphocholine, alkaline phosphatase (AP), glyceraldehydes 3-phospatedehydrogenase (GAPDH) and the other chemicals were purchased from Sigma-Aldrich (St.Louis, MO, US).

Clinical specimensClinical specimens, including OSE cells, were obtained with INT-Milan Review Boardapproval. And informed consent to use leftover biological material for investigation purposeswas obtained from all patients. EOC samples were taken at the time of initial surgery from 21patients with histologically confirmed EOC (stages III–IV according to InternationalFederation Gynecologic Oncology criteria) who underwent exploratory laparatomy between1998 and 2005. Samples were immediately frozen in liquid nitrogen and stored at −80°C untiluse.

Cell extractsAqueous cell extracts were prepared as described [15]. Lipid extracts were obtained anddissolved in CDCl3/CD3OD (2:1, v/v) as described [13].

NMR spectroscopyHigh-resolution NMR experiments (25°C) were performed at either 400 or 700 MHz (BrukerAVANCE spectrometers, Karlsruhe, Germany). 1H and 31P NMR spectra of aqueous cellextracts were obtained using references, acquisition parameters, data processing and dataanalysis as described [15,30]. Fully relaxed 1H NMR spectra of lipid extracts were acquiredusing 30° flip angle, 12.7 s repetition time, 32K time domain data points and 128 transients.Relative PC quantification was performed on the –N+(CH3)3 headgroup signal at 3.22 ppm.

Biochemical assays of PC-cycle enzymes in EOC and EONT cellsEnzyme activities were determined at 25°C by NMR assays specifically developed andvalidated in our laboratory. Due to the difficulty of obtaining sufficient amounts of OSE cells,hTERT cells were used as EOC non-tumoral counterparts. This choice was justified by the nonsignificant differences in the respective contents of PCho, GPC, free Cho and tCho detectedin OSE and hTERT cells [15].

ChoK activity—1H and 31P NMR assays were performed upon addition of exogenous cholinechloride, ATP and Mg++ ions to cytosolic cell preparations in Tris-HCl, pH 8.0, as described[15,30].

PC-specific phospholipases and GPC-phosphodiesterase—Cell pellets (15–20 ×106 cells) were resuspended, sonicated and centrifuged as described [30,31] and the assaysperformed on supernatants using as substrate a monomeric short-chain PC, C6PC, below thecritical micellar concentration [32], 5.0 mM in 10 mM CaCl2, pH 7.2 [33].

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Choline formation was measured by 1H NMR as product of the following catabolic pathways:

1) pld-mediated C6PC hydrolysis

a)

and

2) phosphodiesterase (pd)-mediated hydrolysis of GPC formed by the combined action onC6PC of pla (plA1 and plA2) and lysophospholipases (lpl).

b)

plus

c)

The overall activity rate of Cho released by the pathways in a) and b) plus c) is herereferred to as C6PC-pld*.

The PC-plc activity was determined from the additional increase in Cho productionin the same cell lysates as above, in the presence of exogenous alkaline phosphatase(AP), according to the reactions:

d)

plus

e)

For these assays, each cell lysate was divided into four aliquots (15 × 106 cells each), twoprepared in the presence or absence of C6PC (to determine, by subtraction, the contribution ofendogenous Cho) and two in the presence or absence of AP (to determine the contributions ofeither pld* or PC-plc activity to the final Cho content).

31P NMR analyses allowed evaluation of differential plA2- and plA1-mediated productionfrom C6PC (0.01 ppm) deacylation of 1-hexanoyl-α-glycerophosphocholine (2-lyso-C6PC;0.31 ppm) and 2-hexanoyl-α-glycerophosphocholine (1-lyso-C6PC; 0.47 ppm).

The activity of GPC-pd was determined by adding exogenous GPC (5 mM) to the cell lysate,in the presence of 10 mM MgCl2, as previously described [34].

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No differences in phospholipase assays were observed in cell lysates incubated in the presenceor absence of protease inhibitors, indicating that Cho and PCho were not produced by proteindegradation.

Microarray analysisGene expression of the enzymes and transporters demonstrated or proposed to be involved incholine metabolism was searched using a dataset generated at INT in an independent study(unpublished data). Human genome U133 Plus 2.0 GeneChip (Affymetrix), covering 47,000transcripts and variants was used. Briefly, total RNA from 8 cell lines and OSE cells (4 differentpreparations, 1–3 passages at maximum) was extracted using TRIzol Reagent (Gibco) andcleaned-up on mini columns (RNeasy Mini Kit, Qiagen). A similar protocol was used for 20EOC frozen surgical specimens in some experiments, as specified. Total RNA (3 μg) wasreverse transcribed, labeled and hybridized to the chip at 45°C for 16 h. After washing, chipswere scanned (Affymetrix GeneChip Scanner3000 7G) and images were analyzed usingGeneChip Operating Software v1.4 (GCOS1.4). Probe set intensities were calculated using theMicroarray Analysis Suite (MAS v5.0, Affymetrix). Data analysis and exploration of probeset intensities levels were performed using Bioconductor open source(http://www.bioconductor.org/) under the R software environment [35]. The reading of probelevel data, background correction, normalization by MAS5 “global scaling” procedure andsummarizing the probe set values into one expression measure were performed using theaffy package. All microarray data are available in Gene Expression Omnibus, accession numberGSE19352.

Real Time PCRTotal RNA was extracted from OSE and IGROV1, SKOV3, OVCAR3 cell lines using theRNAspin Mini Isolation Kit (GE Healthcare, Piscataway, NJ) and reverse transcribed usingthe High Capacity cDNA Archive Kit (Applied Biosystems).

Quantitative real-time PCR was performed by an ABI Prism 7900 HT Sequence detectionSystem (Applied Biosystems) using TaqMan® Gold RT-PCR Reagents. Three independentRNA preparations were reverse transcribed and at least two qPCR reactions were done usingeach RT product. The pairs of primers and the TaqMan probes for the target mRNAs werefrom Applied Biosystems, Assay ID: CHKA, Hs00608045_m1; CHKB, Hs00993897_g1;PCYT1A, Hs00192339_m1; PCYT1B, Hs00191464_m1; CHPT1, Hs00220348_m1; PLD1,Hs00160118_m1; PLD2, Hs00160163_m1.

The ΔΔCT method was used to determine the quantity of the target sequences in EOC celllines relative both to OSE cells (calibrator) and to an endogenous control (GAPDH). Analyseswere performed using SDS software 2.2.2 (Applied Biosystems). Expression levels werepresented as the relative fold change and calculated as:

Western blotting in cells and tissuesTotal cell and tissue lysates were obtained as described [36]. Lysates were separated by 10%SDS-PAGE and transferred to nitrocellulose (Hybond C-Super; Amersham) or polyvinylidenefluoride membranes (Immobilon PVDF, Millipore, Bedford, MA). Membranes were incubatedin 1% nonfat dry milk overnight at 4°C with rabbit polyclonal anti–PC-PLC antibody [28] orcustom-made rabbit polyclonal anti-ChoK antibody [27] or the goat polyclonal antibody anti-ChoK (Santa Cruz Biotechnology, Inc.). Mouse monoclonal antibodies against β-actin orGAPDH were used as loading controls.

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Horseradish peroxidase-labeled secondary antibodies (Amersham) were added for 1 h at roomtemperature. Immunoblots were developed using the SuperSignal West Picochemiluminescence substrate kit (Pierce Biotechnology, Inc.). Densitometry analyses wereperformed with a Bio-Rad apparatus (Bio-Rad Laboratories Srl) using the Quantity Onesoftware or using ImageJ (Wayne Rasband, NIH, Washington, DC).

Statistical analysisData were analyzed using GraphPad Software version 3.03 or using JMP software package(Brooks/Cole-Thomson Learning, Belmont, CA). Statistical significance of differences wasdetermined by one-way ANOVA or by Student’s t-test, as specified. Differences wereconsidered significant at P < 0.05.

ResultsLevels of choline-containing metabolites in EONT and EOC cells

In agreement with our previous study [15], the tCho content in EONT cells was 5.4±0.6 nmol/106 cells, comprising a PCho content of 2.6±0.3 nmol/106 cells (Fig. 1B). Significantly highertCho (10.3 to 20.3 nmol/106 cells) and PCho levels (8.0 to 14.0 nmol/106 cells) were detectedin the set of four analyzed EOC cell lines (Fig. 1B , one-way ANOVA P<0.001). Thecontribution of PCho to the tCho resonance increased from 53.2±4 % in EONT to 75±5% inEOC cells (P < 0.001), with a resulting decrease in the GPC/PCho ratio. The overall PC contentincreased 1.3- to 1.6-fold (P<0.04) in EOC compared with EONT cells (Fig. 1C).

Choline transportGene expression analyses of members of the three Cho transporter families [37] showed nodifferential mRNA levels for the high-affinity Cho transporter CHT1 in EOC compared withOSE cells. Among organic cation transporters, OCT1 and OCT2 remained below theexpression threshold, while OCT3, the most highly expressed one, was substantiallydownregulated in cancer cells (Fig. 2A). Among Cho transporter-like proteins (CTL1-5), onlyCTL3 showed some increase in mRNA expression in EOC cells. Choline might also betransported by a Cho/H+ antiport system involving Na+/H+ exchangers (NHE) [37] but, in ouranalysis, the overall expression of NHE1-5 did not reveal any difference between EOC andOSE cells (Fig. 2A).

Overexpression and activation of ChoK in EOC cellsThe mRNA expression analyses of eight EOC cell lines compared with OSE cells showedChoKα (CHKA gene) but not ChoKβ (CHKB gene) upregulation (data not shown). Data wereindependently validated by RT-qPCR on three EOC cell lines (OVCAR3, IGROV1 andSKOV3) compared with three different OSE preparations (Fig. 2B). These results, togetherwith the downregulation of ct (PCYT1A and PCYT1B genes) and pct (CHPT1 gene) in cancercells (Fig. 2B), point to a major role of ChoKα in the build-up of the PCho pool. Indeed, asignificant 2.7±0.3 fold increase in ChoK protein expression was observed by Western blottingof EOC cell lysates (Fig. 3A,B). Also in agreement with our previous study [15], assays oncytosolic preparations (Fig. 3C) showed a 12- to 25-fold ChoK activation in EOC (7.0 to 16.0nmol/106 cells·h, i.e. 28.5 to 62.3 nmol/mg protein·h) compared with hTERT cells (0.6 ± 0.2nmol/106 cells·h, i.e. about 3.0 nmol/mg protein·h).

Activity of PC-specific phospholipases in choline producing PC degradation pathwaysPCho production in cancer cells may also derive from phosphorylation of intracellular Chorelased by PC catabolism.

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The overall rate of Cho production by PC hydrolytic pathways (defined as pld* activity inMethods) measured in cell lysates in CaCl2 10 mM, was 6.7 ± 0.7 nmol/106 cells·h in EONTcells, increased 2- to 3-fold in IGROV1 and OVCAR3, but remained unaltered in CABAI andSKOV3 (Fig. 4A, white bars).

To dissect the individual contributions to the rate of Cho formation (pld*) respectively givenby pld and by the deacylation pathway, we performed two types of experiments. GPC-pd assays[34] carried out on cell lysates under the same conditions of ionic strength (CaCl2 10 mM)showed that this enzyme gave only a minor, if any, contribution (max 5–10%) to the rate ofCho production (Fig. 4A, black bars). Therefore, under these conditions, the pld* and pldactivity rates were practically identical. We could then conclude that pld was significantlyactivated (about 2- to 4-fold) only in some (IGROV1 and OVCAR3) but not in all EOC cells.On the other hand, gene expression analyses (not shown) and RT-qPCR experiments (Fig.4B) performed on pooled EOC compared with EONT cells, did not show differential expressionfor PLD1 and showed only a moderate, if any, overexpression for the PLD2 gene.

When the GPC-pd assay was performed under optimal conditions of ionic strength (MgCl2 10mM, pH 7.2 [34]) the rate of Cho production was about 3.8 nmol/106 cells·h in EONT cells,and to increased 2- to 4-fold in some, but not in all EOC cells (Fig. 4A, grey bars). Moreover,since no PCho was formed in the reaction mixture, we could exclude any additionalcontribution to GPC degradation from the alternative GPC phosphodiesterase.

Regarding the deacylation pathway, 31P NMR analysis of cell lysates in the presence of C6PC(in CaCl2 10mM), showed that the 1-lyso-C6PC level was less than 5% that of 2-lyso-C6PCin both EOC and EONT cells (data not shown) and the content of 2-lyso-C6PC was, at 1 h,significantly lower in cancer cells (Fig. 4C). The concentration of this derivative results fromthe balance between upstream plA2-mediated C6PC deacylation and downstream lpl-mediated2-lyso-C6PC degradation into GPC. Since the latter remained below detection at any time pointof incubating the cell lysates with C6PC (data not shown), and the GPC-pd activity was verylow in CaCl2 10 mM (see above) we concluded that the deacylation pathway was dominatedby plA2 and the activity of this enzyme was lower in EOC than in EONT cells. Indeed, mRNAexpression analyses showed that PLA1A was practically below the expression threshold inboth EONT and EOC cells. Only four out of nineteen PLA2 isoforms appeared to bedifferentially expressed in EOC compared with EONT cells, but the global difference in overallplA2 expression was not significant (Fig. 4D).

Activation of PC-specific phospholipase C in EOC cellsThe activity of PC-plc was about 0.45 ± 0.30 nmol/106 cells·h in EONT cells and increased 5-to 17-fold (one-way ANOVA P<0.03) in the investigated EOC cells (Fig. 5A).

Following exposure of OVCAR3 cells to the PC-plc inhibitor D609, the PC-plc activitydecreased from 4.3±1.2 to 0.3±0.3 nmol/106 cells·h (n=5, P<0.001) and the average PCho leveldecreased by 37%±8% (P<0.04) (Fig. 5B and C), while GPC and Cho were not significantlyaltered. Similar effects were found in SKOV3 cells, in which exposure to D609 induced a PChodecrease of 44% (data not shown). These data provided the first direct demonstration that PC-plc can substantially contribute to PCho accumulation in EOC cells.

Our previous studies showed that cell exposure to D609 induced long-lasting Go/G1 cell cyclearrest in PDGF-stimulated fibroblasts [38] and blocked the S-phase fraction recovery inOVCAR3 cells re-exposed to complete medium after serum depletion [28]. Experiments in thepresent study showed that incubation with D609 induced a long-lasting (at least up to 72 h)cell proliferation arrest in OVCAR3 cells, an effect comparable with that of FCS deprivation(Fig. 5D).

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ChoK and PC-plc detection in clinical specimensSince upregulation of both ChoK and PC-plc was found in all investigated EOC cell lines,preliminary experiments were conducted to evaluate the expression of these enzymes in a setof EOC surgical specimens. The mRNA relative expression levels of ChoKα measured inclinical tissue samples were significantly higher than those in OSE cells. Overall these levelswere, however, lower than those detected in EOC cell lines, (ANOVA analysis for all the threegroups, P<0.001) (Fig. 6A). The increased, albeit heterogeneous expression of ChoK was alsoconfirmed at protein level by Western blot analysis of lysates of surgical specimens randomlyselected from those analyzed for gene expression (Fig. 6B).

Only protein expression data can be at present obtained for PC-plc, whose gene has not yetbeen identified in mammalian cells. Also the expression of this enzyme was higher (althoughvariable) in lysates of surgical specimens compared with those of EONT cells (see Fig. 6C andref 28).

DiscussionBy combining 1H MRS studies with biochemical assays and mRNA and protein expressionanalyses, this study showed that the elevated PCho pool in EOC cells primarily resulted fromupregulation/activation of two enzymes, ChoK and PC-plc, respectively involved in de novobiosynthesis and PC degradation.

Changes in choline transport and ChoK activity may both be responsible for enhancedradioactive choline uptake and PCho accumulation in cancer cells [15,17,39,40]. Thesemechanisms have direct implication on choline-based PET examinations [7,8]. Due to reportedeffects on cell proliferation, choline transport may represent a potential target for therapy [8,17,37]. No differential changes were however observed in mRNA expression of CHT1, OCTor CTL proteins, except for downregulation of OCT2 and moderate upregulation of CTL3 inEOC compared with EONT cells. Further investigations are needed to clarify whether post-transcriptional modifications of choline transporters may contribute to enhance choline uptakeby EOC cells [15].

In the Kennedy pathway, ChoKα was over-expressed at mRNA level in EOC cells. Similar oreven higher ChoKα upregulation was reported in breast and bladder cancer cell lines [17,26].The elevated ChoKα expression in these cancer cells suggests an increase in ChoKα/ChoKαat expenses of ChoKα/ChoKβ and ChoKβ/ChoKβ aggregates, a condition reported in livercarcinogenesis [24]. It is worth noting that rather uniform increases in ChoKα mRNA (3.8±0.2fold) and ChoK protein expression (2.7±0.3-fold) were associated in EOC with a strong andvariable amplification (12- to 25-fold) of ChoK activity, which reached the highest values sofar reported for epithelial cancer cell lines [17,26]. These results indicate that other factors,likely depending upon oncogene-driven signaling alterations, may influence ChoK activity, inagreement with evidence obtained in other tumor systems [23,41].

Accruing evidence points to the role of ChoK in cell proliferation, transformation andcarcinogenesis and supports the use of this enzyme as a novel target for treatment of differenttypes of tumors [22,23,27,41–43]. In fact, a ChoK inhibitor, Mn58b, was found to reduce tumorgrowth and tCho in human carcinoma models [18,41]. Transient ChoKα down-regulation bysmall interfering RNA (siRNA-ChoKα) induced cell differentiation, decreased cellproliferation and reduced PCho and tCho levels in breast cancer cells [27]. A combination ofsiRNA-ChoKα with 5-Fluorouracil resulted in a larger reduction of cell viability/proliferationin breast cancer than in non-tumoral breast epithelial cells [42]. Finally, lentivirus-mediatedsystemic delivery of the analogous short-hairpin RNA resulted in a significant reduction in

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tumor growth along with reduced PCho and tCho levels in breast tumor xenografts in vivo[43]. These findings suggest that EOC may also be a candidate for similar treatment strategies.

Among other PC-cycle pathways possibly contributing to PCho accrual, only PC-plc-mediatedPC degradation was substantially and consistently activated in all investigated EOC cell lines.In fact, in the deacylation pathway the mRNA expression of nineteen plA2 isoforms was eitherunaltered or reduced and the overall PlA2 activity decreased in EOC compared with EONTcells, in agreement with the plA2 group IVA underexpression reported for a breast cancer cellline [13]. At the end of the deacylation pathway, a 2- to 4-fold activation of GPC-pd was limitedto only some EOC cell lines. Moreover, preliminary results in our laboratory showed unalteredmRNA expression in EOC versus EONT for GDPD5, a glycerol-phosphodiesterase recentlyindicated as a plausible candidate for mediating GPC-pd activity [44]. Even the activity of pld,an enzyme playing a critical role in cell proliferation and neoplastic processes [45,46], wasenhanced in only some of the investigated EOC cells and the average mRNA expression levelsof its major isoforms, pld1 and pld2, were substantially unaltered in EOC compared with EONTcells. Similar variable patterns of pld activity and isoforms’ expression were reported in breastcancer cells [17].

A growing evidence indicates a relevant role of PC-plc in mitogenesis, differentiation andapoptosis [38,47–49]. Although isolated from some mammalian cells, this enzyme has not yetbeen cloned. Evidence of possible PC-plc activation was initially reported by Glunde et al inbreast cancer cells [13]. Following detection and characterization of differential subcellularlocalization of a 66 kDa PC-plc [28], the present study shows 5- to 17-fold increases in PC-plc activity in EOC compared with EONT cells. Furthermore, our preliminary experimentsshowed that ChoK silencing in breast cancer cells resulted in compensatory PC-plcupregulation, suggesting links between pathways responsible for activation of these twoenzymes.1

Inhibition of PC-plc by D609 induced a 30-to-40% decrease in the PCho content of OVCAR3and SKOV3 cells, providing direct evidence of the contribution of PC-plc activity to theintracellular PCho pool in these cells. This result, together with the antiproliferative effectsinduced on these cells by D609, point to the possible use of the MRS PCho resonance to monitorthe effects of treatments directed against PC-PLC activity in EOC cells. Additional studies arerequired to further elucidate the molecular mechanisms of increased PC-plc activity in ovarianand other cancers. Although a recent study suggests that PC-plc activity may also be conferredby sphingomyelinases (SMase) [50], this mechanism unlikely contributes to PC-plc activationin EOC cells, since PC-plc activity was abolished in OVCAR3 cells by D609, a very poorSMase inhibitor.

The here reported data on upregulation of ChoK and PC-plc in EOC cells, led us to investigatethe expression levels of these enzymes in EOC surgical specimens. ChoKα overexpression hasbeen reported and proposed as an indicator of reduced patient survival in human lung andbladder carcinomas [25,26]. Our study shows for the first time that ChoKα mRNA expression,as well as ChoK and PC-plc protein expression, are elevated in EOC surgical specimens. Thedifference between in vitro and in vivo gene expression levels of ChoKα may derive fromstromal contamination and/or from in vitro culture conditions. Independently of absolute levelsof gene or protein expression, we can envisage that, upon validation, the parallel detection ofChoKα and PC-plc in the same specimens may facilitate future investigations on their role aspredictive or prognostic factors.

1Glunde K, Mori N, Takagy T, Cecchetti S, Ramoni C, Iorio E, Podo F, Bhujwalla ZM. Choline kinase silencing in breast cancer cellsresults in compensatory upregulation of phosphatidylcholine-specific phospholipase C. Proc Intl Soc Mag Reson Med 2008;16, Abstract244.

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In conclusion, the tCho spectral metabolite provides an endogenous reporter on the activity ofthese enzymes, and can be used to detect and monitor in ovarian cancer the effects of thedownregulation or inhibition of these enzymes as novel therapeutic strategies.

AcknowledgmentsWe thank Mr Massimo Giannini, Ms Paola Alberti, Ms Elena Luison, Ms Tomoyo Takagi and Ms Yelena Mironchikfor experienced technical assistance; Dr Carlo Pini and Dr Bianca Barletta for preparation and purification of anti-PC-plc antibodies; Gynecological and Pathological Units of INT-Milan for providing patient materials.

Financial support: AIRC 2007–2010, Special Oncology Programme RO 06.5/N.ISS/Q09, Ministry of Health, Italyand Accordo di Collaborazione Italia-USA ISS/530F/0F29 (FP); Programma Tumori Femminili 2008, Ministry ofHealth, Italy (SC); NIH P50 CA103175 (KG and ZMB).

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Figure 1. Phosphatidylcholine metabolitesA, The phosphatidylcholine (PC) cycle. Metabolites: CDP-Cho, cytidine diphosphate choline;Cho, choline; DAG, diacylglycerol; FFA, free fatty acid; G3P, sn-glycerol-3-phosphate; GPC,glycerophosphocholine; LPC, lysophosphatidylcholine; PA, phosphatidate; PCho,phosphocholine. Enzymes: Kennedy pathway: Chok, choline kinase (EC 2.7.1.32); ct,cytidylyltransferase (EC 2.7.7.15); pct, phosphocholine transferase (EC 2.7.8.2). Headgrouphydrolysis pathways: plc, phospholipase C (EC 3.1.4.3); pld, phospholipase D (EC 3.1.4.4).Deacylation pathway: plA1, phospholipase A1 (EC 3.1.1.32); plA2, phospholipase A2 (EC3.1.1.4); lpl, lysophospholipase (EC 3.1.1.5); pd, glycerophosphocholine phosphodiesterase(EC 3.1.4.2). B, Quantification of aqueous metabolites (mean values ± SEM) in EOC and

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EONT cells [OSE (n=2); IOSE (n=4); hTERT (n=15)]). C, Relative PC contents (± maximumdeviation for n=2; ± SD for n≥3) in EOC normalized to hTERT cells. In parenthesis, numberof independent experiments.

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Figure 2. Choline transporters and Kennedy pathway enzymes in EOC and EONT cellsA, Microarray analysis of choline transporters (dotted line, sensitivity threshold). *Significance of differences: P ≤ 0.015; B, RT-qPCR analysis of enzymes’ expression in EOC(OSE cells used as internal calibrator; horizontal line at quantification level = 1). Arepresentative experiment of three performed is shown. For each gene the mean value (± SD)of the analyzed EOC cell lines is reported.

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Figure 3. ChoK protein expression and activityA, ChoK protein expression by Western blotting, as detected by the custom-made rabbitpolyclonal antibody. B, Chok densitometric evaluation referred to GAPDH (mean ± SEM, n≥4.* = P<0.05; ** = P<0.01, relative to EONT by one-way ANOVA. C, Chok activity(mean ±SD, n ≥3).

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Figure 4. Enzymes of pld-mediated and deacylation pathways in EOC and EONT cellsA, pld* and GPC-pd activity(mean value ± SD, n ≥6). B, RT-qPCR analysis of PLD1 and PLD2in EOC cells (OSE used as internal calibrator; horizontal line at quantification level = 1). Arepresentative experiment of three performed is shown. For each gene mean value ± SD isreported. C, Relative 2-Lyso-C6PC formation (± SD, n ≥3) in EOC and hTERT cell lysates at1h of exposure to C6PC. D, Gene expression of plA1 and of plA2 isoforms. The dotted linerepresents sensitivity threshold.

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Figure 5. Activation of PC-plc in EOC cellsA, PC-plc activity (± SD, n ≥4). B, PC-plc activity and C, PCho content in OVCAR3 cellsfollowing incubation in absence (CTRL) or presence of D609 (53 μg/mL, 24 h). Inset: 1H NMRtCho profile in aqueous extracts. D, OVCAR3 cell growth in complete medium in absence(CTRL) or presence of D609 and in serum-deprived medium (-FCS). Error bars, ± SD, n ≥ 5(Student’s t-test * P<0.05; ***P< 0.001).

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Figure 6. ChoK and PC-plc detection in EOC surgical specimensA, Gene expression analysis of ChoKα in OSE cells, surgical EOC specimens and EOC celllines. Horizontal bars represent relative median expression for each group. Western blotanalyses of total lysates of EOC surgical specimens and hTERT cells are shown for ChoKprotein expression, as detected by the commercially available goat polyclonal antibody (B) andPC-plc protein expression (C). Actin, internal loading control.

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