Choline-Releasing Glycerophosphodiesterase EDI3 Drives Tumor Cell Migration and Metastasis
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Choline-releasing glycerophosphodiesterase EDI3 drives tumor cell migration and metastasis Joanna D. Stewarta,1, Rosemarie Marchana,1, Michaela S. Lesjaka, Joerg Lambertb, Roland Hergenroederb, James K. Ellisc, Chung-Ho Lauc, Hector C. Keunc, Gerd Schmitzd, Juergen Schillere, Mandy Eibische, Christian Hedbergf, Herbert Waldmannf,g, Ekkehart Lauschh, Berno Tanneri, Jalid Sehoulij, Jens Sagemuellerk, Hagen Staudel, Eric Steinerm,2, and Jan G. Hengstlera,2,3 aLeibniz Research Centre for Working Environment and Human Factors (IfADO), University of Dortmund, 44139 Dortmund, Germany; bInterface Processes, Leibniz-Institut fuer Analytische Wissenschaften (ISAS), 44227 Dortmund, Germany; cBiomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London SW7 2AZ, United Kingdom; dInstitute for Clinical Chemistry and Laboratory Medicine, University Hospital Regensburg, 93053 Regensburg, Germany; eMedical Faculty, Institute of Medical Physics and Biophysics, University of Leipzig, 04107 Leipzig, Germany; fChemical Biology, Max Planck Institute of Molecular Physiology, 44227 Dortmund, Germany; gFakultaet Chemie, Lehrbereich Chemische Biologie, Technische Universitaet Dortmund, 44227 Dortmund, Germany; hDepartment of Pediatrics, Freiburg University Medical Center, 79106 Freiburg, Germany; iDepartment of Obstetrics and Gynecology, Oranienburg Clinic, 16515 Oranienburg, Germany; jDepartment of Gynecology, Charite University Hospital, 13353 Berlin, Germany; kDepartment of Pharmacy, Duesseldorf University Hospital, 40225 Duesseldorf, Germany; lDepartment of Pediatrics, University of Rostock, 18057 Rostock, Germany; and mDepartment of Obstetrics and Gynecology, Gesundheits-und Pflegezentrum Ruesselsheim Gemeinnützige GmbH, 65428 Ruesselsheim, Germany Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved April 4, 2012 (received for review October 26, 2011) Metastasis from primary tumors remains a major problem for tumor ovary (4, 7, 10–12). Targeting CK with inhibitors resulted in therapy. In the search for markers of metastasis and more effective antimitogenic and antiproliferative effects (13). It has also been therapies, the tumor metabolome is relevant because of its impor- reported that an increased rate of choline transport may be re- tance to the malignant phenotype and metastatic capacity of tumor sponsible for the increased PC levels (10, 14, 15). However, cells. Altered choline metabolism is a hallmark of cancer. More gene-expression analysis of members of the three choline specifically, a decreased glycerophosphocholine (GPC) to phosphocho- transporter families in ovarian cancer cells showed no differen- line (PC) ratio was reported in breast, ovarian, and prostate cancers. tial levels for the high-affinity transporter families compared with CELL BIOLOGY Improved strategies to exploit this altered choline metabolism are normal cells (7). A further source of choline is through an in- therefore required. However, the critical enzyme cleaving GPC to crease in phospholipase D (PLD) activity (16, 17). PLD is a produce choline, the initial step in the pathway controlling the GPC/PC phospholipase that hydrolyses phosphatidylcholine (PtdCho) to ratio, remained unknown. In the present work, we have identified the phosphatidic acid (PA) and choline. Increased PLD expression enzyme, here named EDI3 (endometrial differential 3). Purified and activity was reported in the metastatic breast cancer cell line, recombinant EDI3 protein cleaves GPC to form glycerol-3-phosphate MDA-MB-231, and inhibition of activity in these cells rendered and choline. Silencing EDI3 in MCF-7 cells decreased this enzymatic them more sensitive to apoptotic insult (17). activity, increased the intracellular GPC/PC ratio, and decreased down- At present, the only reported mechanism that involves GPC is stream lipid metabolites. Downregulating EDI3 activity inhibited cell the report of increased phospholipase A2 (PLA2) activity (18). migration via disruption of the PKCα signaling pathway, with stable PLA2 is a membrane associated phospholipase, which hydrolyses overexpression of EDI3 showing the opposite effect. EDI3 was origi- the sn-2 fatty acyl ester bond of phosphoglycerides, converting nally identified in our screening study comparing mRNA levels in me- PtdC to 1-acylglycerophosphocholine, a precursor to GPC. Mem- tastasizing and nonmetastasizing endometrial carcinomas. Both brane PLA enzyme levels were significantly higher in breast cancer – 2 Kaplan Meier and multivariate analyses revealed a negative associa- patients with distant metastases compared with patients without tionbetweenhighEDI3expressionand relapse-free survival time in metastases (19). both endometrial (P < 0.001) and ovarian (P = 0.029) cancers. Overall, In this study we provide a direct mechanism for the decreased fi we have identi ed EDI3, a key enzyme controlling GPC and choline GPC levels observed in various cancer cells and tissues. In- metabolism. Because inhibition of EDI3 activity corrects the GPC/PC creased expression and activity of endometrial differential 3 ratio and decreases the migration capacity of tumor cells, it rep- (EDI3), the enzyme responsible for the cleavage of GPC, does resents a possible target for therapeutic intervention. not only provide a source of choline for the Kennedy pathway, resulting in increased PC, but also directly explains the decreased glycerophosphodiesterase 5 | phosphatidic acid | lysophosphatidic acid | GPC levels reported in transformed cells (1, 4). Because GPC is glycerophosphocholine phosphodiesterase GDE1 homolog (Saccharomyces a key component of choline metabolism, we used both NMR and cerevisiae) | glycerophosphodiester phosphodiesterase domain containing 6 mass spectrometry to investigate the role of EDI3 in lipid metabolism and in particular the GPC/PC ratio. We further t is widely accepted that choline metabolism represents a criti- Ical aspect of the tumor metabolome (1–4). As cells undergo malignant transformation, there is an overall increase in total Author contributions: J.D.S., R.M., E.S., and J.G.H. designed research; J.D.S., R.M., M.S.L., choline metabolites and phosphocholine (PC). The elevation of J.L., J.K.E., C.-H.L., and M.E. performed research; R.H., J.K.E., H.C.K., G.S., J. Schiller, C.H., PC and total choline is increasingly used in tumor diagnostics H.W., E.L., B.T., J. Sehouli, J. Sagemueller, H.S., and E.S. contributed new reagents/analytic with detection by 1Hor31P magnetic resonance spectroscopy (5– tools; J.D.S., R.M., M.S.L., J.L., J.K.E., G.S., J. Schiller, and C.H. analyzed data; and J.D.S., R.M., and J.G.H. wrote the paper. 9). In breast and ovarian carcinomas, elevated PC are accom- fl panied by a decrease in glycerophosphocholine (GPC), resulting The authors declare no con ict of interest. in a decreased GPC/PC ratio compared with normal tissue (1, 4). This article is a PNAS Direct Submission. The increased PC levels have previously been reported to occur Freely available online through the PNAS open access option. through several mechanisms; however, the decreased GPC has 1J.D.S. and R.M. contributed equally to this work. not yet been adequately explained. 2E.S. and J.G.H. contributed equally to this work. Choline kinase (CK) is the enzyme responsible for the phos- 3To whom correspondence should be addressed. E-mail: [email protected]. phorylation of available intracellular choline to PC, and is active This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in malignant cells and tumors of lung, colon, breast, cervix, and 1073/pnas.1117654109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1117654109 PNAS | May 22, 2012 | vol. 109 | no. 21 | 8155–8160 Downloaded by guest on September 24, 2021 examined the impact of EDI3 expression on signaling pathways other members of the GDE family, GDE5/GPCPD1/EDI3 (EDI3 known to be involved in cellular migration. for simplicity) contains no transmembrane domains, but is instead localized to the cytoplasm (20, 21). A recent study illustrated Results that the preferred substrate for EDI3 is GPC, and that in mice EDI3 Predicts Metastasis in Endometrial and Ovarian Cancer. A the enzyme may play an important role in skeletal muscle de- screening set of endometrial carcinomas consisting of pairs of velopment (21). However, the presence of EDI3 mRNA in all tumor tissue specimens with identical histopathological features tissues of both mouse and human (Fig. S1 D and E) suggests a more but different metastatic behavior were used to identify differ- generalized physiological function. entially expressed genes by differential display analysis. A 270-bp To specifically investigate the enzymatic action of EDI3, high- fragment was found only in the tumors that went on to form resolution 31P NMR analyses were performed using GPC as metastasis. Furthermore, higher expression of this gene, named a substrate. Incubating lysates from EDI3 overexpressing NIH EDI3, was confirmed in metastasizing carcinomas by quantitative 3T3 cells resulted in a time-dependent increase in glycerol-3- RT-PCR (qRT-PCR) using an independent cohort (P < 0.001) phosphate (G3P) signal (Fig. 2A). Similar results were obtained (Fig. 1A, using alternative primers in Fig. S1A, and Table S1). with recombinant EDI3 protein (Fig. 2B), confirming that EDI3 Both Kaplan–Meier and multivariate analyses revealed a nega- hydrolyses GPC to form G3P. The presence of the second tive association between high EDI3 expression