SUBMICROMOLAR DOSES OF ALKYL-LYSOPHOSPHOLIPIDS INDUCE RAPID INTERNALIZATION, BUT NOT ACTIVATION, OF EPIDERMAL GROWTH FACTOR RECEPTOR AND CONCOMITANT MAPK/ERK ACTIVATION IN A431 CELLS Gerald A. RUITER 1,2 , Marcel VERHEIJ 1,2 , Shuraila F. ZERP 1,2 , Wouter H. MOOLENAAR 1 and Wim J. VAN BLITTERSWIJK 1 * 1 Division of Cellular Biochemistry, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, the Netherlands 2 Department of Radiotherapy, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Ziekenhuis, Amsterdam, the Netherlands Synthetic ALPs, e.g., Et-18-OCH 3 and HePC, are antican- cer agents that accumulate in cell membranes, where they interfere with lipid-mediated signal transduction. We previ- ously reported that ALPs, when added at micromolar con- centrations (5–25 M), inhibit growth factor–induced MAPK/ ERK activation and enhance radiation-induced apoptosis. We now show that, at nanomolar doses (10 –500 nM), ALPs acti- vate the MAPK/ERK pathway in A431 cells without stimulat- ing cell proliferation. Strikingly, ALPs (500 nM) also trigger rapid clustering and internalization of the EGFR in A431 cells. Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, blocks ALP-induced MAPK/ERK activation but not EGFR in- ternalization. We found no evidence for ALPs acting via G protein– coupled receptors and/or transactivation of EGFRs, as determined by calcium mobilization, EGFR phosphoryla- tion and Grb2 binding assays. Since ALPs readily intercalate into the plasma membrane, our data suggest that they in- duce subtle changes in the lipid microenvironment of the EGFR, resulting in clustering and internalization of the EGFR and concomitant MAPK/ERK activation. © 2002 Wiley-Liss, Inc. Key words: alkyl-lysophospholipid; epidermal growth factor recep- tor; internalization; MAPK/ERK Synthetic membrane-permeable ALPs represent a class of anti- cancer drugs that primarily act at the level of the cell membrane, where they accumulate and affect both apoptotic and survival signal-transduction pathways. 1–5 Examples of clinically relevant ALPs include the prototypic compound Et-18-OCH 3 (Edelfosine), HePC (Miltefosine) and octadecyl-(1,1-dimethyl-piperidinio- 4-yl)-phosphate (Zentaris compound D-21266, Perifosine). Sev- eral reports have shown that ALPs disturb phospholipid metabo- lism and thereby affect membrane-localized signaling pathways. 6,7 For example, Et-18-OCH 3 and HePC inhibit de novo PC synthesis at the CTP:phosphocholine citidylyltransferase step. 7 Interest- ingly, when applied at micromolar doses, ALPs act as potent inducers of apoptosis in a variety of tumor cells. In combination with radiation and certain cytostatic drugs, ALPs cause a syner- gistic cytotoxic effect. 4,5,8,9 We have previously shown that, in human leukemic and carcinoma cells, apoptotic concentrations of ALPs inhibit the MAPK/ERK mitogenic pathway and the Akt/ PKB survival pathway, whereas they activate the proapoptotic SAPK/JNK pathway. 4,5 We now report that, unexpectedly, submicromolar concentra- tions of ALPs activate the MAPK/ERK pathway in A431 carci- noma cells. We considered the possibility that ALPs might activate a G protein– coupled receptor since these compounds, Et-18-OCH 3 in particular, are structurally similar to bioactive (lyso)phospho- lipids, such as PAF and LPC, which act on their cognate G protein– coupled receptors. 10,11 However, we found no evidence of this. Instead, ALP triggers rapid internalization but not phosphor- ylation of the EGFR, which is expressed at very high density in A431 cells. 12,13 While the underlying mechanism remains to be elucidated, ALP-induced endocytosis of the EGFR may be suffi- cient to trigger MAPK/ERK signaling in A431 cells. This novel effect of ALPs on EGFR fate may serve as a tool to downregulate overexpressed EGFRs in A431 cancer cells. MATERIAL AND METHODS Material Et-18-OCH 3 , HePC and lyso-PAF were purchased from Biomol (Plymouth Meeting, PA) and diluted in serum-free DMEM. D-21266 (Perifosine) was kindly provided by Zentaris (Frankfurt, Germany). EGF was from Becton Dickinson (Bedford, MA). LPA, LPC and MAbs against activated p42/p44 MAPK/ERK (diphos- phorylated ERK-1, -2) were from Sigma (St. Louis, MO). An- tiphosphotyrosine MAb PY99 was from Santa Cruz Biotechnology (Santa Cruz, CA). The p44/42 MAP Kinase Assay Kit was pur- chased from Cell Signaling (Beverly, MA). MAbs against Akt/ PKB and antiphosphotyrosine (PY-20) were purchased from Transduction Laboratories (Lexington, KY). Polyclonal phospho- Akt (Ser 473 ) antibodies were from New England Biolabs (Beverly, MA). [- 32 P]ATP (3 mCi/mmol) was from Amersham (Aylesbury, UK). PTX was from List Biological Laboratories (Campbell, CA). The EGFR- and PDGFR-specific tyrphostins (AG1478 and AG1296, respectively) were from Calbiochem (San Diego, CA). Rabbit polyclonal serum 282.7 against EGFR was kindly provided by Dr. L.H. Defize (Hubrecht Laboratory, Utrecht, the Nether- lands). Mouse MAb 528 against EGFR 14 was from the ATCC (Rockville, MD). GST-Grb2 fusion protein, isolated from bacterial lysates, was kindly provided by Dr. J. Borst (The Netherlands Cancer Institute). Abbrevations: ALP, alkyl-lysophospholipid; CLSM, confocal laser scanning microscopy; CTP, cytidine triphosphate ; ECL, enhanced chemi- luminescence; EGF, epidermal growth factor; EGFR, EGF receptor; Et- 18-OCH 3 , 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; GST, glutathione S-transferase; HePC, hexadecylphosphocholine; LPA, lyso- phosphatidic acid; LPC, lysophosphatidylcholine; MAb, monoclonal anti- body ; MAPK/ERK, mitogen-activated protein kinase/extracellular signal– regulated kinase; MEK-1/2, MAPK/ERK kinase 1/2; PAF, platelet- activating factor; PC, phosphatidylcholine ; PDGFR, platelet-derived growth factor receptor; PKB, protein kinase B ; PLC, phospholipase C; PTX, pertussis toxin; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase ; TBST, 10 mM TRIS HCl (pH 7.5), 0.5 M NaCl, 0.05% Tween-20. *Correspondence to: Division of Cellular Biochemistry, The Nether- lands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Neth- erlands. Fax: +31-20-5121989. E-mail: w.v.blitterswijk@nki.nl Received 19 February 2002; Revised 28 June, 19 August 2002; Ac- cepted 26 August 2002 DOI 10.1002/ijc.10741 Published online 7 October 2002 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 102, 343–350 (2002) © 2002 Wiley-Liss, Inc. Publication of the International Union Against Cancer