FGF Receptor Phosphotyrosine 766 Is a Target for Grb14 to Inhibit MDA-MB-231 Human Breast Cancer Cell Signaling

ANTICANCER RESEARCH 25: 3877-3882 (2005)

FGF Receptor Phosphotyrosine 766 is a Target for Grb14 to Inhibit MDA-MB-231 Human Breast Cancer Cell Signaling

KATIA CAILLIAU1, DOMINIQUE PERDEREAU2, ARLETTE LESCUYER1, HUIXIONG CHEN3, CHRISTIANE GARBAY3, JEAN PIERRE VILAIN1, ANNE-FRANÇOISE BURNOL2 and EDITH BROWAEYS-POLY1

1Université des Sciences et Technologies de Lille, Laboratoire de Biologie du Développement, UE 1033, IFR 118, Bâtiment SN3, 59 655 Villeneuve d’Ascq Cedex; 2Institut Cochin INSERM U567-CNRS UMR 8104-Université René Descartes, Département d’Endocrinologie, 24 rue du Faubourg Saint-Jacques, 75674 Paris; 3Laboratoire de Pharmacochimie Moléculaire et Cellulaire, CNRS FRE 2718 - INSERM U648, UFR Biomédicale des Saints Pères, 45, Rue des Saints Pères, 75270 - Paris Cedex 06, France

Abstract. Background: Fibroblast growth factors receptors secretes FGF1, which is involved in its proliferation and (FGFRs) are involved in estrogen-independent breast cancer migration (5-7). FGF1 interacts with tyrosine kinase cell growth. Grb14, a member of the Grb7 family of adapters, receptors termed fibroblast growth factor receptors is an inhibitor of FGFR signaling. Materials and Methods: (FGFRs). Four FGFRs have been cloned, but MDA-MB- FGFR from highly invasive MDA-MB-231 cells were expressed 231 cells mainly express FGFR1 (8). Ligand binding results in Xenopus oocyte, a widely used model system to question in receptor dimerisation, activation, and initiates several cascade transduction regulations. The effect of microinjection transduction cascades (9). Phospholipase C gamma (PLCÁ), of Grb14 and various mimetic peptides for FGFR tyrosine which binds to the phosphorylated Y766 residue (10), and residues were analysed by FGFR immunoprecipitation and the phosphatidylinositol-3 kinase (PI-3 kinase)/ AKT Western blot analysis of signaling cascades. Results: PLCÁ, pathways (11) were shown to mediate MDA-MB-231 ERK2, JNK1 and AKT were blocked by Grb14. Only the invasiveness (12), while the MAPK (ERK/JNK) cascade was pY766 phosphopeptide mimetic of the PLCÁ binding site on recruited for cell proliferation (7). FGFR released the inhibitory action of Grb14. Conclusion: The growth factor receptor-bound protein 14 (Grb14) Grb14 binds to the Y766 site of MDA-MB-231-FGFR, belongs to the Grb7 family of adapters, which interact with competing for PLCÁ activation, thus inducing an arrest of the numerous activated tyrosine kinase receptors, including the signaling transduction cascades. insulin-like growth factor-1 receptor (IGF-1R), insulin receptor (IR), platelet-derived growth factor receptor Fibroblast growth factors (FGFs) are involved in cell (PDGFR), epidermal growth factor receptor (EGFR) and division and cell migration. At least two FGFs, FGF1 and FGFR1 (13, 14). Growing evidence is emerging for an FGF2, stimulate the growth of the mammary gland (1). In inhibitory role of Grb14 in insulin and FGFR signaling (14- breast cancers, these FGFs are involved in the stimulation 18). In an estrogen-positive breast cancer cell line, MCF7, of cell proliferation and angiogenesis (2-4). The estrogen- Grb14 overexpression blocked insulin-induced ERK negative human breast cancer cell line, MDA-MB-231, activation and induced a negative feedback mechanism in insulin signaling (19). Interestingly, Grb14 is mainly expressed in normal epithelial breast cells and estrogen- dependent cell lines (19). Estrogen-negative cells, like Correspondence to: Edith Browaeys-Poly, Université des Sciences MDA-MB-231, which represent a model of a more et Technologies de Lille, Laboratoire de Biologie du advanced disease, have lost the expression of Grb14. It Développement, UE 1033, IFR 118, Bâtiment SN3, 59 655 would then be of interest to investigate the molecular Villeneuve d’Ascq Cedex, France. Fax: 33 03 20 43 40 38, e-mail: mechanisms involved in the Grb14-induced FGF1-FGFR [email protected] signaling blockade in these cells. Key Words: MDA-MB-231, fibroblast growth factor receptor, Cancer cell signaling cascades are complex to analyse, Grb14, breast cancer, Xenopus oocyte. partly because they are generated by the superimposed

0250-7005/2005 $2.00+.40 3877 ANTICANCER RESEARCH 25: 3877-3882 (2005) action of many growth factors. To overcome this complexity, synthesiser with ABI small-scale Fmoc chemistry on HMP resin we have used a model system devoid of FGFRs, the and the DCC/HOBt coupling method. Fmoc groups were Xenopus oocyte, where FGFRs from MDA-MB-231 can be removed by piperidine (20% in dichloromethane). The final peptidyl resin was then dried and cleaved with a mixture of expressed and specifically stimulated by the addition of TFA/TIPS/H2O (9.5/0.25/0.25 in volume) for 4 h at room exogenous FGF1 (16, 20). Xenopus oocytes represent a temperature. The filtrate from the cleavage reaction was powerful experimental approach to question cascade precipitated and collected by centrifugation. The crude peptide transduction regulation and their effects on cell growth. was purified by semi-preparative HPLC on a Nucleosil C18 They are physiologically arrested at the G2 stage. Growth column (Vydac, 5 mm, 10 x 250 mm), and the fraction was factors such as insulin (21) or FGF1 (22), when binding to analysed by analytical HPLC on a Nucleosil C18 column (Vydac, appropriate receptors, induce Ras-dependent and Ras- 5 mm, 4.6 x 250 mm). The pure fractions were collected and lyophilised. The structure of the peptides was confirmed by independent cascades, leading to the oocyte G2/M electrospray mass spectrometry (double focusing VG 7Æ-250 transition and oocyte maturation. spectrometer equipped with a FAB gun). In the present study, we showed that Grb14 exerts an inhibitory action on FGF1-stimulated MDA-MB-231- Microinjections. Microinjection of 60 ng of MDA-MB-231 FGFR FGFRs, by blocking the phosphorylation and activation of mRNAs was performed in oocytes 48 h before the addition of ERK2, JNK1 and AKT. Furthermore, we demonstrated that FGF1 to the extracellular medium (5 nM). Seventy-five ng of the target for Grb14 is the FGFR tyrosine phosphorylated Grb14, a value reported to block signal transduction in oocytes 766 site. These results suggest that Grb14 could represent expressing FGFRs from MDA-MB-231 cells (16), 100 ng of tandem SH2 domains of PLCÁ were injected 1 h before FGF1 stimulation. an attractive strategy to inhibit FGF1-induced estrogen- Three or 30 ng of phosphopeptide pY766, 3 ng of pY730, of pY776 negative MDA-MB-231 cell signaling. or of Y776 were injected alone or after incubation with Grb14 (75 ng) for 1 h at room temperature. For each experiment, 20-30 Materials and Methods oocytes were removed from at least 2 animals.

Breast cancer cell culture and RNA preparation. MDA-MB-231 cells Immunoprecipitations of FGFRs. Oocytes, expressing FGFR from were cultured at confluence in a humidified atmosphere of 5% MDA-MB-231 for 48 h, were injected or not with fusion proteins CO2 in Eagle's medium containing 10% fetal calf serum, 20 mM or peptides 1 h before stimulation by FGF1 (5 nM). After 5 min, HEPES, 2 g/L sodium bicarbonate, 2 mM L-glutamine, 100 IU/mL 20 treated oocytes were lysed in 200 Ìl of buffer A (25 mM MOPS penicillin, 100 Ìg/mL streptomycin, 10 Ìg/mL gentamycin sulfate pH 7.2, 60 mM ‚glycerophosphate, 15 mM paranitrophenyl and 5 Ìg/mL insulin. phosphate, 15 mM EDTA, 15 mM MgCl2, 2 mM DTT, 1 mM PolyA mRNAs from MDA-MB-231 cells were extracted by the sodium orthovanadate, 1 mM NaF, 1 mM phenylphosphate, guanidium thiocyanate/cesium chloride gradient, using RNA plus 10 Ìg/mL leupeptin, 10 Ìg/mL aprotinin, 10 Ìg/mL soybean trypsin reagent from Bioprobe followed by polydT columns (Pharmacia, inhibitor, 10 ÌM benzamidine) added with 1% Triton X-100. Biotech, Orsay, France) (20). Supernatants were precleared with protein A-agarose (50%, Sigma) for 1 h at 4ÆC. Immunoprecipitations were performed Oocyte handling. After anaesthesia with MS 222 (1g/L, Sandoz, using anti-FGFR antibodies (anti-FGFR1, clone 19B2, Upstate Vienna, Austria), Xenopus laevis ovarian fragments were surgically Biotechnology, Dundee, UK) for 3 h and Protein A-agarose (50%, removed and placed in ND96 medium (in mM: NaCl 96, KCl 2, Sigma) was added for 1 h at 4ÆC. Immuno-complexes were MgCl2 1, CaCl2 1.8, HEPES 5, adjusted to pH 7.4 with NaOH), collected by centrifugation, rinsed 3 times, resuspended in supplemented with streptomycin/penicillin (50 Ìg/mL, Eurobio, Les Laemmli sample buffer and subjected to a SDS-PAGE analysis. Ulis, France), sodium pyruvate (225 Ìg/mL, Sigma, Saint Quentin Fallavier, France) and soybean trypsin inhibitor (30 Ìg/mL, Sigma). Electrophoresis and Western blot analysis. For Western blot analysis, Stage VI oocytes were harvested by using 1-h treatment with oocytes were electrophoresed as described (22). Proteins were collagenase A (1 mg/mL, Boehringer, France). Complete transfered to a Hybond ECL membrane (Amersham Life Sciences, defolliculation of the oocytes was achieved by manual dissection. Freiburg, Germany) in Tris/NaCl/Tween/BSA pH 8.0 (15 mM Tris The oocytes were kept at 19ÆC in the ND96 medium. HCl, 150 mM NaCl, 0.1% tween, 10% bovine serum albumin, Sigma). The membranes were revealed with the following Fusion proteins and peptide synthesis. GST-Grb14 was produced antibodies: anti-PLCÁ antibody (Upstate Biotechnology), anti- as described before (18). PLCÁ-SH2 was a gift from Dr. S. phospho-PLCÁ antibody (Tyr 783, Upstate Biotechnology), anti- Courtneidge. Peptides pY766 (SNQE(p)YLDLS) and pY730 Grb14 antibodies (18), anti-FGFR1 antibody (clone 19B2, Upstate (TNEL(p)YMMMR) were a gift from Dr. Doherty. Peptides Biotechnology), anti-phospho-AKT antibody (Ser 473, Upstate pY776 (PLDQ(pY)SPSF) and Y776 (PLDQYSPSF) were Biotechnology). ERK2 phosphorylation was revealed by its mobility synthesised as follows: Fmoc-Tyr(PO3-MDPSE2)-OH was shift in Western blotting using an anti-ERK2 antibody (Santa Cruz purchased from Bachem Inc, and HMP resin and the other Biotechnology, Santa Cruz, USA). JNK1 phosphorylation was reagents for solid-phase peptide synthesis were from Applied revealed using an anti-active JNK antibody (pTPpY, Promega, Biosystems, (Foster City, CA, USA). The protected peptide Charbonnieres, France). Antibodies complexes were detected by the chains were performed using the stepwise solid-phase method of advanced enhanced chemiluminescence Western blotting detection Merrifield on an Applied Biosystems (ABI) 431A automated system (Amersham).

3878 Cailliau et al: Grb14 Adapter Blocks FGFR Signaling from MDA-MB-231 Cells

Figure 1. Grb14 blocks AKT, JNK1 and ERK2 activation. Immunodetection of AKT, JNK and ERK2 phosphorylation state. Oocytes expressing FGFRs from MDA-MB-231 for 48 h were stimulated or not by FGF1 (5 nM). One h before stimulation, oocytes were injected with Grb14 (75 ng), peptides pY766 (3 or 30 ng), or Y776, pY776 or pY730 (3 ng), or PLCÁ-SH2 (100 ng) alone or with Grb14 incubated for 1 h with the various peptides. The phosphorylated form of ERK2 is shown by an arrow.

Results 6, 14). However, pre-incubation of Grb14 with pY766 (3 ng) for 1 h before injection into the oocytes, restored MDA- Grb14 inhibits MDA-MB-231-FGFR transduction. Oocytes MB-231-FGFR signaling pathways: AKT, JNK1, ERK2 expressing FGFRs from MDA-MB-231 for 48 h were were phosphorylated, as in control FGF-stimulated oocytes stimulated or not by FGF1 (5 nM) for 5 min. FGF1 (lane 5 vs. 2). When an excess of phosphopeptide (30 ng) stimulation induced phosphorylation of AKT, JNK1 and was incubated with Grb14 prior injection, AKT, JNK1 and ERK2 in oocytes expressing FGFRs from MDA-MB-231 ERK2 remained unphosphorylated (lane 7). On the other cells, whereas unstimulated oocytes displayed no AKT, no hand, pY730 (a minor PLCÁ binding site on FGFR) (23) JNK1 and no ERK2 phosphorylation (Figure 1, lanes 1, 2). was unable to block AKT, JNK1 or ERK2 phosphorylation Injection of Grb14 before stimulation by FGF1 blocked (lane 12). In addition, pre-incubation of pY730 with Grb14 AKT, JNK1 and ERK2 phosphorylation (lane 3). did not prevent Grb14 inhibitory effect on FGFR signaling (lane 13). The tyrosine 766 phosphorylated site of FGF receptors is Y776 was described in fibroblast as another potential targeted by Grb14. We next investigated the potential targets target for Grb14 under its unphosphorylated or of Grb14. Injection of either a phosphopeptide pY766 (3 or phosphorylated form (15). Y776 or pY776 alone were 30 ng, a major binding site for PLCÁ on FGFR (10)) or unable to block FGFR signaling (lanes 8, 10). Their pre- PLCÁ-SH2 domains, 1 h before FGF1 addition, blocked incubation with Grb14 did not impede Grb14 ability to AKT, JNK1 and ERK2 phosphorylation (Figure 1, lanes 4, block AKT, JNK1 and ERK2 phosphorylation (lanes 9, 11).

3879 ANTICANCER RESEARCH 25: 3877-3882 (2005)

Discussion

In the present paper, we attempted to determine how Grb14 blocked FGF-induced signaling in MDA-MB-231 using a model system, the Xenopus oocyte, which is widely used to question cascade transduction regulations (21, 22, 24). We showed that Grb14 blocked the ERK2/JNK1 and PI-3 kinase/AKT pathways from FGF1-stimulated MDA-MB- 231-FGFRs, as no phosphorylation of ERK2, JNK1 or AKT were observed in Grb14-injected oocytes. We observed that PLCÁ coprecipitated with Grb14 and the receptor, but was not phosphorylated on its Y783 site, which is necessary for its activation (25). We further determined whether the FGFR Y766 site could be involved in Grb14-induced PLCÁ inhibition. pY766 is a phosphomimetic peptide encompassing the phosphorylated Y766 FGFR site. When injected into oocytes, it trapped endogenous PLCÁ, which was then no longer available for FGFR binding and subsequent activation of signaling pathways. Previous incubation of pY766 at low dose with Grb14 prevented Grb14 interacting with FGFR. Under these conditions, PLCÁ recruitment and phosphorylation were restored, as Figure 2. Grb14 is recruited to the activated FGFR and inhibits PLCÁ was the activation of the ERK2/JNK1 and AKT signaling phosphorylation. Oocytes expressing FGFRs of MDA-MB-231 for 48 h pathways. When an excess of pY766 (30 ng) was incubated were stimulated or not by FGF1 (5 nM). One h before stimulation, oocytes with Grb14, neither Grb14 nor PLCÁ coprecipitated with were injected with Grb14 alone, pY766 peptides (3 or 30 ng) alone or with the receptor, and no signaling was restored. In this case, the Grb14 preincubated for 1 h with the peptides. After 5-min stimulation, the phosphopeptide pY766 was then likely to bind and trap oocytes were lysed and immunoprecipitated with anti-FGFR antibodies. After SDS-PAGE analysis, the precipitated proteins were successively both Grb14 and PLCÁ. This suggests that Grb14 competes visualized by immunodetection with the indicated antibodies (anti-PLCÁ, with PLCÁ for the pY766 site of FGFR. In the presence of anti-phospho-PLCÁ (anti P-PLCÁ), anti-Grb14, anti-Grb2). The Grb14, PLCÁ still coprecipitated with FGFRs, but was not membrane was also immunorevealed with anti-FGFR1 antibodies to phosphorylated. Grb14 occupies the binding site of PLCÁ, control immunoprecipitation efficiency. which is then anchored directly or indirectly to FGFR but remains inactivated. Other FGFR tyrosine residues, such as phosphorylated or unphosphorylated Y776 (15), have been proposed to be implicated in Grb14 interaction, while To further confirm the involvement of the pY766 PLCÁ Y730 (23) has been suggested to be a minor PLCÁ-binding binding site in Grb14 blocking, we performed immuno- site. However, coprecipitation of Grb14, PLCÁ and precipitations of MDA-MB-231-FGFRs expressed in FGFRs, and phosphorylation of ERK2, JNK1 and AKT oocytes. PLCÁ did not coprecipitate with unstimulated remained unchanged when these peptides were tested, MDA-MB-231-FGFRs (Figure 2, lane 1), but in FGF1- indicating that these sites are not involved in the stimulated oocytes, PLCÁ coprecipitated with the receptors. recruitment of Grb14 to FGFRs. Interestingly, no In that case, PLCÁ was phosphorylated on its Y783 residue inhibition of Grb2 recruitment to the phosphorylated (lane 2). However, when Grb14 was injected before FGF1 FGFR was observed when Grb14 was injected, suggesting stimulation, both Grb14 and PLCÁ coprecipitated with that Grb14 specifically inhibits PLCÁ pathways. FGFRs, but in that case PLCÁ was not phosphorylated (lane A complex network of transduction cascades exists between 3). pY766 alone (3 or 30 ng) prevented PLCÁ precipitation the various effectors activated after ligand-induced FGFR1 with the receptor (lanes 4, 6). When a small amount of stimulation. We have previously shown that FGF1-induced pY766 was pre-incubated with Grb14, PLCÁ coprecipitated stimulation of MDA-MB-231-FGFRs and FGF1-FGFR1 with the receptor and was phosphorylated (lane 5). However, triggered three main transduction pathways, involving Grb2, in the presence of an excess of phosphopeptide, neither PLCÁ and PI-3 kinase that further lead to the downstream PLCÁ nor Grb14 were immunoprecipitated with the receptor activation of ERK2 and AKT (20). Furthermore, ERK2 also (lane 7). Whatever the experimental conditions, Grb2 always induces JNK1 activation after FGF1-FGFR1 interaction (26). coprecipitated with the stimulated receptors (lanes 2-7). In the literature, several results demonstrated the involvement

3880 Cailliau et al: Grb14 Adapter Blocks FGFR Signaling from MDA-MB-231 Cells of these signaling elements in breast cancer estrogen- 5 Renaud F, El Yazidi I, Boilly-Marer Y, Courtois Y and Laurent independent MDA-MB-231 cells: PLCÁ (12, 27) and ERK M: Expression and regulation by serum of multiple FGF1 mRNA (28) are involved in cell migration; ERK (29) and JNK in cell in normal transformed, and malignant human mammary epithelial cells. Biochem Biophys Res Commun 219: 679-685, 1996. proliferation; JNK (30) and PI-3 kinase/AKT (31) maintain 6 Fernig DG, Chen HL, Rahmoune H, Descamps S, Boilly B and anti-apoptosis mechanisms. The first target of Grb14 is the Hondermarck H: Differential regulation of FGF-1 and -2 Y766 FGFR site. Grb14 thus inhibits PLCÁ activation, and mitogenic activity is related to their kinetics of binding to consequently indirectly inhibits downstream effectors involved heparan sulfate in MDA-MB-231 human breast cancer cells. in cell invasiveness and anti-apoptosis. We cannot rule out Biochem Biophys Res Commun 267: 770-776, 2000. other interactions between Grb14 and other effectors. 7 Nurcombe V, Smart CE, Chipperfield H, Cool SM, Boilly B However, this inhibition is specific for the Y766 site, and does and Hondermarck H: The proliferative and migratory activities not affect the tyrosine kinase domain activation as assessed by of breast cancer cells can be differentially regulated by heparan sulfates. J Biol Chem 275: 30009-30018, 2000. Grb2 recruitment. We have shown previously that Grb14 does 8 Penault-Llorca F, Bertucci F, Adelaide J, Parc P, Coulier F, not affect the global tyrosine phosphorylation state of FGFR1 Jacquemier J, Birnbaum D and deLapeyriere O: Expression of under FGF1 stimulation (16). Targeting the Y766 residue of FGF and FGF receptor genes in human breast cancer. Int J FGFR can thus dramatically affect MDA-MB-231 cell growth. Cancer 61: 170-176, 1995. As a conclusion, our results suggest that Grb14 and its 9 Klint P and Claesson-Welsh L: Signal transduction by fibroblast binding site Y766 FGFR residue could represent an attractive growth factor receptors. Front Biosci 4: D165-177, 1999. pharmacological target for the inhibition of FGF1-induced 10 Mohammadi M, Dionne CA, Li W, Li N, Spivak T, Honegger AM, Jaye M and Schlessinger J: Point mutation in FGF estrogen-negative MDA-MB-231 cell signaling. Grb14 could receptor eliminates phosphatidylinositol hydrolysis without serve as a pharmacological inhibitor of these highly invasive affecting mitogenesis. Nature 358: 681-684, 1992. cell lines, which have lost the expression of this repressor. 11 Ong SH, Hadari YR, Gotoh N, Guy GR, Schlessinger J and Lax I: Stimulation of phosphatidylinositol 3-kinase by fibroblast growth Acknowledgements factor receptors is mediated by coordinated recruitment of multiple docking proteins. Proc Natl Acad Sci USA 98: 6074-6079, 2001. We are sincerely indebted to Dr. Doherty (Kings College, London, 12 Piccolo E, Innominato PF, Mariggio MA, Maffucci T, Iacobelli UK) for the Y766 and the Y730 peptides. This work was supported S and Falasca M: The mechanism involved in the regulation of by the "Ministère de l'Education Nationale", and by grants from the phospholipase Cgamma1 activity in cell migration. Oncogene "Ligue Nationale Contre le Cancer, Comité Du Nord" (grant 2005 to 21: 6520-6529, 2002. EBP and KC), from the "Ligue Nationale Contre le Cancer, Comité 13 Daly RJ: The Grb7 family of signalling proteins. Cell Signal 10: de Paris" (labelled team to CG), from the "Ministère de la 613-618, 1998. Recherche" (grant nÆ 01 C 0786 to AFB), and from the "Association 14 Cariou B, Bereziat V, Moncoq K, Kasus-Jacobi A, Perdereau pour la Recherche sur le Cancer" (ARC, grant 5237 to AFB), France. D, Le Marcis V and Burnol AF: Regulation and functional roles of Grb14. 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