A Nodal- and ALK4-Independent Signaling Pathway Activated by Cripto-1 Through Glypican-1 and C-Src1

[CANCER RESEARCH 63, 1192–1197, March 15, 2003] Advances in Brief

A Nodal- and ALK4-independent Signaling Pathway Activated by Cripto-1 through Glypican-1 and c-Src1

Caterina Bianco, Luigi Strizzi, Aasia Rehman, Nicola Normanno, Christian Wechselberger, Youping Sun, Nadia Khan, Morihisa Hirota, Heather Adkins, Kevin Williams, Richard U. Margolis, Michele Sanicola, and David S. Salomon2 Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 [C. B., L. S., A. R., Y. S., N. K., M. H., D. S. S.]; Experimental Oncology B Unit, Istituto Fondazione Pascale, Naples 80131, Italy [N. N.]; Upper Austrian Research GmbH Zentrum, Linz 4020, Austria [C. W.]; Biogen, Inc., Cambridge, Massachusetts 02142 [H. A., K. W., M. S.]; and Department of Pharmacology, New York University Medical Center, New York, New York 10016 [R. U. M.]

Abstract ␤-related peptide ligand Nodal, signaling through the type II and type I (ALK4) serine/threonine kinase receptor complex that subsequently Human Cripto-1 (CR-1) is a member of the epidermal growth factor- induces Smad-2, Smad-3, and Smad-4 activation (5). In agreement Cripto FRL1 Cryptic family that has been shown to function as a core- with these results, we have cloned and identified ALK4 as a receptor ceptor with the type I Activin serine-threonine kinase receptor ALK4 for the transforming growth factor ␤-related peptide Nodal. However, CR-1 for CR-1 in mammalian cells (4). However, we found that activation can also activate the mitogen-activated protein kinase and Akt pathways of the ras/raf/MAPK and PI3K/Akt pathways by CR-1 is independent independently of Nodal and ALK4 by an unknown mechanism. Here, we of Nodal and ALK4 (4). Therefore, an unknown receptor(s) is medi- demonstrate that CR-1 specifically binds to Glypican-1, a membrane- ating the ability of CR-1 to activate these two signaling pathways. associated heparan sulfate proteoglycan, and activates the tyrosine kinase HSPGs are cell membrane proteins containing HSGAGs side chains c-Src, triggering the mitogen-activated protein kinase and Akt signaling and are able to bind and interact with a wide variety of molecules (6). pathways. Finally, an active Src kinase is necessary for CR-1 to induce in Recently, the two main groups of HSPGs, the transmembrane synde- vitro transformation and migration in mouse mammary epithelial cells. cans and the GPI-linked glypicans, have been implicated in the Introduction regulation of several aspects of cancer biology, including tumorigenesis and metastasis (6). The cytoplasmic tyrosine kinase c-Src has also Human CR-13, a GPI-linked membrane protein, is the founding been implicated in cancer development (7). Here, we demonstrate for member of the EGF-CFC family of proteins (1). Several studies have the first time that CR-1 specifically binds to a GPI-linked membrane implicated CR-1 in human carcinogenesis, suggesting that it might HSPG, Glypican-1, inducing activation of the cytoplasmic tyrosine function as an oncogene (2). In this context, overexpression of CR-1 kinase c-Src. can lead to the in vitro transformation of EpH-4 mouse mammary epithelial cells and can increase migration and branching morphogen- Materials and Methods esis of several mouse mammary epithelial cell lines (2, 3). CR-1 is also overexpressed in different types of primary human carcinomas, Cell Culture, Growth Factors, and Inhibitors. EpH-4, NMuMG, and including breast, colon, stomach, pancreas, ovary, and testis (2). HC-11 mouse mammary epithelial cells and COS1 monkey kidney cells were Finally, a direct role for CR-1 in tumorigenesis is supported from grown as described previously (3, 4). Human CR-1⌬C-Fc and Glypican-1-Fc transgenic studies demonstrating that mice which are overexpressing recombinant protein were purified as described previously (4, 8). PD153035, a mouse mammary tumor virus-human CR-1 transgene in the mam- AG1478, H-7, pertussis toxin, and PP2 were purchased from Calbiochem (San Diego, CA), PI-PLC from Sigma (St. Louis, MO), heparitinase from Seikagaku mary gland develop ductal hyperplasias and papillary adenocarcino- 4 Corporation (Falmouth, MA), EGF from Collaborative Research (Bedford, mas. Although a potential role for CR-1 in human carcinogenesis is MA), and GDNF from R&D Systems (Minneapolis, MN). SU-6656 was kindly evident, the signaling pathways activated by CR-1 that are responsible provided by Sugen, Inc. for cellular proliferation and transformation need clarification. In this ELISA. Glypican-1-Fc (200 ng/well), unglycanated Glypican-1-Fc (4 ␮g/ respect, we have shown that CR-1 can induce activation of signaling well), or BSA (1 ␮g/well) were absorbed to 96-well microtiter plates overnight pathways involved in cellular proliferation and survival such as ras/ at 4°C and then incubated with CR-1⌬C-Fc recombinant protein at concen- raf/MAPK and PI3K/Akt pathways (4). Genetic studies have also trations ranging from 6 ng to 1.6 ␮g/well (100 ␮l)for1hatroom temperature. shown that CR-1 functions as an obligatory coreceptor for the TGF- The ELISA was then developed as described previously (4). Coimmunoprecipitation in COS1 Cells. COS1 cells (1 ϫ 106 cells in 60-mm plates) were transiently transfected with Glypican-1-Fc (8), CR-1 (4), Received 10/7/02; accepted 1/31/03. The costs of publication of this article were defrayed in part by the payment of page and/or c-Src (Upstate Biotechnology, Lake Placid, NY) expression vectors charges. This article must therefore be hereby marked advertisement in accordance with either alone or combined using Fugene 6 (Roche, Indianapolis, IN). Forty-eight 18 U.S.C. Section 1734 solely to indicate this fact. h after transfection, the cells were lysed as previously described (4), and 800 1 Supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (to ␮g of total proteins were incubated with protein G Sepharose (Amersham N. N.). 2 To whom requests for reprints should be addressed, at MBTL, National Cancer Pharmacia, Piscataway, NJ), and the bound proteins were eluted with sample Institute, NIH, 10 Center Drive Building 10 5B39, Bethesda, MD 20892. Phone: buffer and analyzed by Western Blot analysis as previously described (4) using (301) 496-9536; Fax: (301) 402-8656; E-mail: [email protected]. a rabbit polyclonal anti-CR-1 antibody (Biocon, Frederick, MD) or a mouse 3 The abbreviations used are: CR-1, Cripto-1; HSPG, heparan sulfate proteoglycan; monoclonal anti-c-Src antibody (GD11; Upstate Biotechnology) or an antihu- HSGAG, heparan sulfate glycosylaminoglycan; GPI, glycosylphospatidylinositol; EGF, epidermal growth factor; CFC, Cripto FRL1 Cryptic; TGF-␤, transforming growth factor man Fc antibody (The Jackson Laboratory, West Grove, PA). ␤; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3Ј kinase; PI- Western Blot Analysis. EpH-4, EpH-4 Nodal (4), NMuMG, and HC-11 PLC, phosphatidylinositol phospholipase C; FGF, fibroblast growth factor; GDNF, glial mouse mammary epithelial cells were seeded in 60-mm plates (8 ϫ 105 cell line-derived neurotrophic factor. 4 cells/plate) and serum starved for 24 h. The cells were pretreated with C. Wechselberger, M. Hirota, N. Kenney, C. Bianco, L. Strizzi, Y. Sun, M. Sanicola, ␮ and D. S. Salomon. Mouse mammary tumor virus-Cripto-1 transgenic mice, manuscript in PD153035 (75 mM), AG1478 (1 M), pertussis toxin (100 ng/ml), H-7 (100 preparation. ␮M), PI-PLC (1 unit/ml), or heparitinase (0.02 units/ml) for1horPP2(1or 1192

10 ␮M) for 15 min at 37°C and then stimulated with EGF (50 ng/ml for 5 min), presence of a biotinilated-c-Src substrate peptide, and the phosphorylated GDNF (50 ng/ml for 5 min), or CR-1⌬C-Fc (200 ng/ml for 5 min for MAPK peptide was absorbed on strepavidin-coated plates (Chemicon, Temecula, CA). and 400 ng/ml for 30 min for Smad-2 and Akt). Western blots for phospho and The amount of phosphorylated peptide was detected with a phosphotyrosine- total MAPK, Akt, and Smad-2 were performed as described previously (4). specific monoclonal antibody (Chemicon), and the absorbance read at 450 nm. Western blot analysis for phospho and total c-Src in serum-starved EpH-4 cells The experiment was done in duplicates and repeated three times. stimulated with EGF (50 ng/ml for 30 min) or CR-1⌬C-Fc (400 ng/ml for 15 and 30 min) was performed using an antiphospho c-Src (Tyr 416) rabbit Results polyclonal antibody (Cell Signaling, Beverly, MA) or GD11 mouse monoclonal anti-c-Src antibody (Upstate Biotechnology). Serum-starved EpH-4 PI-PLC, Heparitinase, and PP2 Block CR-1 Activation of cells were also pretreated with PP2 (1 ␮M) and then stimulated for 30 min with MAPK and Akt but have no Significant Effect on Smad-2 Phos- EGF (100 ng/ml) or CR-1⌬C-Fc (400 ng/ml). Basal levels of phospho-c-Src in phorylation. We tested the effect of the following signaling pathway EpH-4 wild type and CR-1 cells were assessed after 24 h of serum starvation inhibitors on CR-1-induced MAPK phosphorylation in EpH-4 mam- by Western blot analysis as described above. mary epithelial cells: the EGF receptor tyrosine kinase inhibitors Cell Proliferation, Soft-Agar, and Migration Assays. Anchorage-depen- (PD153035 and AG1478); a serine/threonine kinase inhibitor (H-7); a dent cell growth was performed as described previously (3). PP2, SU-6656 (1 G-protein inhibitor (pertussis toxin); the enzymes PI-PLC and hep- or 10 ␮M), or the vehicle DMSO (control samples) was added to the cells together with serum-free medium. For the soft agar assay, EpH-4, and EpH-4 aritinase, which induce cleavage of GPI-linked proteins and digestion CR-1 were seeded in 6-multiwell plates (5 ϫ 104/well) in the presence of PP2, of HSGAG polysaccharide side chains, respectively; and the c-Src SU-6656 (1 or 10 ␮M), or DMSO (control samples), and after 2 weeks, tyrosine kinase inhibitor PP2. EpH-4 mouse mammary epithelial cells colonies were counted as described previously (3). Cell migration assays were were serum-starved for 24 h and then pretreated for different times performed using FluoroBlok 24-multiwell Insert Plates (8-␮m pore size) with various inhibitors followed by stimulation with a soluble, COOH coated with Matrigel Membrane Matrix (Becton Dickinson, Bedford, MA). terminus-truncated CR-1 recombinant protein containing an Fc fusion Five percent FBS was used as chemoattractant in the lower chamber. EpH-4 or tag (CR-1⌬C-Fc). Whereas the EGF receptor tyrosine kinase, G- 6 EpH-4 CR-1 cells (5 ϫ 10 ) were labeled in situ with 10 ␮g/ml of Dil (Becton protein, and serine/threonine kinase inhibitors did not have any sig- ϫ 4 Dickinson) for1hat37°C and then seeded in 12-multiwell plates (25 10 nificant effect on MAPK phosphorylation induced by CR-1 (data not cells/well). After 16 h, the migrated cells were counted with a fluorescent shown), PI-PLC and heparitinase significantly interfered with the microscope (three random fields/well at magnitude ϫ400). All these experiments were done in duplicates and repeated at least three times. ability of CR-1 to induce MAPK activation in EpH-4 cells (Fig. 1, A Src Tyrosine Kinase Assay. Serum-starved EpH-4 cells were stimulated and B). MAPK phosphorylation was also inhibited by PI-PLC when with CR-1⌬C-Fc (400 ng/ml), and c-Src protein was immunoprecipitated using the cells were stimulated with GDNF, which binds to a GPI-linked a rabbit polyclonal anti-c-Src antibody (2 ␮g; Santa Cruz Biotechnology, Santa coreceptor GFR␣1 and signals through the tyrosine kinase receptor Cruz, CA). The immunoprecipitated c-Src protein was then incubated in the c-RET (Fig. 1A; Ref. 9). In contrast, MAPK phosphorylation by EGF

Fig. 1. PI-PLC, heparitinase and PP2 interfere with the ability of CR-1 to activate MAPK and Akt but not Smad-2 in mouse mammary epithelial cells. Serum-starved EpH-4 cells were treated with different inhibitors, stimulated with CR-1⌬C-Fc (CR-1), GDNF, or EGF and analyzed by Western blot analysis using phopsho- and nonphospho-specific anti MAPK (A–C), Smad-2 (D) and Akt (E) antibodies. 1193

1-Fc fusion protein was absorbed to microtiter plates and binding of a CR-1⌬C-Fc recombinant protein was assessed by ELISA. CR- 1⌬C-Fc recombinant protein was able to bind in a specific and ϳ saturable manner to Glypican-1-Fc with a Kd of 10 nM (Fig. 2A). In contrast, an unglycanated Glypican-1-Fc core protein or BSA were unable to bind to CR-1⌬C-Fc recombinant protein. To determine whether CR-1 could bind to Glypican-1 expressed on the surface of mammalian cells, monkey kidney COS1 cells were transiently transfected with full-length CR-1 and with Fc-tagged Glypican-1 expression vectors. Because c-Src is required for CR-1 activation of MAPK and Akt, c-Src was also coexpressed in COS1 cells with Glypican-1 and CR-1. Glypican-1-Fc was immunoprecipitated from transiently transfected COS1 cells using protein G-Sepharose gel that binds to the Fc portion of the Glypican-1-Fc fusion protein, and immunoprecipitated proteins were analyzed for the presence of CR-1 or c-Src using monospecific antibodies. CR-1 was found to directly interact with Glypican-1, and c-Src was present in a complex with Glypican-1 and CR-1 proteins (Fig. 2B). c-Src was also found to interact directly with Glypican-1 in the absence of CR-1 but to a lesser extent.

Fig. 2. CR-1 binds to Glypican-1 in ELISA and in coimmunoprecipitation experiments. A, Glypican-1-Fc, unglycanated Glypican-1-Fc, or BSA were absorbed to 96-well microtiter plates and incubated with various concentrations of CR-1⌬C-Fc recombinant protein ␮ ␮ ( g/100 l). The inset shows the kd of the experiment. B, COS1 cells were transiently transfected with Glypican-Fc, CR-1, and/or c-Src expression vectors, and Fc-tagged immunoprecipitated proteins were analyzed by Western blot analysis with an anti-CR-1, anti-c-Src, or antihuman Fc antibodies.

was not affected by the presence of heparitinase or PI-PLC (Fig. 1B and data not shown). The c-Src inhibitor PP2 also inhibited phosphorylation of MAPK by CR-1 in these cells (Fig. 1C). In contrast, when EpH-4 Nodal cells were treated with PI-PLC, heparitinase, or PP2, CR-1 was still able to activate Smad-2 regardless of the presence of these different inhibitors (Fig. 1D). Interestingly, when the same membrane was reprobed with a specific phospho-Akt antibody, the three compounds specifically inhibited Akt phosphorylation induced by CR-1 in these cells (Fig. 1E). Similar results were obtained in other two mouse mammary epithelial cell lines NMuMG and HC-11 (data not shown). CR-1 Binds to Glypican-1 in ELISA and Coimmunoprecipi- tates with Glypican-1 and c-Src in COS1 Cells. The syndecans and the glypicans contain HSGAG side chains, but only glypicans are Fig. 3. CR-1 induces activation of the tyrosine kinase c-Src. A, serum-starved EpH-4 GPI-linked proteins and therefore are sensitive to both PI-PLC and cells were stimulated with CR-1⌬c-Fc (CR-1) or EGF and analyzed by Western blot heparitinase digestion. We therefore evaluated whether CR-1 could analysis using phospho- and nonphospho-specific anti-c-Src antibodies. B, serum-starved EpH-4 cells were pretreated with PP2 and then stimulated with EGF or CR-1⌬c-Fc interact with Glypican-1, which among the six glypicans identified (CR-1). C, serum-starved EpH-4 cells were stimulated with CR-1⌬c-Fc (CR-1), and the thus far, is the one most involved in tumorigenesis (6). A Glypican- kinase reaction was performed as described under “Materials and Methods.” 1194

CR-1 Induces Activation of c-Src. We next evaluated whether CR-1 Induced in Vitro Transformation and Migration in CR-1 after binding to Glypican-1 could induce c-Src activation in EpH-4 CR-1 Cells Requires Active c-Src. We assessed whether EpH-4 mouse mammary epithelial cells. A 10- and 18-fold in- c-Src is essential for CR-1 to induce in vitro transformation and crease in the phosphorylation of c-Src was detected in EpH-4 cells migration in EpH-4 cells. As shown in Fig. 4B, CR-1-expressing after 15- or 30-min treatment with CR-1⌬C-Fc protein, respec- EpH-4 cells exhibited a 4-fold increase in growth rate after 3 days tively (Fig. 3A). EGF, which was used as a positive control, was under serum-free conditions compared with wild-type EpH-4 cells. also found to induce c-Src phosphorylation. Pretreatment of EpH-4 The enhanced proliferation of EpH-4 CR-1 cells could be reduced cells with PP2 inhibited c-Src phosphorylation induced by CR-1 or to basal levels in a dose-dependent manner by treatment of the cells EGF (Fig. 3B). We then measured c-Src kinase activity directly with the c-Src inhibitor PP2. The proliferation of wild-type EpH-4 using a specific c-Src peptide substrate in an ELISA kinase assay. cells was also inhibited by high concentrations of PP2 but to a CR-1 was found to induce activation of the tyrosine kinase c-Src lesser degree. Likewise, EpH-4 CR-1 cells growth in soft agar was in a time-dependent manner with a peak at 30 min of stimulation also inhibited by treatment with PP2 in a dose-dependent fashion (Fig. 3C). (Fig. 4C). Another c-Src inhibitor, SU-6656, also specifically EpH-4 Cells Expressing CR-1 Exhibit High Levels of Activated inhibited growth in serum-free and in soft agar of EpH-4 CR-1 c-Src. Because CR-1 can activate c-Src in mammary epithelial cells, cells (data not shown). To determine the effect of PP2 on the we investigated the basal tyrosine phosphorylation levels of c-Src in migratory behavior of EpH-4 CR-1 cells, we performed a migra- CR-1-transfected EpH-4 cells. After 24 h of starvation in serum-free tion assay using Matrigel base membrane-coated plates. EpH-4 medium, EpH-4 CR-1 cells showed a ϳ10-fold increase in the basal CR-1 cells exhibited a 6-fold increase in their ability to migrate levels of activated, phosphorylated c-Src as compared with wild-type compared with wild-type cells. PP2 induced a dose-dependent EpH-4 cells (Fig. 4A). inhibition in the migration of EpH-4 CR-1 cells, whereas PP2 did

Fig. 4. Src is essential for CR-1 induced in vitro transformation and migration. A, basal levels of c-Src phosphorylation in serum-starved cells were assessed by Western blot analysis with an antiphospho-src antibody. B, EpH-4 and EpH-4 CR-1 cells were cultured in serum-free medium in the presence of PP2 and counted after the indicated times. C, EpH-4 and EpH-4 CR-1 cells were cultured for 2 weeks in soft agar in the presence of different concentration of PP2. D, migration of EpH-4 and EpH-4 CR-1 cells through Matrigel base membrane-coated filters in the presence of different concentrations of PP2. 1195

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. SIGNALING PATHWAYS ACTIVATED BY CRIPTO-1 not exert any significant effect on migration of wild-type EpH-4 motility has been demonstrated in several types of cells (7). However, cells (Fig. 4D). the mechanism by which CR-1 activates c-Src is unclear. It is possible that CR-1 and c-Src colocalize in lipid rafts, regions of cell membranes Discussion enriched in cholesterol and sphingolipids (19). Lipid rafts serve as a This study describes the identification of Glypican-1 as a corecep- microdomain either for a number of signaling proteins that are GPI- tor for CR-1 and c-Src as a key intracellular regulator of MAPK and linked such as Glypican-1 and CR-1 or lipid-bound intracellular mole- Akt phosphorylation induced by CR-1. Cell surface HSPGs such as cules such as Src family kinases (19). Therefore, CR-1, Glypican-1, and glypicans and syndecans can directly bind growth factors that are c-Src could potentially cluster together in these membrane regions. In this important for tumor development such as FGFs 1 and 2, vascular respect, coimmunoprecipitation of c-Src with Glypican-1 also in the endothelial growth factor, TGF-␤, and Wnt proteins (6, 10). Upon absence of CR-1 suggests that these two molecules probably colocalize. binding to these different growth factors, HSPGs increase the affinity Therefore, interaction of CR-1 with Glypican-1 in the lipid rafts might of these molecules for their signaling receptors and facilitate receptor trigger activation of c-Src kinase. Likewise, GPI-anchored immunore- dimerization and subsequent signaling (11). Therefore, they have been ceptors on T cells upon interaction with their natural ligands or by considered as coreceptors for various growth factors. Our study dem- antibody cross-linking cause a redistribution of Src family kinases on the onstrates for the first time a direct interaction between an HSPG, cytoplasmic site, initiating their autophosphorylation and activation (20). Glypican-1, and CR-1. In fact, CR-1 binds in a specific and saturable Another possibility is that an unknown transmembrane tyrosine kinase manner to Glypican-1 in an ELISA assay, and this interaction is lost receptor is activated by CR-1 and recruits c-Src through the SH2 domain. when HSGAG side chains are removed from the Glypican-1 core Whereas Glypican-1 and c-Src are required for activation of MAPK and protein, suggesting that HSGAGs are mediating the binding of CR-1 Akt pathways by CR-1, Smad-2 phosphorylation induced by CR-1 is to Glypican-1. This result is in agreement with several studies show- independent of these two signaling molecules. These findings are also ing that the specificity of HSPGs interactions with growth factors such supported by our previous biochemical studies in which CR-1 can trigger as FGF is dependent on HSGAG side-chain sequence (6). Further- MAPK/Akt activation in cells that lack Nodal and/or ALK4 expression more, coimmunoprecipitation of Glypican-1 and CR-1 from mamma- (4). Therefore, we propose a model in which CR-1 can function as either lian cells shows that this interaction also occurs in vivo. The impor- a ligand for Glypican-1 or as a coreceptor for Nodal by independently tance of this interaction is demonstrated by the ability of PI-PLC and activating the c-Src/ras/raf/MAPK/PI3K/Akt or the Nodal/ALK4/ heparitinase to significantly interfere with activation of MAPK and Smad-2 signaling pathways. However, these two signaling pathways may Akt signaling pathways by CR-1. Likewise, it has previously been not be mutually exclusive because TGF-␤ can also activate c-Src in shown that treatment of human breast cancer cell lines with PI-PLC epithelial cells and because a ras-GAP binding protein DOK-1 can also blocks the mitogenic response to several heparin binding growth mediate Activin A induced activation of Smad-2 and Smad-3 through factors such as heparin binding-EGF and FGF2 (12). In addition, ALK4 (7, 21). expression of Glypican-1 is elevated in human breast cancer, whereas its expression is low in normal breast tissue (12). Because CR-1 is also Acknowledgments overexpressed in human breast cancer (2), Glypican-1 may act to enhance the growth-promoting effects of CR-1 in breast cancer cells. We thank Matthew Jarpe of Biogen for help in calculating the Kd and Glypicans, such as dally and knypek, also have a well-established role analyzing the kinetics of the ELISA binding. We also thank Brenda Jones for in embryonic development where they serve as coreceptors for TGF-␤ her excellent technical assistance. family members such as dpp as well as other heparin binding growth factors such as the Wnt proteins (13, 14). Therefore, Glypican-1 or a References related HSPG might also regulate CR-1 signaling during embryonic 1. Shen, M. M., and Schier, A. F. The EGF-CFC gene family in vertebrate development. development. Inappropriate activation of MAPK and Akt transduction Trends Genet., 16: 303–309, 2000. pathways by CR-1 could play an essential role in the pathogenesis of 2. Salomon, D. S., Bianco, C., Ebert, A., Khan, N., De Santis, M., Normanno, N., Wecheslberger, C., Seno, M., Williams, K., Sanicola, M., Foley, S., Gullick, W. J., human cancer because these two signaling pathways have been shown and Persico, M. G. The EGF-CFC family: novel epidermal growth factor-related to induce cell growth and survival (15, 16). In this study, we demon- proteins in development and cancer. Endocr. Relat. Cancer, 7: 199–226, 2000. strate that c-Src is required by CR-1 to induce MAPK and Akt 3. Wechselberger, C., Ebert, A., Bianco, C., Khan, N., Sun, Y., Wallace-Jones, B., Monte- sano, R., and Salomon, D. S. Cripto-1 enhances migration and branching morphogenesis activation in mouse mammary epithelial cells. Activated c-Src prob- of mouse mammary epithelial cells. Exp. Cell Res., 266: 95–105, 2001. ably stimulates the ras/MAPK pathway through phosphorylation of 4. Bianco, C., Adkins, H. B., Wechselberger, C., Seno, M., Normanno, N., De Luca, A., the adaptor protein Shc and the Akt pathway by activation of PI3K. In Sun, Y., Khan, N., Kenney, N., Ebert, A. D., Williams, K. P., Sanicola, M., and Salomon, D. S. Cripto-1 activates nodal- and ALK4-dependent and -independent this context, CR-1 has been shown to stimulate Shc phosphorylation signaling pathways in mammary epithelial cells. Mol. Cell. Biol., 22: 2586–2597, and activation of PI3K in mouse mammary epithelial cells (17, 18). 2002. Furthermore, high levels of c-Src activity are present in EpH-4 cells 5. Yeo, Y., and Whitman, M. Nodal signals to Smads through Cripto-dependent and Cripto-independent mechanisms. Mol. Cell, 7: 949–957, 2001. overexpressing CR-1, and an intact c-Src kinase is required by CR-1 6. Sasisekharan, R., Shriver, Z., Venkataraman, G., and Narayanasami, U. Roles of to induce in vitro transformation and to enhance migration of EpH-4 heparan-sulphate glycosaminoglycans in cancer. Nat. Rev., 2: 521–528, 2002. cells. We have also detected high levels of c-Src activity in mammary 7. Frame, M. C. Src in cancer: deregulation and consequences for cell behaviour. Biochim. Bhiophys. Acta, 16: 114–130, 2002. tumors of mouse mammary tumor virus-CR-1 transgenic mice as 8. Ronca, F., Andersen, J. S., Paech, V., and Margolis, R. U. Characterization of Slit compared with the c-Src activity in the mammary gland of multipa- protein interactions with glypican-1. J. Biol. Chem., 276: 29141–29147, 2001. rous animals, suggesting that c-Src may also mediate CR-1 induced 9. Airaksinen, M. S., and Saarma, M. The GDNF family: signalling, biological functions 5 and therapeutic value. Nat. Rev. Neurosci., 3: 383–394, 2002. tumorigenesis in vivo. Elevated c-Src kinase activity, and in some 10. Filmus J. Glypicans in growth control and cancer. Glycobiology, 11: 19R–23R, 2001. cases protein expression, has also been reported in human breast 11. Park, P. W., Reizes, O., and Bernfield, M. Cell surface heparan sulfate proteoglycans: tumors, and a role for c-Src in regulating cellular migration and selective regulators of ligand-receptor encounters. J. Biol. Chem., 275: 29923–29926, 2000. 12. Matsuda, K., Maruyama, H., Guo, F., Kleeff, J., Itakura, J., Matsumoto, Y., Lander, 5 L. Strizzi, C. Bianco, C. Wechselberger, Y. Sun, M. Hirota, N. Kenney, M. Sanicola, A. D., and Korc, M. Glypican-1 is overexpressed in human breast cancer and and D. S. Salomon. Epithelial mesenchimal transition signaling in breast lesions from modulates the mitogenic effects of multiple heparin-binding growth factors in breast Cripto-1 transgenic mice, manuscript in preparation. cancer cells. Cancer Res., 61: 5562–5569, 2001. 1196

13. Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox, B., Humphrey, tyrosine phosphorylation of Shc and activates mitogen-activated protein kinase M., Olson, S., Futch, T., Kaluza, V., Siegfried, E., Stam, L., and Selleck, S. B. The (MAPK) in mammary epithelial cells. J. Biol. Chem., 272: 3330–3335, 1997. cell-surface proteoglycan Dally regulates Wingless signalling in Drosophila. Nature 18. De Santis, M., Kannan, S., Smith, G. H., Seno, M., Bianco, C., Kim, N., Martinez- (Lond.), 400: 276–280, 1999. Lacaci, I., Wallace-Jones, B., and Salomon, D. S. Cripto-1 inhibits ␤-casein expres- 14. Topczewski, J., Sepich, D. S., Myers, D. C., Walker, C., Amores, A., Lele, Z., sion in mammary epithelial cells through a p21ras-and phosphatidylinositol 3Ј-kinase- Hammerschmidt, M., Postlethwait, J., and Solnica-Krezel, L. The zebrafish glypican dependent pathway. Cell Growth Differ., 8: 1257–1266, 1997. knypek controls cell polarity during gastrulation movements of convergent extension. 19. Simons, K., and Toomre, D. Lipid rafts and signal transduction. Nat. Rev., 1: 31–39, Dev. Cell, 1: 251–264, 2001. 2000. 15. Robinson, M. J., and Cobb, M. H. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol., 9: 180–186, 1997. 20. Horejsi, V., Drbal, K., Cebecauer, M., Cerny, J., Brdicka, T., Angelisova, P., and 16. Datta, S. R., Brunet, A., and Greenberg, M. E. Cellular survival: a play in three Akts. Stockinger, H. GPI-microdomains: a role in signalling via immunoreceptors. Immu- Genes Dev., 13: 2905–2927, 1999. nol. Today, 20: 356–361, 1999. 17. Kannan, S., De Santis, M., Lohmeyer, M., Reise, D. J., II, Smith, G., Hynes, N., Seno, 21. Yamakawa, N., Tsuchida, K., and Sugino, H. The rasGAP-binding protein, Dok-1, M., Brandt, R., Bianco, C., Persico, M., Kenney, N., Normanno, N., Martinez-Lacaci, mediates activin signaling via serine/threonine kinase receptors. EMBO J., 21: I., Ciardiello, F., Stern, D., Gullick, W., and Salomon, D. S. Cripto enhances the 1684–1694, 2002.

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FUTURE ANNUAL MEETINGS OF THE AMERICAN AACR-NCI-EORTC INTERNATIONAL CONFERENCE ON ASSOCIATION FOR CANCER RESEARCH MOLECULAR TARGETS AND CANCER THERAPEUTICS November 17–21, 2003 2004 March 27–31, Orlando, FL Hynes Center, Boston, MA 2005 April 16–20, Anaheim, CA Chairpersons Charles L. Sawyers, Los Angeles, CA Edward A. Sausville, Bethesda, MD AACR SPECIAL CONFERENCES IN CANCER RESEARCH Jaap Verweij, Rotterdam, The Netherlands A number of meetings are now being organized in the AACR’s series of smaller scientific meetings. Following are the topics, dates, locations, and CALENDAR OF EVENTS program committees for these meetings. When full details of each meeting are available, AACR members will be the first to receive complete brochures and 16th Annual Meeting of the American Society of Pediatric Hematology & application forms for participation in these important conferences. Nonmem- Oncology, May 1–4, 2003, Sheraton Seattle Hotel and Tours, Seattle, WA. bers may receive this information by sending their names and addresses to Contact: Marilyn Rutkowski. Phone: 847.375.4830; E-mail: mrutkowski@ Meetings Mailing List, American Association for Cancer Research, Public amctec.com; Website: www.aspho.org. Ledger Building, 150 South Independence Mall West, Suite 826, Philadelphia, PA 19106-3483. Up-to-date program information is also available via the Life Sciences Research Office Conference on Acrylamide in Food: Are Chil- Internet at the AACR’s website (http://www.aacr.org). dren a Special Population at Risk? May 29–30, 2003. Sheraton Crystal City, Arlington, VA. Contact: Life Sciences Research Office, 9650 Rockville Pike, Bethesda, MD 20814-3998. Phone: 301.530.7030; Fax: 301.571.1876; E-mail: [email protected]. Website: http://www.Isro.org. SNPs AND CANCER: MOLECULAR EPIDEMIOLOGY IN HUMAN POPULATIONS Thomas L. Petty Aspen Lung Conference 46th Annual Meeting: Lung Cancer: September 13–17, 2003 Early Events, Early Interventions, June 4–7, 2003, Given Institute, Aspen Sonesta Beach Resort, Key Biscayne, FL Colorado. Contact: Jeanne Cleary, University of Colorado Health Sciences Center, Box C272, 4200 East 9th Avenue, Denver, CO 80262. Phone: 303.752.2681; Fax: 303.752.2678; E-mail: [email protected]; Website: Chairpersons www.aspenlungconference.org. Timothy R. Rebbeck, Philadelphia, PA Fred F. Kadlubar, Jefferson, AR 16th International Symposium of the Journal of Steroid Biochemistry & Molecular Biology: Recent Advances in Steroid Biochemistry and Molecular Biology, June 5–8, 2004, Seefeld, Tyrol, Austria. Contact: Prof. J. R. Pas- qualini, Steroid Hormone Research Unit, Institut de Pue´riculture, 26 Boulevard NEW DISCOVERIES IN BREAST CANCER: METHODS, BIOLOGY, Brune, 75014 Paris, France. Phone: 33.1.4539.9109; Fax: 33.1.4542.6121; AND CLINICAL IMPLICATIONS E-mail: [email protected]. October 8–12, 2003 Hyatt Regency Huntington Beach Resort & Spa, Huntington Beach, CA MASCC/ISOO 15th International Symposium of Supportive Care in Cancer, June 18–21, 2003, Berlin, Germany. Contact: Kinga M. Tahy/Julia Boettger, Chairpersons Dachauer Str. 44a, D-80335 Munich, Germany. Phone: 49.89.5490.96.70; Carlos L. Arteaga, Nashville, TN Fax: 49.89.5490.96.75; E-mail: [email protected]; Website: www. Lewis A. Chodosh, Philadelphia, PA symposium-online.de/mascc.

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Correction

In this article by C. Bianco et al., entitled “A Nodal- and ALK4-independent Signaling Pathway Activated by Cripto-1 through Glypican-1 and c-Src,” which appeared in the March 15, 2003 issue of Cancer Research (pp. 1192– 1197), the affiliations of Nicola Normanno appeared incorrectly. The correct affiliations are Division of Hematological Oncology and Department of Experimental Oncology, INT-Fondazione Pascale, 80131 Naples, Italy.

Catarina Bianco Luigi Strizzi Aasia Rehman Nicola Normanno Christian Wechselberger Youping Sun Nadia Khan Morihisa Hirota Heather Adkins Kevin Williams Richard U. Margolis Michele Sanicola David S. Salomon

1997 A Nodal- and ALK4-independent Signaling Pathway Activated by Cripto-1 through Glypican-1 and c-Src

Caterina Bianco, Luigi Strizzi, Aasia Rehman, et al.

Cancer Res 2003;63:1192-1197.

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