[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 tumorigen- esis 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 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. SIGNALING PATHWAYS ACTIVATED BY CRIPTO-1 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 mono- clonal 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.