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[Cancer Research 63, 1192-1197, March 15, 2003]
© 2003 American Association for Cancer Research

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
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Human Cripto-1 (CR-1) is a member of the epidermal growth factor-Cripto FRL1 Cryptic family that has been shown to function as a coreceptor with the type I Activin serine-threonine kinase receptor ALK4 for the transforming growth factor ß-related peptide Nodal. However, CR-1 can also activate the mitogen-activated protein kinase and Akt pathways independently of Nodal and ALK4 by an unknown mechanism. Here, we demonstrate that CR-1 specifically binds to Glypican-1, a membrane-associated heparan sulfate proteoglycan, and activates the tyrosine kinase c-Src, triggering the mitogen-activated protein kinase and Akt signaling pathways. Finally, an active Src kinase is necessary for CR-1 to induce in vitro transformation and migration in mouse mammary epithelial cells.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Human CR-13 , a GPI-linked membrane protein, is the founding member of the EGF-CFC family of proteins (1) . Several studies have implicated CR-1 in human carcinogenesis, suggesting that it might function as an oncogene (2) . In this context, overexpression of CR-1 can lead to the in vitro transformation of EpH-4 mouse mammary epithelial cells and can increase migration and branching morphogenesis of several mouse mammary epithelial cell lines (2 , 3) . CR-1 is also overexpressed in different types of primary human carcinomas, including breast, colon, stomach, pancreas, ovary, and testis (2) . Finally, a direct role for CR-1 in tumorigenesis is supported from transgenic studies demonstrating that mice which are overexpressing a mouse mammary tumor virus-human CR-1 transgene in the mammary gland develop ductal hyperplasias and papillary adenocarcinomas.4 Although a potential role for CR-1 in human carcinogenesis is evident, the signaling pathways activated by CR-1 that are responsible for cellular proliferation and transformation need clarification. In this respect, we have shown that CR-1 can induce activation of signaling pathways involved in cellular proliferation and survival such as ras/raf/MAPK and PI3K/Akt pathways (4) . Genetic studies have also shown that CR-1 functions as an obligatory coreceptor for the TGF-ß-related peptide ligand Nodal, signaling through the type II and type I (ALK4) serine/threonine kinase receptor complex that subsequently induces Smad-2, Smad-3, and Smad-4 activation (5) . In agreement with these results, we have cloned and identified ALK4 as a receptor for CR-1 in mammalian cells (4) . However, we found that activation of the ras/raf/MAPK and PI3K/Akt pathways by CR-1 is independent of Nodal and ALK4 (4) . Therefore, an unknown receptor(s) is mediating the ability of CR-1 to activate these two signaling pathways. HSPGs are cell membrane proteins containing HSGAGs side chains and are able to bind and interact with a wide variety of molecules (6) . Recently, the two main groups of HSPGs, the transmembrane syndecans and the GPI-linked glypicans, have been implicated in the regulation of several aspects of cancer biology, including tumorigenesis and metastasis (6) . The cytoplasmic tyrosine kinase c-Src has also been implicated in cancer development (7) . Here, we demonstrate for the first time that CR-1 specifically binds to a GPI-linked membrane HSPG, Glypican-1, inducing activation of the cytoplasmic tyrosine kinase c-Src.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Culture, Growth Factors, and Inhibitors.
EpH-4, NMuMG, and HC-11 mouse mammary epithelial cells and COS1 monkey kidney cells were grown as described previously (3 , 4) . Human CR-1{Delta}C-Fc and Glypican-1-Fc recombinant protein were purified as described previously (4 , 8) . PD153035, AG1478, H-7, pertussis toxin, and PP2 were purchased from Calbiochem (San Diego, CA), PI-PLC from Sigma (St. Louis, MO), heparitinase from Seikagaku Corporation (Falmouth, MA), EGF from Collaborative Research (Bedford, MA), and GDNF from R&D Systems (Minneapolis, MN). SU-6656 was kindly provided by Sugen, Inc.

ELISA.
Glypican-1-Fc (200 ng/well), unglycanated Glypican-1-Fc (4 µg/well), or BSA (1 µg/well) were absorbed to 96-well microtiter plates overnight at 4°C and then incubated with CR-1{Delta}C-Fc recombinant protein at concentrations ranging from 6 ng to 1.6 µg/well (100 µl) for 1 h at room temperature. The ELISA was then developed as described previously (4) .

Coimmunoprecipitation in COS1 Cells.
COS1 cells (1 x 106 cells in 60-mm plates) were transiently transfected with Glypican-1-Fc (8) , CR-1 (4) , and/or c-Src (Upstate Biotechnology, Lake Placid, NY) expression vectors either alone or combined using Fugene 6 (Roche, Indianapolis, IN). Forty-eight h after transfection, the cells were lysed as previously described (4) , and 800 µg of total proteins were incubated with protein G Sepharose (Amersham Pharmacia, Piscataway, NJ), and the bound proteins were eluted with sample buffer and analyzed by Western Blot analysis as previously described (4) using a rabbit polyclonal anti-CR-1 antibody (Biocon, Frederick, MD) or a mouse monoclonal anti-c-Src antibody (GD11; Upstate Biotechnology) or an antihuman Fc antibody (The Jackson Laboratory, West Grove, PA).

Western Blot Analysis.
EpH-4, EpH-4 Nodal (4) , NMuMG, and HC-11 mouse mammary epithelial cells were seeded in 60-mm plates (8 x 105 cells/plate) and serum starved for 24 h. The cells were pretreated with PD153035 (75 mM), AG1478 (1 µM), pertussis toxin (100 ng/ml), H-7 (100 µM), PI-PLC (1 unit/ml), or heparitinase (0.02 units/ml) for 1 h or PP2 (1 or 10 µM) for 15 min at 37°C and then stimulated with EGF (50 ng/ml for 5 min), GDNF (50 ng/ml for 5 min), or CR-1{Delta}C-Fc (200 ng/ml for 5 min for MAPK and 400 ng/ml for 30 min for Smad-2 and Akt). Western blots for phospho and total MAPK, Akt, and Smad-2 were performed as described previously (4) . Western blot analysis for phospho and total c-Src in serum-starved EpH-4 cells stimulated with EGF (50 ng/ml for 30 min) or CR-1{Delta}C-Fc (400 ng/ml for 15 and 30 min) was performed using an antiphospho c-Src (Tyr 416) rabbit polyclonal antibody (Cell Signaling, Beverly, MA) or GD11 mouse monoclonal anti-c-Src antibody (Upstate Biotechnology). Serum-starved EpH-4 cells were also pretreated with PP2 (1 µM) and then stimulated for 30 min with EGF (100 ng/ml) or CR-1{Delta}C-Fc (400 ng/ml). Basal levels of phospho-c-Src in EpH-4 wild type and CR-1 cells were assessed after 24 h of serum starvation by Western blot analysis as described above.

Cell Proliferation, Soft-Agar, and Migration Assays.
Anchorage-dependent cell growth was performed as described previously (3) . PP2, SU-6656 (1 or 10 µM), 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 CR-1 were seeded in 6-multiwell plates (5 x 104/well) in the presence of PP2, SU-6656 (1 or 10 µM), or DMSO (control samples), and after 2 weeks, colonies were counted as described previously (3) . Cell migration assays were performed using FluoroBlok 24-multiwell Insert Plates (8-µm pore size) coated with Matrigel Membrane Matrix (Becton Dickinson, Bedford, MA). Five percent FBS was used as chemoattractant in the lower chamber. EpH-4 or EpH-4 CR-1 cells (5 x 106) were labeled in situ with 10 µg/ml of Dil (Becton Dickinson) for 1 h at 37°C and then seeded in 12-multiwell plates (25 x 104 cells/well). After 16 h, the migrated cells were counted with a fluorescent microscope (three random fields/well at magnitude x400). All these experiments were done in duplicates and repeated at least three times.

Src Tyrosine Kinase Assay.
Serum-starved EpH-4 cells were stimulated with CR-1{Delta}C-Fc (400 ng/ml), and c-Src protein was immunoprecipitated using a rabbit polyclonal anti-c-Src antibody (2 µg; Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitated c-Src protein was then incubated in the presence of a biotinilated-c-Src substrate peptide, and the phosphorylated peptide was absorbed on strepavidin-coated plates (Chemicon, Temecula, CA). The amount of phosphorylated peptide was detected with a phosphotyrosine-specific monoclonal antibody (Chemicon), and the absorbance read at 450 nm. The experiment was done in duplicates and repeated three times.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
PI-PLC, Heparitinase, and PP2 Block CR-1 Activation of MAPK and Akt but have no Significant Effect on Smad-2 Phosphorylation.
We tested the effect of the following signaling pathway inhibitors on CR-1-induced MAPK phosphorylation in EpH-4 mammary epithelial cells: the EGF receptor tyrosine kinase inhibitors (PD153035 and AG1478); a serine/threonine kinase inhibitor (H-7); a G-protein inhibitor (pertussis toxin); the enzymes PI-PLC and heparitinase, which induce cleavage of GPI-linked proteins and digestion of HSGAG polysaccharide side chains, respectively; and the c-Src tyrosine kinase inhibitor PP2. EpH-4 mouse mammary epithelial cells were serum-starved for 24 h and then pretreated for different times with various inhibitors followed by stimulation with a soluble, COOH terminus-truncated CR-1 recombinant protein containing an Fc fusion tag (CR-1{Delta}C-Fc). Whereas the EGF receptor tyrosine kinase, G-protein, and serine/threonine kinase inhibitors did not have any significant effect on MAPK phosphorylation induced by CR-1 (data not shown), PI-PLC and heparitinase significantly interfered with the ability of CR-1 to induce MAPK activation in EpH-4 cells (Fig. 1, A and B)Citation . MAPK phosphorylation was also inhibited by PI-PLC when the cells were stimulated with GDNF, which binds to a GPI-linked coreceptor GFR{alpha}1 and signals through the tyrosine kinase receptor c-RET (Fig. 1ACitation ; Ref. 9 ). In contrast, MAPK phosphorylation by EGF was not affected by the presence of heparitinase or PI-PLC (Fig. 1BCitation and data not shown). The c-Src inhibitor PP2 also inhibited phosphorylation of MAPK by CR-1 in these cells (Fig. 1C)Citation . 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)Citation . 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)Citation . Similar results were obtained in other two mouse mammary epithelial cell lines NMuMG and HC-11 (data not shown).



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  		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{Delta}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.

 
CR-1 Binds to Glypican-1 in ELISA and Coimmunoprecipitates with Glypican-1 and c-Src in COS1 Cells.
The syndecans and the glypicans contain HSGAG side chains, but only glypicans are GPI-linked proteins and therefore are sensitive to both PI-PLC and heparitinase digestion. We therefore evaluated whether CR-1 could interact with Glypican-1, which among the six glypicans identified thus far, is the one most involved in tumorigenesis (6) . A Glypican-1-Fc fusion protein was absorbed to microtiter plates and binding of a CR-1{Delta}C-Fc recombinant protein was assessed by ELISA. CR-1{Delta}C-Fc recombinant protein was able to bind in a specific and saturable manner to Glypican-1-Fc with a Kd of ~10 nM (Fig. 2A)Citation . In contrast, an unglycanated Glypican-1-Fc core protein or BSA were unable to bind to CR-1{Delta}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)Citation . c-Src was also found to interact directly with Glypican-1 in the absence of CR-1 but to a lesser extent.



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  		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{Delta}C-Fc recombinant protein (µg/100 µl). 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.

 
CR-1 Induces Activation of c-Src.
We next evaluated whether CR-1 after binding to Glypican-1 could induce c-Src activation in EpH-4 mouse mammary epithelial cells. A 10- and 18-fold increase in the phosphorylation of c-Src was detected in EpH-4 cells after 15- or 30-min treatment with CR-1{Delta}C-Fc protein, respectively (Fig. 3A)Citation . EGF, which was used as a positive control, was also found to induce c-Src phosphorylation. Pretreatment of EpH-4 cells with PP2 inhibited c-Src phosphorylation induced by CR-1 or EGF (Fig. 3B)Citation . We then measured c-Src kinase activity directly using a specific c-Src peptide substrate in an ELISA kinase assay. CR-1 was found to induce activation of the tyrosine kinase c-Src in a time-dependent manner with a peak at 30 min of stimulation (Fig. 3C)Citation .



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  		Fig. 3. CR-1 induces activation of the tyrosine kinase c-Src. A, serum-starved EpH-4 cells were stimulated with CR-1{Delta}c-Fc (CR-1) or EGF and analyzed by Western blot 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{Delta}c-Fc (CR-1). C, serum-starved EpH-4 cells were stimulated with CR-1{Delta}c-Fc (CR-1), and the kinase reaction was performed as described under "Materials and Methods."

 
EpH-4 Cells Expressing CR-1 Exhibit High Levels of Activated c-Src.
Because CR-1 can activate c-Src in mammary epithelial cells, we investigated the basal tyrosine phosphorylation levels of c-Src in CR-1-transfected EpH-4 cells. After 24 h of starvation in serum-free medium, EpH-4 CR-1 cells showed a ~10-fold increase in the basal levels of activated, phosphorylated c-Src as compared with wild-type EpH-4 cells (Fig. 4A)Citation .



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  		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.

 
CR-1 Induced in Vitro Transformation and Migration in EpH-4 CR-1 Cells Requires Active c-Src.
We assessed whether c-Src is essential for CR-1 to induce in vitro transformation and migration in EpH-4 cells. As shown in Fig. 4BCitation , CR-1-expressing EpH-4 cells exhibited a 4-fold increase in growth rate after 3 days under serum-free conditions compared with wild-type EpH-4 cells. The enhanced proliferation of EpH-4 CR-1 cells could be reduced to basal levels in a dose-dependent manner by treatment of the cells with the c-Src inhibitor PP2. The proliferation of wild-type EpH-4 cells was also inhibited by high concentrations of PP2 but to a lesser degree. Likewise, EpH-4 CR-1 cells growth in soft agar was also inhibited by treatment with PP2 in a dose-dependent fashion (Fig. 4C)Citation . Another c-Src inhibitor, SU-6656, also specifically inhibited growth in serum-free and in soft agar of EpH-4 CR-1 cells (data not shown). To determine the effect of PP2 on the migratory behavior of EpH-4 CR-1 cells, we performed a migration assay using Matrigel base membrane-coated plates. EpH-4 CR-1 cells exhibited a 6-fold increase in their ability to migrate compared with wild-type cells. PP2 induced a dose-dependent inhibition in the migration of EpH-4 CR-1 cells, whereas PP2 did not exert any significant effect on migration of wild-type EpH-4 cells (Fig. 4D)Citation .


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
This study describes the identification of Glypican-1 as a coreceptor for CR-1 and c-Src as a key intracellular regulator of MAPK and Akt phosphorylation induced by CR-1. Cell surface HSPGs such as glypicans and syndecans can directly bind growth factors that are important for tumor development such as FGFs 1 and 2, vascular endothelial growth factor, TGF-ß, and Wnt proteins (6 , 10) . Upon binding to these different growth factors, HSPGs increase the affinity of these molecules for their signaling receptors and facilitate receptor dimerization and subsequent signaling (11) . Therefore, they have been considered as coreceptors for various growth factors. Our study demonstrates for the first time a direct interaction between an HSPG, Glypican-1, and CR-1. In fact, CR-1 binds in a specific and saturable manner to Glypican-1 in an ELISA assay, and this interaction is lost when HSGAG side chains are removed from the Glypican-1 core protein, suggesting that HSGAGs are mediating the binding of CR-1 to Glypican-1. This result is in agreement with several studies showing that the specificity of HSPGs interactions with growth factors such as FGF is dependent on HSGAG side-chain sequence (6) . Furthermore, coimmunoprecipitation of Glypican-1 and CR-1 from mammalian cells shows that this interaction also occurs in vivo. The importance of this interaction is demonstrated by the ability of PI-PLC and heparitinase to significantly interfere with activation of MAPK and Akt signaling pathways by CR-1. Likewise, it has previously been shown that treatment of human breast cancer cell lines with PI-PLC blocks the mitogenic response to several heparin binding growth factors such as heparin binding-EGF and FGF2 (12) . In addition, 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 overexpressed in human breast cancer (2) , Glypican-1 may act to enhance the growth-promoting effects of CR-1 in breast cancer cells. Glypicans, such as dally and knypek, also have a well-established role in embryonic development where they serve as coreceptors for TGF-ß 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 related HSPG might also regulate CR-1 signaling during embryonic development. Inappropriate activation of MAPK and Akt transduction pathways by CR-1 could play an essential role in the pathogenesis of human cancer because these two signaling pathways have been shown to induce cell growth and survival (15 , 16) . In this study, we demonstrate that c-Src is required by CR-1 to induce MAPK and Akt activation in mouse mammary epithelial cells. Activated c-Src probably stimulates the ras/MAPK pathway through phosphorylation of the adaptor protein Shc and the Akt pathway by activation of PI3K. In this context, CR-1 has been shown to stimulate Shc phosphorylation and activation of PI3K in mouse mammary epithelial cells (17 , 18) . Furthermore, high levels of c-Src activity are present in EpH-4 cells overexpressing CR-1, and an intact c-Src kinase is required by CR-1 to induce in vitro transformation and to enhance migration of EpH-4 cells. We have also detected high levels of c-Src activity in mammary tumors of mouse mammary tumor virus-CR-1 transgenic mice as compared with the c-Src activity in the mammary gland of multiparous animals, suggesting that c-Src may also mediate CR-1 induced tumorigenesis in vivo.5 Elevated c-Src kinase activity, and in some cases protein expression, has also been reported in human breast tumors, and a role for c-Src in regulating cellular migration and motility has been demonstrated in several types of cells (7) . However, 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 enriched in cholesterol and sphingolipids (19) . Lipid rafts serve as a microdomain either for a number of signaling proteins that are GPI-linked such as Glypican-1 and CR-1 or lipid-bound intracellular molecules such as Src family kinases (19) . Therefore, CR-1, Glypican-1, and c-Src could potentially cluster together in these membrane regions. In this respect, coimmunoprecipitation of c-Src with Glypican-1 also in the absence of CR-1 suggests that these two molecules probably colocalize. Therefore, interaction of CR-1 with Glypican-1 in the lipid rafts might trigger activation of c-Src kinase. Likewise, GPI-anchored immunoreceptors on T cells upon interaction with their natural ligands or by antibody cross-linking cause a redistribution of Src family kinases on the cytoplasmic site, initiating their autophosphorylation and activation (20) . Another possibility is that an unknown transmembrane tyrosine kinase receptor is activated by CR-1 and recruits c-Src through the SH2 domain. Whereas Glypican-1 and c-Src are required for activation of MAPK and Akt pathways by CR-1, Smad-2 phosphorylation induced by CR-1 is independent of these two signaling molecules. These findings are also supported by our previous biochemical studies in which CR-1 can trigger MAPK/Akt activation in cells that lack Nodal and/or ALK4 expression (4) . Therefore, we propose a model in which CR-1 can function as either a ligand for Glypican-1 or as a coreceptor for Nodal by independently activating the c-Src/ras/raf/MAPK/PI3K/Akt or the Nodal/ALK4/Smad-2 signaling pathways. However, these two signaling pathways may not be mutually exclusive because TGF-ß can also activate c-Src in epithelial cells and because a ras-GAP binding protein DOK-1 can also mediate Activin A induced activation of Smad-2 and Smad-3 through ALK4 (7 , 21) .


   ACKNOWLEDGMENTS
 
We thank Matthew Jarpe of Biogen for help in calculating the Kd and analyzing the kinetics of the ELISA binding. We also thank Brenda Jones for her excellent technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (to N. N.). Back

2 To whom requests for reprints should be addressed, at MBTL, National Cancer Institute, NIH, 10 Center Drive Building 10 5B39, Bethesda, MD 20892. Phone: (301) 496-9536; Fax: (301) 402-8656; E-mail: davetgfa{at}helix.nih.gov Back

3 The abbreviations used are: CR-1, Cripto-1; HSPG, heparan sulfate proteoglycan; HSGAG, heparan sulfate glycosylaminoglycan; GPI, glycosylphospatidylinositol; EGF, epidermal growth factor; CFC, Cripto FRL1 Cryptic; TGF-ß, transforming growth factor ß; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3' kinase; PI-PLC, phosphatidylinositol phospholipase C; FGF, fibroblast growth factor; GDNF, glial cell line-derived neurotrophic factor. Back

4 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 preparation. Back

5 L. Strizzi, C. Bianco, C. Wechselberger, Y. Sun, M. Hirota, N. Kenney, M. Sanicola, and D. S. Salomon. Epithelial mesenchimal transition signaling in breast lesions from Cripto-1 transgenic mice, manuscript in preparation. Back

Received 10/ 7/02. Accepted 1/31/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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