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[Cancer Research 59, 4502-4505, September 1, 1999]
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[Cancer Research 59, 4502-4505, September 15, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Cripto-1 Induces Phosphatidylinositol 3'-Kinase-dependent Phosphorylation of AKT and Glycogen Synthase Kinase 3ß in Human Cervical Carcinoma Cells

Andreas D. Ebert1, Christian Wechselberger, Stephan Frank, Brenda Wallace-Jones, Masaharu Seno, Isabel Martinez-Lacaci, Caterina Bianco, Marta De Santis, Hans K. Weitzel and David S. Salomon2

National Cancer Institute, Laboratory of Tumor Immunology and Biology, Tumor Growth Factor Section [A. D. E., C. W., B. W-J., I. M-L., C. B., D. S. S.] and National Institute of Neurological Disorders and Stroke, Surgical Neurology Branch [S. F.], National Institutes of Health, Bethesda, Maryland 20892; Free University Berlin, Medical Center Benjamin Franklin, Department of Obstetrics and Gynecology, 12200 Berlin, Germany [A. D. E., H. K. W.]; Faculty of Engineering, Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan [M. S.]; and Walter Reed Army Institute of Research, Department of Molecular Pathology, Washington, D.C. 20307 [M. D. S.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Cripto-1 (CR-1), a member of the epidermal growth factor-CFC peptide family, activates the ras/raf/mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In the present study, the role of CR-1 in the phosphatidylinositol 3'-kinase (PI3K)/AKT (protein kinase B)/glycogen synthase kinase 3ß (GSK-3ß)-dependent signaling pathway was evaluated in human SiHa cervical carcinoma cells. Our data demonstrate that CR-1 can enhance the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and transiently induce the phosphorylation of AKT in a time- and dose-dependent manner. In addition, CR-1 was found to induce the phosphorylation of GSK-3ß. Phosphorylation of AKT and GSK-3ß by CR-1 can be blocked by LY294002, a specific inhibitor of PI3K, thus leading to apoptosis. Finally, the apoptotic effect of LY294002 can be partially rescued by exogenous CR-1. In summary, our data suggest that human CR-1 may function as a survival factor through a PI3K-dependent signaling pathway involving AKT and GSK-3ß.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The EGF3 - CFC family of proteins [CR-1 (human), cripto (Cr-1, mouse), cryptic (mouse), FRL-1 (Xenopus), and one-eyed pinhead (oep, zebrafish)] has recently been recognized as a novel class of cell-associated and extracellular factors that are essential during zebrafish, Xenopus, and mouse development (1, 2, 3) . It has been suggested that EGF-CFC proteins might act as essential cofactors or coreceptors for nodal signaling during vertebrate development (4) . As compared with nontransformed tissue, CR-1 is overexpressed in several human malignancies such as breast, colon, gastric, pancreatic, and testicular cancer (5) . CR-1 was first identified and sequenced from a human embryonal carcinoma cell line NTERA2/D1 cDNA expression library (6) . The human CR-1 gene encodes a major mRNA species of 2.2 kb and a protein of 188 amino acids (7) . Proteins of Mr 24,000, Mr 26,000, and Mr 36,000 that are immunologically related to CR-1 have been detected in several different cell lines and may be due to posttranslational modifications of a Mr 21,000 core protein (7) . CR-1 possesses a highly conserved internal region of 37 amino acids that has a modified EGF-like motif that differentiates CR-1 from other members of the EGF family (7) . Additionally, CR-1 shares a cysteine-rich CFC region in the COOH-terminal region with one-eyed pinhead, FRL-1, and cryptic (4 , 7) . CR-1 does not bind directly to any of the four known type 1 erbB receptor tyrosine kinases but binds to a high affinity receptor that is specific for CR-1 (8 , 9) . In HC-11 mouse mammary epithelial cells, CR-1 induces a rapid and transient increase in tyrosine phosphorylation of Shc (9) . It also facilitates the interaction of Shc with the Grb2-SOS complex. Furthermore, CR-1 can enhance the downstream tyrosine phosphorylation and activation of MAPK, demonstrating that the ras/raf/MEK/MAPK pathway can be activated by CR-1 directly or indirectly through its receptor or by indirect transactivation of erbB-4 (9 , 10) . Because CR-1 can transiently activate PI3K in HC-11 mouse mammary epithelial cells (11) , this study was designed to evaluate the role of CR-1 in the activation of the PI3K/AKT/GSK-3ß signaling pathway, which is involved in regulating apoptosis (12 , 13) . We provide evidence that in human cervical carcinoma cells, CR-1 enhances the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and the serine phosphorylation of AKT and GSK-3ß, one of the principal downstream effectors of AKT. Activation of the PI3K/AKT/GSK-3ß pathway was blocked by LY294002, a specific inhibitor of PI3K, leading to apoptosis. The apoptotic effect of LY294002 was rescued by the addition of exogenous CR-1. Our data suggest that human CR-1 may function as a survival factor through the PI3K/AKT/GSK-3ß signaling pathway.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Culture.
Human SiHa and CaSki cervical cancer cell lines and transient or stable transfected SiHa cell lines (S-pCI, S-AC9, and S-CR-1) were cultured in DMEM/F12 (1:1) supplemented with 5% fetal bovine serum. Twenty-four h before treatment, cells were split, washed three times in PBS, and seeded in serum-free DMEM/F12 in 60-mm dishes at 37°C.

Plasmid Construction and Transfection.
Stable or transient transfected SiHa cell lines were generated using the eukaryotic expression vector pCI-neo (Promega, Madison, WI) containing the human cytomegalovirus promoter region, the full-length cDNA encoding human CR-1, and a neomycin resistance gene. Briefly, using the LipofectAMINE Reagent kit (Life Technologies, Inc.), cells at 60–70% confluence were transfected with 2 µg of either control pCI vector or recombinant pCI/CR-1 vector in Opti-MEM I reduced serum medium for 12 h, followed by incubation in DMEM/5% fetal bovine serum containing G418 (400 µg/ml; Geneticin; Life Technologies, Inc.). After detection of CR-1 expression by Western blotting, clones were cultured in standard medium. Recombinant CR-1 was obtained from conditioned medium of stable transfected SiHa cervical carcinoma cells.

Western Blot Analysis, Immunoprecipitation, and Antibodies.
Whole cell protein extracts were prepared and analyzed by Western blotting or immunoprecipitation, as described previously (8 , 10) . Antibodies were obtained from following sources: (a) rabbit antiphospho-AKT (Ser-473; 1:1000 dilution), New England BioLabs Inc.; (b) rabbit anti-AKT (1:1000 dilution), New England BioLabs Inc.; (c) rabbit antiphospho-GSK-3{alpha}/ß (1:1000 dilution), New England BioLabs Inc.; (d) rabbit anti-GSK-3ß (1:750 dilution), New England BioLabs Inc.; (e) secondary antirabbit or antimouse horseradish peroxidase antibody (1:3000 dilution), Amerhsam; (f) antiphosphotyrosine mouse monoclonal antibody (PY99; 1 µg/200 µg total protein), Santa Cruz Biotechnology; (g) rabbit anti-p85-subunit of PI3K (1:750 dilution), Santa Cruz Biotechnology; and (h) rabbit polyclonal anti-CR-1 {alpha}MS2 (1:750 dilution; Ref. 8 ), .

Assessment of Apoptosis.
Cells were seeded in serum-free medium at a density of 50,000 cells/chamber in two-chamber slides (LabTec Inc.) in the absence or presence of CR-1 (100 ng/ml), CR-1 (100 ng/ml), and LY294002 (30 µM) or LY294002 (30 µM) alone for 30 min. Medium was removed, and cells were grown for 24 h in serum-free DMEM/F12. Cells were fixed in 100% methanol and incubated for 15 min in PBS- blocking buffer containing 1% BSA and 0.1% Tween 20. Cells were then incubated in buffer containing 100 µl of mouse monoclonal M30 CytoDEATH antibody (Boehringer Mannheim) for 60 min at 23°C. After additional washings with PBS, cells were incubated with 10 µg/ml antimouse-immunoglobulin-FITC for 30 min. For Hoechst staining, methanol-fixed cells were incubated in buffer containing Hoechst stain at 10 µg/ml for 10 min at 23°C. Immunofluorescence staining was examined under a fluorescence microscope (Zeiss). Chromatin condensation, nuclear shrinkage, and nucleosomal fragmentation were used as the criteria for apoptosis. Intact or mitotic nuclei were counted as normal. The number of apoptotic cells/1000 vital cells was counted in four separate fields in each chamber.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
CR-1 Induces Phosphorylation of the p85 Regulatory Subunit of PI3K and Phosphorylation of AKT and GSK-3ß.
To ascertain whether CR-1 can enhance the tyrosine phosphorylation of the regulatory subunit of PI3K in human SiHa cervical carcinoma cells, serum-starved cells were treated with CR-1 (100 ng/ml). Cell lysates were immunoprecipitated with an antiphosphotyrosine antibody, and Western blot analysis was performed using an anti-p85 regulatory subunit antibody (Fig. 1A)Citation . A significant increase in the tyrosine phosphorylation of the p85 regulatory subunit of PI3K in CR-1-treated SiHa cells was observed, whereas in LY294002-treated cells, no p85 phosphorylation could be demonstrated. CR-1 also induced hyperphosphorylation of AKT in SiHa cells in a time-dependent manner, with maximum phosphorylation observed after 15–30 min of stimulation (Fig. 1B)Citation . At this time point, CR-1 induced a 3–5-fold increase in the phosphorylation of AKT. There were no significant changes in total AKT levels during this interval. AKT hyperphosphorylation can also be induced in a dose-dependent manner as determined by the treatment of serum-starved SiHa cells with different concentrations of CR-1 for 15 min (Fig. 1C)Citation . A clear dose-dependent increase in the phosphorylation of AKT was detectable, with maximum phosphorylation observed at 100–200 ng/ml CR-1. No changes in total AKT levels were observed. To exclude the possibility that this response might be unique to SiHa cells, CaSki cervical carcinoma cells were also tested for their response to CR-1 (Fig. 2)Citation . In both cell lines, CR-1-induced AKT phosphorylation and basal constitutive AKT phosphorylation were blocked by pretreatment with LY294002 (30 µRM) for 30 min, suggesting that CR-1 is able to activate AKT through PI3K. Finally, a 2.5–3-fold increase in the phosphorylation of GSK-3ß was detected in CR-1-treated SiHa and CaSki cells (Fig. 2)Citation .



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  		Fig. 1. A, serum-starved human SiHa cervical carcinoma cells were treated in the absence or presence of 100 ng/ml CR-1 for 15 min. To inhibit PI3K, cells were pretreated in some cases with 30 µM LY294002 for 30 min. Total cell protein (200 µg) was immunoprecipitated (IP) with an antiphosphotyrosine-specific antibody (PY99), run on a 10% SDS-PAGE gel, and probed with a rabbit polyclonal anti-p85 antibody; WB, Western blot. B, serum-starved SiHa cells were treated with 100 ng/ml CR-1 for various times or (C) treated with various concentrations of CR-1 for 15 min. Phosphorylation of AKT and total AKT levels were analyzed by Western blot analysis using a rabbit polyclonal antiphospho-AKT (Ser-473) antibody or a rabbit polyclonal anti-AKT antibody.

 


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  		Fig. 2. Serum-starved human SiHa and CaSki cervical carcinoma cells were treated with or without 100 ng/ml CR-1 for 15 min. The indicated cells were pretreated with 30 µM LY294002 for 30 min. Cell lysates (10% SDS-PAGE) were probed with a rabbit polyclonal antiphospho-AKT (Ser-473) antibody or antiphospho-GSK-3ß antibody. The membranes have been stripped and reprobed for total AKT and GSK-3ß using rabbit polyclonal anti-AKT and rabbit polyclonal anti-GSK-3ß antibodies.

 
Effect of Long-Time CR-1 Treatment on AKT Phosphorylation.
To ascertain whether AKT phosphorylation could be detected after long-time treatment, SiHa cells were treated with CR-1 for 24 h in the absence or presence of LY294002 (30 µM; Fig. 3ACitation ). LY294002 completely inhibited the constitutive activation of AKT. After 24 h of treatment with CR-1, there were no differences in AKT phosphorylation levels between control and CR-1-treated cells. However, a decrease in total AKT expression in LY294002-treated samples was found, suggesting that inhibition of AKT phosphorylation is a short-term effect of LY294002, whereas down-regulation of total AKT expression occurs after a more prolonged exposure to this compound.



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  		Fig. 3. A, serum-starved human SiHa cervical carcinoma cells were treated for 24 h in the absence or presence of 30 µM LY294002 with or without CR-1 (100 ng/ml). Cell lysates (50 µg/lane; 10% SDS-PAGE) were probed with a rabbit polyclonal antiphospho-AKT (Ser-473) antibody. The same samples were analyzed for total AKT expression using a rabbit polyclonal anti-AKT antibody. B, serum-starved stable CR-1 transfected SiHa cells (S-AC9) or empty vector-transfected SiHa cells (S-pCI) were treated for 30 min in the absence or presence of 30 µM LY294002. Cell lysates (50 µg/lane; 10% SDS-PAGE) were probed with a rabbit polyclonal antiphospho-AKT (Ser-473) antibody. The same samples were analyzed for CR-1 expression using a rabbit polyclonal anti-CR-1 antibody. C, SiHa cells were transfected with CR-1 (S-CR-1) or the empty vector (S-pCI). Twenty-four h after transfection, cells were treated for 30 min in the absence or presence of 30 µM LY294002. Cell lysates were run on a 10% SDS-PAGE and probed with a rabbit polyclonal antiphospho-AKT (Ser-473) antibody. The same samples were analyzed for CR-1 expression (10–20% SDS-PAGE) using a rabbit polyclonal anti-CR-1 antibody.

 
Activation of AKT in CR-1 Stable and Transient Transfected Cells.
To determine whether endogenous CR-1 could also enhance AKT phosphorylation, a stable CR-1-transfected SiHa cell line was produced and treated with LY294002, respectively. No differences in the phosphorylation levels of AKT were observed between control and CR-1-overexpressing cells (Fig. 3B)Citation . Together with the results of the 24-h treatment, these findings suggest that the CR-1-induced hyperphosphorylation of AKT might represent an acute and transient response. To prove this hypothesis, we transiently transfected SiHa cells with an expression vector containing human CR-1 and treated the cells for 30 min with LY294002. Twenty-four h after transfection, an approximately 2-fold increase in AKT phosphorylation was observed in CR-1-expressing cells (Fig. 3C)Citation . AKT phosphorylation was inhibited by LY294002. These data suggest that CR-1 might activate the PI3K/AKT pathway in a transient manner.

Antiapoptotic Effects of CR-1.
Because cleavage of K18 represents a caspase-dependent event during apoptosis, detection of caspase-cleaved K18 fragments has become a useful tool in assessing apoptosis (14) . Using a monoclonal antibody that recognizes proteins resulting from apoptotic K18 fragmentation, a clear reduction in staining was observed in CR-1-treated SiHa cells as compared with untreated or LY294002-treated cells, suggesting that CR-1 is able to inhibit apoptosis and therefore might function as a survival factor (Fig. 4, A–D)Citation . To confirm this antiapoptotic effect of CR-1, Hoechst staining was performed, and apoptotic cells were assessed. When cells were maintained under serum-free conditions, the addition of LY294002 induced apoptosis (Fig. 4E)Citation . In CR-1-treated samples, a significant reduction of apoptotic cells was observed as compared with control samples (13.5 ± 1.9 versus 25.4 ± 1.3; P < 0.002). In comparison to LY294002 treatment alone, the addition of CR-1 to LY294002-treated SiHa cells decreased the number of apoptotic nuclei (51.1 ± 2.7 versus 28.2 ± 2.4; P < 0.001).



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  		Fig. 4. Detection of caspase-cleaved K18 fragments in SiHa cervical carcinoma cells by indirect immunofluorescence. Cells were treated for 30 min in the absence (A) or presence (B) of CR-1 (100 ng/ml) and in the presence of 30 µM LY294002 (C) or in combination with CR-1 (D). After treatment, the cells were grown for 24 h in serum-free medium. The arrows indicate late apoptotic events. E, detection of apoptotic cells by Hoechst staining. Serum-starved SiHa cells were treated for 30 min in the absence (a) or presence (b) of CR-1 (100 ng/ml) and in the presence of 30 µM LY294002 (d) or in combination with CR-1 (c).

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Growth factors, such as nerve growth factor, insulin-like growth factor I, and EGF, are able to activate PI3K, which is followed by AKT phosphorylation (12 , 15) . AKT has been identified as a key effector of PI3K-mediated cell survival (12 , 16) . AKT can block apoptosis and promote cell survival in response to growth factor stimulation through the ability of AKT to promote phosphorylation of GSK-3 and BAD (13 , 16, 17, 18) . However, there are no data demonstrating an involvement of the EGF-related CFC protein family in regulating PI3K signaling and blocking apoptosis. The present data provide evidence that human CR-1 is able to induce phosphorylation of AKT in a time- and dose-dependent manner. This effect is due to the enhanced phosphorylation of the p85 regulatory subunit of PI3K that is associated with the activation of PI3K (19) . In addition, after CR-1 treatment, the downstream effector of AKT, GSK-3ß, is phosphorylated in human cervical carcinoma cells. These CR-1 effects can be inhibited by LY294002, a specific inhibitor of PI3K. Although prior treatment with LY294002 leads to complete inhibition of CR-1-induced as well as basal constitutive AKT phosphorylation within 30 min, the antiapoptotic effect of CR-1 could still be observed in serum-starved carcinoma cells, suggesting the involvement of additional survival pathways (20 , 21) . Although CR-1 induces phosphorylation of AKT and GSK-3ß, our data are merely correlative and suggest that GSK-3ß may be a substrate that is phosphorylated by AKT, as gleaned from other studies (13 , 18) . In this regard, GSK-3ß has a central role in the wnt-1 signaling pathway and in facilitating the turnover of ß-catenin, a cell adhesion protein (22) . Inactivation of GSK-3ß by phosphorylation leads to an increase in soluble ß-catenin that can then interact with the transcription factor LEF-1/TCF. Preliminary data demonstrate that CR-1 treatment leads to a modulation of the level of free, soluble ß-catenin in human SiHa and Caski cervical carcinoma cells. Because ß-catenin can modulate apoptosis (23) , changes in its expression or interaction with E-cadherin may also contribute to the antiapoptotic effects of CR-1.

CR-1 can activate the ras/raf/MAPK pathway (9) that may also contribute to the survival-promoting effects (24) of CR-1 in cervical carcinoma cells. In this respect, LY294002 has been reported to modulate MEK1/MAPK phosphorylation (25) . In agreement, a small decrease in CR-1-stimulated MAPK phosphorylation was observed in cells that had been treated with LY294002 (data not shown). Recently, it has been demonstrated that apoptosis induced by PI3K inhibition can be attenuated by EGF-like growth factors such as EGF, transforming growth factor {alpha}, and HB-EGF in human prostate carcinoma cells (26) . Therefore, at least in part, the antiapoptotic effect of CR-1 might also be due to activation of the MEK1/MAPK pathway (9 , 10) . Nevertheless, the present data suggest that activation of the PI3K/AKT pathway by CR-1 is an acute and transient effect. Prolonged CR-1 treatment or stable transfection does not alter the steady-state levels of AKT phosphorylation, whereas in cells transiently transfected with CR-1, an increase in AKT phosphorylation is detectable. Collectively, these data demonstrate that in human carcinoma cells, a member of the EGF-related CFC protein family, human CR-1, is involved in modulating the PI3K/AKT/GSK-3ß pathway.


   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 A. D. E. was supported by Deutsche Forschungsgemeinschaft Grants EB-152/4-1 and EB 152/4-2. Back

2 To whom requests for reprints should be addressed, at National Cancer Institute, National Institutes of Health, Laboratory of Tumor Immunology and Biology, Building 10, Room 5B39, 10 Center Drive, MSC-1750, Bethesda, MD 20892. Phone: (301) 496-9536; Fax: (301) 402-8656; E-mail: davetgfa{at}helix.nih.gov Back

3 The abbreviations used are: EGF, epidermal growth factor; CR-1, Cripto-1; PI3K, phosphatidylinositol 3'-kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; GSK-3ß, glycogen synthase kinase 3ß; F12, Ham’s F-12 medium; K18, keratin 18. Back

Received 5/27/99. Accepted 8/ 5/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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