Cripto-1 Alters Keratinocyte Differentiation via Blockade of Transforming Growth Factor-B1 Signaling: Role in Skin Carcinogenesis

Cripto-1 is an epidermal growth factor-Cripto/FRL1/ Cryptic family member that plays a role in early embryogenesis as a coreceptor for Nodal and is overexpressed in human tumors. Here we report that in the two-stage mouse skin carcinogenesis model, Cripto-1 is highly up-regulated in tumor promoter– treated normal skin and in benign papillomas. Treatment of primary mouse keratinocytes with Cripto-1 stimulated proliferation and induced expression of keratin 8 but blocked induction of the normal epidermal differentiation marker keratin 1, changes that are hallmarks of tumor progression in squamous cancer. Chemical or genetic blockade of the transforming growth factor (TGF)-B1 signaling pathway using the ALK5 kinase inhibitor SB431542 and dominant negative TGF-B type II receptor, respectively, had similar effects on keratinocyte differentiation. Our results show that Cripto-1 could block TGF-B1 receptor binding, phosphorylation of Smad2 and Smad3, TGF-B–responsive luciferase reporter activity, and TGF-B1–mediated senescence of keratinocytes. We suggest that inhibition of TGF-B1 by Cripto-1 may play an important role in altering the differentiation state of keratinocytes and promoting outgrowth of squamous tumors in the mouse epidermis. (Mol Cancer Res 2008;6(3):509–16) Introduction Mouse Cripto-1 is a member of the epidermal growth factorCripto/FRL1/Cryptic (EGF-CFC) protein family that includes human Cripto-1 and cryptic, mouse cryptic, chicken Cripto-1, Xenopus FRL1, and zebrafish one-eyed pinhead (oep; refs. 1, 2). These proteins are glycosylphosphatidylinositol linked and contain an EGF-like domain and a cysteine-rich CFC domain. A short COOH terminus contains consensus sequences for glycosylphosphatidylinositol linkage to the cell membrane (3). EGF-CFC proteins have activity both when expressed as soluble proteins and when secreted from the cell surface following enzymatic cleavage of their glycosylphosphatidylinositol anchor (4, 5). Cripto-1 is expressed in early development and plays an important role in differentiation of heart muscle cells, formation of germ layers, formation of the organizer, and specification of the anterior-posterior and left-right axes and of the embryonic midline (6-10). During embryonic development, Cripto-1 functions as a coreceptor for Nodal, a transforming growth factor h (TGF-h) family ligand, to initiate mesoderm formation. Nodal signals through the activin receptor ALK4 but requires Cripto-1 to bind to ALK4 and signal (11). On the other hand, Cripto forms a complex with activin and a type II receptor, ActRII or ActRIIB, and prevents association of activin with ALK4 (12). Activin, in the absence of Cripto-1, binds and strongly activates ALK4. Thus, Cripto-1 inhibits activin signaling but potentiates Nodal signaling. Apart from activin signaling, in a recent report, Cripto was also shown to suppress TGF-h signaling by reducing association of TGF-h with its receptor in 293T cells (13). Cripto-1 is overexpressed in a wide variety of human carcinomas such as gastric, pancreatic, colorectal, gall bladder, breast, bladder, ovarian, and basal cell carcinomas, and may play a role in cancer pathogenesis (14). Overexpression of Cripto-1 increases cell proliferation, anchorage-independent growth, and transformation of mammary epithelial cells in vitro and causes mammary gland hyperplasia and tumor formation in transgenic MMTV-Cripto-1 mice (15, 16). We previously showed that mouse skin tumors caused by overexpression of Smad7 had elevated expression of Cripto-1 (17). Here we show that Cripto-1 expression is elevated in chemically induced mouse skin tumors compared with normal skin. Cripto-1 causes impaired TGF-h1 signaling in primary mouse keratinocytes, resulting in modification of their differentiation status and a phenotype associated with tumor progression. Our results suggest a mechanism by which Cripto1 may enhance skin tumor progression. Results Cripto-1 Expression Is Up-Regulated in Multistage Skin Carcinogenesis A number of human cancers are associated with enhanced Cripto-1 expression. We wanted to determine if Cripto-1 expression was altered during chemically induced skin tumor Received 8/22/07; revised 10/22/07; accepted 11/30/07. Grant support: NIH grant CA122109 (A.B.Glick) and the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH. 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. Note: Current address for X. Liu: Penn State Cancer Institute, 500 University Drive H072, Room C6815B, Hershey, PA-17033. Requests for reprints: Adam B. Glick, Center for Molecular Toxicology and Carcinogenesis, Pennsylvania State University, University Park, PA 16802. Phone: 814-865-7170; Fax: 814-863-1357. E-mail: [email protected] Copyright D 2008 American Association for Cancer Research. doi:10.1158/1541-7786.MCR-07-0396 Mol Cancer Res 2008;6(3). March 2008 509 on April 19, 2017. © 2008 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from formation and if there was a tumor stage–specific response. We examined Cripto-1 expression by quantitative reverse transcription-PCR in RNA isolated from samples of normal skin, skin treated chronically with the tumor promoter 12-Otetradecanoyl-phorbol-13-acetate (TPA), benign papillomas, and squamous cell carcinomas generated by a 7,12-dimethylbenz(a)-anthracene-TPA chemical skin carcinogenesis protocol. Interestingly, relative to normal skin where it was nearly undetectable, Cripto-1 mRNA was elevated in TPA-treated normal skin (an average expression value of 2.7). Cripto-1 expression was further enhanced in papillomas (an average expression value of 4), but with further progression in tumor stage, the levels decreased in carcinomas (an average expression value of 0.9; Fig. 1A). To validate these results and to determine the skin compartment where Cripto-1 is expressed, tissue sections from papillomas, carcinomas, and normal skin were immunostained using an anti–Cripto-1 antibody. Figure 1B shows that, similar to mRNA expression results, Cripto-1 protein expression is higher in squamous papillomas compared with normal skin, whereas the squamous cell carcinoma tissue is completely devoid of Cripto-1. Figure 1B (middle), depicting a papilloma section, shows that the protein is localized to the differentiating suprabasal layers of the papilloma. Cripto-1 Stimulates Keratinocyte Proliferation and Blocks Differentiation To determine the significance of the tumor stage–specific and suprabasal expression pattern of Cripto-1, we analyzed the effect of Cripto-1 on keratinocyte proliferation and differentiation in vitro . Primary mouse keratinocytes were treated with varying concentrations of recombinant Cripto-1 for 24 hours and the effect on keratinocyte proliferation was measured by a radioactive thymidine incorporation assay. Figure 2 shows that Cripto-1 treatment caused a 2-fold increase in DNA synthesis at 400 ng/mL, indicating that Cripto-1 can enhance keratinocyte proliferation. High-calcium media has been shown to induce differentiation of keratinocytes (18). To examine the effects of Cripto-1 on differentiation, we pretreated primary keratinocytes in growth medium with 200 ng/mL Cripto-1 for 24 hours before switching to high-calcium media (0.12 mmol/L Ca) to induce differentiation. Whole-cell protein lysates were analyzed for keratin 1, an early differentiation marker that is induced in 0.12 mmol/L Ca media (18). Pretreatment with Cripto-1 before switching to 0.12 mmol/L Ca media blocked the induction of keratin 1, whereas treatment with Cripto-1 after the cells had been switched to the 0.12 mmol/L Ca media reduced, but did not completely block, the expression of keratin 1 (Fig. 3A). Keratin 8, a marker of simple epithelium and malignant conversion, is not expressed in normal mouse keratinocytes. Interestingly, Cripto-1 strongly induced expression of keratin 8 in these cells, suggesting that it was altering the differentiation state of normal keratinocytes (Fig. 3B). TGF-b Superfamily Signaling Blockade Alters Keratinocyte Differentiation Because previous studies have shown that Cripto-1 inhibits activin signaling, we tested if activin signaling induced or inhibited keratin 8 expression in keratinocytes. We determined that although primary mouse keratinocytes do not express detectable levels of nodal, they do express activin A, activin B, ActRI, ActRIB, and ActRII/ALK4 (data not shown), suggesting that Cripto-1 was acting to inhibit activin signaling. When preneoplastic keratinocytes were treated with activin A or FIGURE 1. Cripto-1 expression is up-regulated in chemical ly induced skin tumors. A. cDNAs from multiple sections of normal skin, papillomas, squamous cell carcinomas, and skin chronically treated with TPA were analyzed for Cripto-1 expression by quantitative real-time PCR. Data are presented as Cripto-1 expression normalized to respective GAPDH levels used as an internal control (2 ). Normal skin (control) samples had a 2 (DDCt) of 0.0000. The values are statistically different from control; chronic TPA-treated skin, P = 0.0003; papillomas, P = 0.02; and squamous cell carcinoma (SCC ), P = 0.003. B. Formalin-fixed tissue sections from normal skin (a), papillomas (b), and squamous cell carcinomas (c) were immunostained for Cripto-1 as described in Materials and Methods. bl, basal layer; sb, suprabasal layer. b, inset, section of papilloma stained with secondary antibody alone. Bar, 15 Am. Shukla et al. Mol Cancer Res 2008;6(3). March 2008 510 on April 19, 2017. © 2008 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from activin B in culture conditions that normally induce keratin 8, the expression of this keratin was blocked (Fig. 4A). To test the hypothesis that TGF-h1 superfamily members regulated keratinocytes differentiation more directly, we treated primary mouse keratinocytes with the ALK5 inhibitor SB431542. Figure 4B shows that treatment with 5 Amol/L SB431542 induced expression of keratin 8 in keratinocytes, whereas induction of keratin 1 by elevated calcium was suppressed by pretreatment with SB431542 (Fig. 4C). Blocking TGF-h signaling with an adenovirus expressing a dominant negative TGF-h type II receptor also induced keratin 8 expression, suggesting the importance of TGF-h1 signaling for this pathway (Fig. 4D). We isolated RNA from keratinocytes under basal or elevated calcium conditions in the presence or absence of SB431542 and examined changes in keratin 1 and keratin 8 expression by quantitative PCR. As expected, keratin 1 mRNA levels were induced by 0.12 mmol/L Ca (Fig. 4E). Inclusion of SB431542 at the same time as 0.12 mmol/L Ca completely blocked the calcium-dependent induction of keratin 1 transcript, whereas pretreatment with SB431542 and its inclusion during the calcium switch significantly suppressed expression. In contrast, treatment of keratinocytes in 0.05 mmol/L Ca media with SB431542 caused a 50% increase in keratin 8 expression compared with untreated control. Thus, the changes in keratin 1 and keratin 8 protein levels caused by SB431542 are, at least in part, due to changes in mRNA expression. Cripto-1 Inhibits TGF-b1 Signaling in Keratinocytes The similar effects of inhibition of TGF-h1 signaling and Cripto-1 treatment on keratinocyte differentiation markers suggested that Cripto-1 may be acting through inhibition of TGF-h1 signaling. To test this, we pretreated mouse keratinocytes with Cripto-1 for 30 minutes before addition of TGF-h1 for 1 hour, and protein lysates were analyzed for phosphoSmad2 and phospho-Smad3. Under these conditions, Cripto-1 abolished the rapid induction of phospho-Smad2 and phosphoSmad3 by TGF-h1, although this was more apparent at lower concentrations of TGF-h1 (Fig. 5A). As expected, activin A– and activin B– induced phosphorylation of Smad2 was also blocked (data not shown). Cripto-1 doses as low as 25 to 50 ng/mL showed inhibition of Smad2 and Smad3 phosphorylation with greater suppression occurring in a dose-dependent manner (Fig. 5B). The effect of Cripto-1 on TGF-h–dependent gene transcription was analyzed using pGL-SBE4-luc, a Smad binding element–containing luciferase reporter (19). Activation of SBE4-luciferase reporter construct with 200 pg/mL TGF-h1 was reduced by 50% following pretreatment with 200 ng/mL Cripto-1 (Fig. 5C). To test if Cripto-1 blocked Smad phosphorylation by interfering with TGF-h1 interaction with its receptor, we crosslinked I-TGF-h1 to keratinocyte surface TGF-h1 receptors in the presence or absence of Cripto-1. Figure 5D shows that in the absence of Cripto-1, I-TGF-h1 bound the expectedf70-kDa and f280-kDa proteins representing the type II and type III TGF-h1 receptors (lane 4). In this case, the type I receptor, although present on keratinocytes (20), was not efficiently cross-linked. A 30-minute pretreatment with Cripto-1 abolished detectable cross-linking of I-TGF-h1 to TGF-h1 receptors on the primary keratinocytes similar to competition with unlabeled TGF-h1. These data suggest that Cripto-1 interferes with ITGF-h1 binding to its receptor, although it cannot be determined if this occurs through Cripto-1 binding to TGF-h1 or through blocking a binding site on the receptor. Cripto-1 Blocks TGF-b1–Induced Senescence of RasExpressing Keratinocytes One potential mechanism of tumor suppression by TGF-h1 is induction of senescence in premalignant keratinocytes (21). We examined the effect of Cripto-1 on TGF-h1–mediated senescence using senescence-associated h-galactosidase as a marker, which is shown to be strongly correlated with an irreversible G1 arrest in keratinocytes (22). Primary FVB/n keratinocytes were infected with v-ras retrovirus treated with FIGURE 3. Cripto-1 alters keratinocyte differentiation. Primary BALB/c keratinocytes were treated as indicated and total protein extracts isolated for detection of keratin 1 or keratin 8 expression by immunoblotting, with h-actin as a loading control. A. Cripto-1 suppresses induction of keratin 1. Keratin 1 expression in keratinocytes cultured in media containing 0.05 mmol/L Ca (lane 1 ), switched to 0.12 mmol/L Ca media for 24 h (lanes 2 and 4) to induce differentiation, treated with Cripto-1 (200 ng/mL) in either 0.05 or 0.12 mmol/L Ca media (lanes 3-5 ), or pretreated with Cripto-1 (200 ng/mL) in 0.05 mmol/L Ca for 24 h before being switched to 0.12 mmol/L Ca F Cripto-1 (lanes 6 and 7). B. Cripto-1 induces keratin 8 expression. Keratin 8 expression in keratinocytes treated with indicated concentrations of Cripto-1 for 24 h. 400 200 0 50 0 100 200 Cripto-1 (ng/ml) 3 H -t h ym id in e In co rp o ra ti o n