(2005) 24, 5731–5741 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc

Cripto-1: a multifunctional modulator during embryogenesis and oncogenesis

Luigi Strizzi1, Caterina Bianco1, Nicola Normanno2 and David Salomon*,1

1Tumor Section, Mammary Biology & Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA; 2Division of Haematological Oncology and Department of Experimental Oncology, ITN-Fondazione Pascale, 80131 Naples, Italy

It is increasingly evident that known to perform tions within stem cells or alterations in signaling critical roles during early embryogenesis, particularly pathways within the stem cell niche are almost certainly during stem cell renewal, pluripotentiality and survival, central to the early appearance of premalignant cells. are also expressed during the development of cancer. In Stem cells, therefore, represent desirable targets for the this regard, oncogenesis may be considered as the development of future cancer therapies (Reya et al., recapitulation of embryogenesis in an inappropriate 2001; Beachy et al., 2004). Growth factors that are temporal and spatial manner. The epidermal growth important in development, oncogenesis and -Cripto-1/FRL1/cryptic family of consists self-renewal include members of the Hedgehog (Hh), of extracellular and cell-associated proteins that have Wnt, Notch, transforming growth factor beta (TGFb/ been identified in several vertebrate species. During early Activin/Nodag/BMP) and - embryogenesis, epidermal growth factor-Cripto-1/FRL1/ Cripto-1/FRL1/cryptic (EGF-CFC) superfamilies cryptic proteins perform an obligatory role as coreceptors (Salomon et al., 2000; Shen and Schier, 2000; Kodja- for the transforming growth factor-beta subfamily of bachian, 2001; Kleber and Sommer, 2004; Nelson and proteins, which includes Nodal. Cripto-1 has also been Nusse, 2004; Tabata and Takei, 2004). shown to function as a ligand through a Nodal/Alk4- independent signaling pathway that involves binding to Identification and structure of the EGF-CFC family glypican-1 and the subsequent activation through src of phosphoinositol-3 kinase/Akt and ras/-activated Human Cripto-1 (CR-1), also known as teratocarcino- kinase intracellular pathways. Expression of ma-derived growth factor-1 (TDGF-1), is a member of Cripto-1 is increased in several human cancers and its the EGF-CFC protein family (Salomon et al., 2000). overexpression is associated with the development of Orthologous genes have been identified in the mouse mammary tumors in mice. Here, we review the role of (Cr-1) (Dono et al., 1993), chicken (Colas and Schoen- Cripto-1 during embryogenesis, cell migration, invasion wolf, 2000), zebrafish (Zhang et al., 1998) and Xenopus and angiogenesis and how these activities may relate to (FRL1) (Kinoshita et al., 1995). Related genes include cellular transformation and tumorigenesis. We also briefly mouse cryptic (Cfc1) and human cryptic (CFC1) (Shen discuss evidence suggesting that Cripto-1 may be involved et al., 1997; Bamford et al., 2000; Shen and Schier, 2000; in stem cell maintenance. de la Cruz et al., 2002). EGF-CFC family members Oncogene (2005) 24, 5731–5741. doi:10.1038/sj.onc.1208918 contain an NH2-terminal signal peptide, a modified EGF-like region, a conserved cysteine-rich domain Keywords: Cripto-1; embryogenesis; oncogenesis; EHT; (CFC motif) and a short hydrophobic COOH-terminus engiogenesis; therapy that contains additional sequences for glycosyl- phosphatidylinositol (GPI) cleavage and attachment (Minchiotti et al., 2000) (Figure 1). In addition, all EGF-CFC proteins contain a consensus O-linked Introduction fucosylation site within the EGF-like motif that is necessary for their ability to function as coreceptors for Embryonic genes are important in regulating asym- the TGFb-related proteins, Nodal or Vg1/GDF-1 metric cell division, pluripotentiality and/or in main- (Schiffer et al., 2001; Yan et al., 2002). Most of the taining cellular and tissue-specific niches that are EGF-CFC proteins are cell-associated glycoproteins necessary for stem cell survival (Kopper and Hajdu, that contain from 171 to 202 amino acids and that 2004; Ohlstein et al., 2004; Sell, 2004). Genetic muta- range from 18 to 21 kDa in size. However, the native mouse and human Cripto-1 proteins are 24, 28 and 36 kDa in size and additional proteins ranging from 14 *Correspondence: DS Salomon, Tumor Growth Factor Section, Mammary Biology & Tumorigenesis Laboratory, National Cancer to 60 kDa have also been identified and are the result of Institute, National Institutes of Health, Buillding 37, Room 118B, differential glycosylation at N- and O-linked asparagine Bethesda, MD 20892, USA; E-mail: [email protected] and serine residues (Brandt et al., 1994; Kenney et al., Cripto-1: a modulator during embryogenesis and oncogenesis L Strizzi et al 5732 O-fucose (Thr 88)

1 169 188 NH2 Signal sequence EGF-like domain CFC-domain GPI site COOH

O-linked N-linked O-linked (Ser 40) (Asn 79) (Ser 161) Figure 1 Schematic diagram illustrating the different domains composing the Cripto-1 glycoprotein

1996; Seno et al., 1998; Niemeyer et al., 1999; Minchiotti absence of head and trunk mesoderm, loss of prechordal et al., 2000; Schiffer et al., 2001). The mouse Cr-1 plate and ventral neuroectoderm, impairment of gas- protein can be released by treatment of cells with trulation movements, loss of A/P axis positioning and phosphatidylinositol-specific phospholipase C (PI-PLC) left/right laterality defects (Zhang et al., 1998; Gritsman (Minchiotti et al., 2000). Removing the COOH-terminal et al., 1999). One-eyed pinhead, like mouse Cr-1, is stretch of residues where GPI linkage occurs generates absolutely required for the migration of mesendoderm soluble forms of biologically active mouse and human cells through the primitive streak (Warga and Kane, Cripto-1 (Xu et al., 1999; Minchiotti et al., 2001; Bianco 2003). Mutations of oep effectively block the ability of et al., 2002b, 2003; Yan et al., 2002). Full activity epiblast cells to migrate and induce a more cohesive requires the presence of a peptide containing only an interaction between these cells. This effect is indepen- intact EGF domain and CFC domain (Minchiotti et al., dent of the two zebrafish Nodal-related genes, squint 2001; Yan et al., 2002). However, whether there are (Sqt) and Cyclops (Cyc), suggesting that these pheno- significant qualitative and/or quantitative differences in types are unique to oep. the biological activities that are regulated by Cripto-1 Mouse Cr-1 is expressed in the precardiac mesoderm between the soluble versus cell-associated forms is not and performs an essential role during the early stages of yet known since Cripto-1 can function both as a cardiac lineage specification and differentiation (Dono coreceptor in cis (autocrine/cell-autonomous) and as et al., 1993). Homozygous knockout of the Cr-1 gene ligand in trans (paracrine/cell-nonautonomous) (Yan (Cr-1À/À) in pluripotential embryonic stem (ES) cells et al., 2002). impairs their ability to spontaneously differentiate in vitro into cardiomyocytes (Parisi et al., 2003) without Function of Cripto-1 during embryonic development affecting the ability of ES cells to differentiate into other cell types (Xu et al., 1998). In fact, Cr-1À/À ES cells Genetic studies in zebrafish and mice have defined an actually show extensive neural differentiation in vitro essential role for Nodal that functions through Cripto-1 and in vivo suggesting that neuroectodermal differentia- in the formation of the primitive streak, patterning of tion is the preferred default pathway that occurs in the the Anterior/Posterior axis (A/P) and specification of absence of Cr-1 expression (Parish et al., 2005; Sonntag mesoderm and endoderm (mesoendoderm) during gas- et al., 2005). Chimeric embryos that were generated trulation, whereas later in embryogenersis Nodal func- between wild-type and Cr-1À/À ES cells in vitro were tions through cryptic in the establishment of Left/Right normal indicating that Cr-1 produced by the wild-type (L/R) asymmetry of developing organs (Ding et al., host cells could rescue the mutant phenotype in a 1998; Yan et al., 1999; Schier and Shen, 2000; Hamada paracrine manner (Xu et al., 1998). Germline disruption et al., 2002; Schier, 2003). Cr-1-null mice (Cr-1À/À) die at of Cr-1 in Cr-1À/À embryos results in the formation of day 7.5 due to their inability to gastrulate and form embryos that possess a head without a trunk demon- appropriate germ layers (Liguori et al., 1996; Ding et al., strating that there is a severe deficiency in embryonic 1998). Nodal, which functions through Cripto-1, co- mesoderm and endoderm without a loss of anterior operates with wnt3 in the mouse in initiating the neuroectoderm formation (Xu et al., 1999). specification of mesoderm and endoderm formation and in regulating the development of dorsal and anterior mesoderm and head structures (Harland and Gerhart, 1997; Beddington and Robertson, 1999; Liu et al., 1999; Intracellular pathways that mediate Thisse et al., 2000; Kodjabachian, 2001; Robb and Tam, Cripto-1 activity 2004). In contrast, Cripto-1 in conjunction with Fgf8 and brachyury are important for initiating and main- Cripto-1 is involved in the activation of several different taining the migration of mesodermal cells through the signaling pathways during embryonic development and primitive streak (Conlon and Smith, 1999; Ciruna and cellular transformation (Bianco et al., 2004). The major Rossant, 2001; Griffin and Kimelman, 2003; Warga and signaling pathways activated by Cripto-1 are a Nodal/ Kane, 2003). Mutations in zebrafish of the Cripto-1 ALK4/ALK7/Smad-2 signaling pathway and a Glypican-1/ ortholog, one-eyed pinhead (oep) result in cyclopia, an c-Src/MAPK/AKT signaling pathway.

Oncogene Cripto-1: a modulator during embryogenesis and oncogenesis L Strizzi et al 5733 Nodal/ALK4/ALK7/Smad-2 signaling pathway signaling, CR-1 and Nodal may have distinct functional roles independently of one another. For example, During embryogenesis, EGF-CFC proteins function as zebrafish oep regulates cell motility independently of cell-surface coreceptors for Nodal through activation of Zebrafish Nodal-related proteins Cyc and Sqt, whereas the serine threonine kinase type I (ALK4) and type II Nodal can inhibit BMP signaling by binding directly to (ActRIIB) receptor complex in Xenopus (Schier, 2003). BMP independently of CR-1 (Yeo and Whitman, 2001; Biochemical evidence has demonstrated a direct inter- Warga and Kane, 2003). action between mouse Cr-1 and ALK4 in Xenopus oocytes and this interaction is necessary for Nodal to bind to the ALK4/ActRIIB receptor complex and to Glypican-1/c-Src/MAPK/AKT signaling pathway stimulate Smad-2 phosphorylation and activation (Yeo Our lab has previously demonstrated that CR-1 can and Whitman, 2001; Bianco et al., 2002a). Cripto-1 can activate the ras/raf/MAPK and PI3-K/AKT/GSK-3b also directly interact with serine threonine kinase signaling pathways (De Santis et al., 1997; Kannan receptor ALK7 enhancing the ability of ALK7 to et al., 1997; Ebert et al., 1999; Bianco et al., 2004). respond to Nodal (Reissmann et al., 2001). However, Interestingly, activation of these signaling pathways can Nodal can directly bind ALK7 in the absence of Cripto- be achieved with a soluble GPI-truncated CR-1 recom- 1, although Cripto-1 significantly enhances its signaling binant protein (Kannan et al., 1997; Bianco et al., activity (Reissmann et al., 2001). Site-directed mutagen- 2002b). Since soluble CR-1 has been found to be esis experiments have shown that Cripto-1 interacts with secreted in the conditioned medium of several human Nodal through the EGF-like domain, while the CFC carcinoma cell lines, it is possible that a functional domain mediates binding of Cripto-1 to ALK4 (Yeo soluble isoform of CR-1 is naturally cleaved from the and Whitman, 2001). In addition to functioning as a cell membrane (Brandt et al., 1994; Minchiotti et al., coreceptor for Nodal, Cr-1 has been shown to mediate 2000; Normanno et al., 2004). Activation of these two signaling of other TGFb ligands, such as Activin and intracellular pathways is independent of Nodal and Xenopus Vg1 and its ortholog in mouse GDF1 (Cheng ALK4, since CR-1 can stimulate MAPK and AKT et al., 2003). Similar to Nodal, Vg1 and GDF1 can bind phosphorylation in EpH4 mouse mammary epithelial to ALK4/ActRIIB receptor complex only in the cells and MC3T3-C1 osteoblast cells that lack ALK4 presence of EGF-CFC coreceptors. In contrast, binding and Nodal expression, respectively (Bianco et al., of CR-1 to Activin can inhibit Activin signaling in 2002a). Furthermore, activation of these two intracel- mammalian cells (Adkins et al., 2003; Gray et al., 2003). lular pathways occurs through a direct binding of CR-1 In this regard, Adkins et al. have demonstrated that CR- to Glypican-1, a membrane-associated heparan sulfate 1 interacts with Activin B through the CFC domain, proteoglycan (HSPG) that functions as a coreceptor for sequestering Activin B from a functional ALK4/ several different growth factors (i.e. Wnts, FGF, HB- ActRIIB receptor complex and blocking its ability to EGF) (Bianco et al., 2003). Binding of CR-1 to function as an inhibitor of cell growth in cancer cells. Glypican-1 activates the cytoplasmic c- Therefore, antagonism of Activin signaling by CR-1 Src, triggering the MAPK and AKT signaling pathways may represent an additional mechanism used by CR-1 (Bianco et al., 2003). Since MAPK and AKT signaling to promote tumorigenesis. In addition to being EGF- pathways have been involved in regulating cell pro- CFC-dependent, Nodal signaling is regulated by several liferation and survival, CR-1 might be involved in the antagonists that function at different levels. Tomoregu- pathogenesis of human cancer through the inappropri- lin-1, A and B and Antivin can antagonize Nodal ate activation of these two signaling pathways indepen- signaling through EGF-CFC coreceptors. In this regard, dently of Nodal and ALK4. CR-1 has also been shown the transmembrane protein Tomoregulin-1 inhibits to function during development through an FGFR-1 Nodal signaling in Xenopus embryos through interac- signaling pathway. In Xenopus oocytes and in yeast, tion with the CFC domain of CR-1 (Harms and Chang, Xenopus FRL1 can indirectly increase the phosphoryla- 2003). Furthermore, Tomoregulin-1, through its EGF- tion of Xenopus FGFR-1 (Kinoshita et al., 1995). like domain, can specifically bind with low affinity to the However, no direct binding between FGFR-1 and tyrosine kinase receptor erbB-4 (Uchida et al., 1999). FRL1 could be detected. Finally, several findings Since CR-1 can indirectly increase the tyrosine phos- suggest a potential interaction of the CR-1 signaling phorylation of erbB-4 in several mouse and human pathway with the wnt/b-catenin/Lef-1 signaling path- mammary epithelial cells, interaction between CR-1 and way. In fact, Cr-1 has been identified by microarray Tomoregulin-1 might explain erbB-4 trans-phosphory- analysis as a primary target gene in the wnt/b-catenin lation by CR-1 in mammalian cells (Bianco et al., 1999). signaling pathway during embryonic development The Lefty/Antivin subfamily has also been shown to (Morkel et al., 2003). More recently, FRL1 and mouse antagonize Nodal and Vg1/GDF1 signalings through a Cr-1 have been found to function as coreceptors for direct interaction with EGF-CFC coreceptors (Chen wnt-11 by binding directly to Wnt-11, providing a link and Shen, 2004). Similarly, Xantivin, a Lefty-related between the EGF-CFC proteins and wnt family protein in Xenopus, interacts directly with Xenopus members (Tao et al., 2005). Interestingly, in Xenopus, FRL-1 and CR-1, leading to inhibition of Nodal glypican-4 physically interacts with Wnt-11 suggesting signaling (Tanegashima et al., 2004). Although EGF- that both glypican-4 and FRL1 are involved in CFC proteins play an essential role in mediating Nodal regulating Wnt signaling (Ohkawara et al., 2003).

Oncogene Cripto-1: a modulator during embryogenesis and oncogenesis L Strizzi et al 5734 Biological effects of Cripto-1 inhibited with a blocking anti-CR-1 monoclonal anti- body (A8.G3.5), suggesting that CR-1 directly mod- Regulation of milk protein expression ulates angiogenesis. Finally, overexpression of CR-1 in MCF-7 human breast cancer cells enhances tumor It has been shown that CR-1 can modulate milk protein neovascularization in a xenograft model. expression in HC-11 mouse mammary epithelial cells. HC-11 mouse mammary epithelial cells express the milk protein b-casein at confluency after exposure to the Regulation of cellular migration and invasion by Cripto-1 lactogenic hormones , and pro- Overexpression of Cr-1 in the mouse EpH4 mammary lactin (DIP) (De Santis et al., 1997). Like other EGF- epithelial cells caused not only an increase in cell related peptides, pretreatment of HC-11 cells with CR-1 proliferation and anchorage-independent growth in soft during logarithmic growth induces an optimal response agar but formation of duct-like structures when grown to DIP with respect to the induction of the milk protein in collagen type I matrix and increased chemotaxis and b-casein (De Santis et al., 1997). In contrast, the migration when these cells were cultured on plastic or on simultaneous treatment of HC-11 cells with CR-1 in porous filters coated with extracellular matrix proteins the presence of DIP inhibits b-casein expression. In (Wechselberger et al., 2001). The effects of Cr-1 in EpH4 addition, HC-11 cells that overexpress Cr-1 protein also cells was the first study which suggested that Cr-1 may exhibit low levels of b-casein expression following DIP play a role in inducing epithelial to mesenchymal treatment (De Santis et al., 1997). This inhibitory effect transition (EMT) of mammary epithelial cells. Similarly, of CR-1 on b-casein expression in response to DIP is not human MCF-7 breast cancer cells, which overexpress unique to this mouse mammary epithelial cell line, since CR-1 in addition to acquiring higher proliferation a similar inhibitory effect on b-casein expression in the rates in serum-free medium, increased colony formation presence of DIP is observed in CID-9 overexpressing in soft agar and increased resistance to anoikis QJ;Cr-1 (Niemeyer et al., 1998). In addition, CR-1 also showed increased invasion through matrix-coated modulates expression of other milk proteins in response membranes as demonstrated by Boyden chamber to lactogenic hormones in primary mouse mammary invasion assay (Normanno et al., 2004). Further explant cultures established from midpregnant mice evidence of the role that CR-1 might play in the (De Santis et al., 1997). In this regard, concurrent induction of EMT comes from a recent study where the treatment with DIP plus CR-1 significantly impairs expression of markers that characteristically define DIP-induced expression of b-casein and whey acidic EMT was investigated in hyperplasias protein (WAP) in mammary explant cultures. The and tumors from MMTV-CR-1 transgenic mice and ability of CR-1 to block lactogenic hormone-induced in HC-11/Cr-1 cells (Strizzi et al., 2004). During expression of b-casein is mediated through a ras- EMT, intercellular contacts between epithelial cells are dependent, PI3-K-dependent pathway (De Santis lost due to decreased E-cadherin expression and et al., 1997). This inhibitory effect of CR-1 on milk disruption of the adherens junction complex as cells protein expression may be biologically significant since become less adhesive and potentially more motile endogenous Cr-1 expression is upregulated in mouse (Boyer et al., 2000; Thiery, 2002). E-cadherin expression mammary epithelial cells during pregnancy and lacta- was significantly decreased in tissue extracts from the tion and soluble CR-1 protein is secreted in human milk mammary tumor lesions that express the CR-1 trans- (Bianco et al., 2001). gene and in extracts from HC-11/Cr-1 cells. Further- more, the zinc-finger repressor transcription factor Regulation of angiogenesis snail, which is known to downregulate or interfere with the normal expression of E-cadherin (Savagner, We have recently demonstrated that human CR-1, in 2001), was detected, by RT–PCR and Western addition to regulating cell proliferation, survival and blot analysis, at significantly higher levels in MMTV- transformation, plays an essential role also in the CR-1 mammary tumor lesions and HC-11/Cr-1 cells as multistep process of tumor angiogenesis (Bianco et al., compared to control nontumorigenic mammary tissue 2005). In fact, CR-1 has a strong angiogenic activity in and HC-11 wild-type cells, respectively (Strizzi et al., vitro in cultured human umbilical endothelial cells 2004). Expression of the mesenchyme cell cytoskeleton (HUVECs). CR-1 enhances proliferation of HUVECs component, vimentin, is also increased in epithelial cells in a dose-dependent manner, stimulates their migration and tumors during EMT (Casaroli-Marano et al., 1999; and invasion and induces formation of vascular-like Dandachi et al., 2001; Ackland et al., 2003). Over- structures on Matrigel. In addition, the chemotactic expression of CR-1 in human cervical carcinoma activity of CR-1 in HUVEC is mediated by c-Src and cells was associated with the increased expression of PI3-K/AKT signaling pathways. The angiogenic activity vimentin in these cells as they exhibited increased of CR-1 in endothelial cells is not dependent on the migration and invasion through matrix-coated mem- autocrine production of VEGF, since a specific VEG- branes suggesting that CR-1-induced vimentin expres- FRI inhibitor does not block CR-1 angiogenic activity sion may contribute to a more aggressive phenotype in vitro in HUVECs. In vivo, recombinant CR-1 protein of cervical carcinoma (Ebert et al., 2000). Expression induces microvessel formation in silicone cylinders filled for vimentin was also increased in extracts from with Matrigel and microvessel formation is strongly mammary gland hyperplasias and tumors from

Oncogene Cripto-1: a modulator during embryogenesis and oncogenesis L Strizzi et al 5735 Cripto-1 Role of Cripto-1 in transformation and cancer LRP5/6 Glypican-4

Wnt3 Wnt5A Glypican-1 In vitro transformation of cells by Cripto-1

Apical Lipid Raft The first in vitro study suggesting a potential role for Src Frz Frz Cripto-1 during cell transformation involved transfec- tion of human CR-1 into NIH3T3 cells which induced Dsh RhoDsh Rac PI-3K MEK morphological changes and colony formation of these cells when grown in monolayer (Ciccodicola et al., ROCK JNK Akt MAPK 1989). When full-length CR-1 DNA was transfected into GSK-3β immortalized mouse NOG-8 mammary epithelial cells, those cells that expressed CR-1 were able to form β-Catenin Axin Apc colonies in soft agar and exhibited anchorage-indepen- Trans-Golgi dent growth in soft agar as compared to the neo- βTrCP Lef-1/Tcf Jun Cripto-1 transfected NOG-8 cells (Ciardiello et al., 1991). Similarly, overexpression of CR-1 cDNA in normal rat fibroblasts was shown to induce growth of these cells in soft agar (Brandt et al., 1994) and exogenous recombinant CR-1 protein was shown to stimulate cell Basolateral proliferation of differentiated and undifferentiated embryonal carcinoma cells (Su et al., 1993; Baldassarre Figure 2 Cripto-1 is a target gene in a canonical Wnt3/b-catenin/ et al., 1997). Further support for transforming potential Lef-1 (Tcf) signaling pathway and also a coreceptor for Wnt and glypican-1. Wnt3 binds to a frizzled (Frz) receptor and coreceptors of Cripto-1 stems from studies showing increased LRP5/6 and Cripto-1. Activation of Dishevelled (Dsh) results in expression of CR-1 in cells transformed by different the inactivation of the b-catenin degradation complex (GSK-3b/ . For instance, overexpression of Cr-1 mRNA Axin/Apc), which promotes ubiquination and proteosomal degra- was detected in tumorigenic and metastatic Ha-ras- dation of b-catenin. b-Catenin accumulates and can bind to the DNA-binding proteins of the transcription factor family Lef-1/Tcf transformed rat CREF embryo fibroblasts (Su et al., after translocation to the nucleus. Cripto-1 and glypican-4 can also 1993). Interestingly, in these cells, reversion of the function as coreceptors for Wnts such as Wnt5A with Frz to transformed phenotype of the Ha-ras-transformed cells activate the planar cell polarity pathway (PCP). This pathway is by overexpression of a Krev-1 ras suppressor gene also regulated through Dsh which can stimulate RhoA/ROCK and resulted in a loss of expression of Cr-1. This suggests Rac/JNK to activate the transcription factor c-Jun and compo- nents of the cytoskeleton and cell motility. Finally, Cripto-1 can that Cr-1 may play a role in ras-induced CREF cell bind to glypican-1 in lipid rafts and activate c-src which can then transformation. Cripto-1 was also found to be over- enhance the ras/MAPK and/or PI-3 kinase/Akt pathways. PI3K/ expressed in K-ras-transformed FRLT-5 rat thyroid Akt signaling molecules are also known to be capable of epithelial cells which show TSH-independent growth inactivating GSK-3b causing similar cellular modifications that occur during Wnt signaling capability (Mincione et al., 1998). Also v-ras/Smad-7- transformed keratinocytes develop skin tumors that overexpress Cr-1 (Liu et al., 2003), suggesting that Smad-7-induced tumor formation may require upregu- MMTV-CR-1 transgenic mice and from HC-11/Cr-1 lation of Cr-1 and other EGF-related peptides. cells. In addition, mammary tumors from the MMTV- In concert with Cripto-1 cellular transforming poten- CR-1 transgenic mice and HC-11/Cr-1 cells showed tial, it appears that Cripto-1 levels may affect cell increased expression of active c-Src, focal adhesion differentiation. Mouse CID-9 mammary epithelial cells kinase (FAK) and AKT (Strizzi et al., 2004). These are derived from mammary glands of 14.5-day pregnant signaling molecules can be activated during EMT and mice and normally express Cr-1 (Niemeyer et al., 1998). may play a role in increasing tumor cell migration and When these cells are treated with lactogenic hormones invasion (Thiery and Chopin, 1999). Interestingly, and prolactin and are cultured on extracellular matrix mammary tumors from the MMTV-CR-1 transgenic substrates, they form differentiated polarized epithelial mice and HC-11/Cr-1 cells were found to express the structures called mammospheres and express the milk inactive form of glycogen synthease kinase 3 beta (GSK- protein b-casein and Cr-1. This suggests that hormones 3b) as well as the active form of b-catenin. During wnt released during pregnancy may be involved in the signaling, GSK-3b is inactivated by phosphorylation regulation of Cr-1 expression. When CID-9 cells over- causing accumulation of nonphosphorylated b-catenin express Cr-1, they show significantly higher proliferation which translocates to the nucleus in a complex with Tcf/ rates, increased colony formation in soft agar and cell Lef-1 and functions as a transcription factor activating survival, but no longer form mammospheres and show genes involved in increased cell survival, proliferation reduced expression of b-casein. Conversely, if undiffer- and migration such as, c-myc, cyclin-D1, snail and slug entiated human or mouse teratocarcinoma cells are (Polakis, 2000). These data suggest that Cripto-1 may induced to differentiate by treatment with retinoic play a role in the regulation of cellular migration and acid, expression of Cripto-1 is significantly reduced invasion possibly through crosstalk with the Wnt (Ciccodicola et al., 1989). These data suggest that signaling pathway (Figure 2). increased CR-1 levels may negatively affect cell

Oncogene Cripto-1: a modulator during embryogenesis and oncogenesis L Strizzi et al 5736 differentiation. However, the effects of Cripto-1 ob- enhanced ductal branching and dilatation, intraductal served in vitro may depend on the type of cells used and hyperplasia and hyperplastic alveolar nodules. Mam- their responsiveness to variations in growth conditions, mary glands from 12- to 20-month-old multiparous reflecting the fact that the mammary gland epithelium MMTV-CR-1 mice showed multifocal hyperplasias and cyclically goes through phases of proliferation and approximately 33% of these mice subsequently devel- involution depending on the variable levels of growth oped papillary adenocarcinomas (Figure 3). The long factors and hormones present in the environment in latency period suggests that additional genetic altera- which they grow. For instance, normal HC-11 cells tions are required to facilitate mammary tumor forma- cultured in low serum and treated with CR-1 show an tion in conjunction with CR-1. The WAP promoter can increase in apoptosis through activation of caspase-3 be used to drive high levels of transgene expression in a protease and downregulation of expression of the significantly greater number of mammary epithelial antiapoptotic protein Bcl-xl (De Santis et al., 2000). cells, predominantly during pregnancy and lactation (Kordon et al., 1995). This allows assessment of any Cripto-1 is upregulated in transgenic mouse mammary significant effect on mammary gland development or tumors formation of premalignant or malignant lesions in the pregnant and lactating mammary gland of multiparous Transgenic mice overexpressing either neu (erbB-2), mice. Mice expressing the human CR-1 transgene under TGF-a, int-3, polyoma middle T (PyMT) or simian the regulation of the WAP promoter in FVB/N mice virus 40 large T antigens have been shown to develop have been recently developed (Sun et al., 2005, mammary gland tumors (Jhappan et al., 1990, 1992; submitted). Higher levels of phosphorylated AKT and Guy et al., 1992; Maroulakou et al., 1994; Muller et al., MAPK were detected in mammary glands of multi- 1998). All of these tumors were found to express parous WAP-CR-1 mice as compared to multiparous significant levels of Cr-1 mRNA and protein as FVB/N mice, suggesting increased cell proliferation and determined by RT–PCR, Western blot analysis and survival of the transgenic mammary gland. In addition, immunohistochemistry (Kenney et al., 1996). In addi- over half of the WAP-CR-1 multiparous female mice tion to expression in mammary gland tumors of developed multifocal mammary tumors of mixed transgenic mice, Cr-1 was also consistently expressed histological subtypes. These tumors were histologically in hyperplastic lesions in mammary glands of PyMT, distinct from the mammary tumors that developed in neu and TGF-a mouse transgenic models suggesting a the MMTV-CR-1 transgenic mouse. The mammary role for Cr-1 in deregulation of mammary epithelial tumors from the WAP-CR-1 mice contained foci of proliferation (Niemeyer et al., 1999). The insertion epithelial hyperplasia mixed with glandular, papillary of Elvax pellets containing the EGF-like motif of and solid carcinomas. Areas of adenosquamous and CR-1 protein into the mammary gland of ovariecto- myoepithelial differentiation were also identified within mized virgin mice produced a dramatic increase in these tumors (Figure 4). These tumors were similar to DNA synthesis in mammary epithelial cells imme- the mixed histotype of mammary tumors that arise in diately adjacent to the pellets (Kenney et al., 1997). In addition, increased ductal branching and hyperplasia was observed when FVB/N or C57B1/6 mouse mammary epithelial cells, containing retroviral-driven overexpression of Cr-1 cDNA, were subsequently transplanted into cleared mammary fat pad of syngenic ovariectomized virgin mice (Kenney et al., 1997). Although mammary carcinomas were not observed to arise from the transplanted hyperplasias, the hyperpla- sias could be serially transplanted, suggesting the presence of a Cr-1-induced immortalized population of mammary epithelial cells. Similarly, when NOG-8 or CID-9 Cripto-1-transformed cells were subsequently transplanted in vivo, these cells were unable to form tumors (Ciardiello et al., 1991; Niemeyer et al., 1998). This suggests that other genetic alterations must take place in addition to Cripto-1 overexpression for tumors to develop.

In vivo tumorigenesis induced by Cripto-1 The first in vivo study that showed CR-1-dependent hyperplasia and tumor growth in mouse mammary Figure 3 A histological section (20 Â ) of a mammary tumor from the MMTV-CR-1 transgenic mice shows the papillary morphology glands used the mouse mammary tumor virus (MMTV) of this adenocarcinoma. The same figure also shows evidence of to drive CR-1 overexpression (Wechselberger et al., high-grade proliferation by immunohistochemical staining of the 2005). Virgin MMTV-CR-1 transgenic mice exhibited tumor cell nuclei with the cell proliferation marker PCNA

Oncogene Cripto-1: a modulator during embryogenesis and oncogenesis L Strizzi et al 5737 D’Antonio et al., 2002). Expression of CR-1 in normal tissues has been examined in some of these studies. CR-1 protein is usually expressed with low frequency and at low levels in normal mucosae, whereas an increase in CR-1 expression has been observed in the normal mucosa adjacent to either colon or breast carcinoma (Saeki et al., 1992; Panico et al., 1996). Furthermore, CR-1 expression is significantly increased in premalig- nant lesions, such as colon adenomas, intestinal metaplasia of the gastric mucosa and ductal carcinoma in situ (DCIS) of the breast. In this respect, the frequency of CR-1 expression in the adenomas was found to correlate directly with the degree of dysplasia, and with the size and the histological subtype of colon adenomas (Saeki et al., 1994b). CR-1 expression was also detected in 62% of normal colon mucosa specimens from individuals that belong to families with a high incidence of colorectal carcinomas, whereas only 20% of colon mucosa from low-risk patient specimens was Figure 4 A representative H&E-stained histological section (20 Â ) positive for CR-1 (De Angelis et al., 1999). In an of a mammary tumor from the WAP-CR-1 transgenic mice shows additional study, expression of CR-1 immunoreactivity aspects of the different lesion types identifiable within the same in normal rectal mucosa adjacent to carcinoma was tumor. Green arrow ¼ ductal hyperplasia; red arrow ¼ tumor cells associated with an increase in the incidence of bowel with mesenchyme-like morphology; yellow arrow ¼ squamous metaplasia; black arrow ¼ epithelial tumor cells in solid or cord- wall penetration, lymph node involvement and a like arrangement recurrence rate of 100% (Gagliardi et al., 1994). Therefore, the gradual increase in CR-1 expression that occurs in the multistage process that evolves from normal mucosa to premalignant lesions, and from MMTV-Wnt1 mice or mammary tumors that develop in premalignant lesions to carcinoma, suggests that CR-1 mice expressing stabilized mutant forms of b-catenin may be involved in the early events of carcinogenesis in with either the MMTV or WAP promoters (Miyoshi different tissues. Although no significant correlations et al., 2002a, b; Rosner et al., 2002; Li et al., 2003; have been found between CR-1 expression and patient Teuliere et al., 2005). In fact, increased expression of prognosis in human carcinomas, several findings suggest dephosphorylated b-catenin in WAP-CR-1 mammary that CR-1 has an important role in tumor progression. tumors suggests that a canonical For example, a statistically significant higher expression may be activated in these tumors. In addition, as of CR-1 was found in extra-ovarian specimens as described for Wnt pathway mammary tumors (Miyoshi compared with ovarian primary tumors, in a subset of et al., 2002a), evidence of epidermal transdifferentiation, patients for which both type of tissues were available as determined by positive immunostaining for the (D’Antonio et al., 2002). This observation suggests that epidermal cell cytokeratins CK-1 and CK-6 and for overexpression of CR-1 might be associated with an hair and nail cytokeratin, AE-13 (Weiss et al., 1984; increased ability to form metastasis. This hypothesis is Fuchs and Byrne, 1994) was also found in the mammary in agreement with recent in vitro findings discussed tumors from the WAP-CR-1 transgenic mice. These above that have demonstrated that expression of CR-1 results demonstrate that overexpression of CR-1 during is associated with increased motility and invasiveness of pregnancy and lactation can lead to the development of tumor cells (Wechselberger et al., 2001; Bianco et al., mammary tumors in multiparous mice and that CR-1 2003; Normanno et al., 2004; Strizzi et al., 2004). may be a significant mediator of some of the oncological Interestingly, CR-1 is frequently coexpressed with other effects of an activated canonical Wnt signaling pathway EGF-related peptides, such as TGF-b, amphiregulin in the mammary gland. and heregulin in human breast and mouse epidermal carcinomas, implying that different growth factors Expression of Cripto-1 in human carcinomas and might cooperate in supporting the autonomous prolif- premalignant lesions eration of cancer cells (Saeki et al., 1992; Qi et al., 1994; Normanno et al., 1995; Panico et al., 1996; De Angelis Expression of CR-1 mRNA and/or immunoreactive et al., 1999; D’Antonio et al., 2002; Liu et al., 2003). protein has been demonstrated to occur with high Finally, expression of CR-1 has been reported in other frequency in colorectal, breast, ovarian and gastric carcinoma types, including lung carcinoma (Fontanini cancers in different studies that have examined a et al., 1998), nonseminomatous testicular tumors relevant number of tumor specimens (Kuniyasu et al., (Baldassarre et al., 1997), pancreatic ductal adenocarci- 1991; Saeki et al., 1992, 1994a, b; Qi et al., 1994; noma (Friess et al., 1994; Tsutsumi et al., 1994), basal Stromberg et al., 1994; Normanno et al., 1995; Panico cell carcinoma (Welss et al., 2003) and bladder et al., 1996; Niikura et al., 1997; De Angelis et al., 1999; carcinoma (Byrne et al., 1998).

Oncogene Cripto-1: a modulator during embryogenesis and oncogenesis L Strizzi et al 5738 Cripto-1 and Wnt in stem cell renewal and maintenance region for Wnt11 in conjunction with the frizzled receptor Xfz7 and glypican-4 to activate a canonical Recently, mouse Cr-1 and human CR-1 have been Wnt pathway that is dependent upon b-catenin and that identified as direct target genes in a canonical Wnt/b- is necessary for dorsal axis specification during early catenin/Tcf signaling pathway (Morkel et al., 2003). Cr- embryogenesis (Tao et al., 2005). Finally, Wnt8c and 1 expression was found to be downregulated in either b-catenin, which are expressed in Hensens node and b-cateninÀ/À or Wnt3À/À mutant mouse embryos, which regulate L/R axis formation in the chick, are able whereas a constitutively active form of b-catenin was to directly induce Nodal expression (Rodriguez-Esteban able to upregulate Cr-1 expression during early embryo- et al., 2001). genesis. Likewise in ApcÀ/À Min mice, Cr-1 expression In addition to maintaining stem cell identity, other was elevated in intestinal adenomas that develop in significant biological activities that are shared by Wnts these postnatal mice. Reciprocally, in human colon and Cripto-1 are their ability to stimulate cell motility, carcinoma cells that express high levels of CR-1, invasion and EMT, as previously discussed in this induction of a dominant-negative Tcf4 abolished CR-1 review (Wechselberger et al., 2001; Bianco et al., 2003; expression. Therefore, in contrast to transient activation Kemler et al., 2004; Strizzi et al., 2004; Endo et al., 2005; of Wnts and Cripto-1 which is obligatory for the Yook et al., 2005). Loss of cell–cell adhesion due to a promotion of stem cell self-renewal, pluripotency and reduction in E-cadherin expression, possibly through fixation of stem cell identity in ES cells and in stem Wnt/Cripto-1 crosstalk, may also affect the integrity of cells from normal adult tissues of the haematopoietic the stem cell niche since cell adhesion between support- system, skin, central nervous system, pancreas, ing niche cells and stem cells is required for stem cell intestine and breast (Beachy et al., 2004; Besser, 2004; survival and asymmetric cell division (Matsui and Kanatsu-Shinohara et al., 2004; Kleber and Sommer, Okamura, 2005; Yamashita et al., 2005). Reciprocally, 2004; Nelson and Nusse, 2004; Sato et al., 2004; Zhang a loss of cell–cell adhesion due to a repression of et al., 2004; James et al., 2005; Wei et al., 2005), E-cadherin expression may also be important for the continuous activation of these genes is associated with movement of differentiated intestinal and skin epithelial the initiation and/or progression of cancer in mouse and cells from the niche as occurs in the intestinal crypts and human tissues such as in the hematopoietic system, skin, in the bulge region of the papillae of the hair follicles. In colon, stomach, liver, ovary and breast (Polakis, 2000; addition, in the intestinal epithelium, expression of Salomon et al., 2000). In addition to being a direct target guidance proteins such as, the Wnt target gene EphB3, is gene in a canonical Wnt signaling pathway, in Xenopus, also necessary for directing migration of the Paneth cells the Cripto-1 ortholog, FRL1 functions as a coreceptor out of and down from the crypts (Batlle et al., 2002; through the EGF-like domain and the juxtamembrane Escaffit et al., 2005).

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