Oncogene (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 Growth Factor 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 genes 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 stem cell factor-Cripto-1/FRL1/cryptic family of proteins 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 epidermal growth factor- 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 gene family glypican-1 and the subsequent activation through src of phosphoinositol-3 kinase/Akt and ras/mitogen-activated Human Cripto-1 (CR-1), also known as teratocarcino- protein 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 signal transduction 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.