The EGF-CFC gene family encodes a group of structurally related proteins that serve as important competence factors during early embryogenesis in Xenopus, zebrafish, mice and humans. This multigene family consists of Xenopus FRL-1, zebrafish one-eyed-pinhead (oep), mouse cripto (Cr-1) and cryptic, and human cripto (CR-1) and criptin. FRL-1, oep and mouse cripto are essential for the formation of mesoderm and endoderm and for correct establishment of the anterior/ posterior axis. In addition, oep and cryptic are important for the establishment of left-right (L/R) asymmetry. In zebrafish, there is strong genetic evidence that oep functions as an obligatory co-factor for the correct signaling of a transforming growth factor-β (TGFβ)-related gene, nodal, during gastrulation and during L/R asymmetry development. Expression of Cr-1 and cryptic is extinguished in the embryo after day 8 of gestation except for the developing heart where Cr-1 expression is necessary for myocardial development. In the mouse, cryptic is not expressed in adult tissues whereas Cr-1 is expressed at a low level in several different tissues including the mammary gland. In the mammary gland, expression of Cr-1 in the ductal epithelial cells increases during pregnancy and lactation and immunoreactive and biologically active Cr-1 protein can be detected in human milk. Overexpression of Cr-1 in mouse mammary epithelial cells can facilitate their in vitro transformation and in vivo these Cr-1-transduced cells produce ductal hyperplasias in the mammary gland. Recombinant mouse or human cripto can enhance cell motility and branching morphogenesis in mammary epithelial cells and in some human tumor cells. These effects are accompanied by an epithelial-mesenchymal transition which is associated with a decrease in β-catenin function and an increase in vimentin expression. Expression of cripto is increased several-fold in human colon, gastric, pancreatic and lung carcinomas and in a variety of different types of mouse and human breast carcinomas. More importantly, this increase can first be detected in premalignant lesions in some of these tissues. Although a specific receptor for the EGF-CFC proteins has not yet been identified, oep depends upon an activin-type RIIB and RIB receptor system that functions through Smad-2. Mouse and human cripto have been shown to activate a ras/raf/MAP kinase signaling pathway in mammary epithelial cells. Activation of phosphatidylinositol 3-kinase and Akt are also important for the ability of CR-1 to stimulate cell migration and to block lactogenic hormone-induced expression of β-casein and whey acidic protein.In mammary epithelial cells, part of these responses may depend on the ability of CR-1 to transactivate erb B-4 and/or fibroblast growth factor receptor 1 through an src-like tyrosine kinase.

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor ␣, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin ␤1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185-and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42 erk-2 mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Growth and morphogenesis in the mammary gland depend on locally derived growth factors such as those in the epidermal growth factor (EGF) superfamily. Cripto‐1 (CR‐1, human; Cr‐1, mouse)—also known as teratocarcinoma‐derived growth factor‐1—is a novel EGF‐related protein that induces branching morphogenesis in mammary epithelial cells both in vitro and in vivo and inhibits the expression of various milk proteins. In the mouse, Cr‐1 is expressed in the growing terminal end buds in the virgin mouse mammary gland and expression increases during pregnancy and lactation. Cr‐1/CR‐1 is overexpressed in mouse and human mammary tumors and inappropriate overexpression of Cr‐1 in mouse mammary epithelial cells can lead to the clonal expansion of ductal hyperplasias. Taken together, this evidence suggests that Cr‐1/CR‐1 performs a role in normal mammary gland development and that it might contribute to the early stages of mouse mammary tumorigenesis and the pathobiology of human breast cancer. BioEssays 1999;21:61–70. Published 1999 John Wiley & Sons, Inc.

Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that fails to directly bind to any of the four known erb B type 1 receptor tyrosine kinases. The present study demonstrates that CR-1 indirectly induces tyrosine phosphorylation of erb B-4 but not of the epidermal growth factor-related receptors erb B-2 and erb B-3 in different mouse and human mammary epithelial cell lines. In addition, downregulation of erb B-4 in NMuMG mouse mammary epithelial cells and in T47D human breast cancer cells, using an anti-erb B-4 blocking antibody or a hammerhead ribozyme vector targeted to erb B-4 mRNA, impairs the ability of CR-1 to fully activate mitogen-activated protein kinase. Finally, chemical cross-linking of 125 I-CR-1 to mouse and human mammary epithelial cell membranes results in the labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor structurally unrelated to any of the known type I receptor tyrosine kinases. In conclusion, these data demonstrate that CR-1, upon binding to an unknown receptor, can enhance the tyrosine kinase activity of erb B-4 and that a functional erb B-4 receptor is required for CR-1-induced MAPK activation.The human CR-1 gene encodes for an EGF 1 -related peptide that was isolated and sequenced from a human NTERA2/D1 embryonal carcinoma cDNA expression library (1). A homologous gene has also been identified in the mouse from an F-9 mouse embryonal carcinoma cDNA expression library (2). More recently, additional Cripto-related genes have been identified in Xenopus laevis (FRL1), in mouse embryonic stem cell-derived mesoderm cells (Cryptic), and in Zebrafish (One-eyed pinhead) (3-5). Based on the strong sequence similarities, CR-1, FRL1, Cryptic, and One-eyed pinhead represent a new family of growth factor-like molecules named CFC (CR-1, FRL1, and Cryptic) family. These genes share a potential Nterminal leader sequence, a modified EGF-like domain, a second cysteine-rich region, and a C-terminal hydrophobic domain (5, 6). The modified EGF-like domain found in the CFC family is highly conserved. All EGF-like motifs contain six cysteines, which form three disulfide bonds in the case of EGF and the other related peptides in this family. In CR-1, FRL1, Cryptic, and One-eyed pinhead, the EGF-like domain is quite unusual. In fact, the first two cysteines are adjacent, thereby eliminating the A loop that is normally found between these residues. In addition, the spacing between the third and fourth cysteines is reduced relative to other EGF-like repeats, resulting in a smaller B loop (6). Because these three disulfide-linked loops are important in the ability of these proteins to assume a specific secondary structure that is essential for binding to specific erb B type I receptor tyrosine kinases, it has been proposed that the presence of an unusual EGF-like domain in the CFC family may indicate that this subfamily of peptides binds to a unique receptor. In fact, we have previously shown th...