Endocrine-Related Cancer (2000) 7 199–226

The EGF-CFC family: novel epidermal growth factor-related proteins in development and cancer

D S Salomon1, C Bianco1, A D Ebert 2, N I Khan1, M De Santis1, N Normanno 3, C Wechselberger1, M Seno 4, K Williams 5, M Sanicola 5, S Foley 5, W JGullick 6 and G Persico7

1Tumor Growth Factor Section, Laboratoryof Tumor Immunologyand Biology,National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA 2Freie Universita¨t Berlin, UK Benjamin Franklin, Frauenklinik, Berlin, Germany 3Instituto Oncologie Sperimentale, Napoli, Italy 4Okayama University, Okayama, Japan 5Biogen Inc, Cambridge, Massachusetts 02142, USA 6Department of Biological Sciences, Universityof Kent, Canterbury,UK 7Instituto Internationale di Genetica e Biofisica, Napoli, Italy (Requests for offprints should be addressed to D S Salomon)

Abstract The EGF-CFC gene familyencodes a group of structurallyrelated proteins that serve as important competence factors during earlyembryogenesisin Xenopus, zebrafish, mice and humans. This multigene familyconsists 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 mammarygland. In the mammarygland, expression of Cr-1 in the ductal epithelial cells increases during pregnancyand lactation and immunoreactive and biologicallyactive Cr-1 protein can be detected in human milk. Overexpression of Cr-1 in mouse mammaryepithelial cells can facilitate their in vitro transformation and in vivo these Cr-1–transduced cells produce ductal hyperplasias in the mammarygland. Recombinant mouse or human cripto can enhance cell motilityand branching morphogenesis in mammaryepithelial cells and in some human tumor cells. These effects are accompanied byan epithelial-mesenchymaltransition 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 pathwayin mammaryepithelial 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 wheyacidic protein. In mammaryepithelial cells, part of these responses maydepend on the abilityof CR-1 to transactivate erb B-4 and/or fibroblast growth factor receptor 1 through an src-like tyrosine kinase.

Endocrine-Related Cancer (2000) 7 199–226 Online version via http://www.endocrinology.org 1351-0088/00/007–199  2000 Societyfor Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 05:47:06AM via free access Salomon et al.: The EGF-CFC family

The EGF-CFC gene family is frequently deleted or exhibits loss of heterozygosity (LOH) in a subpopulation of head and neck, renal, gastric, bladder, A new family of genes, the EGF-CFC family, that encodes breast and lung carcinomas (Dono et al. 1991, Saccone et al. extracellular growth factor-like and/or co-receptor-like 1995, Todd et al. 1996, Sekido et al. 1998, Cuthbert et al. proteins has been identified in the human, mouse, frog and 1999). Interestingly, the β-catenin gene (CTNNB1) is located zebrafish. Members of this family include human cripto-1 at the chromosomal region 3p21.3–p22 while the activin (CR-1) (also known as teratocarcinoma-derived growth receptor IIB is located at 3p22 (Kraus et al. 1994, Bondestam factor-1 (TDGF-1)) and criptin (unpublished Human et al. 1999). These two genes may be involved in Genome Sciences Inc. patent number S5981215), mouse intracellular signaling by EGF-CFC proteins (see below). cripto-1 (Cr-1/tdgf-1) and cryptic, Xenopus FRL-1 and The mouse and human cripto genes consist of six exons and zebrafish one-eyed pinhead (oep) (Table 1) (Ciccodicola et five introns and possess inverted Alu and B1 sequence al. 1989, Dono et al. 1993, Kinoshita et al. 1995, Shen et al. elements respectively, and AUUU(A)-type Kamen-like 1997, Zhang et al. 1998). CR-1 was serendipitously isolated sequences in a large 3′ untranslated region, suggesting that in a screen for glucose-6–phosphate dehydrogenase as a they encode relatively short-lived mRNA species (Fig. 1) chimeric cDNA from a human NTERA2/D1 embryonal (Dono et al. 1991, 1993). The human CR-1 coding sequence carcinoma cDNA library (Ciccodicola et al. 1989). The containing the six exons is 4.8 kb in length. There is an mouse gene, Cr-1, was subsequently identified and cloned excellent conservation of the exon-intron structure in the from a mouse embryo cDNA library (Dono et al. 1993). The region of exon 4 which contains the EGF-like motif, while closely related mouse cryptic gene was cloned by differential exons 1 and 3 of the mouse are 15 and 33 nucleotides shorter RT-PCR from a mouse embryoid body-derived mesoderm respectively than the corresponding human exons (Dono et cDNA library, and criptin from a human pancreatic al. 1991, 1993). The 5′ upstream genomic sequences of carcinoma cDNA library (Shen et al. 1997). Similarly, mouse Cr-1 and human CR-1 between -610 and -1 from the Xenopus laevis FRL-1 was isolated in a functional screening most distal translation start sites are quite dissimilar in yeast with the intent of identifying fibroblast growth factor (Baldassarre et al. 2000). Several TATA and CAAT boxes receptor-1 (FGFR-1) activating genes from a Xenopus are present in the mouse Cr-1 promoter region while in the embryonic mesoderm cDNA library (Kinoshita et al. 1995). human CR-1 promoter these sequences are missing. These More recently, the zebrafish oep gene has been positionally data suggest that regulation of expression of the mouse and cloned and identified (Zhang et al. 1998). human cripto genes may not be entirely similar. Multiple copies of cripto-specific sequences are present in the human Genomic organization of mouse and human and mouse genomes (Dono et al. 1991, Saccone et al. 1995, cripto Liguori et al. 1996, 1997, Scognamiglio et al. 1999). At least CR-1 maps centromerically to a region on chromosome five other human CR-1–related pseudogenes and two mouse 3p21.3 that is adjacent to or possibly part of a region which Cr-1 pseudogenes have been identified (Scognamiglio et al. 1999). The CR-2, CR-4 and CR-5 genes are truncated at the ′ Table 1 EGF-CFC familyand their function. 5 -end and have accumulated point mutations, deletions and insertions (Scognamiglio et al. 1999). These genes map to EGF-CFC proteins Function chromosomes 2q37, 6p25 and 3q22 respectively while CR-6 Human maps to 19q13.1. The CR-3 pseudogene which maps to the Cripto-1 (CR-1) Gastrulation/mammarymorphogen Xq21–q22 region and the mouse Cr-2 pseudogene are Cryptin ? intronless genes that have many characteristics of a Mouse retroposon but nevertheless have the potential to code for cripto-1 (Cr-1) Primitive streak A/P axial formation and positioning functional proteins that differ from the proteins encoded by Mesoderm and endoderm formation either CR-1 or Cr-1 by only five amino acids. Cardiomyocyte development CR-1 and Cr-1 encode major mRNA species of cryptic L/R symmetry approximately 2.2 kb. In some cases, less abundant transcripts Cardiac defects/right isomerization of about 1.7, 3.0, 3.2 and 3.5 kb have been detected in Postnatal lethality midgestation mouse embryos and in primary and metastatic Xenopus FRL-1 Mesoderm formation human colon and hepatic carcinomas, suggesting that they may Neuroectoderm formation arise by the use of different polyadenylation sequences, by Zebrafish alternative splicing or by the use of an alternative initiation site one-eyed pinhead A/P axial formation and positioning for transcription (Ciccodicola et al. 1989, Dono et al. 1993, (oep) mesoderm and endoderm formation Johnson et al. 1994, Baldassarre et al. 2000). In this regard, a Ventral neuroectoderm truncated CR-1 protein of 145 amino acids may be expressed L/R symmetry from the 1.7 kb mRNA transcript in metastatic human

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Figure 1 (A) Schematic diagram of the human CR-1 gene and mRNA. Hatched areas represent the six exons, with the red exon encoding EGF-like motif. Numbers represent the number of bases in each region (obtained from Baldassarre et al. 2000). (B) Amino acid sequence alignment of cripto-related proteins, zebrafish oep, mouse cripto, mouse cryptic and Xenopus FRL-1. Green and yellow bars indicate amino acid identity among 4/4 and 3/4 proteins respectively. Blue and red underline represent EGF-like motif and cysteine-rich domains respectively (data obtained from Zhang et al. 1998 and reproduced with permission of the author). (C) Sequence alignment of EGF-like motifs. Green and red shaded areas represent amino acid identity in three or more proteins and in six proteins respectively. Conservation of six cysteines noted in yellow with disulfide bonds between cysteines (Cys) 1 and 3, 2 and 4, and 5 and 6 constitute loops A, B and C respectively. Note absence of residues between Cys 1 and 2 in cripto-related proteins compared with mouse EGF. colorectal carcinomas and in hepatic colon metastases due to pancreas, heart, stomach, small intestine, mammary gland, the use of a second CUG initiation codon (at leucine 44 in the skeletal muscle and liver. Likewise, mouse cryptic contains coding sequence) that eliminates the use of the first two exons two in-frame potential translation initiation AUG start sites in and thereby deletes 43 amino acids from the N-terminus of the which the second AUG start site would code for a protein that protein. In the mouse, low levels of Cr-1 mRNA expression is truncated at the NH2-terminus by 13 amino acids (Shen et al. can be detected by RNAse protection assays in the adult 1997). spleen, heart, lung and in distinct regions of the brain (Dono et al. 1993, Johnson et al. 1994). Adult tissues in the human that Physiochemical properties of the EGF-CFC express low levels of mRNA transcripts for the 188 amino acid proteins isoform of CR-1 as detected by RT-PCR include lung, kidney, brain, testis, ovary and spleen (Baldassarre et al. 2000). The EGF-CFC family of proteins has a general profile

Tissues expressing only the truncated form of CR-1 include consisting of a potential NH2-terminal signal peptide, a www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021201 05:47:06AM via free access Salomon et al.: The EGF-CFC family modified epidermal growth factor (EGF)-like motif, a transforming growth factor α (TGFα), amphiregulin (AR), conserved cysteine-rich motif (CFC region) and a short heparin-binding EGF-like growth factor (HB-EGF), hydrophobic COOH-terminus which in some cases contains betacellulin (BTC), epiregulin (EPI), tomoregulin (TR) and additional consensus sequences for potential glycosyl- three distinct genes in the neuregulin (NRG) family each phosphatidylinositol (GPI) cleavage and attachment (Fig. 1 coding for at least 15 distinct isoforms of α, β or γ heregulin and Table 2) (Ciccodicola et al. 1989, Dono et al. 1993, (HRG) (Uchida et al. 1999, Klapper et al. 2000). The Kinoshita et al. 1995, Shen et al. 1997, Zhang et al. 1998, solution structure as determined by nuclear magnetic Gritsman et al. 1999). An overall sequence identity of 22– resonance (NMR) of the EGF-like domains from these 32% exists between mouse and human cripto, mouse cryptic, EGF-related peptides is very similar since the B loop human criptin, Xenopus FRL-1 and zebrafish oep. Within the contains two anti-parallel β-sheets and a β-turn with a hinge EGF-like domain of the EGF-CFC proteins, there is nearly a region separating this region from the C loop which contains 60–70% sequence similarity, while in the CFC region the an additional smaller anti-parallel β-sheet and a β-turn. All similarity ranges from 35 to 48%. The variant EGF-like motif these peptides bind to and function through homo- and/or is a region of approximately 40 amino acids containing six heterodimers of the erb B type 1 tyrosine kinase family of cysteine residues that can form three intramolecular disulfide cell surface receptors which includes the epidermal growth bonds (Fig. 1). This region has the thermodynamic potential factor receptor (EGFR/c-erb B), c-erb B-2/HER-2, c-erb to refold into a tertiary structure. While in the canonical EGF B-3/HER-3 and c-erb B-4/HER-4 (Klapper et al. 2000). motif, three loops (A, B and C) can be recognized, the variant Conserved amino acids within the A loop of these peptides EGF-like motif in the EGF-CFC proteins lacks the A loop, are essential for erb B receptor binding. Since the EGF-CFC possesses a truncated B loop and has a complete C loop peptides lack the A loop, this suggests that these proteins which differentiates the EGF-CFC proteins from other probably do not bind directly to any of the known erb members in the EGF superfamily of peptides (Fig. 2). B-related tyrosine kinase receptors. Molecular modeling of Members of the EGF superfamily of peptides include EGF, this modified EGF-like motif in the EGF-CFC proteins has

Table 2 Physiochemical properties of EGF-CFC proteins. Cripto (CR) cripto (Cr) Cryptic FRL-1oep Amino acids 188 171 202 190 183

Mr 21 169 18 753 21 792 21 462 20 404 pl 8.5 8.2 8.6 8.97 8.5 GPI 150–152 133–135 173–175 — 161–163 DGL CDG SGA SGS 169–171 139–141 PSA DQD Glycosylation N-(Asn) 79–82 63–66 65–68 54–57 — NRTC NKTC NSSR NATH O-(T/S) S40,S161 T151,T155,T7 S64 — S169 T156 S51 Phosphorylation PKC 64–66 — 11–13 13–15 36–38 169–171 66–68 64–66 103–105 118–120 76–78 163–165 143–145 103–105 PKA 126–129 110–113 — — — CK2 — 16–19 38–41 — 23–26 34–37 57–60 81–84 87–90 163–166 Myristylation 26–31 55–60 31–36 28–33 24–29 71–76 76–81 174–179 72–77 93–98 92–97 120–125 118–123 128–133 136–141 128–133 137–142 179–184 137–142 162–167 Amino acids are represented byletters.

202 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:47:06AM via free access Endocrine-Related Cancer (2000) 7 199–226 shown a closely packed three-disulfide stacked arrangement GPI-modification within the COOH-terminal region that is with a disulfide β-cross motif for the first and second involved in anchoring Cr-1 to the cell membrane (Minchiotti disulfides (Lohmeyer et al. 1997). The EGF-motif contains et al. 2000), and that removing this COOH-terminal stretch two β-hairpins of six residues in the NH2- and of residues generates Cr-1 forms that are soluble. On COOH-terminus respectively. In general, the EGF-like motif SDS-PAGE under reducing conditions, the human CR-1∆C in the EGF-CFC proteins exhibits less homology to the protein migrated as two bands with molecular masses of ȁ28

EGFR ligands and appears to be somewhat more related in kDa. NH2-terminal sequencing revealed that CR-1∆C starts structure to the EGF motif in the HRGs that bind to erb B-3 with Leu-31, consistent with predictions for processing of the and/or erb B-4. signal peptide using the SIGNALP program (SWISS PROT EGF-CFC proteins range from 171 to 202 amino acids DATA BASE). Biochemical characterization by peptide and have a slightly basic pI (Table 2). The unmodified core mapping, mass spectrometry and glycosidase treatment proteins within this family range from 18 to 21 kDa. Native identified Asn-79 as being an N-linked glycosylation site mouse and human cripto proteins are 24, 28 and 36 kDa in with >90% occupancy, and Ser-40 and Ser-161 as being size but additional proteins ranging from 14 to 60 kDa have O-linked glycosylation sites with 80% and 40% occupancy also been identified by electrophoretic mobility (Brandt et al. respectively. In addition, we found that Thr-88 is modified 1994, Kenney et al. 1996, Seno et al. 1998, Niemeyer et al. with a single O-linked fucose (Foley S, Williams K & 1998, 1999, Minchiotti et al. 2000). The cryptic protein Sanicola M, unpublished data). The O-linked fucose exists as 20, 21.8 and 23 kDa isoforms while 18 and 20 kDa modification is rare and has been shown to occur exclusively forms of the oep protein have been found (Shen et al. 1997, within EGF-like modules (Harris & Spellman 1993), and a Zhang et al. 1998, Gritsman et al. 1999). The variation in consensus site has been identified (Harris et al. 1993) as size of these proteins as calculated on the basis of C2XXGGS/TC3 where the site is located between the second electrophoretic mobility may be due to the removal of the and third conserved cysteines of the EGF module. This hydrophobic signal peptide and to additional consensus site is present in all EGF-CFC proteins, but not in posttranslational modifications of the core protein. In this the soluble EGF/erb B-ligands such as EGF, HRG-α or respect, with the exception of oep all the other members of TGFα that bind to members of the erb B-related type I the EGF-CFC family are glycoproteins that contain a single growth factor receptor family (Fig. 4). There is growing N-glycosylation site and potential O-glycosylation sites. evidence that the presence of fucose groups within Human cripto-1 was initially expressed in CHO cells as the functionally important EGF modules is unique to these full-length protein (comprising residues 1–188 of the cDNA domains and is a biologically significant feature. EGF-like sequence (Fig. 1) (Brandt et al. 1994)). Treatment of CR-1– modules are usually found on secreted proteins involved in expressing CHO cells with tunicamycin or treatment of cell blood coagulation or clot dissolution such as blood lysates or conditioned medium with N-glycanase resulted in coagulation factor VII (Kao et al. 1999) and urokinase a shift in the size of the 28 kDa recombinant CR-1 protein plasminogen activator (uPA) (Rabbani et al. 1992). to a protein of approximately 21 kDa, the expected size of O-Fucosylation of uPA within its EGF-module does not the core protein (Brandt et al. 1994). A similar effect of affect the affinity of uPA for binding to its receptor but is N-glycanase was observed with the soluble Cr-1 protein critical for signaling (Rabbani et al. 1992). Recently, this (Minchiotti et al. 2000). Although CR-1 was initially O-linked fucose modification was shown to be present on the expressed in CHO cells as the full-length protein (comprising EGF-modules of mammalian Notch 1, and this protein was residues 1–188 of the cDNA sequence) (Fig. 1), we found identified as the first membrane-associated protein with an that the majority of this protein was retained in the cell as an O-linked fucose (Moloney et al. 2000). Indeed, in one case insoluble form and was subsequently difficult to purify. of the human disease CADASIL, a mutation in Notch 3 Studies on the zebrafish CR-1 ortholog, one-eyed pinhead disrupts the O-linked fucosylation site, which may influence (oep) indicated that the COOH-terminal region may contain receptor-ligand interactions (Joutel et al. 1996). Although a putative GPI-anchorage site (Zhang et al. 1998, Gritsman EGF-CFC family members and the EGF ligands both belong et al. 1999) and removal of the COOH-terminal stretch to the EGF superfamily, the presence of a consensus generated a soluble form of oep (Gritsman et al. 1999). fucosylation site within all EGF-CFC family members and Consequently, we transiently expressed in CHO cells a the identification of a fucose modification on CR-1 suggests COOH-terminally truncated form of CR-1 (CR-1C) (Fig. 3) that the EGF domain in CR-1 more closely resembles the comprising residues 1–169 of the protein sequence. The EGF module subfamily and is a biologically important human CR-1∆C protein was efficiently secreted into the modification that is functionally distinct from the supernatant and purified from the conditioned media by EGF-ligands that bind to the type 1 growth factor receptors. chromatography on SP Sepharose, Superose 12 gel- Assessment of the predicted protein products using the filtration and heparin-Sepharose. Subsequently, the recent PROSITE data base of protein motifs suggests that all of the characterization of mouse Cr-1 confirmed the presence of a EGF-CFC proteins also contain several potential www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021203 05:47:06AM via free access Salomon et al.: The EGF-CFC family

Figure 2 Ribbon schematic of the predicted and known NMR solution structures of human CR-1 (CRIPTO) and human EGF. Cysteine intramolecular disulfide bridges (yellow) contain three loops: A, red; B, pink; and C, green.

Figure 3 Sequence of recombinant human CR-1 expressed in CHO cells. Human CR-1 (comprising residues 1–169) was expressed in CHO cells. Cysteines are shown in bold. The fucosylation consensus site is underlined. Domains are color coded to sequences.

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Figure 4 Sequence comparisons of EGF-domain containing proteins. Hu, human; Mu, murine. myristylation sites which may contribute to their membrane antagonists (Table 3) (Kimelman et al. 1992, Beddington & localization (Brandt et al. 1994, Minchiotti et al. 2000). Smith 1993, Beddington & Robertson 1999, Vogel & Gerster Native or recombinant mouse Cr-1 is a membrane-bound 1999, Fraser & Harland 2000, Thisse et al. 2000). Members protein anchored by a GPI-linkage to the lipid bilayer of the TGFβ (nodal, activin and bone morphogenic proteins (Minchiotti et al. 2000). CR-1, Cr-1, cryptic and oep but not (BMPs)), Wnt, Hedgehog (Sonic Hedgehog (Shh)) and FGF FRL-1, also contain potential GPI sites for cleavage and (fgf-4 and fgf-8) families are important in controlling attachment that are present in the COOH-terminus. In this positional information, differentiation, patterning and cell regard, COOH-truncation by mutagenesis of Cr-1 by 16 movements during early embryonic development in amino acids or of oep by 15 amino acids results in proteins vertebrates (Kimelman et al. 1992, Beddington & Smith that are secreted and, as shown by oep, still retain their 1993, Cornell & Kimelman 1994, Ungar et al. 1995, biological activity (Zhang et al. 1998, Gritsman et al. 1999, Furthauer et al. 1997, Isaacs 1997, McGrew et al. 1997, Minchiotti et al. 2000). In addition, Cr-1 protein can also be Rossant et al. 1997, Burdsal et al. 1998, Dale & Jones 1999, released by treatment of cells with phosphatidylinositol- Kofron et al. 1999, McDowell & Gurdon 1999, Miller et al. specific phospholipase C (PI-PLC) suggesting that the 1999, Vogel & Gerster 1999, Wicking et al. 1999, Agius et EGF-CFC proteins may function both in a cell-autonomous al. 2000, Christian 2000, Gritsman et al. 2000, Nishita et al. fashion as GPI-linked, juxtacrine factors and, under certain 2000, Schier & Shen 2000, Thisse et al. 2000). Antagonistic conditions in which PI-PLC might be activated as soluble, binding proteins such as follistatin, noggin, chordin, antivin autocrine or paracrine factors in a non-autonomous manner and cerberus for specific members within the TGFβ family (Gritsman et al. 1999, Minchiotti et al. 2000). Finally, all of and antagonists for the Wnt family such as cerberus, the EGF-CFC proteins have the potential to be modified at frizzled-related protein (FRP) and dickkopf-1 (dkk-1) are also specific serine and threonine residues by phosphorylation expressed during early embryogenesis (Piccolo et al. 1996, through the action of either protein kinase (PK) C, PKA or 1999, Moon et al. 1997, Capdevila & Belmonte 1999, Meno casein kinase-2 (CK2). et al. 1999, Smith 1999, Thisse & Thisse 1999, Belo et al. 2000, Hashimoto et al. 2000). These antagonists are essential EGF-CFC genes in embryonic in the generation of morphogenic gradients which are instrumental in controlling different spatial and temporal development aspects of various developmental processes. Finally, Vertebrate embryonic development is specified after transcription factors such as tcf-4, Smad-2 and T-box genes fertilization by both maternal and zygotic genes. This results (e.g. Brachyury and Eomesodermin) are either part of a in an orchestrated set of morphogenic movements and regulatory pathway engaged by these growth factors/ differentiation to generate specific sets of cell lineages morphogens (e.g. tcf-4/wnt-1–β-catenin signaling pathway; (Beddington & Robertson 1999, Fraser & Harland 2000). Smad-2/activin and nodal signaling pathway) or their Inductive interactions between cells of different lineages are expression is regulated by these factors (e.g. Brachyury/ equally important for the generation of tissues and are activin and fgf-8) (Smith et al. 1991, Tadano et al. 1993, mediated by soluble or cell-associated growth factors and Yasuo & Satoh 1993, Herrmann & Kispert 1994, morphogens and intracellular (e.g. sprouty) or extracellular Schulte-Merker et al. 1994, Haegel et al. 1995, Huber et al. www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021205 05:47:06AM via free access Salomon et al.: The EGF-CFC family

Table 3 Factors regulating early vertebrate gastrulation and left-right asymmetry. Growth factors and Function morphogens Activins (A and B) Dorsalization of mesoderm, L/R symmetry BMP-4 Ventralization of mesoderm, L/R symmetry Nodal (Squint and Cyclops) Anterior/posterior patterning and axis positioning Mesoderm and endoderm formation, L/R symmetry Fgf-3 Ventralization of mesoderm, EMT and mesoderm Fgf-4 migration, L/R symmetry Fgf-8 Modifiers EGF–CFC peptides Required for nodal signaling, mesoderm migration wnt-1 Cooperate with activins for dorsalization of mesoderm and with FGF for ventralization of wnt-3 mesoderm wnt-8 Sonic Hedgehog (Shh) Induces wnts and nodal, L/R symmetry Co-stimulators DallyTGF β and wnts GPI-linked co-receptors (Glypicans) HSPGs (Syndecans) Antagonists Noggin BMP and activin Chordin Dorsalization of mesoderm Follistatin Antivin (Lefty-1 and -2) Nodal Cerberus (Caronte, Dan, Nodal, BMP and wnts Gremlin) FRPs wnts dkk-1 SproutyFgfs Transcriptional effectors Lef/tcf-4 Induces nodal, c-myc, cyclin-D1 Smad-2/Smad-4 Mesoderm formation, L/R symmetry T-box genes (Brachyury, Posterior mesoderm formation and migration, induces activin and cadherins Eomesodermin and Tbx6) Cubitus interruptus (Ci) Shh signalling

1996, Knezevic et al. 1997, Latinkic et al. 1997, Smith 1997, (D/V) axis are established, the left/right (L/R) axis is formed Miller et al. 1999, Arnold et al. 2000, Nishita et al. 2000, and this eventually leads to the asymmetric development of Russ et al. 2000, Schier & Shen 2000). A number of these different tissues and organ systems. These complex events proteins that are important for early development can also are regulated by Spemann’s organizer (e.g. node/mouse) contribute to the pathogenesis of cancer in different adult which temporally expresses different morphogens that can tissues by their inappropriate overexpression (e.g. wnt-1, initiate regional differentiation, cell movements and axial fgf-8, tcf-4) or by genetic mutation or loss of expression (e.g. positioning (Camus & Tam 1999, Kodjabachian et al. 1999). Smad-2 and β-catenin) (Miller et al. 1999, Wicking et al. The first inductive event is the formation of the mesoderm 1999, Massague & Chen 2000). from adjacent ectoderm along the primitive streak. As posterior ectodermal epiblast cells converge towards the Gastrulation and germ layer formation primitive streak, they ingress and migrate between the ectoderm and endoderm. Presumptive mesodermal cells Gastrulation establishes the body plan of the embryo through migrate through and away from the primitive streak and the formation of the ectoderm, mesoderm and endoderm undergo an epithelial to mesenchymal transition (EMT) (Hay germ layers (Beddington & Robertson 1999, Camus & Tam 1995, Viebahn 1995, Camus & Tam 1999). During EMT, 1999, Kodjabachian et al. 1999, Fraser & Harland 2000). cell-cell contacts are disrupted with a loss of expression of Spatial positioning of the germ layers results in the the adherens junction protein, E-cadherin (Hay 1995, establishment of polarity along the anterior/posterior (A/P) Guarino et al. 1999). Concomitantly, cytokeratin expression axis of the embryo. After the A/P axis and the dorsal/ventral is reduced and the expression of intermediate filament

206 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:47:06AM via free access Endocrine-Related Cancer (2000) 7 199–226 proteins such as vimentin is up-regulated (Thiery & Chopin essential component for sqt and cyc signaling and functions 1999). These events are quite analogous to events which in a cell-autonomous role as a possible co-receptor or occur during the invasive stages of tumor cell metastasis competence factor for nodal-related proteins but not for where tumor epithelial cells become motile and assume more activin (Gritsman et al. 1999, Schier & Shen 2000). mesenchymal characteristics (Guarino et al. 1999, Thiery & Nodal-related proteins and oep subsequently depend upon Chopin 1999). and/or signal through the downstream serine/threonine kinase Differentiation of posterior epiblast cells into mesoderm activin type IIB and type IB receptors that engage cells and their migration away from the primitive streak are intracellular Smad-2 and Smad-4 phosphorylation and controlled by an integrative and synergistic effect of transcriptional activation (Peyrieras et al. 1998, Schier & overlapping extracellular signals such as activin, BMP-4, Shen 2000). Antivin, a lefty-related factor, and cerberus are nodal, wnt-8 and fgf-8, in conjunction with the appropriate competitive antagonists of activin and nodal for binding to spatial expression of receptors and intracellular signaling the type IIB activin receptor in this pathway (Thisse et al. proteins for these growth factors and morphogens (Table 3) 2000). Mutations in oep result in cyclopia, absence of head (Kimelman et al. 1992, Beddington & Smith 1993, Cornell & and trunk mesoderm, loss of prechordal plate and ventral Kimelman 1994, Ungar et al. 1995, Furthauer et al. 1997, neuroectoderm, impairment of gastrulation movements and Isaacs 1997, McGrew et al. 1997, Rossant et al. 1997, loss of A/P axis patterning and positioning (Zhang et al. Dale & Jones 1999, Kofron et al. 1999, McDowell & Gurdon 1998, Gritsman et al. 1999). Rescue of the oep mutant 1999, Vogel & Gerster 1999, Schier & Shen 2000). phenotype can be achieved by expression of full-length or Disruption of any of these signaling pathways can impair secreted COOH-terminal truncated oep protein suggesting gastrulation and is therefore embryonically lethal (Deng et that oep can function under certain conditions as a paracrine al. 1994, Yamaguchi et al. 1994, Ciruna et al. 1997, Gu et effector. Ectopic expression of Xenopus FRL-1 or mouse al. 1998, 1999, Sirard et al. 1998, Wacker et al. 1998, Cr-1 or overexpression of activin or activation of Waldrip et al. 1998, Heyer et al. 1999, Song et al. 1999, Sun downstream components in an activin-like signaling pathway et al. 1999). In general, nodal, activin and wnt-1 are involved such as the activin type IB receptor or Smad-2 can also in initiating overall mesoderm and endoderm formation and rescue the mutation (Gritsman et al. 1999). However, rescue in the development of dorsal and anterior mesoderm and of the mutant phenotype cannot be achieved with BMP-2 or head structures whereas BMP-4, wnt-8 and fgf-8 are more -4, activated ras, mitogen-activated protein kinase (MAPK) involved in controlling ventral and posterior mesoderm, tail or with different members in the wnt/β-catenin signaling and trunk development (Kimelman et al. 1992, Beddington & pathway (Gritsman et al. 1999, Schier & Shen 2000). Smith 1993, Furthauer et al. 1997, Harland & Gerhart 1997, In Xenopus, there is some indication that FRL-1 function McGrew et al. 1997, Beddington & Robertson 1999, during development may depend upon a functional FGFR Camus & Tam 1999, Kodjabachian et al. 1999, Thisse et signaling pathway since this gene was initially identified al. 2000). Fgf-8, FGF receptor-1 (FGFR-1) and Brachyury T using a functional screen in yeast to detect novel ligands of expression which is regulated by both activin and fgf-8 are the FGFR-1 (Kinoshita et al. 1995). In Xenopus oocytes and important for initiating and maintaining the migration of in yeast (Saccharomyces cerevisiae), simultaneous ectopic mesodermal cells through the primitive streak (Rossant et al. overexpression of FRL-1 and the Xenopus FGFR-1 kinase 1997, Conlon & Smith 1999, Sun et al. 1999). Brachyury or leads to enhanced Ca2+ influx and to an increase in the another T-related gene such as eomesodermin or Tbx6 are indirect transphosphorylation of tyrosine on and also involved in the regulation of E-cadherin or transactivation of the FGFR-1. However, no direct binding of protocadherin expression which, in turn, are necessary for FRL-1 to the Xenopus FGFR-1 could be detected. In isolated gastrulation (Burdsal et al. 1993, Huelsken et al. 2000, Russ animal caps from Xenopus embryos, FRL-1 similar to et al. 2000). FGF-2, activin, Xenopus-nodal (Xnr) or the Xwnt-8 induces In zebrafish, two mouse nodal-related genes squint (sqt) the formation of mesoderm and the expression of and cyclops (cyc) are essential for mesoderm and endoderm mesoderm-related genes. In the case of FRL-1, this occurs in formation and for A/P axis development (Feldman et al. a paracrine, non-cell-autonomous manner through an 1998, Rebagliati et al. 1998, Sampath et al. 1998, Gritsman FGFR-1-mediated pathway. Overexpression of FRL-1 in et al. 2000, Schier & Shen 2000). Expression of zygotic Xenopus embryos leads to the preferential development genes such as nodal/sqt and cyc and fgf-8 are, in turn, of posterior mesoderm-related structures at the expense of regulated by a maternally-derived T-box gene (e.g. VegT) anterior head structures. FRL-1 expression in Xenopus is (Kofron et al. 1999, Agius et al. 2000, Schier & Shen 2000). detected during gastrulation and during the early stages Similar to sqt and cyc, zebrafish oep is also necessary for of neurulation. initiating mesoderm, endoderm and A/P axis formation During early mouse embryogenesis, Cr-1 expression can (Schier et al. 1997, Zhang et al. 1998, Gritsman et al. 1999, first be detected by RT-PCR in the inner cell mass (ICM) of 2000, Schier & Shen 2000). In this context, oep is an the blastocyst at day 4 of development, which seems logical www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021207 05:47:06AM via free access Salomon et al.: The EGF-CFC family since CR-1 and Cr-1 were first identified in undifferentiated the same signaling molecules are utilized in multiple human NTERA2/D1 and mouse F9 embryonal carcinoma developmental pathways (Meno et al. 1997, 1998, Oh & Li cells respectively (Ciccodicola et al. 1989, Dono et al. 1993, 1997, Varlet & Robertson 1997, Harvey 1998, Bisgrove et Johnson et al. 1994, Ding et al. 1998, Xu et al. 1999). al. 1999, Boettger et al. 1999, Gaio et al. 1999, Mercola Expression of both genes is lost in NTERA2/D1 and F9 cells 1999, Meyers & Martin 1999, Tsang et al. 1999, Capdevila following retinoic acid-induced differentiation (Ciccodicola et al. 2000, Saijoh et al. 2000, Schier & Shen 2000). In this et al. 1989, Dono et al. 1993, Minchiotti et al. 2000). This respect, members of the nodal family and lefty-2 exhibit expression pattern is clearly distinct from that of cryptic laterality since they are always expressed in the left but not which is only found in differentiated mesoderm cells the right lateral plate mesoderm (Meno et al. 1997, 1998, following retinoic acid treatment and not in embryonal Bisgrove et al. 1999). The asymmetric expression of these carcinoma or ICM stem cells (Shen et al. 1997). Cr-1 mRNA genes ultimately controls left/right asymmetry with respect to expression in the embryo is found in the embryonic ectoderm the later development of organs such as the heart and spleen following implantation of the blastocyst. Expression (Mercola 1999, Capdevila et al. 2000). In the mouse, fgf-8 increases on day 6.5 of gestation which is the point where induces left side expression of nodal which, in turn, functions gastrulation begins with the formation of the primitive streak through an activin type IIB receptor to induce lefty-2 (Dono et al. 1993, Johnson et al. 1994, Ding et al. 1998, Xu expression (Oh & Li 1997, Varlet & Robertson 1997, Harvey et al. 1999). Expression then decreases dramatically by day 1998, Boettger et al. 1999, Meyers & Martin 1999, Tsang et 7. By in situ hybridization, expression of Cr-1 mRNA in the al. 1999, Saijoh et al. 2000). Nodal signaling in zebrafish midgestation mouse embryo is temporally and spatially and mouse during L/R asymmetry development also requires restricted to the epiblast cells of the primitive streak, to the functional oep and cryptic genes (Gaio et al. 1999, Yan et developing mesoderm during gastrulation and in later fetal al. 1999, Saijoh et al. 2000, Schier & Shen 2000). Mutation stages to the myocardium of the truncus arterious of the and partial rescue of oep in the zebrafish or targeted developing heart. In the developing heart, Cr-1 mRNA can disruption of cryptic in the mouse results in laterality be detected in the epimyocardium but not in the defects including randomization of cardiac looping endocardium. With the exception of the developing heart, and atrial-ventricular septal defects, pulmonary right little if any expression of Cr-1 mRNA can be detected in the isomerization, inverted situs of the spleen, pancreas and remainder of the embryo after day 8. Expression of Cr-1 stomach, hyposplenia and heterotaxia (Gaio et al. 1999, Yan mRNA in adult tissues is generally several-fold less than in et al. 1999, Saijoh et al. 2000, Schier & Shen 2000). undifferentiated F-9 mouse embryonal carcinoma cells Germline deletion of cryptic eventually leads to postnatal (Dono et al. 1993, Minchiotti et al. 2000). Cr-1 protein death at approximately 2 weeks because of severe cardiac expression in the developing embryo was also assessed by malfunction (Gaio et al. 1999, Yan et al. 1999). immunocytochemistry using a rabbit antibody generated Mouse Cr-1 may also perform an essential role during against the full-length mouse Cr-1 protein (Minchiotti et al. earlier stages of cardiac lineage specification and 2000). Cr-1 mRNA and immunoreactive protein were found differentiation. Homologous recombination that leads to to co-localize in the developing mesoderm of the 6.5 day homozygous knockout of the Cr-1 gene (Cr-1−/−)in embryo while at day 8.5 Cr-1 protein expression was pluripotential embryonic stem cells (ES cells) impairs their detected in the myocardium of the developing heart ability spontaneously to differentiate in vitro into (Minchiotti et al. 2000). The expression pattern for cryptic is distinct from the cardiomyocytes without affecting the ability of ES cells to expression pattern for Cr-1 since it is not expressed in adult differentiate into other cell types (Xu et al. 1998). mouse tissues and has a partially overlapping yet distinct Interestingly, a similar embryonically lethal phenotype due expression pattern to Cr-1 in the midgestation mouse embryo to myocardial and endocardial defects is found in day 10 (Shen et al. 1997, Yan et al. 1999). Cryptic expression can embryos from erb B-4, erb B-2 and NRG1 knockout mice first be detected in the axial mesoderm and then becomes (Klapper et al. 2000). This may have functional significance more restricted to the anterior end of the primitive streak and since the human CR-1 protein can indirectly increase the the head process. Expression is absent in the prechordal plate tyrosine transphosphorylation of erb B-4 and may depend but is present in the lateral plate mesoderm, node and floor upon erb B-4 for signaling in certain cells via plate of the neural tube. After day 8.5, little expression of heterodimerization with or indirect cross-talk to the CR-1 cryptic mRNA can be detected in the embryo. receptor (Bianco et al. 1999). Germline knockout of the mouse Cr-1 gene is embryonically lethal as embryos die in utero between 8.5 and 9.5 days of development due to their Left-right axis formation and cardiac inability to gastrulate and form appropriate germ layers as a development consequence of the complete absence of the primitive streak The nodal signaling pathway is also involved in the (Ding et al. 1998, Xu et al. 1999). Specifically, disruption of establishment of the L/R embryonic axis, demonstrating that Cr-1 in Cr-1−/− embryos results in the formation of embryos

208 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:47:06AM via free access Endocrine-Related Cancer (2000) 7 199–226 that possess a head without a trunk demonstrating that there mouse Cr-1 protein in primary FVB/N or C57Bl/6 mouse is a severe deficiency in embryonic mesoderm and endoderm mammary epithelial cells using a recombinant amphotropic without a loss of anterior neuroectoderm formation. retroviral expression vector containing the Cr-1 cDNA Therefore, Cr-1 signaling is necessary for cell movements facilitated lateral ductal branching and clonal expansion of that correctly orient the embryo along the A/P axis and for mammary ductal hyperplasias after these retrovirally cell migration of the presumptive developing mesoderm and transduced cells were reintroduced into the cleared mammary for reorientation of the endoderm (Ding et al. 1998, Xu et fat pad of syngeneic, ovariectomized virgin mice (Kenney et al. 1999, Schier & Shen 2000). Chimeric embryos that were al. 1997a). These ductal hyperplasias could be serially generated between wild-type and Cr-1−/− ES cells were transplanted in the mammary fat pad, demonstrating that they normal indicating that Cr-1 produced by the wild-type host represent an immortalized population of mammary epithelial cells could rescue the mutant phenotype in a paracrine stem cells. However, frank mammary carcinomas have not manner (Xu et al. 1998). Fibroblasts that were derived from been observed in these hyperplastic outgrowths even after Cr-1−/− embryos were found in vitro to exhibit a ȁ50% several in vivo transplantations. reduction in growth rate as compared with embryonic When inserted into the fourth inguinal mammary gland fibroblasts from wild-type embryos. In addition, Cr-1−/− of ovariectomized virgin mice, slow-release Elvax pellets fibroblasts exhibited negligible migration in a wound healing containing the p47 EGF-like motif of the CR-1 protein assay and were impaired in their ability to migrate over or produce a 20–fold increase in DNA synthesis in the adjacent chemotactically towards either fibronectin or type 1 collagen ductal epithelial cells. The same increase is seen in terminal as compared with embryonic fibroblasts from wild-type end buds (TEBs) that immediately surround these pellets embryos (Xu et al. 1999). relative to the labeling index and branching in epithelial cells that surround Elvax pellets containing only bovine serum Biological effects of cripto in albumin (Kenney et al. 1997a). In addition, a threefold differentiated mammalian cells increase in secondary and tertiary lateral ductal branching in areas surrounding these CR-1 pellets occurs in nearly 70% of the mammary outgrowths. A comparable in vitro-related In vitro effects of transduced Cr-1or effect has been observed with recombinant CR-1 or with recombinant CR-1in mammary epithelial cells cells transduced with a Cr-1 cDNA expression vector in In rat CREF embryo fibroblasts or rat FRLT-5 thyroid mouse NMuMG, TAC-2 and EpH4 mammary epithelial cells epithelial cells that have been transformed by either an that are grown in type 1 collagen gels (C Wechselberger, activated c-Ha-ras or c-Ki-ras proto-oncogene respectively, A D Ebert, C Bianco, B Wallace-Jones, N Khan, Y Sun & expression of rat Cr-1 is substantially up-regulated (Su et al. D S Salomon, unpublished data). Despite having a 1993, Mincione et al. 1998). Reversion of the transformed growth-promoting effect when overexpressed, Cr-1 also phenotype of several of the Ha-ras-transformed CREF clones reduced contact inhibition in monolayer culture. In addition, by overexpression of the Krev-1 ras suppressor gene results induction of anchorage-independent growth was achieved in in a loss of expression of rat Cr-1, suggesting that Cr-1 might the Cr-1-transfected EpH4 cell line. More importantly, all function as a proximal effector of p21ras transformation in the transfected, Cr-1 overexpressing cell lines exhibited an this system and might contribute to cellular transformation. increase in migratory potential as assessed in various assays. In fact, the first indication that cripto had any biological The cells showed increased scattering on matrigel and activity in a mammalian cell was demonstrated by the ability enhanced chemotaxis in Boyden chamber and migration in of a transduced human CR-1 or mouse Cr-1 cDNA to wound healing assays. Recombinant CR-1 is also able to transform mouse NIH/3T3 fibroblasts, mouse NOG-8 and induce the scattering of NOG-8 mouse mammary epithelial mouse CID-9 mammary epithelial cells when the genes were cells grown at low densities as colonies on plastic. This effect overexpressed in these cells in vitro (Ciardiello et al. 1991a, is probably related to an increase in cell motility that is Niemeyer et al. 1998). Cripto-transduced mammary induced by this peptide either through chemokinesis or epithelial cells were able to grow in soft agar. In monolayer through chemotaxis. When cultured in a three-dimensional culture, the cripto-transduced cells exhibited an enhanced type-1 collagen gel matrix, the Cr-1–transduced cells formed proliferation rate, clonogenic potential and a loss of contact branching tubules. growth inhibition. However, NOG-8 or CID-9 transformants The ability of Cr-1 to stimulate both scattering and were unable to form tumors in nude mice, suggesting that branching morphogenesis in collagen gels is reminiscent of additional genetic alterations were necessary to complete the the effects induced by hepatocyte growth factor (HGF) or tumorigenic phenotype in vivo (Ciardiello et al. 1991a, TGFβ1 in mammary epithelial cells (Santos & Nigam 1993, Niemeyer et al. 1998). Reciprocally, knockout of the Cr-1 Montesano et al. 1997). The scattering effect is preceded by gene in CID-9 cells reduced growth and increased apoptosis a change in morphology of these epithelial cells to a more in these cells in vitro and in vivo. Overexpression of the fibroblastic-like phenotype. A similar effect on motility and www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021209 05:47:06AM via free access Salomon et al.: The EGF-CFC family

EMT can be produced in these cells by HGF but not by impair to different degrees DIP-induced expression of HRGβ1, which is only mitogenic. EMT is often characterized β-casein in primary mouse mammary explant cultures. by a decrease in cell-cell adhesion which coincides with Expression of whey acidic protein (WAP) in these primary enhanced migration. In this respect it was reported that mammary epithelial explant cultures was also induced by tyrosine phosphorylation of β-catenin can inhibit its DIP, and this response to DIP is also significantly attenuated association with E-cadherin and facilitate cell scattering and to different degrees by these various EGF-related peptides migration (Shibamoto et al. 1994). Cr-1 overexpressing cell including CR-1. In contrast to growth factors that activate lines exhibit an enhanced basal level of tyrosine the EGFR or the CR-1 receptor, HRGβ1 stimulates the phosphorylation of β-catenin, resulting in a decreased expression of β-casein and WAP in these DIP-treated association of β-catenin to E-cadherin. This effect could only midgestation mammary explant cultures. α-Lactalbumin be observed in the presence of the tyrosine phosphatase expression following DIP treatment was generally less inhibitor, sodium orthovanadate, which indicates a very sensitive to the modulatory effects of these peptides. The stringent regulation of this process in mammary epithelial differential regulatory effects of these structurally similar cells. peptides may be related to the normal temporal sequence of HC-11 mouse mammary epithelial cells express the milk lactogenic hormone-induced activation of early (β-casein) protein β-casein at confluency after exposure to the and late (WAP and α-lactalbumin) milk protein genes that lactogenic hormone mixture of dexamethasone, insulin and occurs during pregnancy in mammary alveolar epithelial prolactin (DIP) (Ball et al. 1988, Hynes et al. 1990). Similar cells and/or to the combination or sequence of activation of to other EGF-related peptides that bind to the EGF receptor different members of the erb B type 1 receptor tyrosine (EGFR) or peptides that can bind to either c-erb B-3 or c-erb kinase family expressed in these cells. B-4, CR-1 can function as a competence factor and can prime The inhibitory effect of exogenous CR-1 on β-casein and HC-11 cells that have been pretreated during logarithmic WAP expression may be biologically significant since growth to respond optimally to subsequent treatment with endogenous Cr-1 expression is up-regulated in alveolar cells DIP for the induction of expression of β-casein (De Santis et during pregnancy and lactation and therefore may function al. 1997). In contrast, the continuous presence of EGF, as a negative modulator to initiate a reduction in milk protein TGFα,orHRGβ1 can actually inhibit DIP-mediated expression as the mammary gland enters involution when the transcription of the β-casein gene presumably due to the secretory epithelial cells begin to undergo apoptosis (Kenney inactivation of the transcription factor Stat-5, which is et al. 1995, Herrington et al. 1997). This may be the case activated by prolactin receptor engagement (Hynes et al. since we have found recently that CR-1 can induce apoptosis 1990, Taverna et al. 1991, Yang et al. 1994, Marte et al. in several nontransformed mouse mammary epithelial cell 1995). Similar to EGF, the simultaneous presence of CR-1 lines. Apoptosis could be observed after 2 days exposure of with DIP was found to effectively block DIP induction of confluent HC-11 cells to CR-1 when cells were deprived of β-casein protein expression in a dose-dependent fashion (De the survival factors, EGF and insulin (De Santis et al. 2000). Santis et al. 1997). In addition, HC-11 cells which are Apoptosis was mediated through the induction of a infected with a recombinant amphotropic retroviral caspase-3-like protease and down-regulation in the expression vector containing the mouse Cr-1 cDNA and expression of the anti-apoptotic protein Bcl-xL.The induction which continuously overexpress the Cr-1 protein also exhibit of apoptosis under reduced serum by CR-1 was preceded by low levels of β-casein protein expression following DIP an inhibition in cell growth and a reduction in treatment. The inhibitory effect of Cr-1 is not unique to poly(ADP-ribose) polymerase (PARP) protein and an HC-11 cells since a similar inhibitory effect on β-casein increase in β-catenin cleavage. A reduction in β-catenin expression in the presence of DIP was also observed in levels due to caspase-3-mediated degradation may result in a CID-9 cells that are overexpressing the Cr-1 gene (Niemeyer loss of cell-cell and cell-matrix interactions with an increase et al. 1998). in anoikis (De Santis et al. 2000). Cr-1 has also been To ascertain if CR-1 could alter the expression of demonstrated to act as a survival factor in CID-9 cells additional milk proteins in response to lactogenic hormones, transfected with a Cr-1 expression vector when cells were mammary explant cultures from midpregnant mice were propagated under sparse, subconfluent conditions in medium maintained in serum-free medium with various peptides in containing low serum and insulin (Niemeyer et al. 1998). the absence or presence of DIP (De Santis et al. 1997). There was little β-casein expression in primary explants prior to Expression of mouse Cr-1in the mammary culture or in explant cultures maintained in the absence or gland and in mammary tumors presence of different EGF-related peptides. However, inclusion of DIP alone into the medium was able fully to Expression of Cr-1 in the developing mouse mammary gland induce β-casein expression. Concurrent treatment with EGF, has been examined by RT-PCR, in situ hybridization, TGFα, HB-EGF, AR, BTC or CR-1 was able significantly to immunocytochemistry and Western blot analysis (Kenney et

210 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:47:06AM via free access Endocrine-Related Cancer (2000) 7 199–226 al. 1995, Herrington et al. 1997, Niemeyer et al. 1998). mammary gland that expresses only 18 and 26 kDa isoforms Different levels of Cr-1 expression are found in the virgin, of Cr-1, mammary tumors that arise in different trans- pregnant, lactating, involuting and aging mammary gland. In genic animals overexpress several distinct and specific the virgin prepubescent mammary gland, a 26 kDa immunoreactive isoforms of the Cr-1 protein ranging from immunoreactive Cr-1 protein and Cr-1 mRNA are observed 14 to 60 kDa (Kenney et al. 1996). These proteins may arise primarily but not exclusively in the cap stem cells of the by alternative mRNA splicing, posttranslational modi- growing terminal end buds (Kenney et al. 1995). As assessed fications (e.g. glycosylation or myristylation) or proteolytic by Western blot analysis, immunocytochemistry and in situ processing. hybridization, expression of Cr-1 in both ductal and lobuloalveolar cells is enhanced by approximately three- to Effect of CR-1on human cervical carcinoma fivefold during pregnancy and lactation and is totally lost cells in vitro during involution (Kenney et al. 1995, Herrington et al. 1997, Niemeyer et al. 1998). Enhanced expression of Cr-1 Alterations in the behavior of tumor cells such as cell may be driven by lactational hormones. In this respect CID-9 migration and invasion correlate with changes in the cells, which like HC-11 cells, were established from composition or structure of the intermediate filament network Comma-1D cells that were isolated from a mid-pregnant (Goldman et al. 1996, Hendrix et al. 1996). The intermediate Balb/c mouse express Cr-1 (Niemeyer et al. 1998). type-III filament protein, vimentin, is generally expressed in Expression of Cr-1 in these cells can be enhanced when the mesenchymal cells and is overexpressed in several different cells are stimulated to grow and differentiate in response to human carcinomas (Raymond & Leong 1989, Fuchs & DIP on a reconstituted basement membrane (Niemeyer et al. Weber 1994). Vimentin expression is associated with 1998). This may be of physiological significance since increased invasiveness and motility of primary human immunoreactive and bioactive CR-1 has recently been cervical cancer cells in vitro (Gilles et al. 1994, 1996, 1999). detected in the milk from several different human donors Caski cervical carcinoma cells which normally express only (Bianco et al. 2000). Interestingly, Cr-1 expression is low levels of vimentin were stably transfected with an elevated to an even greater extent in ductal epithelial cells expression vector containing the human CR-1 cDNA. in the aging (2–year-old) mammary gland of nulliparous or Changes in the expression pattern of several genes were multiparous Balb/c mice which exhibit spontaneous analyzed with the cDNA expression array technique (Ebert mammary tumor development (Herrington et al. 1997, et al. 2000). Genes found to be either up- or down-regulated Kenney et al. 1997b). in the CR-1 overexpressing Caski cells relative to Caski cells Cr-1 mRNA and protein are generally overexpressed in transfected with the vector alone are summarized in Table 4. a majority of mouse mammary carcinoma cells relative to Gene expression changes of more than threefold are indicated noninvolved adjacent mammary epithelium in several in bold letters. As compared with vector-transfected Caski different spontaneous and carcinogen-induced mouse cells, a threefold increase in vimentin gene expression was mammary tumors (Herrington et al. 1997). In addition, Cr-1 observed in the CR-1-transfected Caski cells as verified by overexpression has also been observed in mammary tumors Northern blot analysis. In cell lysates of CR-1–transfected and in their lung metastases arising in transgenic mice that Caski cells, a sixfold increase in vimentin protein expression overexpress different transforming oncogenes such as TGFα, was detected by Western blot analysis as compared with c-neu (the rodent homolog of erb B-2), erb B-2, int-3, vector-transfected cells which correlated with the cDNA polyoma middle T antigen or SV40 large T antigen (Kenney expression array data. These results were verified by treating et al. 1996, Niemeyer et al. 1999). In mouse mammary tumor wild-type Caski cells with exogenous, recombinant CR-1 for virus (MMTV)-polyoma middle T transgenic FVB/N mice, 24 h. At this time, a 3.5-fold upregulation of vimentin protein Cr-1 protein expression in the mammary gland could first be expression was detected by Western blot analysis. To assess detected in day 44 virgin mice where levels were seven- to the migratory potential of CR-1-overexpressing and tenfold higher than in the mammary gland from comparably CR-1-treated Caski cells, cell motility was quantified in the aged control FVB/N virgin female mice (Niemeyer et al. Boyden chamber assay. CR-1-transfected Caski cells 1999). At 44 days, this expression is detected in ductal exhibited a significant increase in their invasive capacity with hyperplasias. Tumors developing from these hyperplasias respect to migration through collagen- or gelatin-coated showed levels of Cr -1 protein 25–fold higher than in the polycarbonate membranes. mammary gland from non-transgenic mice. Enhanced CR-1 may also function as a survival factor in human expression of Cr-1 was also detected in hyperplasias from carcinoma cells. For example, transfection and over- MMTV-erb B-2 and metallothionein (MT)-TGFα transgenic expression of human CR-1 in SiHa and Caski human cervical mice suggesting that expression of Cr-1 is an early marker carcinoma cells can enhance the survival of these cells under for premalignancy in the mammary gland of these animals serum-restricted culture conditions (Ebert et al. 1999). (Niemeyer et al. 1999). Compared with the lactating Apoptosis can be initiated by exposure of these cervical www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021211 05:47:06AM via free access Salomon et al.: The EGF-CFC family

Table 4 Genes that are modulated byCripto-1 in human oep is cell-associated and acts predominantly in a cervical cancer. Genes that are highlighted in bold represent cell-autonomous manner, oep might function as a co-receptor an increase or decrease in expression byat least threefold as for an activin type IIB receptor complex (Schier et al. 1997, detected byuse of a Clontech Human Tumor Atlas Array. Zhang et al. 1998, Gritsman et al. 1999). Although activin Induced Repressed type IIB receptors are expressed in adult mammalian cells, p21CIP/WAF IGFBP-3 nodal expression has not yet been detected in a fully GADD153 IGFBP-5 differentiated mammalian cell. In addition, there is no source GADD45 p16INK for recombinant nodal protein to ascertain its potential Cyclin D2 p14 CDK inhibitor interaction with other EGF-CFC proteins in various GDN Keratin 2 Vimentin Keratin 13 mammalian cell types. Nevertheless, recent experiments with Integrin α5 Integrin β4 mouse mammary HC-11 cells have demonstrated that Integrin α6 γ-Catenin recombinant CR-1 is able to enhance the tyrosine Tenascin FGF-5 phosphorylation of the SH2–adapter protein Shc leading to Rho 8 FGF-6 the subsequent downstream activation of the ras/raf/MAPK Laminin β1 MMP-2 signaling pathway (Kannan et al. 1997). It was shown that tPA uPA exogenous CR-1 protein binds to high affinity, saturable Plasminogen binding site(s) on HC-11 cells as well as on several different VEGF human breast cancer cell lines. This interaction was highly HB-EGF specific and could not be blocked by ligands for the various Fibronectin erb B receptors such as EGF, TGFα, AR, BTC or HRGβ1. PAI-1 Exogenous CR-1 induced a rapid increase in the tyrosine PAI-2 MMP-14 phosphorylation of p66, p52, and p46 Shc in various cell lines. Furthermore, CR-1 promoted an increased association IGFBP, insulin like growth factor binding protein; VEGF, of the adapter Grb2–SOS signaling complex with tyrosine vascular endothelial growth factor; GDN glial cell-derived neurotropinin; PAI, plasminogen activator inhibitor; MMP, phosphorylated Shc in HC-11 cells. This cascade led to an metalloprotease protein. increase of MAPK activity demonstrating that CR-1 can function through a receptor which activates intracellular carcinoma cells to the phosphatidylinsitol 3–kinase (PI-3 effectors in the ras/raf/MEK/MAPK pathway. In addition to kinase) inhibitor, LY294002. This drug-induced apoptosis various isoforms of Shc, CR-1 also induced an increase in can be partially rescued by treatment of SiHa and Caski cells the tyrosine phosphorylation of other unidentified proteins at with recombinant CR-1 suggesting that the PI-3 kinase/Akt 120 kDa, 80 kDa and 60 kDa. pathway may be responsible for this survival activity. In fact, The inhibitory response of CR-1 on β-casein expression CR-1 expression and enhanced survival is correlated with the after treatment of HC-11 cells with DIP could be attenuated ability of CR-1 to induce the tyrosine phosphorylation of the by B-581 which is a specific peptidomimetic farnesyl p85 regulatory subunit of PI-3 kinase in these cells and to the transferase inhibitor that blocks p21ras farnesylation and subsequent activation of PKB/Akt as assessed by an increase activation, or by the PI-3 kinase inhibitors, wortmannin or in the phosphorylation of Akt and as measured by an increase LY-294002 but not by PD-98059, a mitogen-activated, in the phosphorylation of the Akt substrate, glycogen synthase erk-activating kinase (MEK) inhibitor that blocks MAPK kinase-3β (GSK-3β). Since phosphorylation of GSK-3β activation (De Santis et al. 1997). These data suggest that the inactivates the enzyme, which in the wnt signaling pathway ability of CR-1 to block lactogenic hormone-induced results in a reduction in β-catenin degradation, CR-1 may also expression of β-casein at least in HC-11 cells is mediated contribute to the activation of this pathway. This may be through a p21ras-dependent, PI-3 kinase-mediated pathway. significant since cyclin-D1 and c-myc are two oncogenes This is further substantiated by the observation that CR-1 is that are up-regulated by the wnt-activated β-catenin/Tcf able transiently to stimulate the tyrosine phosphorylation of transcription complex (Brabletz et al. 2000). the p85 PI-3 kinase regulatory subunit and transiently to increase the activity of PI-3 kinase in HC-11 cells. These Mechanism of action of EGF-CFC data collectively demonstrate that CR-1 can function as a proteins in mammalian cells growth factor but more importantly as a differentiation factor in mammary epithelial cells during pregnancy in the Activation of ras/raf/MAPK and PI-3 kinase/ regulation of milk protein expression. The elevated Akt/GSK-3β pathways by cripto expression of Cr-1 during pregnancy and its presence in milk In zebrafish, oep functions in a nodal signaling pathway supports a potential in vivo role for this growth factor at this during embryonic development (Gritsman et al. 1999). Since stage in mammary gland development.

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Transactivation of erb B-4 and FGFR-1by neutralizing antibody or inhibiting expression with an erb CR-1 B-4 antisense expression vector was able to inhibit MAPK activation in response to treatment with recombinant CR-1 in Since EGF-CFC proteins contain a modified EGF-like motif, MDA-MB-453 and T47–D human breast cancer cells. These it seemed possible that these peptides might bind to one or data suggest that erb B-4 although not a direct receptor for several of the erb B type 1 tyrosine kinase receptors. this growth factor is required for the full activation of MAPK However, recombinant CR-1 in radioreceptor competition in a signal transduction cascade that is engaged by CR-1 in assays with 125I-EGF in human mammary MCF-10A cells or some human breast cancer cells. A similar effect on the A431 epidermoid carcinoma cells failed to demonstrate any enhancement of tyrosine transphosphorylation occurs in binding to the EGFR on these cells (Kannan et al. 1997). response to FRL-1 for the FGFR-1 receptor in Xenopus Reciprocally, 125I-CR-1 binding to several different human (Kinoshita et al. 1995). In addition, recombinant CR-1 breast carcinoma cells lines could be blocked by CR-1 but produces a similar effect on specifically enhancing FGFR-1 not by EGF, TGFα, BTC, AR or HRGβ1. Finally, but not FGFR-2, FGFR-3 or FGFR-4 tyrosine recombinant CR-1 using BAF/3 mouse lymphoid cells that phosphorylation in trans in human breast cancer were ectopically expressing the human EGFR, erb B-2, erb MDA-MB-134 and T47–D cells (C Bianco, C B-3, or erb B-4 either alone or in various pairwise Wechselberger & D S Salomon, unpublished data). This combinations was unable to activate directly the tyrosine stimulatory effect occurs in trans since a specific FGFR-1 phosphorylation of these tyrosine kinase receptors, tyrosine kinase inhibitor, SU-5460, is able to block FGF-2– demonstrating that CR-1 was not able to bind directly to induced tyrosine phosphorylation but not CR-1–induced these type 1 receptor kinases either as homodimers or as phosphorylation of the FGFR-1. heterodimers (Kannan et al. 1997). Ligand binding to various tyrosine kinase receptors can induce tyrosine transphosphorylation between different Expression of cripto in human receptor subtypes leading to an enormous degree of signal carcinomas and premalignant lesions diversification through dimerization both within receptors of Several studies have demonstrated that growth factors are the same family and in some cases between receptors of involved in the pathogenesis of human cancer (Goustin et different families (Carraway & Cantley 1994, van Weering & al. 1986). In fact, tumor cells generally exhibit a reduced Bos 1998). This has been shown to be a common event requirement for exogenous growth factors when compared within the erb B family of receptor tyrosine kinases. with normal cells (Aaronson 1991, Sporn & Roberts 1992). However, this has also been documented to occur in the This phenomenon may be due to an increase in the synthesis FGFR and platelet-derived growth factor (PDGF) receptor by transformed cells of various growth factors that can families (Bellot et al. 1991, Turck & Edenson 1994). erb B-2 regulate tumor growth, angiogenesis and metastasis. CR-1 is undergoes transactivation and an enhanced tyrosine overexpressed in a number of human breast, colon, gastric phosphorylation in response to both EGF and neu and pancreatic cell lines, suggesting that CR-1 may function differentiation factor/HRGs which do not bind directly to this as an autocrine growth factor in these tumor cells (Ciardiello receptor but bind either to the EGFR or to the erb B-3 or erb et al. 1991a, Kuniyasu et al. 1991, Friess et al. 1994). To B-4 receptors respectively (King et al. 1988, Sliwkowski et assess the role of CR-1 in human malignancies, different al. 1994, Beerli et al. 1995). Another example of primary human carcinomas, premalignant lesions and heterodimerization is the interaction with erb B-3. erb B-3 adjacent noninvolved tissues have been analyzed for the has an impaired tyrosine kinase activity, but it can undergo frequency and relative expression of CR-1 mRNA and/or transactivation and phosphorylation by other erb B members immunoreactive protein (Table 5). (Guy et al. 1994, Riese et al. 1995). These cross-receptor interactions within the erb B family raised the possibility that although CR-1 does not bind directly to any of the erb B Expression of CR-1in gastrointestinal receptors, it may be able indirectly to transactivate members malignancies of the erb B receptor family by heterodimerization and transphosphorylation by either its receptor or indirectly Gastric cancer through a soluble, cytoplasmic src-like or JAK-like tyrosine CR-1 can be detected in normal gastric mucosa and in the kinase. This possibility was investigated in detail and it was mature regenerative epithelium of gastric ulcers where it may shown that CR-1 was able specifically to transactivate erb be involved in ulcer healing, probably through a paracrine B-4 in several mouse and human mammary epithelial cell mechanism (Abe et al. 1997). An increase in CR-1 lines whereas it failed to increase the tyrosine expression has been observed in 53% of premalignant lesions phosphorylation of the other three erb B members (Bianco of the stomach, such as intestinal metaplasia of the gastric et al. 1999). Blocking of the erb B-4 receptor with a specific mucosa, in gastric adenomas, and in 46% of early and www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021213 05:47:06AM via free access Salomon et al.: The EGF-CFC family

Table 5 Expression of immunoreactive CR-1 in human lesions. Data represent number of positive samples/total number of samples analyzed. Numbers in parentheses represent % positive samples. Tissue Noninvolved epithelium Premalignant lesion Carcinoma TA TVA Colon 25/193 (13) 26/65 (40) 10/13 (77) 122/168 (73)** IM Stomach 1/37 (3) 16/30 (53) 17/37 (46)** Pancreas 10/58 (17) 58/98 (59)** Hyperplasias Adenomas Gall baldder N.D. 6/9 (67) 4/7 (58) 89/132 (68)** DCIS Breast 5/33 (15) 26/55 (47) 497/631 (79)** Non small cell lung 178/195 (91)** Cystadenomas Serous Mucinous Serous Mucinous Ovary6/7 (86) post-menopausal 6/14 (43) 0/7 4/10 (40) 4/5 (80) 23/40 (58) 25/48 (52) 3/9 (33) 3/8 (38) 4/8 (50) 9/10 (90) Borderline: 10/10 Endometrium 10/28 (36) 53/91 (58)* post-menopausal Hyperplasias 18/30 (60) 68/96 (71)* Cervix 4/25 (17) 40/74 (54)* Testis 0/3 29/51 (57)** Embryonal carcinomas Seminomas 19/19 (100)** 10/32 (31) Adrenal cortex 1/3 (33) Bladder 0/6 23/39 (60)** Renal 0/18 Prostate 0/9 TA = tubular adenoma; TVA = tubulovillous adenoma; IM = intestinal metaplasia; N.D. = **Statisticallysignificant expression in carcinomas compared with noninvolved tissues. advanced gastric carcinomas (Kuniyasu et al. 1991, Saeki et Tsutsumi et al. 1994). The presence of CR-1 in pancreatic al. 1994a). Interestingly, the incidence of CR-1 expression cancer cells has been found to correlate with advanced tumor was found to correlate with the degree of dysplasia in the stage, but not with the tumor grade or with the post-operative intestinal metaplasia and with tumor stage and patient survival period of the cancer patients. prognosis in gastric cancer (Kuniyasu et al. 1994, Allgayer et al. 1997). In fact, the incidence of CR-1-positive cases Colon cancer was more frequent in late-stage, locally invasive tumors than CR-1 mRNA and protein are expressed at high levels in 73% in early-stage, noninvasive cancers. CR-1 expression in both of primary and metastatic colorectal cancers. CR-1 mRNA groups was associated with a poorer patient prognosis. expression by Northern blot analysis was detected in 68% of Finally, CR-1 expression has recently been reported in primary or metastatic human colorectal cancers but only in patients with long-term Helicobacter pylori infection, a risk 3% of noninvolved adjacent colon mucosa (Ciardiello et al. factor for gastric carcinogenesis (Haruma et al. 2000). 1991b). Elevated CR-1 protein expression was also observed Collectively, these studies suggest that CR-1 overexpression using immunohistochemistry in 79% of colon tumors, in 57% in gastric premalignant lesions such as in the advanced stage of tubulovillous or tubular adenomas and only in 12% of the of gastric cancer may be essential for the development and noninvolved normal colonic mucosa adjacent to tumors or progression of this neoplasia. adenomas (Saeki et al. 1992). In another study, CR-1 expression was analyzed by immunocytochemistry in 41 Pancreatic cancer colorectal carcinomas, 57 adenomas, 9 hyperplastic polyps CR-1 mRNA and protein have been detected by Northern and 98 normal mucosa samples (Saeki et al. 1995). No blot analysis and immunohistochemistry in normal pancreas staining for CR-1 was observed in normal mucosa, whereas and in chronic pancreatitis, and are overexpressed in 43% of CR-1 expression was detected with an increasing proportion pancreatic ductal adenocarcinomas (Friess et al. 1994, in 22% of hyperplastic polyps, 42% of adenomas and 78%

214 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:47:06AM via free access Endocrine-Related Cancer (2000) 7 199–226 of carcinomas. Within the adenomas, the frequency of CR-1 Finally, a recent study has reported a positive correlation expression was found to correlate directly with the degree of between nuclear erb B-4 expression and CR-1 expression in dysplasia, and with the size and the histological subtype of primary human breast carcinomas (Srinivasan et al. 2000). colon adenomas (Saeki et al. 1994b). Nonpolypoid flat This finding is intriguing since it has been shown that CR-1 adenomas had a higher frequency of CR-1 expression than can indirectly enhance the tyrosine phosphorylation of erb polypoid lesions. The gradual increase in CR-1 expression B-4 through an unknown receptor (Bianco et al. 1999). that is observed in the multistage process that evolves from adenoma to carcinoma suggests that CR-1 may be a tumor Endometrial cancer marker for human colorectal cancer and may be involved in CR-1 mRNA has been detected in normal human the early events of colon carcinogenesis (Saeki et al. 1995). endometrium and in 19 endometrial tumor samples (Pfeirrer To support this hypothesis, a recent study has detected CR-1 et al. 1997). CR-1 expression was higher in the endometrial expression in 62% of high risk normal colon mucosa carcinomas compared with normal endometrium but no specimens from individuals that belong to families with a correlation was observed with tumor stage and tumor high incidence of colorectal carcinomas, whereas only 20% differentiation. In another study, immunoreactivity for CR-1 of colon mucosa from low risk patients specimens were was demonstrated in 67% of normal endometrium samples, positive for CR-1 (De Angelis et al. 1999). in 30% of hyperplastic endometrium and in 65% of tumor samples (Niikura et al. 1995). In this study, CR-1 Rectal cancer immunoreactivity in the tumor samples was correlated with CR-1 immunoreactivity has been detected in 71% of primary surgical stage and progression of the disease. In addition, rectal carcinomas and in 37% of normal rectal mucosa coexpression of CR-1 and EGFR, and CR-1 and TGFα, and adjacent to carcinoma (Gagliardi et al. 1994). Interestingly, not their single expression, was significantly associated with tumors with CR-1 immunoreactivity extending to the surgical stage, depth of myometrial invasion and peritoneal adjacent normal mucosa showed an increase in the incidence washing cytology. Finally, strong CR-1 immunoreactivity of bowel wall penetration, lymph node involvement and a has been detected in endometrial atypical hyperplasia, an recurrence rate of 100%, suggesting a correlation between early event in endometrial carcinogenesis (Ayhan et al. CR-1 immunoreactivity in the normal adjacent colon mucosa 1998). and prognosis and survival of rectal tumors. Cervical cancer Gall bladder CR-1 has been detected using Western blot analysis in 38% CR-1 expression has been detected in 90% of gall bladder of primary human keratinocytes, in 94.1% of HPV-16 carcinomas without any correlation with tumor stage or immortalized keratinocytes and in 84.6% of cervical patient prognosis (Fujii et al. 1996). carcinoma cell lines (A D Ebert, J Stieler, M Nees, C Wechselberger, S Steinberg, C Woodworth, C Bianco, W Gullick, G Schaller & D S Salomon, unpublished data). Expression of CR-1in reproductive tract CR-1 is also overexpressed in human cervical cancer samples malignancies compared with normal or premalignant samples. No statistically significant correlations were found between Breast cancer CR-1 expression and FIGO (International Federation of CR-1 mRNA and protein can be detected in several human Gynecology and Obstetrics) stage, grading, lymph node breast cancer cell lines and in approximately 80% of human involvement or patient age. However, Ertoy et al. (2000) primary infiltrating breast carcinomas, in 47% of ductal recently found that CR-1 was overexpressed in a carcinoma in situ (DCIS) and in only 13% of noninvolved subpopulation of 26% of cervical carcinomas and was adjacent breast tissue samples (Normanno et al. 1993, 1995, correlated with tumor size, lymphovascular space Qi et al. 1994, Panico et al. 1996). However, no significant involvement, endometrial and parametrial involvement, and correlations have been observed between CR-1 mRNA lymph node metastasis. expression or immunoreactivity and various clini- copathological parameters such as tumor stage, estrogen Ovarian cancer receptor status, lymph node involvement, histologic grade, CR-1 was initially detected by immunohistochemistry in proliferative index as assessed by Ki-67 staining or flow 52% of ovarian adenocarcinomas (90% mucinous of high cytometry, LOH on chromosome 17p or overall patient malignant potential and 50% serous), in 33% of mucinous survival (Dublin et al. 1995). CR-1 is frequently coexpressed tumors of low malignant potential and in 35% of with other EGF-related peptides in human breast cancer cells, cystadenomas (Niikura et al. 1997). In addition, a significant such as TGFα, AR and HRGα, suggesting the possibility that correlation was observed with advanced tumor stage when an autocrine growth regulation in the cancer cells may exist. CR-1 was coexpressed with TGFα and AR or with the www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021215 05:47:06AM via free access Salomon et al.: The EGF-CFC family

EGFR, suggesting that coexpression of these EGF-related expression of specific proteins by binding to the growth factors and the EGFR in a single tumor represents a corresponding mRNA and preventing translation. This growth advantage for the cancer cells through potential approach has also been successfully utilized to impair CR-1 autocrine or paracrine mechanisms. In a more recent study, expression in several different types of human carcinoma a large number of human ovarian carcinomas of different cells. Inhibition of CR-1 expression in human GEO and CBS histologic types have been analyzed by immuno- colon cancer cells by using either an amphotropic histochemistry for the expression of CR-1, TGFα and AR (A recombinant CR-1 AS mRNA retroviral expression vector D’Antonio, S Losito, S Pignata, M Grassi, F Perrone, A De or CR-1 AS phosphorothioate oligonucleotides results in a Luca, G Botti, W J Gullick, G R Johnson, D S Salomon & significant growth inhibition in vitro (Ciardiello et al. 1994). N Normanno, unpublished data). Immunoreactive CR-1 was More importantly, GEO cells that were infected with the detected in 46% of ovarian carcinomas (83% mucinous and CR-1 AS retroviral vector exhibited a reduced tumorigenicity 53% serous), whereas no expression of CR-1 was found in in nude mice in which tumors were smaller and appeared endometrioid tumors. In addition, CR-1 expression did not after a longer latency period as compared with noninfected correlate with lymph node involvement, presence of GEO cells. Similar results were obtained in NTERA2 human calcification or necrosis in the tumor, or number of mitoses. embryonal carcinoma cells. Treatment of NTERA2 cells with Interestingly, a significant increase in CR-1 expression was 3 different CR-1 AS oligonucleotides or transfection with a observed in tumor specimens obtained after chemotherapy CR-1 AS mRNA expression vector resulted in an inhibition when compared with tumors obtained from the same patient of monolayer and soft agar growth in vitro (Baldassarre et before the first surgery. Finally, the expression of CR-1 was al. 1996). Interestingly, the inhibition in CR-1 expression significantly associated with short progression-free survival that was achieved in NTERA2 cells resulted in a significant (PFS) and overall survival (OS) in a univariate analysis. reduction in the expression of a differentiation marker on However, this correlation was not observed in a multivariate these tumor cells. In another study, a superadditive effect was analysis. observed on reducing the growth of GEO cells in vitro when a CR-1 AS oligonucleotide was combined with a TGFα AS or AR AS oligonucleotide, suggesting that a combination of Expression of CR-1in other cancers different growth factors is contributing to regulating the CR-1 has been detected by immunohistochemistry in 60% proliferation of colon cancer cells (Normanno et al. 1996). of benign and malignant bladder tumors but no correlation In fact, the anchorage-independent growth (AIG) of GEO between expression of CR-1 and tumor stage, tumor grade cells was inhibited by 50% by each of the AS or clinical outcome has been found (Byrne et al. 1998). oligonucleotides at 5 µM but not with a scrambled missense In a large group of 195 stage I-IIIA non-small-cell lung oligonucleotide. However, the combination of the three AS carcinomas, CR-1 was found to be expressed at high levels oligonucleotides each at 1 µM resulted in 60% inhibition of in 91% of the adenocarcinomas (Fontanini et al. 1998). the AIG of the GEO cells. Similarly, an additive growth In testicular carcinomas, CR-1 expression has been inhibitory effect was observed in MDA-MB-468 human detected by Northern blot analysis and by immunostaining breast cancer cells and in several different human ovarian in all of the non-seminomatous tumors such as embryonal carcinoma cell lines that had been treated with a combination carcinomas and malignant undifferentiated teratocarcinomas of a CR-1 AS oligonucleotide and either a TGF AS or AR that have been surveyed and in only 33% of the seminomas AS oligonucleotide (De Luca et al. 1999, Casamassimi et (Baldassarre et al. 1997). The preferential association of al. 2000). Provocative data have also been obtained using CR-1 expression with non-seminomatous tumors, that are combinations of CR-1 AS oligonucleotides with more clinically aggressive, suggests a correlation between conventional chemotherapeutic drugs in colon cancer cells CR-1 and a more malignant phenotype in germ cell tumors. (De Luca et al. 1997). In a clonogenic assay, pretreatment of GEO cells with different concentrations of 5-fluorouracil, adriamycin, mitomycin C or cis-platinum induced an additive Cripto-1as target for therapy in human growth inhibitory effect when the cells were subsequently cancer treated with a CR-1 AS oligonucleotide. More recently, CR-1 Since CR-1 is expressed at high levels in several different AS oligonucleotides have been used in combination with types of human malignancies, it may represent a useful target agents that block different but related intracellular signal for experimental therapies in human cancer patients. transduction pathways (Normanno et al. 1999). A novel Therapeutic antisense approaches have been successfully mixed backbone CR-1 AS oligonucleotide has been used in used to inhibit the growth of several human cancer cell lines combination with a humanized anti-human epidermal growth in vitro and their growth as xenografts in vivo in nude mice factor receptor antibody MAb C225 and with 8-Cl-cAMP, a (Neckers et al. 1992). Sequence specific antisense (AS) specific analog that inhibits type I protein kinase A. Low oligonucleotides or AS expression vectors can block the doses of each agent produced only a 15–35% growth

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inhibition in GEO cells in vitro but when the CR AS VEGF165 to the VEGF tyrosine kinase receptor-2, Flk-1/KDR oligonucleotide was combined with either the MAb C225 or (kinase insert-domain-containing receptor) (Miao et al. 1999, with 8-Cl-cAMP a synergistic antiproliferative effect Gagnon et al. 2000). In addition to NRP-1, VEGF165 but not occurred. Moreover, when the three agents were added VEGF121 also binds to glypican-1, a cell-associated protein together, a nearly complete suppression in the ability of GEO that is GPI-anchored to the membrane (Gengrinovitch et al. cells to grow in soft agar occurred. Finally, treatment with 1999). Glypican-1 potentiates the binding of VEGF165 to the all three compounds induced apoptosis in GEO cells whereas VEGF receptor Flk-1/KDR. Likewise, dally is a single treatment or a combination of only two agents failed heparin-sulphate containing proteoglycan in Drosophila that to induce any apoptosis. resembles a glypican (Jackson et al. 1997, Lin & Perrimon 1999, Tsuda et al. 1999). Dally can function as a co-receptor β Future perspectives for both Wingless (Wg)/wnt-1 and a TGF -related protein, decapentaplegic (dpp), presenting these proteins to either the The recent identification of the EGF-CFC proteins as a new wnt receptor, frizzled or to a TGFβ receptor (Jackson et al. family of embryonic morphogens and as potential growth or 1997, Lin & Perrimon 1999). differentiation factors in mammary epithelial cells raises a EGF-CFC proteins might also serve as antagonists for a large number of important mechanistic questions. For signaling pathway(s). For example, cerberus is a example, the EGF-CFC proteins oep, Cr-1 and FRL-1 in multifunctional antagonist that can bind and neutralize nodal, zebrafish have been demonstrated through genetic analysis to BMP-4 and wnt-1 (Piccolo et al. 1999, Belo et al. 2000). In act as competence or co-factors for nodal signaling during addition, tomoregulin, a peptide that specifically binds to the gastrulation while oep and cryptic function in a similar role type 1 erb B-4 tyrosine kinase receptor with low affinity, during L/R axis formation (Ciccodicola et al. 1989, Dono et also possesses two follistatin-related domains that have the al. 1993, Kinoshita et al. 1995, Shen et al. 1997, Ding et al. potential to bind and neutralize activin (Uchida et al. 1999). 1998, Zhang et al. 1998, Gritsman et al. 1999, Xu et al. Therefore, oep or other EGF-CFC proteins might function 1999, Yan et al. 1999, Gritsman et al. 2000, Schier & Shen by binding to and subsequently neutralizing an antagonistic, 2000). Since nodal but not activin requires oep function and nodal-specific binding protein, thereby permitting nodal to since both nodal and activin presumably activate the same activate an activin type IIB-type IB receptor-Smad-2 set of activin type II and type I B receptors, it is not entirely signaling pathway. In this context, it has recently been clear as to the nature of the protein interactions that control demonstrated that overexpression of either a wild-type or a these processes since direct binding of nodal or activin to secreted, COOH-terminal truncated form of oep can oep or to other EGF-CFC proteins has not formally been antagonize either BMP or nodal signaling in zebrafish and demonstrated (Gritsman et al. 1999, Schier & Shen 2000). It Xenopus (Kiecker et al. 2000). Finally, oep or EGF-CFC is possible that oep and other EGF-CFC proteins might be proteins might have the capacity to co-activate a parallel functioning as co-receptors for nodal but not for activin by signaling pathway after binding to nodal that is necessary, in facilitating the presentation of nodal to an activin type IIB conjunction with the activin type II and type I B receptor in a manner analogous to the glial cell-derived receptor-Smad-2 signaling pathway, for eliciting a biological neurotrophic factor (GDNF)/c-ret tyrosine kinase receptor response. This co-stimulatory pathway may depend, in part, signaling system (van Weering & Bos 1998, Saarma & upon activation of downstream MAPK by an upstream Sariola 1999, Trupp et al. 1999). In this system, GDNF and tyrosine kinase(s) (e.g. erb B-4, FGFR-1 or src-like kinase) other proteins such as artemin, neuturin and persephin that (LaBonne & Whitman 1984, Weinstein et al. 1998). In this are distantly related to TGFβ do not bind directly to c-ret but respect, TGFβ1 has been shown to activate the ras/raf/ bind to a family of four GPI-anchored co-receptors, GDNF MAPK pathway which in epithelial cells is obligatory for family receptor α 1–4 (GFRα) (Saarma & Sariola 1999, eliciting some TGF responses (Hartsough & Mulder 1997, Trupp et al. 1999). Ligand-bound GFRα proteins then utilize Mulder 2000). These latter two pathways may not be either c-ret or in some cases c-src as signaling components mutually exclusive in a mechanistic framework since there in a tertiary complex. Two additional examples are also is experimental evidence demonstrating that the cloned and noteworthy and illustrate the fact that proteins may be purified mammalian TGFβRII receptor not only has a multifunctional and capable of interacting with multiple serine-threonine kinase activity but also an associated signaling pathways. Neuropilin-1 (NRP-1) is a trans- tyrosine kinase activity that is not directly activated by membrane glycoprotein that is a receptor for several TGFβ1 (Lawler et al. 1997). In fact, there is strong evidence members of the semaphorin/collapsin family that are in zebrafish, Xenopus and mouse that co-activation of a involved in neuronal guidance (Miao et al. 1999, Gagnon et FGFR-1 signaling pathway is also necessary to complete al. 2000, Raper 2000). NRP-1 is also a co-receptor for a gastrulation fully and for L/R asymmetry development specific isoform of vascular endothelial growth factor (Kimelman et al. 1992, Beddington & Smith 1993, Cornell &

(VEGF) VEGF165, but not for VEGF121, which presents Kimelman 1994, Harland & Gerhart 1997, Hartsough & www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/2021217 05:47:06AM via free access Salomon et al.: The EGF-CFC family

Mulder 1997, Rossant et al. 1997, Weinstein et al. 1998). activin, TGFβ1 and/or FGF may be obligatory for or The mechanism by which activin, nodal and fgf-8 cooperate accentuate these biological responses to different members in these processes is not completely defined. However, it is of the EGF-CFC family. These biological effects may be intriguing that both human CR-1 and Xenopus FRL-1 can in functionally important since cripto is an oncofetal protein. In some manner indirectly transactivate FGFR-1 (Kinoshita et this respect, mouse Cr-1 and human CR-1 are differentially al. 1995, C Bianco, C Wechselberger & D S Salomon, overexpressed in a number of different types of carcinomas unpublished data). Likewise, the ability of CR-1 specifically and in premalignant lesions from these same tissues. to transactivate erb B-4 may also be of developmental Identification of factors activating transcription factors that significance since HRGβ1, a ligand for erb B-4, is expressed can bind to the promoter regions of these genes will therefore in the gastrula of Xenopus and can induce mesoderm be important in determining the mechanism by which the formation in animal cap explants in vitro (Chung & Chung expression of these genes are up-regulated in tumor cells. 1999). In addition, HRGβ1 and erb B-4, like Cr-1, are Reciprocally, identification of downstream genes that might important determinants in early cardiogenesis (Dono et al. be regulated by EGF-CFC proteins will be useful in 1993, Xu et al. 1998, 1999, Klapper et al. 2000). These ascertaining the mechanism by which EGF-CFC proteins examples merely serve to illustrate the potential complexity function. by which the EGF-CFC proteins might function as either Development of animal models in which mouse or human co-receptors for one or several distinct groups of structurally cripto genes are transcriptionally regulated by tissue-specific unrelated proteins and receptor signaling pathways and/or as promoters will be necessary to determine if misexpression and/ antagonistic binding proteins or modulators for multiple or overexpression of these genes can contribute to abnormal ligands. The expression and purification of a biologically tissue development and neoplasia. For example, the use of the active form of an EGF-CFC protein will be useful for the WAP or MMTV promoters for directing expression of cripto identification of a ligand(s) and/or receptor for this family of to the epithelial cells of the mammary gland in mice will also proteins and its potential coupling to other signal be useful in assessing the in vivo effects of cripto transduction pathways. overexpression at various stages of mammary gland Irrespective of the molecular mechanism by which development and involution. Studies to generate such mice are EGF-CFC proteins might function, the diversity of biological currently in progress. The observation that Cr-1 expression is effects that can be induced by these proteins is large and elevated during pregnancy and lactation in the mouse and that similar to the pleiotropic spectrum of responses induced by exogenous CR-1 protein can inhibit β-casein and WAP other growth factor families. In this respect, the ability of expression in response to lactogenic hormones in vitro these proteins to induce cell motility in embryonic epiblast suggests that ectopic expression of this gene is likely to cells of the primitive streak, in mouse mammary epithelial produce a mammary phenotype and possibly perturb normal cells and in human carcinoma cells is noteworthy (Xu et al. mammary differentiation and development (DeSantis et al. 1998, 1999, Ebert et al. 2000, C Wechselberger, A D Ebert, 1997, Niemeyer et al. 1998). In addition, the development of C Bianco, B Wallace-Jones, N Khan, Y Sun & D S Salomon, appropriate transgenic mice in which cripto expression is unpublished data). In all cases, this is preceded by an EMT directed to the mammary gland will facilitate assessment of the since cells assume a more mesenchymal phenotype. In the potential function that cripto might perform at various stages case of some of the epithelial cells, there is a loss of in mammary epithelial cell transformation and tumor E-cadherin function and an up-regulation in vimentin progression. The development of ductal hyperplasias in the expression during this conversion (Ebert et al. 2000, C cleared mammary fat pad of virgin mice that harbor Wechselberger, A D Ebert, C Bianco, B Wallace-Jones, N Cr-1-transduced mammary epithelial cells portends such a Khan, Y Sun & D S Salomon, unpublished data). Loss of possibility (Kenney et al. 1997b, Niemeyer et al. 1999). E-cadherin function may be particularly relevant to Finally, Cr-1 can potentially be ablated in the mammary gland facilitating changes in soluble, cytoplasmic β-catenin which by crossing WAP-Cre mice with mice that have had the could then potentially interact through the wnt signaling endogenous Cr-1 gene flanked by two loxP sites. F1–hybrid pathway by binding to Lef/tcf-4. This transcription complex mice that contain the WAP-Cre gene and the floxed Cr-1 gene might then serve to activate genes such as the protease should possess cells expressing the Cre recombinase gene matrilysin that are necessary for morphogenic patterning under the transcriptional control of the WAP promoter which which occurs during branching morphogenesis of mammary should then lead to the selective excision and inactivation of epithelial cells in vitro in type I collagen gels, and in vivo in the Cr-1 gene in the mammary gland during pregnancy where the cleared mammary fat pad in Cr-1–transduced cells the WAP promoter is normally expressed (Wagner et al. 1997). (Crawford et al. 1999). Additionally, these cellular This approach will be necessary to study the role of the Cr-1 alterations may contribute to the increased migratory and gene in postnatal mammary gland development since invasive behavior of tumor cells overexpressing cripto. It is homozygous, germline deletion of the Cr-1 gene in the mouse more than likely that other growth factors such as nodal, is embryonically lethal.

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