Caterina Bianco

Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo

Cripto, a cell surface-associated protein belonging to the EGF-CFC family of growth factor-like molecules, is overexpressed in many human solid tumors, including 70-80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-beta ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-beta ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells. This result suggests a novel mechanism for antagonizing Activin signaling that could promote tumorigenesis by deregulating growth homeostasis. We show that an anti-CFC domain antibody, A8.G3.5, both disrupts Cripto-Nodal signaling and reverses Cripto blockade of Activin B-induced growth suppression by blocking Criptos association with either Alk4 or Activin B. In two xenograft models, testicular and colon cancer, A8.G3.5 inhibited tumor cell growth by up to 70%. Both Nodal and Activin B expression was found in the xenograft tumor, suggesting that either ligand could be promoting tumorigenesis. These data validate that functional blockade of Cripto inhibits tumor growth and highlight antibodies that block Cripto signaling mediated through its CFC domain as an important class of antibodies for further therapeutic development.

Cripto-1 Activates Nodal- and ALK4-Dependent and -Independent Signaling Pathways in Mammary Epithelial Cells

ABSTRACT Cripto-1 (CR-1), an epidermal growth factor-CFC (EGF-CFC) family member, has a demonstrated role in embryogenesis and mammary gland development and is overexpressed in several human tumors. Recently, EGF-CFC proteins were implicated as essential signaling cofactors for Nodal, a transforming growth factor β family member whose expression has previously been defined as embryo specific. To identify a receptor for CR-1, a human brain cDNA phage display library was screened using CR-1 protein as bait. Phage inserts with identity to ALK4, a type I serine/threonine kinase receptor for Activin, were identified. CR-1 binds to cell surface ALK4 expressed on mammalian epithelial cells in fluorescence-activated cell sorter analysis, as well as by coimmunoprecipitation. Nodal is coexpressed with mouse Cr-1 in the mammary gland, and CR-1 can phosphorylate the transcription factor Smad-2 in EpH-4 mammary epithelial cells only in the presence of Nodal and ALK4. In contrast, CR-1 stimulation of mitogen-activated protein kinase and AKT in these cells is independent of Nodal and ALK4, suggesting that CR-1 may modulate different signaling pathways to mediate its different functional roles.

Cripto-1: A multifunctional modulator during embryogenesis and oncogenesis

It is increasingly evident that genes known to perform critical roles during early embryogenesis, particularly during stem cell renewal, pluripotentiality and survival, are also expressed during the development of cancer. In this regard, oncogenesis may be considered as the recapitulation of embryogenesis in an inappropriate temporal and spatial manner. The epidermal growth factor-Cripto-1/FRL1/cryptic family of proteins consists of extracellular and cell-associated proteins that have been identified in several vertebrate species. During early embryogenesis, epidermal growth factor-Cripto-1/FRL1/cryptic proteins perform an obligatory role as coreceptors for the transforming growth factor-beta subfamily of proteins, which includes Nodal. Cripto-1 has also been shown to function as a ligand through a Nodal/Alk4-independent signaling pathway that involves binding to glypican-1 and the subsequent activation through src of phosphoinositol-3 kinase/Akt and ras/mitogen-activated protein kinase intracellular pathways. Expression of Cripto-1 is increased in several human cancers and its overexpression is associated with the development of mammary tumors in mice. Here, we review the role of Cripto-1 during embryogenesis, cell migration, invasion and angiogenesis and how these activities may relate to cellular transformation and tumorigenesis. We also briefly discuss evidence suggesting that Cripto-1 may be involved in stem cell maintenance.

Epithelial mesenchymal transition is a characteristic of hyperplasias and tumors in mammary gland from MMTV-Cripto-1 transgenic mice.

Epithelial‐mesenchymal transition (EMT) facilitates migration and invasion of epithelial tumor cells. Cripto‐1 (CR‐1), a member of the epidermal growth factor‐CFC protein family increases migration of cells in vitro. Here the expression of molecular markers and signaling molecules characteristic of EMT were assessed in mammary gland hyperplasias and tumors from mice expressing the human CR‐1 transgene by the MMTV promoter (MMTV‐CR‐1) and in mouse mammary epithelial cell line HC‐11 overexpressing CR‐1 (HC‐11/CR‐1). Western blot analysis showed decreased expression of E‐cadherin in MMTV‐CR‐1 tumors and in HC‐11/CR‐1 cells. The expression of N‐cadherin, vimentin, cyclin‐D1, and of the zinc‐finger transcription factor, snail, was increased in MMTV‐CR‐1 tumors. Increased snail mRNA was also found in HC‐11/CR‐1 cells. Expression of phosphorylated (P)‐c‐Src, P‐focal adhesion kinase (FAK), P‐Akt, P‐glycogen synthease kinase 3β (GSK‐3β), dephosphorylated (DP)‐β‐catenin, and various integrins such as, alpha 3, alpha v, beta 1, beta 3, and beta 4 was also increased in MMTV‐CR‐1 tumors. Immunohistochemistry showed positive staining for vimentin, N‐cadherin, cyclin‐D1, smooth muscle actin, fibronectin, snail, and β‐catenin in MMTV‐CR‐1 tumor sections. HC‐11/CR‐1 cells treated with the c‐Src inhibitor PP2 reduced the expression of P‐c‐Src and of P‐FAK, P‐Akt, P‐GSK‐3β, DP‐β‐catenin all known to be activated by c‐Src. Migration of HC‐11/CR‐1 cells was also reduced by PP2 treatment. These results suggest that CR‐1 may play a significant role in promoting the increased expression of markers and signaling molecules associated with EMT.

The RIα subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-Receptor

Functional interactions between protein kinase A (PKA) and epidermal growth factor receptor (EGF-R) signalling pathways have been suggested. Unlike the type II isoform of PKA (PKAII), the type I (PKAI) and/or its regulatory subunit RIα are generally overexpressed in cancer cells and are induced following transforming growth factor α (TGFα)/EGF-R-dependent transformation. Downregulation of RIα/PKAI inhibits TGFα expression and EGF-R-dependent signalling. We have previously shown that addition of EGF to quiescent human normal epithelial MCF-10A cells determines PKAI expression and cell membrane translocation before cells enter S phase, while PKAI inhibition prevents S phase entry. Constitutive overexpression of PKAI confers the ability to grow in serum free medium, bypassing EGF requirement. Here we demonstrate a direct interaction of PKAI, but not of PKAII, with the activated EGF-R, that occurs within 5 min following EGF treatment of MCF-10A cells. Moreover, induction of mitogen-activated protein kinase (MAPK) activity following EGF-R activation is mimicked by PKAI overexpression and inhibited by downregulators of PKAI. Finally, the PKAI – EGF-R association occurs through the binding of RIα to the SH3 domain(s) of Grb2 adaptor protein, thus allowing the recruitment of the PKAI holoenzyme to the activated EGF-R. This is the first demonstration of a direct interaction of PKAI with the activated EGF-R macromolecular signalling complex.

Cripto Enhances the Tyrosine Phosphorylation of Shc and Activates Mitogen-activated Protein Kinase (MAPK) in Mammary Epithelial Cells

Cripto-1 (CR-1), a recently discovered protein of the epidermal growth factor (EGF) family, was found to interact with a high affinity, saturable binding site(s) on HC-11 mouse mammary epithelial cells and on several different human breast cancer cell lines. This receptor exhibits specificity for CR-1, since other EGF-related peptides including EGF, transforming growth factor α, heparin-binding EGF-like growth factor, amphiregulin, epiregulin, betacellulin, or heregulin β1 that bind to either the EGF receptor or to other type 1 receptor tyrosine kinases such as erb B-3 or erb B-4 fail to compete for binding. Conversely, CR-1 was found not to directly bind to or to activate the tyrosine kinases associated with the EGFR, erb B-2, erb B-3, or erb B-4 either alone or in various pairwise combinations which have been ectopically expressed in Ba/F3 mouse pro-B lymphocyte cells. However, exogenous CR-1 could induce an increase in the tyrosine phosphorylation of 185- and 120-kDa proteins and a rapid (within 3-5 min) increase in the tyrosine phosphorylation of the SH2-containing adaptor proteins p66, p52, and p46 Shc in mouse mammary HC-11 epithelial cells and in human MDA-MB-453 and SKBr-3 breast cancer cells. CR-1 was also found to promote an increase in the association of the adaptor Grb2-guanine nucleotide exchange factor-mouse son of sevenless (mSOS) signaling complex with tyrosine-phosphorylated Shc in HC-11 cells. Finally, CR-1 was able to increase p42erk-2 mitogen-activated protein kinase (MAPK) activity in HC-11 cells within 5-10 min of treatment. These data demonstrate that CR-1 can function through a receptor which activates intracellular components in the ras/raf/MEK/MAPK pathway.

Cripto: a novel epidermal growth factor (EGF)‐related peptide in mammary gland development and neoplasia

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

Cripto-1 indirectly stimulates the tyrosine phosphorylation of erb B-4 through a novel receptor.

Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that fails to directly bind to any of the four known erb B type 1 receptor tyrosine kinases. The present study demonstrates that CR-1 indirectly induces tyrosine phosphorylation of erb B-4 but not of the epidermal growth factor-related receptors erb B-2 anderb B-3 in different mouse and human mammary epithelial cell lines. In addition, down-regulation of erb B-4 in NMuMG mouse mammary epithelial cells and in T47D human breast cancer cells, using an anti-erb B-4 blocking antibody or a hammerhead ribozyme vector targeted to erb B-4 mRNA, impairs the ability of CR-1 to fully activate mitogen-activated protein kinase. Finally, chemical cross-linking of 125I-CR-1 to mouse and human mammary epithelial cell membranes results in the labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor structurally unrelated to any of the known type I receptor tyrosine kinases. In conclusion, these data demonstrate that CR-1, upon binding to an unknown receptor, can enhance the tyrosine kinase activity oferb B-4 and that a functional erb B-4 receptor is required for CR-1-induced MAPK activation.

Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-beta signaling

We present evidence that Notch4ICD attenuates TGF-β signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-β. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-β/Activin reporter luciferase assays, RT–PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 δRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-β. Blockage of Notch4 processing to ICD4 by γ-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-β. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.

Role of Cripto-1 in Stem Cell Maintenance and Malignant Progression

Cripto-1 is critical for early embryonic development and, together with its ligand Nodal, has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Like other embryonic genes, Cripto-1 performs important roles in the formation and progression of several types of human tumors, stimulating cell proliferation, migration, epithelial to mesenchymal transition, and tumor angiogenesis. Several studies have demonstrated that cell fate regulation during embryonic development and cell transformation during oncogenesis share common signaling pathways, suggesting that uncontrolled activation of embryonic signaling pathways might drive cell transformation and tumor progression in adult tissues. Here we review our current understanding of how Cripto-1 controls stem cell biology and how it integrates with other major embryonic signaling pathways. Because many cancers are thought to derive from a subpopulation of cancer stem-like cells, which may re-express embryonic genes, Cripto-1 signaling may drive tumor growth through the generation or expansion of tumor initiating cells bearing stem-like characteristics. Therefore, the Cripto-1/Nodal signaling may represent an attractive target for treatment in cancer, leading to the elimination of undifferentiated stem-like tumor initiating cells.