David S. Salomon

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.

Epidermal growth factor receptor, but not c-erbB-2, activation prevents lactogenic hormone induction of the beta-casein gene in mouse mammary epithelial cells.

The HC11 cell line was isolated from mammary gland cells of pregnant mice. The cells displayed a normal phenotype and retained some characteristics of mammary epithelial cell differentiation. After treatment with the lactogenic hormones prolactin and glucocorticoids, the HC11 cells expressed the milk protein beta-casein. Various oncogenes were transfected and expressed in HC11 cells. The oncogenes were tested for their transformation ability and for their effects upon the differentiation of the HC11 cells. All of the oncogenes tested, including activated human Ha-ras, human transforming growth factor-alpha, activated rat neuT, and human c-erbB-2 activated by a point mutation in the transmembrane domain, caused transformation of the HC11 cells, as shown by tumor formation in nude mice. HC11 cells expressing the neuT and activated c-erbB-2 genes synthesized beta-casein in response to lactogenic hormones, whereas those expressing the Ha-ras or transforming growth factor-alpha oncogenes were no longer able to respond to the lactogenic hormones. This inhibition of beta-casein production occurs at the transcriptional level and in the transforming growth factor-alpha-transformed cells is due to an autocrine mechanism involving the activation of the epidermal growth factor receptor. This suggests that, although the c-erbB-2 and epidermal growth factor receptors are structurally quite similar, their activation has different effects upon mammary epithelial cell differentiation.

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.

Differential immunohistochemical detection of transforming growth factor α, amphiregulin and CRIPTO in human normal and malignant breast tissues

The expression of growth factors, such as transforming growth factor α (TGFα), amphiregulin (AR) and CRIPTO, a type‐1 tyrosine‐kinase growth factor receptor‐(erbB‐2), and a tumor‐suppressor gene (p53), that have been implicated in the development and/or the progression of breast cancer, was evaluated by immunohistochemistry in 100 human primary infiltrating breast carcinomas (IBC). AR and CRIPTO immunoreactivity was also assessed in 55 human breast ductal carcinomas in situ (DCIS). Within the 100 IBC, 80, 50, 73, 17, and 34 tumors expressed moderate to high levels of TGFα, AR, CRIPTO, erbB‐2, and p53 respectively. In addition, AR and CRIPTO immunoreactivity were found in 11 and in 26 out of 55 DCIS respectively. In contrast, only 4, 3, and 2 out of 10 normal mammary‐gland samples were weakly positive for TGFα, AR, and CRIPTO expression, respectively, whereas none was positive for erbB‐2 or p53. Within the 100 IBC, expression of erbB‐2 significantly correlated with high histologic and nuclear grading, with high growth fraction, and with estrogen‐receptor(ER)‐ and progesterone‐receptor(PgR)‐negative tumors. A statistically significant correlation was also observed between p53 expression and high histologic grading, high growth fraction, and PgR‐negative tumors. In contrast, no significant correlations were found between TGFα, AR, and CRIPTO immunoreactivity and various clinicopathological parameters, with the exception of a positive correlation between TGFα and ER expression. These data demonstrate that TGFα, AR, and CRIPTO expression are significantly increased in malignant mammary epithelium relative to normal epithelium. In particular, the differential expression of CRIPTO may serve as a potential tumor marker for breast carcinogenesis.

ErbB/EGF Signaling and EMT in Mammary Development and Breast Cancer

Activation of the ErbB family of receptor tyrosine kinases via cognate Epidermal Growth Factor (EGF)-like peptide ligands constitutes a major group of related signaling pathways that control proliferation, survival, angiogenesis and metastasis of breast cancer. In this respect, clinical trials with various ErbB receptor blocking antibodies and specific tyrosine kinase inhibitors have proven to be partially efficacious in the treatment of this heterogeneous disease. Induction of an embryonic program of epithelial-to-mesenchymal transition (EMT) in breast cancer, whereupon epithelial tumor cells convert to a more mesenchymal-like phenotype, facilitates the migration, intravasation, and extravasation of tumor cells during metastasis. Breast cancers which exhibit properties of EMT are highly aggressive and resistant to therapy. Activation of ErbB signaling can regulate EMT-associated invasion and migration in normal and malignant mammary epithelial cells, as well as modulating discrete stages of mammary gland development. The purpose of this review is to summarize current information regarding the role of ErbB signaling in aspects of EMT that influence epithelial cell plasticity during mammary gland development and tumorigenesis. How this information may contribute to the improvement of therapeutic approaches in breast cancer will also be addressed.

Epidermal growth factor-related peptides in the pathogenesis of human breast cancer

SummaryA number of different epidermal growth factor (EGF)-related peptides such as EGF, transforming growth factor α (TGFα), amphiregulin (AR), heregulin (HRG), and cripto-1 (CR-1), are coexpressed to varying degrees in both normal and malignant mammary epithelial cells. However, in general the frequency and level of expression of TGFα, AR, and CR-1 are higher in malignant breast epithelial cells than in normal mammary epithelium. In addition, several of these peptides such as TGFα and AR can function as autocrine and/or juxtacrine growth factors in mammary epithelial cells, and their expression is stringently regulated by mammotrophic hormones such as estrogens, activated proto-oncogenes that have been implicated in the pathogenesis of breast cancer, and other growth factors. The redundancy of expression that is observed for a number of these structurally related peptides in both normal and malignant mammary epithelial cells suggests that some of these peptides may be involved in regulating other aspects of cellular behavior such as differentiation in addition to proliferation.

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.

Identification of Stem Cell Units in the Terminal End Bud and Duct of the Mouse Mammary Gland

The mouse mammary gland may undergo cycles of proliferation, terminal differentiation, tissue remodeling, and more importantly malignant transformation. Mammary epithelial stem cells and their progeny participate in these processes. Mammary epithelial stem cells are multipotent, exhibit properties of self renewal (up to 7 divisions) and may exist either as long-lived nondividing cells or as proliferating-differentiating cells. The focus of this study was to locate stem cells by identifying them as long-lived, label-retaining mammary epithelial cells (LRCs) in growth active (developing) or growth static (aged) mammary ducts. Initially, primary epithelial cells were pulse labeled with either fluorescent tracker dye and/or BrdU. Cells were then transplanted into cleared juvenile syngeneic mammary fat pads and held for 5 weeks or 8 weeks. In this study, we demonstrate that LRCs are stem cells and their progeny (transitional cells) are arranged as transitional units (TUs). Additionally, TUs are located every 250 ± 75 μm in ducts or in the terminal end bud 200–600 μm in diameter. Molecules expressed in TUs were Zonula Occludens-1 and α-catenin proteins which were significantly detected in 75%–91% (P < 0.001) of the LRCs cells that make up the TU. These data suggest that transitional units may be a group of label-retaining stem cells and maybe involved in the developmental or cancer process.