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

Abstract

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-β ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-β 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 Binduced growth suppression by blocking Cripto's 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.

Molecular evolution of the EGF–CFC protein family

“…It is possible that the critical mediators of functional differences between Cripto and Cryptic evolved by selection of paralog specific residues in the CFC domain. Although available experimental data suggest that both EGF and CFC domains are involved in the formation of multiprotein complexes between distinct multiple partners in the Nodal signaling pathway, the CFC domain in particular, appears to have a decisive role in this process (Adkins et al, 2003;Calvanese et al, 2008;Foley et al, 2003;Gray et al, 2003;Shani et al, 2008). Overall, the analysis supports the hypothesis that the biological differences between Cripto and Cryptic are more likely due to changes in the interactions of the CFC domain with its other cell-signaling partners.…”

“…Mutations in human Cryptic are associated with heterotaxic phenotypes including congenital laterality defects of the heart (Bamford et al, 2000;Goldmuntz et al, 2002). Strikingly, the homolog Cripto can function as an oncogene in human carcinoma cells and is thus indicated as an important target of tumor-suppressive therapeutic interventions (Adkins et al, 2003;Strizzi et al, 2004;Xing et al, 2004). At least two proposed mechanisms suggest the primary involvement of the CFC domain in the tumourigenic activity of Cripto (Adkins et al, 2003;Gray et al, 2003;Shani et al, 2008).…”

“…Strikingly, the homolog Cripto can function as an oncogene in human carcinoma cells and is thus indicated as an important target of tumor-suppressive therapeutic interventions (Adkins et al, 2003;Strizzi et al, 2004;Xing et al, 2004). At least two proposed mechanisms suggest the primary involvement of the CFC domain in the tumourigenic activity of Cripto (Adkins et al, 2003;Gray et al, 2003;Shani et al, 2008). Consistently, monoclonal antibodies (mAbs) against the CFC domain of Cripto are able to prevent or reduce tumor formation in xenograft models (Bianco et al, 2004;Shen, 2003).…”

Nodal signals pattern vertebrate embryos

“…In contrast, Activin signal transduction does not require Cripto. In fact, Cripto can prevent Activin signaling; Activin bound to ActRIIA/IIB can form a complex with Cripto that prevents binding of the Activin-ActRIIA/IIB complex to ALK4 [20,21].…”

Exploiting the Convergence of Embryonic and Tumorigenic Signaling Pathways to Develop New Therapeutic Targets

“…Efforts have also been made to target the effects of Nodal cell surface binding by the administration of small molecule or immunotherapeutic inhibitors of Cripto-1. To date, antibodies which block Cripto-1 association with ALK and Nodal have been shown to have promising effects in colon, breast and testicular cancers [46][47][48]. Likewise, chemical inhibitors of the ALK4/5/7 receptor, namely SB-431542, have anti-tumorigenic effects in other malignancies [49,50].…”