Abstract
Nodal is a potent embryonic morphogen belonging to the TGF-β superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44–67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (KD 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.
Highlights
Nodal ligands are potent morphogens, belonging to the TGF-β superfamily [1,2,3,4]
On the basis of previous docking and binding studies [21,31] the region of human Nodal (Uniprot Q96S42) including the H3-wrist helix and the pre-helix loop was chosen as hNodal antigen (Figure 1)
After immunization and hybridoma generation, cell supernatants were screened using in parallel a mutated peptide named hNodal(44–67)E49A–E50A, in which E49 and E50 were replaced with two alanines
Summary
Nodal is rarely expressed during late development and adulthood. Aberrant re-activation of Nodal expression in adults is associated with a number of tumors such as metastatic melanoma as well as breast, colon, prostate, and ovarian carcinomas [5,6,7,8]. During the last years, increased attention has been devoted to the correlation existing between the expression levels of Nodal and melanoma development. These studies have associated the hyper-activation of Nodal signalling to melanoma invasiveness, aggressiveness, plasticity, and tumorigenicity [5,9,10,11,12,13]
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