Abstract 3493: Soluble ALK4 extracellular domain antagonizes the cancer/stem cell phenotype caused by Nodal and Cripto signaling in mammary epithelia
Authors: Spike Benjamin T. , Kelber Jonathan A. , Kalathur Madhuri , Booker Evan , Lipianskaya Julia , Klemke Richard , Vale Wylie , Wahl Geoffrey , Gray Peter C. 
Published: August 2012

We recently determined that a fraction of cells in a highly enriched fetal mammary stem cell pool expresses the oncofetal protein Cripto and its receptor/co-factor GRP78 on their surfaces. Cripto is a GPI-anchored protein important for embryonic stem cell self-renewal and developmental plasticity. It is also selectively overexpressed in multiple human tumors including ∼80% of human breast tumors. Cripto overexpression increases cellular proliferation, invasion and migration in vitro and promotes mammary tumor growth in mice. Cripto functions as an obligatory co-receptor for Smad2/3 activation by the TGF-β superfamily member Nodal but attenuates activin-A and TGF-β1 signaling and the cytostatic effects of these ligands on human mammary epithelial cells. Cripto can be secreted from cells and soluble forms activate Src, ras/raf/MAPK and PI3K/Akt pathways. We recently identified the HSP70 family member GRP78 as a cell surface receptor/co-factor that is required for Cripto signaling via TGF-β and Src/MAPK/PI3K pathways. Here, we developed a soluble and highly specific Cripto antagonist based on the extracellular domain of the Activin/Nodal Type I serine/threonine kinase receptor ALK4 (sALK4-ECD). We tested the ability of this antagonist to block Cripto signaling via TGF-β and Src/MAPK/PI3K pathways in oncogenic and stem cell phenotypes in mammary epithelial cells and breast cancer cell lines. The sALK4-ECD protein dose-dependently and specifically inhibited 125I-Cripto binding to 293T cells transfected with full length ALK4. In addition, sALK4-L75A-Fc blocked 125I-Cripto binding to native human mammary epithelial MCF10A cells. sALK4-ECD inhibited Cripto-dependent Nodal signaling in 293T cells and inhibited Nodal-induced Smad2 phosphorylation in native NCCIT cells. sALK4-ECD also blocked Cripto-induced PI3K activation, cell proliferation and migration in MCF10A cells. Cripto and Nodal signaling had profound effects on MCF10A growth in suspension and mammosphere reseeding capacity that were also inhibited by sALK4-ECD. We evaluated Nodal/Cripto signaling in cancer cell lines and primary fetal mammary stem cells and tested the efficacy of sALK-ECD mediated suppression of Nodal and Cripto effects in vitro and in vivo. These studies were designed to test if Cripto represents a functional cell surface marker of normal stem cells and/or the cells that initiate or perpetuate breast cancers. Our results point to distinct roles for Cripto and Nodal/Cripto signaling in promoting the oncogenic phenotype via activation of Smad2/3 and PI3K pathways, and support a role for Cripto antagonism such as that afforded by sALK4-ECD as a potential therapeutic strategy for the treatment of human disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3493. doi:1538-7445.AM2012-3493