Tumor suppressor Eaf 1 and Eaf 2 negatively regulate canonical Wnt / β-catenin signaling

INTRODUCTION Eaf1 and Eaf2 were originally identified as partners of ELL (elevennineteen lysine-rich leukemia), a fusion protein frequently associated with myeloid leukemia (Simone et al., 2001; Simone et al., 2003). Studies suggest that Eaf2 serves as a novel tumor suppressor, especially in prostate cancer (Xiao et al., 2003; Xiao et al., 2008; Xiao et al., 2009); however, the molecular mechanisms underlying this tumor suppression activity remain largely unknown. The Wnt/β-catenin pathway plays crucial roles in embryonic development and tissue regeneration (Klaus and Birchmeier, 2008), and dysregulation of the pathway can result in cancer. Several groups have shown that inhibition of Wnt/β-catenin signaling constitutes a common mechanism for tumor suppressor activity (Morin et al., 1997; Sadot et al., 2001; Major et al., 2007; Meani et al., 2009). Furthermore, Wnt/β-catenin signaling participates in the formation of embryonic axes and neuroectoderm in vertebrates, and perturbation of this pathway often leads to defects in neuroectodermal and mesodermal patterning. Overexpression of chick Wnt8c in the mouse results in anterior neuroectoderm truncation (Pöpperl et al., 1997). Consistently, mice lacking functional Axin or Dkk1, both negative regulators of Wnt signaling, exhibit anterior neuroectoderm truncation (Zeng et al., 1997; Glinka et al., 1998; Mukhopadhyay et al., 2001). In zebrafish, enhanced Wnt signaling also leads to loss of rostral neural domains. Forebrain defects and small eyes are observed in two zebrafish mutants: masterblind (mbl), which carries a mutation in axin1 with high Wnt/β-catenin activities (Heisenberg et al., 2001), and headless (hdl), which carries a mutation abolishing the repressor function of tcf3 (tcf7l1a – Zebrafish Information Network) on Wnt/β-catenin signaling (Kim et al., 2000). By contrast, zebrafish wnt8 mutants and morphants display significant expansion of forebrain markers (Lekven et al., 2001). In mesoderm patterning, maternal Wnt/β-catenin signaling is essential for the induction of sqt (ndr1), boz (dharma) and gsc during Nieuwkoop center formation, which then induces formation of the dorsal mesodermal organizer. After the mid-blastula transition, zygotic Wnt/β-catenin signaling is activated by Wnt ligands to antagonize the organizer, and is involved in ventralposterior mesoderm and neuroectoderm induction by directly regulating ventral mesoderm tbx6 (Szeto and Kimelman, 2004) and posterior ectoderm/mesoderm cdx4, respectively (Pilon et al., 2006). Zebrafish wnt8 mutants show a significant expansion of the organizer and almost absent expression of ventrolateral mesoderm markers, similar to the phenotypes observed in wnt8 morphants (Lekven et al., 2001). Multiple molecules at several different points in the Wnt/β-catenin signaling pathway can modulate the output of the pathway. Extracellular proteins, including members of the Frzb or Dickkopf families, inhibit Wnt signaling by competitively binding to Wnt ligands (Leyns et al., 1997) or the LDL receptor-related protein 5/6 (Mao et al., 2001; Mao et al., 2002), respectively. By contrast, BIO, a bona fide drug that inhibits Gsk3, enhances Wnt/β-catenin signaling (Notani et al., 2010). In the cytoplasm, several proteins, including Gsk3, Apc and Axin, form a destruction complex that phosphorylates β-catenin, leading to its degradation (Barker and Clevers, 2000). As such, apc mutant fish have constitutively active Wnt/β-catenin signaling (Hurlstone et al., 2003; Haramis et al., 2006; North et al., 2007; Goessling et al., 2008). In the nucleus, tcf3 is essential for forebrain formation by repressing Wnt caudal target genes (Kim et al., 2000). The dominant-negative form of Tcf3, dn-Tcf, which lacks the β-catenin-binding domain and acts as a repressor, can efficiently promote anterior brain formation (Kim et al., 2000). Given the potential association between cancer and development, we have explored eaf1 and eaf2 activity in vertebrate 1Key Laboratory of Biodiversity and Conservation of Aquatic Organisms, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China. 2State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China.