Anti-PYGO2 (aa 1-368) polyclonal antibody

This study was undertaken to define whether differences in the expression of Wnt pathway components are present between normal colonic mucosa, early (tubular) adenomas and villous adenomas which have a higher malignant potential. Normal mucosa, tubular adenomas and villous adenomas were obtained from twelve patients. RNA was isolated and utilized for Wnt pathway-specific membrane array expression analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and fluorescent immunohistochemistry (IHC) were utilized for confirmatory analyses. Fifteen Wnt pathway-related genes showed differential expression between villous adenomas and normal mucosa and villous and tubular adenomas at a significance level of p<0.01. Genes involved in canonical Wnt (B-catenin) signaling with increased expression in villous adenomas included wnt1, fz2, csnk2A2, pygo2, pygo1, frat2 and myc, the latter confirmed by qRT-PCR and IHC. Myc protein expression was confined primarily to stromal components of villous adenomas. Genes involved in non-canonical Wnt signaling with increased expression in villous adenomas included rho-u, daam1, damm2, cxxc4 and nlk. Successive increases in the expression of ctnnb1 (B-catenin) from normal to tubular adenomas to villous adenomas was seen. The Wnt pathway gene expression profile can differentiate between tubular and villous adenomas. These data suggest that Wnt signaling regulation changes during the progression from normal mucosa to tubular adenomas to villous adenomas. Expression of Myc in adenoma stroma suggests a dynamic signaling network within adenomas between mucosal and stromal elements. Inhibition of the Wnt pathway may provide a novel approach for cancer prevention in patients with benign tubular adenomas.

Resent studies have identified Pygopus as a core component of the beta-catenin/T-cell factor (TCF)/lymphoid-enhancing factor 1 (LEF) transcriptional activation complex required for the expression of canonical Wg/Wnt target genes in Drosophila. However, the biochemical involvement of mammalian Pygopus proteins in beta-catenin/TCF/LEF gene activation remains controversial. In this study, we perform a series of molecular/biochemical experiments to demonstrate that Pygo2 associates with histone-modifying enzymatic complexes, specifically the MLL2 histone methyltransferase (HMT) and STAGA histone acetyltransferase (HAT) complexes, to facilitate their interaction with beta-catenin and to augment Wnt1-induced, TCF/LEF-dependent transcriptional activation in breast cancer cells. We identify a critical domain in Pygo2 encompassing the first 47 amino acids that mediates its HMT/HAT interaction. We further demonstrate the importance of this domain in Pygo2's ability to transcriptionally activate both artificial and endogenous Wnt target genes and to expand breast cancer stem-like cells in culture. This work now links mechanistically Pygo2's role in histone modification to its enhancement of the Wnt-dependent transcriptional program and cancer stem-like cell expansion.