Data from Rad6B Is a Positive Regulator of β-Catenin Stabilization

Mutations in β-catenin or other Wnt pathway components that cause β-catenin accumulation occur rarely in breast cancer. However, there is some evidence of β-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of β-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces β-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, β-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus–Wnt-1 mammary tumor—derived cells, implicating Rad6B in physiologic regulation of β-catenin stability and activity. Ubiquitinated β-catenin was detectable in chromatin immunoprecipitations performed with β-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of β-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular β-catenin but with minimal effects on cell membrane–associated β-catenin. Measurement of β-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular β-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates β-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of β-catenin, reveal a positive regulatory feedback loop between Rad6B and β-catenin and a novel mechanism of β-catenin stabilization/activation in breast cancer cells. [Cancer Res 2008;68(6):1741–50]