NDRG1 promotes growth of hepatocellular carcinoma cells by directly interacting with GSK-3β and Nur77 to prevent β-catenin degradation

The N-myc downstream regulated gene 1 (NDRG1) is significantly associated with advanced tumor stages and poor survival of hepatocellular carcinoma (HCC), thereby implicating it as a potential target for HCC treatment. We aim to further understand its biological roles in hepatocarcinogenesis, as a means to exploit it for therapeutic purposes. By screening using the ProtoArray (R) Human Protein Microarrays, we identified glycogen synthase kinase 3 beta (GSK-3 beta) and the orphan nuclear receptor (Nur77) as potential interaction partners of NDRG1. These interactions were confirmed in HCC cell lines in vitro by co-immunoprecipitation; and co-localizations of NDRG1 with GSK-3 beta and Nur77 were observed by immunofluorescence staining. Additionally, high levels of NDRG1 competitively bind to GSK-3 beta and Nur77 to allow beta-catenin to escape degradation, with consequent elevated levels of downstream oncogenic genes. In vivo, we consistently observed that NDRG1 suppression in HCC xenografts decreased beta-catenin levels and its downstream target Cyclin D1, with concomitant tumor growth inhibition. Clinically, the over-expression of NDRG1 in HCC patient samples is positively correlated with GSK-3 beta-9ser (vertical bar R vertical bar = 0.28, p = 0.01), Nur77 (vertical bar R vertical bar = 0.42, p < 0.001), and beta-catenin (vertical bar R vertical bar = 0.32, p = 0.003) expressions. In conclusion, we identified GSK-3 beta and Nur77 as novel interaction partners of NDRG1. These protein-protein interactions regulate the turnover of beta-catenin and subsequent downstream signaling mediated by beta-catenin in HCC cells, and provides potential targets for future therapeutic interventions.