Multi-modal mechanisms of the metastasis suppressor NDRG1: Inhibition of WNT/b-catenin signaling by stabilization of protein kinase Cα

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/(3-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased (3- catenin and downregulated glycogen synthase kinase-3(3 (GSK3(3) protein levels and its activation. However, (3-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3(3 was significantly increased after NDRG1 overexpression, suggesting a GSK-3(3-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Ca (PKCa), with PKCa silencing preventing (3-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing (3- catenin expression. Further, NDRG1 and PKCa were demonstrated to associate, with PKCa stabilization occurring after NDRG1 overexpression. PKCa half-life increased from 1.5 +/- 0.8 h (3) in control cells to 11.0 +/- 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCa and PKCa stabilization, resulting in (3-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCa, NDRG1, and (3-catenin was identified, with the formation of a potential metabolon that promotes the latter (3-catenin phosphorylation. This antioncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleocytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in (3-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased (3-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCa and inhibit WNT signaling.