Hypoxia-induced NDRG1 C-terminal poly-phosphorylation impairs its tumor suppressor function in renal cell carcinoma

Hypoxia is a critical factor driving tumor invasion and metastasis. N-myc Downstream Regulated Gene 1 (NDRG1), a known suppressor of invasion and metastasis in various cancers including clear cell renal cell carcinoma (ccRCC), remains poorly understood in the context of hypoxic regulation. Notably, the carboxy terminus (C-terminus) of NDRG1 contains multiple phosphorylation sites within a unique three-tandem-repeat sequence that responds to hypoxic conditions. However, the precise regulatory mechanisms and functional significance of phosphorylation in this region remain unexplored. Our research uncovered that hypoxia triggers poly-phosphorylation at the C-terminus of NDRG1, thereby facilitating epithelial-mesenchymal transition (EMT) in ccRCC. NDRG1 knockdown alone was sufficient to induce EMT, augmenting the invasive and metastatic capabilities of ccRCC cells. Specifically, hypoxia-induced phosphorylation at the C-terminus (sites 328/330 and 346/356/366, phosphorylated by SGK1) of NDRG1 enhances its SUMOylation and ubiquitination, leading to NDRG1 degradation. NDRG1 typically forms a complex with E-cadherin and β-catenin to suppress WNT signaling; however, this complex is disrupted by phosphorylation at the 346/356/366 sites. In vivo studies demonstrated that NDRG1 knockdown expedited tumor growth and pulmonary metastasis, but the re-expression of phosphorylation-deficient mutants, particularly at sites 328/330 and 346/356/366, significantly mitigated these tumor-promoting effects. Our study demonstrates that hypoxia-induced C-terminal poly-phosphorylation of NDRG1 promotes its degradation, activating the WNT pathway and driving ccRCC malignancy. These findings underscore the role of NDRG1 phosphorylation in ccRCC progression under hypoxic conditions and highlight potential therapeutic targets for intervention.