A non-metabolic function of hexokinase 2 in colorectal cancer: HK2 promotes malignant phenotypes by facilitating UBE3A-Dependent ZNF366 degradation

Hexokinase 2 (HK2), a key enzyme in the Warburg effect, is associated with poor prognosis in colorectal cancer (CRC). HK2 is known to exert glycolysis-independent functions, yet the underlying targets and mechanisms remain unclear. This study aims to investigate the functional role of ZNF366 as a direct target of HK2 in colorectal cancer and reveal a non-metabolic function of HK2 in CRC. HK2-binding proteins were identified by immunoprecipitation and mass spectrometry. ZNF366 expression was assessed by western blotting and qRT-PCR. Cell proliferation, invasion, and migration were evaluated by functional assays. In vivo tumor growth was examined using a xenograft model. Molecular mechanisms were dissected by co-immunoprecipitation and ubiquitination assays. ZNF366 is downregulated in CRC tissues and cells. Overexpression of ZNF366 suppresses cell proliferation, invasion, and migration in vitro, and inhibits tumor growth in vivo, accompanied by decreased levels of pERK1/2, Cyclin D1, and MMP9, whereas ZNF366 knockdown increases their expression, indicating a negative regulatory role of ZNF366 on malignant phenotypes. HK2 and ZNF366 protein levels are inversely correlated in CRC specimens. HK2 reduces ZNF366 protein levels without affecting its mRNA expression. Mechanistically, UBE3A is identified as the E3 ubiquitin ligase for ZNF366. HK2 enhances the interaction between ZNF366 and UBE3A, leading to increased polyubiquitylation and degradation of ZNF366, which in turn correlates with enhanced malignancy of CRC cells. Our findings suggest that HK2 facilitates UBE3A-dependent ZNF366 degradation, which may partially contribute to the tumor-promoting effect of HK2, revealing a novel HK2-UBE3A-ZNF366 regulatory axis that provides insights into the non-metabolic functions of HK2 in CRC.