Function of DHX33 in Promoting Warburg Effect and Regulation of DHX33 by GSK-3β

Cancer cells metabolize glucose through glycolysis to promote cell proliferation even with abundant oxygen. Multiple glycolysis genes are deregulated during cancer development. Despite the intensive effort, the cause of the deregulation of multiple glycolysis remains partially understood. In the present study, we demonstrate that DEAH-box helicase 33 (DHX33) plays a critical role in promoting the Warburg effect in cancer cells. DHX33 deficient cells have markedly reduced glycolysis activity. Through RNA-seq analysis, we found that multiple critical genes in the Warburg effect were downregulated after DHX33 deficiency. These genes include lactate dehydrogenase A (LDHA), pyruvate dehydrogenase kinase 1 (PDK1), pyruvate kinase muscle isoform 2 (PKM2), enolase 1 (ENO1), enolase 2 (ENO2), and hexokinase 1/2 (HK1/2). Using the LDHA, PDK1, and PKM2, we revealed that DHX33 altered the epigenetic marks around the glycolytic gene's promoter, through DHX33 in complex with growth arrest and DNA-damage-inducible 45 alpha (Gadd45a). DHX33 is required for loading the Gadd45a and tet methylcytosine dioxygenase 1 (TET1) at the promoter sites of glycolytic genes, leading to active DNA demethylation and enhancing histone H4 acetylation. We conclude that DHX33 changes local epigenetic marks in favor of the transcription of glycolysis genes, thereby promoting cancer cell proliferation. Our study highlights the significance of RNA helicase DHX33 in the Warburg effect and therapeutic role in human cancers. Elevated DHX33 protein plays a pivotal role in cancer cells. However, its post-translational regulation is still poorly understood. In this study, we report that knockdown of GSK-3β can cause DHX33 protein accumulation, and inhibition of GSK-3β activity prolonged DHX33 degradation time. We also confirm that GSK-3β interacts with DHX33, and the phosphorylation sites on mouse DHX33 are identified at T482. When the phosphorylation site T482 is altered by mutagenesis, DHX33 degradation times are changed. The T482D mutation DHX33, which mimics the phosphorylation state at T482, has enhanced the ubiquitination level. This further confirms that GSK-3β regulates DHX33 through phosphorylation mediated ubiquitination. The ubiquitinated level of DHX33 in helicase-defective K94R mutant cells has reduced compared to the WT group, which has confirmed that K94 is a ubiquitination site on DHX33 helicase. In summary, our result provides the first evidence for post-translational regulation of DHX33 by GSK-3β through phosphorylation mediated ubiquitination, thereby providing new therapeutic targets for cancer patients.