HOXD9 Enhances the Release of HMGB1 and Boosts Glycolysis in Glioblastoma under Hypoxic Conditions, Leading to Tumor Growth by Activating the Transcription of PFKFB3

Background: Glioblastoma (GBM) is an aggressive primary brain tumor. The HOX gene family has been implicated in the pathogenesis of different types of tumors. This research aimed to examine the impact of homeobox D9 (HOXD9) in GBM under hypoxic conditions, as well as to elucidate its underlying molecular mechanisms. Methods: The study assessed the differential expression of nine HOXD genes in GBM using the Mann-Whitney U test and identified genes with high correlation with the cancer genome atlas (TCGA)-GBM dataset using receiver operating characteristic (ROC) curves. Prognostic genes of GBM patients were identified through a combination of prognostic Kaplan-Meier (KM) curve and Cox analysis. In vitro experiments were conducted using U87-MG and U251-MG cells, and an animal GBM model was constructed. The study also measured the secretion level of high mobility group box 1 (HMGB1) using enzyme-linked immunosorbent assay (ELISA). Glucose uptake and lactate production levels in cells and tissues were analyzed using kits. The expressions of HOXD9 and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) were detected by immunofluorescence, and chromatin immunoprecipitation (ChIP) validated their relationship. Results: HOXD9 was identified as the target gene, showing a significant correlation between HOXD9 expression and prognostic clinical outcomes. Overexpression of HMGB1 enhanced cell proliferation, migration, and the expression levels of HOXD9 and PFKFB3 and promoted HMGB1 secretion, glucose uptake, and lactate generation. HOXD9 bound to the PFKFB3 promoter region in U87-MG and U251-MG cells. Furthermore, PFKFB3 overexpression partially counteracted the suppressive effects of HOXD9 silencing on tumor formation. Conclusion: HOXD9 promoted hypoxia-induced HMGB1 secretion and glycolysis in GBM through the transcriptional activation of PFKFB3, which in turn promoted tumorigenesis.