HIF-1α silencing downregulates SLC37A2, SLC37A3, and G6PC3 gene expression and impacts glioblastoma stemness features...

Glioblastoma (GBM), a highly aggressive brain tumor, exhibits a notable capacity for metabolic reprogramming, which may support tumor cell adaptation to the rapid growth and survival demands within the hypoxic solid tumor microenvironment (TME). Hypoxia has been implicated in promoting features associated with cancer stem cell (CSC) phenotypes and chemoresistance. Recent preclinical studies have employed three-dimensional (3D) neurosphere models to investigate these phenotypes, although the metabolic changes associated with such models remain incompletely characterized.

GBM cells were cultured as two-dimensional (2D) monolayers or as neurospheres using the 3D hanging drop culture model. Total RNA was extracted using TRIzol, and gene expression was assessed via cDNA arrays and RT-qPCR. Gene silencing was performed using specific siRNAs. Transcriptomic analysis of clinical sample expression profiles was conducted using data from the Genotype-Tissue Expression (GTEx) database.

We observed that neurospheres from human GBM-derived cell lines (U87, U118, U138, and U251) were associated with increased expression of hypoxia-inducible factor (HIF)-1α compared to 2D monolayers. This was accompanied by elevated transcript levels of genes involved in adaptive and angiogenic responses, including GLUT1, VEGF, SLC37A2, SLC37A3, and G6PC3. Silencing HIF-1α correlated with reduced neurosphere size and a decrease in markers commonly associated with CSC phenotypes. Similarly, knockdown of SLC37A2, SLC37A3, and G6PC3 was linked to smaller spheroids and reduced expression of GLUT1 and VEGF, without affecting HIF-1α levels. Differential regulation of CSC markers was observed, with PROM1, ABCB5, CD44, and FGFR2 appearing to be influenced by G6PC3/HIF-1α, while ABCG2, DLL1, GATA3, PTCH1, and FLOT2 were more closely associated with SLC37A2/HIF-1α.

Our findings suggest that GBM-derived 3D neurosphere cultures exhibit HIF-1α-regulated molecular features consistent with CSC and a potential mimicry of chemoresistance-associated traits rather than a direct demonstration of chemoresistance. These correlative observations support a potential link between metabolic reprogramming and the emergence of such phenotypes in response to hypoxic stress. Further investigation is needed to establish causal relationships and to better understand the underlying mechanisms.