Lowering of Cholesterol Hampers Glioblastoma Stem Cell Proliferation in Spheroids Through impaired Hh signaling: upregulation of epigenetic chromatin modifiers and down regulation CAV1 and Stem Cell markers

Glioblastoma multiform (GBM) harbours tumor heterogeneity and shows little efficacy to the current treatment strategies. Persistence of glioma stem cells (GSCs) are the root cause of tumor recurrence and drug resistance. So, targeting GSCs can be a better therapeutic strategy to tackle GBM. To mimic the tumor microenvironment, we have developed tumor spheroids by hanging drop method using U87MG and Ln229 cells with an enriched GSC population. Compared to monolayer cells spheroids had higher expression of stemness markers like CD133, CD44, PAX6 and reduced expression of differentiation marker. Cancer cells rewire the metabolic pathways to sustain high proliferation. Among the metabolic pathways, cholesterol biosynthetic pathways are mostly dysregulated in cancers including GBM. The spheroids showed high expression of cholesterol biosynthetic gene (HMGCR, DHCR24), and Caveolin1 (Cav1). Targeting cholesterol metabolism by lovastatin resulted in depletion of cellular cholesterol levels, including in plasma membrane. Lowering of cholesterol affected membrane fluidity and hampered Hh signaling by lowering Gli1; consequently, causing downregulation of HMGCR, DHCR24, Cav1, and IDH3A, as well as loss of the stemness factors. However, there are enhanced expression of epigenetic chromatin modification enzymes, including DNMT1 and KDM5A. Tracking into the root cause of silencing of Cav1 gene, we found CAV1 gene promoter is methylated by DNMT1, and H3K4me3 level depleted due to enhanced KDM5A mediated demethylation. CAV1 gene silencing by siRNA validated its role in stemness maintenance and metabolic reprogramming of GSCs. Our findings suggest that, lovastatin has therapeutic potential and illustrates the importance of tumor spheroid models better understanding molecular mechanisms of GBM.