Cholesterol in glioblastoma: Impaired Hh signaling enhances epigenetic modifiers and decreases CAV1.

Glioblastoma multiform (GBM) exhibit heterogeneity. 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. Compared to monolayer cells spheroids had higher expression of stemness markers like CD133, CD44, PAX6 and reduced expression of differentiation marker. Cancer cells modulate 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 genes (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, along with the loss of the stemness factors. However, there is enhanced expression of epigenetic chromatin modification enzymes, including DNMT1 and KDM5A. Tracking into the root cause of silencing of CAV1 gene, we found that CAV1 gene promoter is methylated by DNMT1, and H3K4me3 level was depleted due to enhanced KDM5A mediated demethylation. CAV1 gene silencing by siRNA validated its role in maintenance of stem-like phenotype and metabolic alterations of GBM spheroids. Collectively, this study demonstrated the regulatory role of Caveolin1 and cholesterol in maintaining stem-like characteristics of GBM spheroids and the importance of tumor models in better understanding of the molecular mechanism of GBM.Key words: Glioblastoma, cholesterol biosynthesis, stemness, Caveolin1, DNMT1, KDM5A, Gli1.