P01.20 * EPIGENETIC TREATMENT AND INDUCTION OF DIFFERENTIATION IN GLIOBLASTOMA MULTIFORME NEUROSPHERE CELLS LEADS TO DOWNREGULATION OF EGFR, EGFRVIII AND NESTIN TOGETHER WITH REDUCED COLONY FORMATION IN VITRO

Abstract

It is suggested that Glioblastoma Multiforme (GBM) tumors contain a subpopulation of cells with stem cell features, termed brain cancer stem-like cells (bCSC), which are thought to be involved in tumorigenesis, treatment resistance and recurrence. The epidermal growth factor receptor (EGFR) and the constitutively active deletion variant, termed EGFRvIII, are frequently expressed in GBM and EGFRvIII is correlated to chemo- and radiation resistance and poor patient prognosis. Studies suggest that EGFRvIII is linked to the bCSC subpopulation and that its expression can be regulated by epigenetic treatment. The bCSC are capable of differentiating into more mature cells, and it has been proposed differentiation therapy of bCSC could reduce the self-renewing capacity and decrease the production of clones otherwise able to populate the tumor. Here we wanted to elucidate how the EGFRvIII expression is regulated by epigenetic events during induced differentiation, and which role it plays in the tumorigenic potential. A GBM neurosphere cell culture containing bCSC and expressing endogenous EGFR and EGFRvIII under serum-free conditions was used. When inducing differentiation of the GBM neurospheres by treatment with serum this led to a downregulation of EGFR and EGFRvIII expression. However, upon transfer of the neurospheres back to NB serum-free media the EGFRvIII expression was re-gained. When treating the cells over time with a HDAC inhibitor (TSA) the expression of EGFR, EGFRvIII and the stem cell marker Nestin was decreased. Furthermore, clonal growth in vitro was reduced in a dose-dependent manner by treatment with TSA as shown by a reduction of neurosphere colonies formed in soft agar. Treatment of the neurospheres with the DNA methyl-transferase inhibitor (AZA) reduced their expression of Nestin, whereas the expression of both EGFR and EGFRvIII seemed to be unaffected. When combining serum and TSA treatment, no further downregulation of EGFR/EGFRvIII could be observed. However, GFAP, CNPase and βIII-tubulin were all downregulated as compared to treatment with serum alone, suggesting that TSA impairs serum-induced differentiation of the neurospheres. In addition, treatment with a combination of AZA and serum, for 24 hours, did not affect the expression of EGFR/EGFRvIII but led to increased expression of bIII tubulin, GFAP and CNPase, suggesting induction of neurosphere differentiation. In conclusion these results indicate that treatment with drugs that induce differentiation or modify epigenetic gene expression leads to a decrease in the bCSC subpopulation, possibly via downregulation of signaling pathways, including EGFR/EGFRvIII, involved in bCSC maintenance.