Abstract
Glioblastoma (GBM) is the most commonly diagnosed brain tumor that exhibit high mortality rate and chemotherapy resistance is a major clinical problem. Recent studies suggest that estrogen receptor beta (ERβ), may function as a tumor suppressor in GBM. However, the mechanism(s) by which ERβ contributes to GBM suppression and chemotherapy response remains unknown. We examined the role of ERβ in the DNA damage response of GBM cells, and tested whether ERβ sensitizes GBM cells to chemotherapy. Cell viability and survival assays using multiple epitope tagged ERβ expressing established and primary GBM cells demonstrated that ERβ sensitizes GBM cells to DNA damaging agents including temozolomide (TMZ). RNA-seq studies using ERβ overexpression models revealed downregulation of number of genes involved in DNA recombination and repair, ATM signaling and cell cycle check point control. Gene set enrichment analysis (GSEA) suggested that ERβ–modulated genes were correlated negatively with homologous recombination, mismatch repair and G2M checkpoint genes. Further, RT-qPCR analysis revealed that chemotherapy induced activation of cell cycle arrest and apoptosis genes were attenuated in ERβKO cells. Additionally, ERβ overexpressing cells had a higher number of γH2AX foci following TMZ treatment. Mechanistic studies showed that ERβ plays an important role in homologous recombination (HR) mediated repair and ERβ reduced expression and activation of ATM upon DNA damage. More importantly, GBM cells expressing ERβ had increased survival when compared to control GBM cells in orthotopic GBM models. ERβ overexpression further enhanced the survival of mice to TMZ therapy in both TMZ sensitive and TMZ resistant GBM models. Additionally, IHC analysis revealed that ERβ tumors had increased expression of γH2AX and cleaved caspase-3. Using ERβ-overexpression and ERβ-KO GBM model cells, we have provided the evidence that ERβ is required for optimal chemotherapy induced DNA damage response and apoptosis in GBM cells.
Highlights
Glioblastoma (GBM) is one of the most commonly diagnosed and aggressive form of primary malignant brain tumors in adults[1,2]
The ingenuity pathway analysis (IPA) of differentially expressed genes between U87-empty vector (EV) vs U87-ERβ cells revealed that the ERβ–modulated genes were related to DNA damage check point regulation, DNA damage response, DNA repair, ATM signaling pathways and cell cycle (Fig. 1E)
ERβ overexpression attenuated the DNA damage response genes, we further examined the effect of ERβ expression on the response of GBM cells to various genotoxic agents commonly used for treating GBM (TMZ, lomustine, cisplatin and carboplatin)
Summary
Glioblastoma (GBM) is one of the most commonly diagnosed and aggressive form of primary malignant brain tumors in adults[1,2]. Recent studies showed GBM cells uniquely express ERβ18 and using knock out models it was demonstrated that ERβ has tumor suppression function in GBM19. ERβ agonists affect the sensitivity of malignant pleural mesothelial cells to cisplatin toxicity[21] and the inhibition of ERβ, increases DNA repair, which in turn contributes to developing cisplatin resistance in medulloblastoma cells[22]. Our earlier and other in vitro studies have shown that ERβ agonists increases the sensitivity of GBM cells to chemotherapeutic agents that are currently used such as, TMZ and lomustine[23,24]. We examined the mechanisms by which ERβ sensitizes GBM cells to standard chemotherapy. We have provided evidence in vivo demonstrating the tumor suppressor potential of ERβ and that ERβ sensitizes GBM to TMZ therapy. Our results suggest that ERβ is required for optimal chemotherapy induced DNA damage response and apoptosis in GBM cells
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