Glioblastomas (GBM) are the most frequent and aggressive brain tumors. 17β-estradiol (E2) increases proliferation, migration, and invasion of human GBM cells; however underlying mechanisms are no fully understood. Zeste 2 Enhancer Homologous enzyme (EZH2) is a methyltransferase part of Polycomb 2 repressor complex (PRC2). In GBM, EZH2 is overexpressed and involved in the cell cycle, migration, and invasion processes. We studied the role of EZH2 in the pro-oncogenic actions of E2 in human GBM cells. EZH2 gene silencing and pharmacological inhibition of EZH2 blocked proliferation, migration, and invasion of GBM cells induced by E2. We identified in silico additional putative estrogen response elements (EREs) at the EZH2 promoter, but E2 did not modify EZH2 expression. In silico analysis also revealed that among human GBM samples, EZH2 expression was homogeneous; in contrast, the heterogeneous expression of estrogen receptors (ERs) allowed the classification of the samples into groups. Even in the GBM cluster with high expression of ERs and those of their target genes, the expression of PCR2 target genes did not change. Overall, our data suggest that in GBM cells, pro-oncogenic actions of E2 are mediated by EZH2, without changes in EZH2 expression and by mechanisms that appear to be unrelated to the transcriptional activity of ERs.

Glioblastoma (GB) is the most frequent primary brain tumor with a very poor prognosis. Sex hormones are crucial players in the development of GBs. 17 β-estradiol (E2) signaling is involved through its corresponding intracellular receptors [estrogen receptor α (ERα) and β (ERβ)] in GB cell proliferation and progression. E2 activates G-protein coupled estrogen receptor (GPER), leading to rapidly occurring effects, independently of gene transcription. GPER activation is involved in tumor progression in various cancer types. Currently, available data concerning the occurrence and role of GPER in GB are very limited. In the present study, it was observed that GPER was expressed in human brain tumor cell lines [U251 (astrocytoma-derived cell line), U87, LN229 and T98 (glioblastoma-derived cell line)]. Immunofluorescence assays revealed that GPER localizes in the plasma membrane, cytoplasm and nucleus. An in silico analysis identified two potential E2 response elements in the promoter region of the GPER gene. E2 increased GPER expression in the U251, U87 and LN229 cell lines. Molecular modeling data derived from in silico analysis predicted the three-dimensional conformation of GPER, and docking analysis identified potential binding sites of E2 and its specific agonist, G1. Taken together, these results indicate that GPER may be differentially expressed in human GB cell lines with E2 possibly upregulating GPER expression. The present study raises further questions about the implications of GPER-mediated E2 signaling in the biology of GBs.

Glioblastomas are the most aggressive and common primary brain tumors in adults. Glioblastoma cells have a great capacity to migrate and invade the brain parenchyma, often reaching the contralateral hemisphere. Progesterone (P4) and its metabolite, allopregnanolone (3α-THP), promote the migration and invasion of human glioblastoma-derived cells. P4 induces migration in glioblastoma cells by the activation of the proto-oncogene tyrosine-protein kinase Src (cSrc) and focal adhesion kinase (Fak). In breast cancer cells, cSrc and Fak promote invasion by increasing the expression and activation of extracellular matrix metalloproteinases (MMPs). However, the mechanism of action by which P4 and 3a-THP promote invasion in glioblastoma cells remains unclear. The effects of P4 and 3α-THP on the protein expression levels of MMP-2 and -9 and the participation of cSrc in progestin effects in U251 and U87 human glioblastoma-derived cells were evaluated. It was determined by western blotting that the P4 increased the protein expression level of MMP-9 in U251 and U87 cells, and 3α-THP increased the protein expression level of MMP-9 in U87 cells. None of these progestins modified MMP-2 protein expression levels. The increase in MMP-9 expression was reduced when the intracellular progesterone receptor and cSrc expression were blocked with small interfering RNAs. Cell invasion induced by P4 and 3α-THP was also blocked by inhibiting cSrc activity with PP2 or by cSrc gene silencing. These results suggest that P4 and its metabolite 3α-THP induce the invasion of glioblastoma cells by increasing MMP-9 expression through the cSrc kinase family. The results of this study provide information of interest in the context of targeted therapies against molecular pathways involved in glioblastoma invasion.