Desert Hedgehog Downregulation Mediates Proliferative Inhibition by γ-Glutamylcyclotransferase Knockdown in Murine Glioblastoma Stem Cells

Glioblastoma is the most frequent type of adult-onset malignant brain tumors and has a very poor prognosis. A cell population called glioblastoma stem cells has been shown to be one of the mechanisms by which glioblastoma acquires therapy resistance. Therefore, there is a need to establish novel therapeutic strategies useful for inhibiting the cell population. γ-glutamylcyclotransferase (GGCT) is an enzyme involved in the synthesis and metabolism of glutathione, which is highly expressed in a wide range of cancer types including glioblastoma, and inhibition of its expression has been reported to have antitumor effects on various cancer types. In this study, we searched for pathways affected by the growth-promoting effect of GGCT overexpression in mouse embryonic fibroblasts NIH3T3 by comprehensive gene expression analysis and found the hedgehog pathway. Knockdown of GGCT inhibited proliferation of glioblastoma stem cells derived from a mouse glioblastoma model and caused decreased expression of desert hedgehog (Dhh), a representative ligand of the pathway, and the downstream target Gli1. To analyze the contribution of reduced Dhh, we activated the hedgehog pathway by forced expression of Dhh. We found that Dhh overexpression significantly restored the growth suppressive effect of GGCT knockdown. The results of this study indicate that high GGCT expression is important for Dhh expression and activation of the hedgehog pathway, which is required to maintain glioblastoma stem cell proliferation. Therefore, inhibition of GGCT function may be useful in suppressing stemness of glioblastoma stem cells accompanied with activation of the hedgehog pathway. To test the effect of overexpression of GGCT on the gene expression, mouse embryonic fibroblast cell line NIH3T3 was transfected with GGCT expression retroviral vector or control vector. Three independent experiments were performed and GGCT-overexpressing NIH3T3 cells were compared to control cells.