Inhibition of Gli2 suppresses tumorigenicity in glioblastoma stem cells derived from a de novo murine brain cancer model

Prognosis of glioblastoma remains poor despite a great deal of research. In glioblastoma, the existence of glioblastoma stem cells (GSCs) has been shown, which are responsible for tumorigenesis, invasive capacity, and therapy resistance. One of cancer stem cell markers, Leucine-rich repeat-containing G-protein coupled receptor (Lgr5) plays a role in maintenance of GSCs, however, properties of the Lgr5 positive GSCs have not been fully understood. We applied the Sleeping-Beauty transposon-induced glioblastoma model to the Lgr5-GFP transgenic mice and sorted the GFP-positive cells from the neurosphere cultures derived from the mouse glioblastoma tissues. We found that the GFP-positive GSCs exhibited higher expression of Gli2 using a global gene expression analysis. Gli2 knockdown using lentiviral-mediated shRNA downregulates both the Hedgehog- and Wnt signaling pathway-related genes including Lgr5, suppresses tumor cell proliferation and invasion capacity, and induces apoptosis. Pharmacological Gli inhibition by GANT61 also suppresses tumor cell proliferation. Furthermore, the orthotopic transplantation model revealed that silencing of Gli2 suppresses tumorigenicity of GSCs in vivo. These findings demonstrate that Gli2 affects not only Hh pathway but also Wnt pathway and plays an important role in the GSC maintenance, suggesting that Gli2 may be a potential therapeutic target for glioblastoma treatment. To test the effect of suppression of Gli2 on the gene expression, murine GSC was tranducted with lentiviral non-target (control) or Gli2 shRNA. Gli2-knockdown GSCs were compared to control cells.

尽管已有大量研究投入，胶质母细胞瘤（glioblastoma）的预后仍然不佳。研究已证实胶质母细胞瘤中存在胶质母细胞瘤干细胞（glioblastoma stem cells, GSCs），这类细胞与肿瘤发生、侵袭能力及治疗抵抗密切相关。作为癌症干细胞标志物之一的富含亮氨酸重复序列G蛋白偶联受体（Leucine-rich repeat-containing G-protein coupled receptor, Lgr5），在GSCs的维持中发挥作用，但目前对于Lgr5阳性GSCs的特性仍未完全阐明。我们将睡美人转座子（Sleeping-Beauty transposon）诱导的胶质母细胞瘤模型应用于Lgr5-GFP转基因小鼠，并从源自小鼠胶质母细胞瘤组织的神经球培养物中分选得到GFP阳性细胞。通过全基因表达分析，我们发现GFP阳性GSCs的Gli2表达水平更高。利用慢病毒介导的短发夹RNA（short hairpin RNA, shRNA）进行Gli2敲低，可下调包括Lgr5在内的Hedgehog信号通路与Wnt信号通路相关基因，抑制肿瘤细胞增殖与侵袭能力，并诱导细胞凋亡。使用GANT61进行药理学Gli抑制也可抑制肿瘤细胞增殖。此外，原位移植模型实验证实，Gli2沉默可在体内（in vivo）抑制GSCs的致瘤能力。上述研究结果表明，Gli2不仅可调控Hh通路，还可影响Wnt通路，且在GSC的维持中发挥重要作用，提示Gli2或可成为胶质母细胞瘤治疗的潜在靶点。为验证Gli2抑制对基因表达的影响，我们使用靶向非特异性（对照）或Gli2的shRNA慢病毒转导小鼠GSCs，并将Gli2敲低的GSCs与对照细胞进行对比分析。