MELK Inhibition Effectively Suppresses Growth of Glioblastoma and Cancer Stem-Like Cells by Blocking AKT and FOXM1 Pathways

Abstract

Glioblastoma multiforme (GBM) is a devastating disease yet no effective drug treatment has been established to date. Glioblastoma stem-like cells (GSCs) are insensitive to treatment and may be one of the reasons for the relapse of GBM. Maternal embryonic leucine zipper kinase gene (MELK) plays an important role in the malignant proliferation and the maintenance of GSC stemness properties of GBM. However, the therapeutic effect of targeted inhibition of MELK on GBM remains unclear. This study analyzed the effect of a MELK oral inhibitor, OTSSP167, on GBM proliferation and the maintenance of GSC stemness. OTSSP167 significantly inhibited cell proliferation, colony formation, invasion, and migration of GBM. OTSSP167 treatment reduced the expression of cell cycle G2/M phase-related proteins, Cyclin B1 and Cdc2, while up-regulation the expression of p21 and subsequently induced cell cycle arrest at the G2/M phase. OTSSP167 effectively prolonged the survival of tumor-bearing mice and inhibited tumor cell growth in in vivo mouse models. It also reduced protein kinase B (AKT) phosphorylation levels by OTSSP167 treatment, thereby disrupting the proliferation and invasion of GBM cells. Furthermore, OTSSP167 inhibited the proliferation, neurosphere formation and self-renewal capacity of GSCs by reducing forkhead box M1 (FOXM1) phosphorylation and transcriptional activity. Interestingly, the inhibitory effect of OTSSP167 on the proliferation of GSCs was 4-fold more effective than GBM cells. In conclusion, MELK inhibition suppresses the growth of GBM and GSCs by double-blocking AKT and FOXM1 signals. Targeted inhibition of MELK may thus be potentially used as a novel treatment for GBM.

Keywords: OTSSP167; glioblastoma multiforme; glioblastoma stem-like cells; maternal embryonic leucine-zipper kinase; targeted therapy.

Figure 1

OTSSP167 inhibits GBM cell proliferation and colony formation. (A) CCK-8 viability analysis of cells treated with six OTSSP167 concentrations, including 0 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM for 72 h. (B–E) The U87 and LN229 cells were treated with indicated concentrations of OTSSP167 for 24 h, and the EdU assay was performed to assess cell proliferation. Panels (B, C) show the results of the quantitative analysis of the EdU test; panels (D, E) show the representative images of EdU analysis after OTSSP167 treatment of the U87 and LN229 cells. (F–I) OTSSP167 inhibits colony formation in U87 and LN229 cells in a dose-dependent manner. Quantitative analysis of the results of the colony formation experiment was performed. All the Data are presented as means ± SEM. **P < 0.01, ***P < 0.001 compared with the 0.1% DMSO treated group.

Figure 2

OTSSP167 induces cell cycle arrest at the G2/M phase. (A, B) The U87 and LN229 cells were stained with PI after 24 h of OTSSP167 treatment. The results were assessed by flow cytometry. (C, D) Quantitative analysis of cell cycle phase distribution of cells in the control and the OTSSP167 treatment groups. (E) Western blotting analysis of cell cycle-related protein levels in U87 and LN229 cells treated with OTSSP167 for 24 h with indicated antibodies. All the Data are presented as means ± SEM. ***P<0.001.

Figure 3

OTSSP167 inhibit GBM cell migration and invasion. U87 and LN229 cells were treated with 0–100 nM OTSSP167 for 24 h, followed by Transwell migration assay (A–D, no Matrigel) and invasion assay (E–H, addition of Matrigel). All the Data are presented as means ± SEM. **P < 0.01, ***P < 0.001 compared with the 0.1% DMSO treated group.

Figure 4

OTSSP167 reduces MELK protein expression and blocks AKT pathway activation, thereby inhibiting the proliferation and invasion of GBM cells. (A) U87 and LN229 cells were treated with OTSSP167 for 24 h. Western blotting showing the expression levels of MELK, p-AKT(Ser473), AKT, p-mTOR(Ser2448), and p-S6(Thr389) proteins. (B) U87 cells transfected with myr-AKT plasmid were treated with OTSSP167, followed by western blotting to assess changes in p-AKT(Ser473), AKT, and p-S6(Thr389) expression. (C) CCK-8 assay shows the effects of OTSSP167 treatment on U87 cells transfected with myr-AKT plasmid compared to the control group. (D) CCK-8 assay showing the viability of U87 cells treated with 50 nM OTSSP167 and 1 μM MK-2206 (AKT inhibitor) alone or combined OTSSP167 and MK-2206 treatment for 72 h. (E) Measurement of cell proliferation after treating with 50 nM OTSSP167 and 1 μM MK-2206 alone or their combinations by EdU incorporation assay. (F, H) Quantitative analysis of proliferative and invading cell numbers. The numbers of proliferative and invading cells were normalized to that of the control group. (G) U87 cells were incubated with 50 nM OTSSP167 and 1 μM MK-2206 alone or their combinations. Cell invasive abilities were evaluated by transwell assay. Results were expressed as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control group.

Figure 5

OTSSP167 decreases GSCs proliferation and neurosphere formation by inhibiting the phosphorylation and transcriptional activation of FOXM1. (A, B) Effects of OTSSP167 on cell viability in GSC1 (A) and GSC2 (B). Cells treated with OTSSP167 were plated in triplicate wells in stem cell media, and then cell viabilities were assessed at the indicated times (day 0–day 12). In vitro limiting dilution assays of GSC1 (C) and GSC2 (D) treated with indicated does of OTSSP167 or 0.1% DMSO. (E–H) Effects of OTSSP167 on GSCs neurosphere formation. Representative images of GSC1 (E) and GSC2 (G) neurosphere and the quantification of sphere numbers of GSC1 (F) and GSC2 (H) treated with indicated doses of OTSSP167 or DMSO. (I) Immunoblot analysis of MELK, p-FOXM1(Ser35), FOXM1 and β-actin in GSC1 treated with indicated doses of OTSSP167. (J) OTSSP167 inhibits the transactivation ability of FOXM1. Relative FOXM1 luciferase activity normalized with respect to corresponding renilla luciferase activity is shown. (K) The expression levels of FOXM1 were detected by western blot to check the FOXM1 cDNA plasmid transfection efficacy. (L) In vitro cell viability assay of FOXM1 overexpression in GSC1 cells treated with 25 nM OTSSP167 or the vehicle control. All the Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the 0.1% DMSO treated group.

Figure 6

OTSSP167 suppresses tumor growth in vivo and increases the survival of animals bearing intracranial GBM. (A) Representative tumors isolated from the control and OTSSP167-treated groups of subcutaneous tumor model. (B) The mean tumor volumes were assessed at the indicated numbers of days after tumor implantation. (C) Mice bearing U87 xenograft tumor were treated with OTSSP167 (5 μL of 1 μM (dose 1) or 2 μM (dose 2) OTSSP167 in 1% DMSO in PBS per mouse) or vehicle control by intratumoral injection once a week for 4 weeks. Representative images of H&E staining of whole brain sections from control group and OTSSP167 treatment group. (D) Kaplan-Meier survival curves of mice implanted with U87 cells (n=10, **P < 0.01, ***P < 0.001). In vivo animal studies to investigate the effect of OTSSP167 administration on the growth of GSC-driven tumor. Tumor size (E) and survival time (F) were analyzed by using the above same treatment. The survival time of tumor-bearing mice was counted by the end of the 55th day after tumor implantation. (G) Representative IHC staining images of p-AKT(Ser473) and Ki67 expression in U87 xenograft tumor of control and OTSSP167 treatment groups. Sections were counterstained with hematoxylin.