Forkhead Box M1 Is Essential for Nuclear Localization of Glioma-associated Oncogene Homolog 1 in Glioblastoma Multiforme Cells by Promoting Importin-7 Expression

Abstract

The transcription factors glioma-associated oncogene homolog 1 (GLI1), a primary marker of Hedgehog pathway activation, and Forkhead box M1 (FOXM1) are aberrantly activated in a wide range of malignancies, including glioma. However, the mechanism of nuclear localization of GLI1 and whether FOXM1 regulates the Hedgehog signaling pathway are poorly understood. Here we found that FOXM1 promotes nuclear import of GLI1 in glioblastoma multiforme cells and thus increases the expression of its target genes. Conversely, knockdown of FOXM1 expression with FOXM1 siRNA abrogated its nuclear import and inhibited the expression of its target genes. Also, genetic deletion of FOXM1 in mouse embryonic fibroblasts abolished nuclear localization of GLI1. We observed that FOXM1 directly binds to the importin-7 (IPO7) promoter and increases its promoter activity. IPO7 interacted with GLI1, leading to enhanced nuclear import of GLI1. Depletion of IPO7 by IPO7 siRNA reduced nuclear accumulation of GLI1. In addition, FOXM1 induced nuclear import of GLI1 by promoting IPO7 expression. Moreover, the FOXM1/IPO7/GLI1 axis promoted cell proliferation, migration, and invasion in vitro. Finally, expression of FOXM1 was markedly correlated with that of GLI1 in human glioblastoma specimens. These data suggest that FOXM1 and GLI1 form a positive feedback loop that contributes to glioblastoma development. Furthermore, our study revealed a mechanism that controls nuclear import of GLI1 in glioblastoma multiforme cells.

Keywords: FOXM1; GLI1; Hedgehog signaling pathway; Importin-7; brain tumor; gene regulation; molecular cell biology; signal transduction.

FIGURE 1.

FOXM1 promotes nuclear import of GLI1 in a DNA binding-dependent manner. A, IB analysis of nuclear FOXM1 and GLI1 protein expression in Hs683, SW1783, HFU251, and U87 cells. B, attenuation of nuclear import of GLI1 by knockdown of FOXM1 expression. Left: U87 cells were transfected with siFOXM1 or control siRNA (siCTRL). After 24 h of transfection, cells were treated with 5 nm LMB for 8 h. Cells were then stained with an anti-GLI1 antibody and a FITC-conjugated anti-rabbit secondary antibody (green, GLI1), and the nuclei were visualized via staining with DAPI (blue). Scale bars, 20 μm. Right: results are indicated as the percentage of cells with mostly cytoplasmic fluorescence, mostly nuclear fluorescence, or both cytoplasmic and nuclear fluorescence. C, inhibition of nuclear import of GLI1 by FOXM1 deficiency. Left: Foxm1^fl/fl and Foxm1^−/− mouse embryonic fibroblasts were treated with 5 nm LMB for 8 h and then stained. Scale bars, 20 μm. Right: results are indicated as the percentage of cells with mostly cytoplasmic fluorescence, mostly nuclear fluorescence, or both cytoplasmic and nuclear fluorescence. D, promotion of nuclear import of GLI1 by FOXM1 in a DNA binding-dependent manner. Hs683 cells were transfected with the indicated plasmids, nuclear extracts from the cells were prepared, and the GLI1 expression in the extracts was measured. Lamin B was used as a loading control for nuclear fractions. E, induction of decreased nuclear GLI1 expression by FOXM1 depletion. U87 cells were transfected with siFOXM1 or control siRNA (siCTRL). After 36 h of transfection, nuclear cell lysates were subjected to SDS-PAGE and IB analysis using anti-GLI1 or anti-FOXM1 antibodies. F, enhancement of GLI1 target gene expression by FOXM1. Expression of Ptch1, Ptch2, CCND2, and IL-6 mRNA from Hs683 cells transfected with a control vector or FLAG-FoxM1 was measured using quantitative real-time RT-PCR. The mean ± S.D. values for triplicate samples from a representative experiment are presented. *, p < 0.05; **, p < 0.01. G, induction of decreased GLI1 target gene expression by knockdown of FOXM1 expression. U87 cells were transfected with siFOXM1 or control siRNA (siCTRL). The experiment was performed as described in F. The mean ± S.D. values for triplicate samples from a representative experiment are presented.***, p < 0.001.

FIGURE 2.

IPO7 is a direct transcriptional target of FOXM1. A, increased IPO7 expression induced by FOXM1. Hs683 cells were transfected with an empty vector (CTRL) or FOXM1 expression vector. Cell lysates were collected at 48 h after transfection and subjected to IB analysis with the indicated antibodies. B, schematic of the human IPO7 promoter. The sequences of the FOXM1-binding elements are shown. C, results of a ChIP assay performed with U87 cells. Chromatin fragments of the cells were immunoprecipitated with an anti-FOXM1 antibody or control IgG and subjected to PCR using primers for three FOXM1-binding sites. One percent of the total cell lysates was subjected to PCR before IP as an input. D and E, HEK 293T (D) and Hs683 (E) cells were transfected with the human IPO7 promoter and indicated plasmids. The luciferase activity of the cells was then determined. Each error bar indicates that the variation in the mean results from three independent experiments. ***, p < 0.001. F and G, U87 (F) and HFU251 (G) cells were transfected with the human IPO7 promoter and siFOXM1 or a control siRNA (siCTRL). The luciferase activity of the cells was then determined. Each error bar indicates the variation in the mean results from three independent experiments. **, p < 0.01; ***, p < 0.001.

FIGURE 3.

IPO7 is a key nuclear transporter for GLI1 by interacting with GLI1 protein. A, expression of FLAG-GLI1 in HEK 293T cells and IP of it using an anti-FLAG antibody. GLI1-bound IPO7 protein was detected in the cells using an IB with an anti-IPO7 antibody. Whole-cell lysates were directly subjected to immunoblotting with FLAG or IPO7 antibody as an input. B, co-IP of endogenous GLI1 with IPO7 in U87 cells. IPO7 was immunoprecipitated, and the amount of IPO7 bound to GLI1 was determined using an IB with an anti-GLI1 antibody. C, attenuation of nuclear import of GLI1 by knockdown of IPO7 expression. Top: U87 cells were transfected with siRNAs targeting IPO7 (siIPO7) or control siRNA (siCTRL). After 24 h of transfection, cells were treated with 5 nm LMB for 8 h. Cells were then stained with an anti-GLI1 antibody and an FITC-conjugated anti-rabbit secondary antibody (green, for GLI1), and the nuclei were visualized via staining with DAPI (blue). Scale bars, 20 μm. Bottom: results are indicated as the percentage of cells with mostly cytoplasmic fluorescence, mostly nuclear fluorescence, or both cytoplasmic and nuclear fluorescence. D, knockdown of IPO7 expression decreased GLI1 target gene expression. U87 cells were transfected with siRNAs targeting IPO7 or control siRNA. Expression of Ptch1, Ptch2, CCND2, and IL-6 mRNA was measured using quantitative real-time RT-PCR. The mean ± S.D. values for triplicate samples from a representative experiment are presented. **, p < 0.01; ***, p < 0.001.

FIGURE 4.

FOXM1 induces nuclear import of GLI1 by promoting IPO7 expression. A, left: after 24 h of transfection with the indicated siRNA and/or plasmids, U87 cells were treated with 5 nm LMB for 8 h. Cells were then stained with an anti-GLI1 antibody and an FITC-conjugated anti-rabbit secondary antibody (green, for GLI1), and the nuclei were visualized via staining with DAPI (blue). Scale bars, 20 μm. Right: results are indicated as the percentage of cells with mostly cytoplasmic fluorescence, mostly nuclear fluorescence, or both cytoplasmic and nuclear fluorescence. B, U87 cells were transfected with the indicated siRNA and/or plasmids, nuclear extracts were prepared, and the GLI1 expression was measured. Lamin B was used as a loading control for nuclear fractions. C, U87 cells were transfected with the indicated siRNA and/or plasmids. Expression of Ptch1, Ptch2, CCND2, and IL-6 mRNA was measured using quantitative real-time RT-PCR. The mean ± S.D. values for triplicate samples from a representative experiment are presented. **, p < 0.01; ***, p < 0.001. NS, not significant.

FIGURE 5.

The FOXM1/IPO7/GLI1 axis promotes cell proliferation, migration, and invasion. A, U87 cells were transfected with the indicated siRNA and/or plasmids, and their proliferation was analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are presented as the mean ± S.D. values from three independent experiments. siCTRL, control siRNA. ***, p < 0.001. B, left: U87 cells were transfected with the indicated siRNA and/or plasmids, and their growth was examined using a monolayer colony formation assay. Scale bars, 10 mm. Right: results of quantitative analysis of colony numbers shown as the mean ± S.D. values from three independent experiments. ***, p < 0.001. C, U87 cells were transfected with the indicated siRNA and/or plasmids, and their migration was detected using a wound healing assay. The cell motility was quantified by measuring the distance between the invading front of cells in six randomly selected microscopic fields for each condition and time point. The degree of motility is expressed as the percentage of wound closure as compared with the zero time point. The mean ± S.D. values from three independent experiments are presented. **, p < 0.01. D, left: U87 cells were transfected with the indicated siRNA and/or plasmids and subjected to an in vitro invasion assay. Scale bars, 200 μm. Right: quantitative analysis of invasive U87 cells shown as the mean ± S.D. results from three independent experiments. ***, p < 0.001.

FIGURE 6.

FOXM1 expression positively correlates with GLI1 expression in human GBM specimens. A, immunohistochemical staining of 40 human GBM specimens with anti-FOXM1 and anti-GLI1 antibodies was carried out. Photographs of two representative specimens are shown. Scale bars, 200 μm. B, semiquantitative scoring was carried out (r = 0.803; p < 0.001 (Pearson correlation coefficient)) for all 40 GBM specimens. Note: Some of the dots on the graph represent more than one specimen (that is, some scores overlapped). C, model for FOXM1-mediated nuclear localization of GLI1. FOXM1 transcriptionally activates IPO7. In return, IPO7 induces nuclear localization of GLI1. Nuclear GLI1 then enhances FOXM1 protein expression, forming a positive feedback loop and promoting GBM development.