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. 2017 Feb 2;36(1):23.
doi: 10.1186/s13046-017-0491-7.

Aberrant Activation of Hedgehog Signaling Promotes Cell Proliferation via the Transcriptional Activation of Forkhead Box M1 in Colorectal Cancer Cells

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Free PMC article

Aberrant Activation of Hedgehog Signaling Promotes Cell Proliferation via the Transcriptional Activation of Forkhead Box M1 in Colorectal Cancer Cells

DeJie Wang et al. J Exp Clin Cancer Res. .
Free PMC article

Abstract

Background: Recent evidence suggests that the aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas, including colorectal cancer (CRC). Forkhead box M1 (FoxM1) controls the expression of a number of cell cycle regulatory proteins, and FoxM1 expression is elevated in a broad range of human malignancies, which suggests that it plays a crucial role in tumorigenesis. However, the mechanisms underlying FoxM1 expression are not fully understood. Here, we aim to further investigate the molecular mechanism by which Gli1 regulates FoxM1 in CRC.

Methods: Western blotting and immunohistochemistry (IHC) were used to evaluate FoxM1 and Gli1 protein expression, respectively, in CRC tissues and matched adjacent normal mucosa. BrdU (5-bromo-2'-deoxyuridine) and clone formation assays were used to clarify the influence of FoxM1 on CRC cell growth and proliferation. Chromatin immunoprecipitation (ChIP) and luciferase experiments were performed to explore the potential mechanisms by which Gli1 regulates FoxM1. Additionally, the protein and mRNA expression levels of Gli1 and FoxM1 in six CRC cell lines were measured using Western blotting and real-time PCR. Finally, the effect of Hh signaling on the expression of FoxM1 was studied in cell biology experiments, and the effects of Hh signaling activation and FoxM1 inhibition on the distribution of CRC cells among cell cycle phases was assessed by flow cytometry.

Results: Gli1 and FoxM1 were abnormally elevated in human CRC tissues compared with matched adjacent normal mucosa samples, and FoxM1 is a downstream target gene of the transcription factor Gli1 in CRC and promoted CRC cell growth and proliferation. Moreover, the aberrant activation of Hh signaling promoted CRC cell proliferation by directly binding to the promoter of FoxM1 and transactivating the activity of FoxM1 in CRC cells.

Conclusion: The dysregulation of the Hh-Gli1-FoxM1 axis is essential for the proliferation and growth of human CRC cells and offers a potent target for therapeutic intervention in CRC.

Keywords: Colorectal cancer; FoxM1; Gli1; Hedgehog; Proliferation.

Figures

Fig. 1
Fig. 1
FoxM1 and Gli1 are highly expressed in CRC tissues. a The FoxM1 and Gli1 protein expression levels were analyzed in eight pairs of randomly selected human CRC tissues and matched adjacent non-tumor tissues by Western blotting. b Quantification of the Western blot (a). Significance was assessed using a paired samples t-test. c Immunohistochemistry staining of FoxM1 and Gli1 protein expression in a representative human CRC sample and the matched adjacent normal tissue sample in the same section. The areas of carcinoma and adjacent tissues are marked. The subcellular locations of Gli1 and FoxM1 are indicated by red arrows. d, e and f The mRNA expression level of FoxM1 analyzed in three published human CRC sample expression profiling studies using the R2 data sheet (http://r2.amc.nl). p < 0.05 was considered significant
Fig. 2
Fig. 2
Gli1 transactivates the FoxM1 promoter. a Schematic diagram of four potential Gli1 binding sites (BS1, BS2, BS3, and BS4) in the FoxM1 promoter. The 9-base pair sequence of the Gli1 binding site and the sequences of four Gli1 binding sites identified in the FxoM1 promoter are shown. b Chromatin was isolated from HT29 cells, and ChIP assays were performed with goat IgG control, Gli1-specific and Gli2-specific antibodies. DM, DNA marker. c Schematic diagram of a series of FoxM1-luciferase constructs. BS4-Mut, binding site 4 mutation; △BS4, binding site 4 deletion. d FoxM1 luciferase constructs, as indicated, were transfected into LoVo cells together with full-length V5-Gli1 plasmid or control vector for 48 h and subjected to a luciferase reporter assay. The results were normalized to the Renilla luciferase activity and are expressed as the fold change in relative luciferase activity compared with the control. Error bars represent the mean and S.D. of three independent experiments. **, p < 0.01
Fig. 3
Fig. 3
The expression of FoxM1 is regulated by Hh-Gli1 signaling. a Western blot analysis of the Gli1, FoxM1, and CCNB1 protein levels in six CRC cell lines. b Real-time PCR analysis of the Gli1, FoxM1, and CCNB1 mRNA expression levels in six CRC cell lines. The HT29 cells’ mRNA expression level was used as the normalized control. c Detection of Gli1, FoxM1, and CCNB1 protein expression in Caco2 cells after treatment with Hh signaling pathway activator. Caco2 cells were treated with 1 μM or 2 μM purmorphamine for 48 h, lysed and subjected to a Western blot analysis. d-f The inhibition of the Hh signaling pathway inhibited the protein expression of Gli1, FoxM1, and CCNB1, as demonstrated by the Western blot. HCT116 cells were transfected with Gli1-miRNAi or control miRNAi constructs for 48 h (d) or treated with the Gli inhibitor GANT61 (e) or Smo inhibitor cyclopamine (f) at the indicated time and concentrations. The cells were lysed and subjected to Western blotting. g Real-time PCR analysis of Gli1, FoxM1, and CCNB1 mRNA expression in HCT116 cells after treatment with GANT61 or cyclopamine. The mRNA expression levels were normalized to that of β-actin and expressed as fold change compared with the DMSO control. Error bars represent the mean and S.D. of three independent experiments. miR-Gli1: miRNAi-Gli1. *, p < 0.05; **, p < 0.01
Fig. 4
Fig. 4
Gli1 regulates CRC cell proliferation depending on FoxM1. a-d) LoVo cells were separately or simultaneously treated with 1 μM purmorphamine and 1 μM thiostrepton for the indicated time. a The Gli1, FoxM1, and CCNB1 protein expression levels were examined by immunoblotting after drug treatment for 48 h. b Cell viability was detected after 6 days using an MTT assay. c LoVo cells treated with indicated drugs were cultured for 2 weeks, and outgrowth colonies were stained with crystal violet. d The matched colony count of (c). Error bars represent the mean and S.D. of three independent experiments. **, p < 0.01. e and f The cell cycle profile of LoVo cells was examined by fluorescence-activated cell sorting (FACS) with propidium iodine (PI) staining after 48 h of drug treatment (e), and the percentages of multinucleate cells were quantified and are shown as a histogram (f). Purm: purmorphamine; Thios: thiostrepton. Error bars represent the mean and S.D. of three independent experiments. *, p < 0.05

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