Dysregulation of Fra1 expression by Wnt/β-catenin signalling promotes glioma aggressiveness through epithelial-mesenchymal transition

Abstract

Aberrant expression of Fos-related antigen-1 (Fra1) is commonly elevated in various malignant cancers and is strongly implicated in invasion and metastasis. However, the molecular mechanisms underlying its dysregulation in human glioma remain poorly understood. In the present study, we demonstrate that up-regulation of Fra1 plays a crucial role in the glioma aggressiveness and epithelial-mesenchymal transition (EMT) activated by Wnt/β-catenin signal pathway. In glioma cells, activation of Wnt/β-catenin signalling by Wnt3a administration obviously induced EMT and directly activated the transcription of Fra1. Phenotype experiments revealed that up-regulation of Fra1 induced by Wnt/β-catenin signalling drove the EMT of glioma cells. Furthermore, it was found that the cisplatin resistance acquired by Wnt/β-catenin signalling activation depended on increased expression of Fra1. Analysis of clinical specimens verified a positive correlation between Fra1 and β-catenin as well as a poor prognosis in glioma patients with double-high expressions of them. These findings indicate that an aberrant Wnt/β-catenin signalling leads to the EMT and drug resistance of glioma via Fra1 induction, which suggests novel therapeutic strategies for the malignant disease.

Keywords: EMT; Fra1; Wnt; glioma; β-catenin.

Figure 1. Activation of Wnt/β-catenin signalling induces EMT in glioma cells

(A, B) Relative mRNA levels of EMT markers in the U-251 (A) and U-87 (B) cells treated with Wnt3a at different concentrations (50 and 200 ng/ml) for 48 h; *P<0.05. (C) Western blots of EMT markers with specific antibodies in the U-251 cells exposed to Wnt3a (200 ng/ml) for 48 h. (D) Quantificative analysis of (C) by Image J; *P<0.05. (E) Immunofluorescent staining for Vimentin in the U-251 cells treated with Wnt3a (200 ng/ml) or transfected with si-β-catenin for 48 h (nuclei stained with DAPI) (Scale bar 20 μm). (F, G) The protein (F) and mRNA (G) levels of Vimentin in the U-251 cells with si-β-catenin transfection and Wnt3a (200 ng/ml) treatment; *P<0.05. (H) Relative mRNA levels of EMT-TFs in the U-251 cells treated with Wnt3a (200 ng/ml) for 48 h. Lef1 served as a negative control; *P<0.05. (I) Western blots of EMT-TFs with specific antibodies in the U-251 cells exposed to Wnt3a (200 ng/ml) for 48 h. (J) Quantificative analysis of (I) by Image J; *P<0.05.

Figure 2. Wnt/β-catenin signalling activates the transcription of Fra1 in glioma cells

(A) Relative mRNA levels of Fra1 in the U-251 cells treated with the GSK-3β inhibitors, LiCl (20 mM) or CHIR (3 μM) for 48 h; *P<0.05. (B) Protein levels of Fra1 and β-catenin in the U-251 cells treated with the GSK-3β inhibitors, LiCl (20 mM) or CHIR (3 μM) for 48 h; *P<0.05. (C) The transcriptional activity of Fra1 promoter (–2500/+1) in response to different concentrations (20, 50 and 200 ng/ml) of Wnt3a for 48 h was measured by luciferase assay in U-251 and U-87 cells (internal control, pRL-SV40). * Compared with 0 ng/ml; ^# Compared with 20 ng/ml; ^@ Compared with 50 ng/ml; P<0.05. (D) The protein level of Fra1 in response to different concentrations (20, 50 and 200 ng/ml) of Wnt3a was detected by Western blot. (E) Schematic representation of Fra1 promoter with three potential Tcf/Lef-binding sites and the primer pair used in ChIP-PCR assays. The truncated and mutated derivatives are also shown. (F) Relative luciferase activities were measured with a series of truncated constructs of Fra1P in the U-251 cells exposed to Wnt3a (200 ng/ml) for 48 h (internal control, pRL-SV40, *P<0.05). (G) Relative luciferase activity in the U-251 cells transfected with wild-type Fra1P (–1500/+1) or Tcf/Lef-binding site mutated Fra1P (–1500/+1) in response to 48 h Wnt3a (200 ng/ml) treatment (internal control, pRL-SV40, *P<0.05). TOPflash containing wild-type TCF-4-binding sites were used as a positive control, whereas FOPflash with mutant-binding sites served as negative control. The results were normalized to the control group (without Wnt3a). (H, I) The U-251 and U-87 cells treated with Wnt3a (200 ng/ml) for 48 h were analysed by ChIP assays using β-catenin antibody and IgG. QPCR was performed with the immunoprecipitated DNAs or soluble chromatin using the specific primer pairs for Fra1 promoter. Cmyc or control served as a positive or a negative control. Error bars represent S.D. of three individual experiments.

Figure 3. Fra1 promotes Wnt3a-induced migration and invasion of glioma cancer cells

(A) Western blots of EMT markers with specific antibodies in the U-251 cells transfected with Fra1 siRNA or expresssion vector. (B) Left panels: representative images from wound healing assays with the U-251 cells transfected with indicated siRNA. Right panels: representative images of the U-251 cells penetrating the Matrigel in invasion assays after transfection with indicated siRNA (Scale bar 200 μm). (C) Up panel: percentage wound closure 48 h after scratch in (B). Down panel: numbers of invasive cells in (B); *P<0.05. (D) Left panels: representative images from wound healing assays with the U-87 cells transfected with β-catenin siRNA and exposed to Wnt3a (200 ng/ml) for 48 h. Right panels: representative images of the U-87 cells penetrating the Matrigel in invasion assays after transfection with β-catenin siRNA and exposure to Wnt3a (200 ng/ml) for 48 h (Scale bar 200 μm). (E) Up panel: percentage wound closure in (D). Down panel: numbers of invasive cells in (D); *P<0.05. (F) Left panels: representative images from wound healing assays with the U-251 cells transfected with Fra1 siRNA and exposed to Wnt3a (200 ng/ml) for 48 h. Right panels: representative images of the U-251 cells penetrating the Matrigel in invasion assays after transfection with Fra1 siRNA and exposure to Wnt3a (200 ng/ml) for 48 h (Scale bar 200 μm). (G) Up panel: percentage wound closure in (F). Down panel: numbers of invasive cells in (F); *P<0.05. (H) Relative mRNA levels of EMT-TFs in the U-251 cells transfected with Fra1 siRNA and treated with Wnt3a (200 ng/ml) for 48 h. Lef1 served as a negative control; *P<0.05. (I, J) The protein levels of EMT-TFs in the U-251 cells transfected with Fra1 siRNA and treated with Wnt3a (200 ng/ml) for 48 h; *P<0.05.

Figure 4. Fra1 determines the cisplatin resistance of giloma cells induced by Wnt/β-catenin signalling in vitro and in vivo

(A–C) The viability of U-251 cells treated with Wnt3a (200 ng/ml) for 48 h (A) or transfected with Fra1 expression plasmids (B) or siRNA (C) were determined by the CCK-8 assay under different concentrations of cisplatin. (D) Apoptosis levels of the U-251 cells with stable knockdown of Fra1 and overexpression of Wnt3a were measured 48 h after 2 μg/ml cisplatin addition. (E) Analysis of three separate experiments in (D); *P<0.05. (F) Apoptosis levels of the U-87 cells with Fra1 siRNA transfection and 200 ng/ml Wnt3a treatment were measured 48 h after 2 μg/ml cisplatin addition; *P<0.05. (G) Representative images of the tumour specimens dissected on day 28. (H) Time course of tumour volume progression, as determined by caliper measurements; *P<0.05. (I) Tumour weights measured 18 days after the beginning of cisplatin treatment; *P<0.05.

Figure 5. Abnormal Wnt/β-catenin/Fra1 signalling axis indicates a poor prognosis in glioma patients

(A) Investigation of Fra1 mRNA levels in glioblastoma cancer microarray data sets (Lee Brain and Sun Brain sets) using Oncomine (www.oncomine.org). Higher Fra1 mRNA levels were found in glioblastoma (n=78) when compared with normal tissues (n=3) in Lee Brain data set. Higher Fra1 mRNA levels were found in giloblastoma (n=81) compared with normal brain tissues (n=23) in Sun Brain data set. (B) Analysis of the TCGA Glioblastoma database (TCGA, Nature 2008) using cBioPortal showing the correlation between Wnt3a and Fra1 mRNA levels. (C) Linear regression between immunostaining intensity of Fra1 and β-catenin. (D) The association of Fra1 and β-catenin levels with prognosis of glioma patients. Kaplan–Meier OS curves indicated low expressions of Fra1 and β-catenin in glioma tissues were significantly associated with a better OS rate. The ‘low’ or ‘high’ of Fra1 and β-catenin levels was defined according to its cut-off value, which was defined as the median value of the cohort of patients tested; P<0.05 by log-rank test.