E2A attenuates tumor-initiating capacity of colorectal cancer cells via the Wnt/beta-catenin pathway

Abstract

Background: The E2A gene, which encodes two basic helix-loop-helix transcription factors, E12 and E47, regulates colorectal cancer progression and epithelial-mesenchymal transition. However, whether E2A regulates the tumor-initiating capacity of colorectal cancer is unclear. Thus, we have studied E2A expression in the initiation of colorectal cancer in vivo and in vitro.

Methods: Immunohistochemistry and immunoblot were performed to determine protein levels of E2A in colorectal cancer specimens and cells. RNAi was employed to downregulate E2A expression, and the subsequent change in protein level was evaluated by immunoblot. Sphere-forming assay and enumeration of liver metastasis in mouse models were used to identify the tumor formation ability of colorectal cancer cells.

Results: E2A expression in colorectal cancer clinical specimens was inversely associated with patients' progression-free survival. Functional studies demonstrated that E2A significantly decreased tumor formation in vitro and in vivo. Furthermore, nuclear translocation of beta-catenin and activation of the Wnt/beta-catenin pathway occurred after suppression of E2A in colorectal cancer cells. FoxM1 was identified as a down-stream target by mRNA microarray, implying that FoxM1 plays a main role in determining how E2A regulates the tumor-initiating capacity of colorectal cancer.

Conclusion: E2A suppresses tumor-initiating capacity by targeting the FoxM1-Wnt/β-catenin pathway.

Keywords: Epithelial-mesenchymal transition; FoxM1 protein; Helix-loop-helix protein e47; Helix–loop–helix protein e12; Human; Transcription factors; Tumor suppressor gene.

Fig. 1

E2A expression correlates with progression-free survival of colorectal cancer. a E2A expression (upper panel) and Lgr5 expression (lower panel) in representative immunohistochemistry images. 200×. b Progression-free survival of patients with high and low E2A expression (P < 0.05). c Progression-free survival of patients with respective E2A and Lgr5 expression (P < 0.05)

Fig. 2

E2A suppressed the tumor-initiating capacity of CRC cells in vitro. a Effect of shE2A on SW480 cell sphere-formation ability. The sphere-formation ability of SW480 cells was enhanced by shE2A. P < 0.05, Scale bars: 50 μm. b Transfection of plasmids pEZ-M29-E12 and pEZ-M29-E47 to Caco-2 cells decreased the number of spheres per field. Left panel: Representative images of sphere formation assay are shown with original magnification of 40×. Scale bars: 50 μm. Right panel: Data in the histograms is expressed as means ± SD from three separate experiments. c Knockdown of E2A induced Lgr5 expression. Left panel: Immunoblot analysis showed that cancer stem-cell marker Lgr5 was increased in the shE2A-expressing SW480 clone, indicating induction of tumor-initiating ability. GAPDH was used as loading control. Right panel: Densitometric analysis of left panel normalized to GAPDH. d E12 and E47 suppressed Lgr5 expression. Left panel: Immunoblot analysis of Lgr5 and GAPDH expression with or without transfection of E12 or E47. Right panel: Densitometric analysis of left normalized to GAPDH. Data in the histograms is expressed as the means ± SD from three separate experiments. e Immunofluorescence analysis with anti-Lgr5 antibodies as indicated. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). Magnification: 400×; Scale: 50 μm. *, P < 0.05

Fig. 3

E2A suppress CRC liver metastasis in vivo. a Representative images of liver metastasis in SW480 cells in vivo. Scale bar: 100 μm. Magnification: 40×. Six mice are included in each group; (b) The number of metastatic nodules in each group. Data are presented as mean ± SD. *, P < 0.05

Fig. 4

Wnt/β-catenin pathway is crucial for E2A in CRC cell tumor-initiating capacity. a Effect of E2A on the expression of β-catenin in cell nuclei. shE2A expression enhanced the β-catenin expression in SW480 cell nuclei, and E12/E47 attenuated the β-catenin expression in Caco-2 cell nuclei. Lower panel: Densitometric analysis of beta-catenin normalized to SP1. Data in the histograms is expressed as means ± SD from three separate experiments. b, c E2A suppressed the Wnt pathway in CRC cells. shE2A increased expression of the Wnt pathway effectors TCF-1 and cyclin D1 in SW480 cells, whereas E12 and E47 had the opposite effect in Caco-2 cells. Right panel: Densitometric analysis of TCF-1 and cyclin D1 normalized to GAPDH. Data in the histograms is expressed as mean ± SD from three separate experiments. d Wnt pathway is crucial for E2A suppressing tumor-initiating ability. SW480/shE2A cells treated with 5 μM CGP049090, which is a small-molecular compound inhibiting Wnt/β-catenin pathway activity, had decreased tumor-initiating ability compared with that of SW480/shE2A without CGP049090 (upper panel). wnt3a, at 100 ng/mL, added Caco-2 restored the tumor-initiating ability inhibited by E12/E47 to a level like that in Caco-2/NC cells (lower panel). e Lgr5 expression in SW480/shE2A cells treated with 5 μM CGP049090 was like that in SW480/shNC, which was lower than that in SW480/shE2A without CGP049090. wnt3a, at 100 ng/mL, added to Caco-2 cells, restored the Lgr5 expression inhibited by E12/E47 to a level like that in Caco-2/NC cells (lower panel). Data are the mean ± SD from at least three separate experiments. *, P < 0.05

Fig. 5

Foxm1 is a downstream target through which E2A suppressed CRC cell tumor-initiating capacity. a, b Heat map and volcano plot displaying the hierarchical clustering of the mRNA expression profiles. c PPI protein network generated by Genemania showing the relationship between E2A, FoxM1 and β-catenin. d Downregulation of E2A by shE2A resulted in increased FoxM1 mRNA expression in SW480 cells, whereas transfection of plasmids encoding E12 or E47 decreased FoxM1 mRNA in Caco-2 cells, as revealed by semi-qRT-PCR. e E2A decreased FoxM1 protein expression, as determined by immunoblot analysis. Lower panel: Densitometric analysis of FoxM1 normalized to GAPDH. Data in the histograms is expressed as the mean ± SD from three separate experiments. f Immunoblot analysis of FoxM1, with GAPDH as loading control. g shFoxM1 decreased tumor-initiating ability of SW480/shE2A cells to a level like that of SW480/shE2A cells. h Lgr5 expression in SW480/shE2A cells treated with shFoxM1 is like that in SW480/shNC, which is lower than in SW480/shE2A. Lower panel: Densitometric analysis of Lgr5 normalized to GAPDH. Data in the histograms is expressed as mean ± SD from three separate experiments. i Schematic diagram illustrates that E2A inhibited FoxM1 transcription to attenuate FoxM1, depending on β-catenin translocation to cell nuclei, which decreased the Wnt pathway in CRC cells