Vitamin D receptor prevents tumour development by regulating the Wnt/β-catenin signalling pathway in human colorectal cancer

Abstract

Background: Colorectal cancer (CRC) is a common disease threatening human lives worldwide, and vitamin D receptor (VDR) contributes protective roles in this disease. However, the molecular mechanisms underlying VDR protection in CRC progression require further investigation.

Methods: In this study, we statistically analyzed the relationship between VDR expression and CRC development in patients and detected invasion and apoptosis in CRC cells with VDR overexpression and interference. We also detected the expression of key genes involved in Wnt/β-catenin signalling (β-catenin, lymphoid enhancer factor (LEF)-1 and cyclin D1) in SW480 cells and nude mice injected with VDR-overexpressing SW480 cells and observed tumour development. Additionally, we performed Co-immunoprecipitation (Co-IP) and glutathione-S-transferase (GST) pull-down assays to identify the protein interactions of VDR with β-catenin, dual luciferase (LUC) and chromatin immunoprecipitation (ChIP) to detect the activation of LEF-1 by VDR.

Results: The VDR level was closely related to the development and prognosis of CRC patients. VDR overexpression inhibited invasion but promoted apoptosis in cancer cells. β-catenin shRNA contributed oppositely to cancer cell activity with VDR shRNA. Additionally, VDR interacted with β-catenin at the protein level and blocked its nuclear accumulation. VDR regulated the expression of β-catenin, cyclin D1 and LEF-1 and directly activated LEF-1 transcription in vitro. Furthermore, nude mice injected with VDR-overexpressing SW480 cells revealed suppression of tumour growth and decreased expression of β-catenin, cyclin D1 and LEF-1.

Conclusions: This study indicated that VDR protected against CRC disease in humans by inhibiting Wnt/β-catenin signalling to control cancer cell invasion and apoptosis, providing new evidence to explore VDR biomarkers or agonists for CRC patient diagnosis and treatment.

Keywords: Apoptosis; Colorectal cancer; Cyclin D1; Invasion; Vitamin D receptor; Wnt/β-catenin.

Fig. 1

VDR expression is closely related to the development and prognosis of CRC. a and b VDR expression in normal and CRC tissues. c Percentage of patients with low and high VDR expressions respectively in normal and CRC tissues. d VDR immunoreactivity scores in normal tissues and stage I + II and III + IV CRC tissues. e Percentage of patients with different VDR expression scores in normal tissues and stage I + II and III + IV CRC tissues. f Survival rates of patients with different VDR immunohistochemical scores (IS). g Curn hazard analysis of the patients with different VDR ISs. The VDR immunoreactivity scores were calculated according to the percentage of normal and CRC tissues with high VDR expression. The data are represented as means ± SD, n ≥ 100，***P < 0.001

Fig. 2

Detection of cell invasion ability by Transwell experiments. a-c Invasion of SW480 cells using lentivirus-delivered VDR overexpression (b) and VDR interference (c) compared with the controls (a). d and e Invasion of SW480 cells with VDR and β-catenin interference simultaneously (e) compared with the VDR interference plus scramble groups (d). f Quantitative analysis of the migration number of SW480 cells in a to e. The data are presented as means ± SD, n ≥ 3, **P < 0.01

Fig. 3

Detection of apoptosis of SW480 cells by flow cytometry analysis. a-c Early and late apoptosis of SW480 cells with lentivirus-delivered VDR overexpression (b) and VDR interference (c) compared with the controls (a). d and e Early and late apoptosis of SW480 cells with VDR and β-catenin interference simultaneously (e) compared with the VDR interference plus scramble groups (d). f Quantitative analysis of the percentage of early and late apoptosis of SW480 cells in a to e. The data are presented as means ± SD, n ≥ 3, *P < 0.05, **P < 0.01

Fig. 4

Protein expression of β-catenin, cyclin D1 and LEF-1 under VDR overexpression and interference conditions in SW480 cells. a Western blotting analysis revealed the protein expression of β-catenin, cyclin D1 and LEF-1 under VDR overexpression and interference conditions normalized to GAPDH. b-d Quantitative analysis of β-catenin, cyclin D1 and LEF-1 protein expression in a. The data are presented as means ± SD, n ≥ 3, *P < 0.05, **P < 0.01

Fig. 5

mRNA expression of β-catenin (a), cyclin D1 (b) and LEF-1 (c) under VDR overexpression and interference conditions in SW480 cells. The data are represented as means ± SD, n ≥ 3, *P < 0.05, **P < 0.01

Fig. 6

VDR interacts with β-catenin at the protein level, and VDR overexpression promotes the nuclear accumulation of β-catenin. a The Co-IP assay showed that the Myc antibody efficiently immunoprecipitated the β-catenin proteins controlled by the input. The initial biomasses were normalized to beta-Actin. b Western blotting analysis showed the accumulation of β-catenin in the cytoplasm and nucleus with VDR overexpression. GAPDH and CREB were used as protein controls. c The GST pull-down assay showed that the GST-β-catenin fusion proteins could pull down Myc-VDR efficiently. Beta-Actin was used as the protein control

Fig. 7

VDR directly activates LEF-1 expression in SW480 cells. a The diagram represents the LUC reporter construction containing three putative VDR binding sites (WRE-1, WRE-2 and WRE-3) in the LEF-1 promoter region (2000 bp upstream of the ATG) and four mutated LEF-1 promoters with only one WRE binding site or none. b The relative LUC activity of the constructs in A from the dual LUC reporter assay. c The percentage relative to input DNA for VDR ChIP was quantified. d The relative DNA levels of the three WRE binding sites from VDR ChIP were quantified and controlled by IgG. The data are represented as means ± SD, n ≥ 3, *P < 0.05, **P < 0.01

Fig. 8

VDR overexpression suppresses tumour growth and inhibits the expression of β-catenin, cyclin D1 and LEF-1. a Tumours in the nude mice injected with VDR-overexpressing SW480 cells compared with the controls. b Quantitative analysis of the tumour volume within 13 days. c Quantitative analysis of tumour weight in A. d Western blotting analysis showed the protein levels of β-catenin, cyclin D1 and LEF-1 in the mice of A. e Quantitative analysis of the relative protein levels of d. The data are presented as means ± SD, n ≥ 3, *P < 0.05, **P < 0.01