Odd-skipped related 1 is a novel tumour suppressor gene and a potential prognostic biomarker in gastric cancer

Abstract

We report that the odd-skipped related 1 (OSR1) gene encoding a zinc-finger transcription factor was preferentially methylated in gastric cancer by genome-wide methylation screening. OSR1 expression was frequently silenced or down-regulated in gastric cancer cell lines. OSR1 expression was also significantly down-regulated at both mRNA and protein levels in primary gastric cancer tissues compared with adjacent normal tissues. The silencing or down-regulation of OSR1 was closely associated with promoter hypermethylation. Overexpression of OSR1 significantly inhibited cell growth, arrested the cell cycle, and induced apoptosis in the gastric cancer cell lines AGS, MKN28, and MGC803. Conversely, knockdown of OSR1 by OSR1-short hairpin RNA significantly enhanced cell growth, promoted the cell cycle, and inhibited apoptosis in the normal gastric epithelial cell line GES1. The dual-luciferase reporter assay revealed that OSR1 activated p53 transcription and repressed the T-cell factor (TCF)/lymphoid enhancer factor (LEF). Complementary DNA expression array and western blotting showed that OSR1 increased the expression of nuclear p53, p21, Fas, and death receptor-5, and suppressed the expression of cyclin D1 and cyclin-dependent kinase 4 in the p53 signalling pathway. In addition, OSR1 suppressed the expression of cytoplasmic β-catenin, TCF-1, and LEF1 in the Wnt/β-catenin signalling pathway. OSR1 methylation was detected in 51.8% of primary gastric cancer patients (85 of 164) by bisulphite genomic sequencing. Multivariate Cox regression analysis showed that OSR1 methylation was an independent predictor of poor survival. Kaplan-Meier survival curves revealed that OSR1 methylation was associated with shortened survival in TNM stage I-III patients. In conclusion, OSR1 acts as a functional tumour suppressor through the transcriptional activation of p53 and repression of TCF/LEF in gastric cancer. Detection of OSR1 methylation may serve as a potential biomarker of the early stage of gastric cancer.

Keywords: gastric cancer; methylation; odd-skipped related 1 (OSR1); prognosis; tumour suppressor.

Figure 1

OSR1 is silenced or down-regulated by promoter methylation in gastric cancer cells. (A) Expression of OSR1 mRNA in human organ tissues was determined by semi-quantitative reverse transcriptase (RT)-PCR. (B1) Expression of OSR1 mRNA in gastric cancer cell lines and normal gastric epithelial cell line. (B2) Expression of OSR1 protein in gastric cancer cell lines and normal gastric epithelial cell line. (C1) Treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza) restored OSR1 expression in gastric cancer cell lines. (C2) Combination treatment with 5-Aza and the histone deacetylase inhibitor trichostatin A (TSA) restored OSR1 expression in MKN45 and SNU16. (D) A typical CpG island spans the promoter region of OSR1. The transcription start site (TSS) and the regions of bisulphite genomic sequencing (BGS) and methylation-specific PCR (MSP) are indicated. Methylation status of OSR1 in the gastric cancer cell lines was determined by BGS and MSP (M, methylated; U, unmethylated).

Figure 2

OSR1 is down-regulated in primary gastric cancer tissues. (A) Expression levels of OSR1 mRNA in 20 paired primary gastric cancer tissues were determined by quantitative real-time PCR. (B) Expression levels of OSR1 protein in seven paired primary gastric cancer tissues were determined by western blotting. Quantitative analysis of the relative expression density is shown in the lower panel. Each column represents the mean ± standard deviation (SD). ^**p < 0.01 (N, normal; C, cancer). (C1) Expression levels of OSR1 protein in 13 paired primary gastric cancer tissues were determined by immunohistochemistry. Expression of OSR1 protein in normal gastric tissues and gastric cancer tissues is shown. The lower three images indicate cancer cell infiltration under the adjacent normal epithelial mucosa. (C2) Immunohistochemistry scoring was performed according to the percentage of positive tumour cells (0, none; 1, < 20%; 2, 20–50%; 3, > 50%). Each bar represents the mean ± SD.

Figure 3

Overexpression of OSR1 inhibits cell growth. (A1) Overexpression of OSR1 mRNA was revealed by semi-quantitative RT-PCR. (A2) Overexpression of OSR1 protein was confirmed by western blotting. (B) Effect of OSR1 overexpression on cell viability was determined by MTS assay. Each column represents the mean ± SD. ^**p < 0.01. (C1) Effect of OSR1 overexpression on colony numbers was determined by colony formation assay. (C2) Quantitative analysis of colony formation efficiency (%) is shown. Each column represents the mean ± SD. ^**p < 0.01.

Figure 4

Knockdown of OSR1 by OSR1-shRNA-1 (A–D) and OSR1-shRNA-2 (E–G) enhances cell growth. (A1) Knockdown of OSR1 mRNA was revealed by semi-quantitative RT-PCR (shCt, shRNA control vector; shOSR1-1, OSR1-shRNA-1). (A2) Knockdown of OSR1 protein was confirmed by western blotting. (A3) Knockdown efficiency of OSR1 mRNA was examined by quantitative real-time PCR. Each column represents the mean ± SD. ^**p < 0.01. (B) Effect of OSR1 knockdown on cell viability was determined by MTS assay. Each column represents the mean ± SD. ^**p < 0.01. (C) Effect of OSR1 knockdown on cell proliferation was determined by xCELLigence analysis. Data are shown as mean ± SD. (D) Effect of OSR1 knockdown on colony numbers was determined by colony formation assay. Quantitative analysis of colony formation efficiency (%) is shown in the right panel. Each column represents the mean ± SD. ^**p < 0.01. (E1) Knockdown of OSR1 mRNA was revealed by semi-quantitative RT-PCR (shCt, shRNA control vector; shOSR1-2, OSR1-shRNA-2). (E2) Knockdown of OSR1 protein was confirmed by western blotting. (E3) Knockdown efficiency of OSR1 mRNA was examined by quantitative real-time PCR. Each column represents the mean ± SD. ^**p < 0.01. (F) Effect of OSR1 knockdown on cell viability was determined by MTS assay. Each column represents the mean ± SD. ^**p < 0.01. (G) Effect of OSR1 knockdown on colony numbers was determined by colony formation assay. Quantitative analysis of colony formation efficiency (%) is shown in the right panel. Each column represents the mean ± SD. ^**p < 0.01.

Figure 5

OSR1 arrests the cell cycle and induces apoptosis. (A) Effects of OSR1 overexpression and knockdown on the cell cycle were determined by fluorescence activated cell sorting (FACS) analysis. Quantitative analysis of cell proportion (%) is shown in the right panel. Each column represents the mean ± SD. ^*p < 0.05; ^**p < 0.01 (shCt, shRNA control vector; shOSR1-1, OSR1-shRNA-1; shOSR1-2, OSR1-shRNA-2). (B) Effects of OSR1 overexpression and knockdown on apoptosis were determined by FACS analysis after the dual staining with annexin V-APC and 7-aminoactinomycin (7-AAD). Quantitative analysis of apoptotic cells (%) is shown in the right panel. Each column represents the mean ± SD. ^*p < 0.05; ^**p < 0.01.

Figure 6

OSR1 activates the transcription of p53 and represses the transcription of T-cell factor (TCF). (A1) Effect of OSR1 overexpression and knockdown on p53 luciferase reporter activity was determined in gastric cancer cell lines (WT, wild type; Mut, mutant). Each column represents the mean ± SD. ^**p < 0.01. (A2) Effect of OSR1 overexpression on p53 luciferase reporter activity was determined in a colon cancer cell line (WT, wild type; KO, knockout). Each column represents the mean ± SD. ^**p < 0.01. (A3) Effect of OSR1 overexpression on TCF luciferase reporter activity was determined in gastric cancer cell lines (WT, wild type; Mut, mutant). Each column represents the mean ± SD. ^**p < 0.01. (B) Effect of OSR1 overexpression on the expression levels of candidates in the p53 and Wnt/β-catenin signalling pathway was validated by western blotting. (C) Schematic diagram showing the mechanism of OSR1 tumour suppressive function.

Figure 7

OSR1 methylation is associated with poor survival of patients at the early stage of gastric cancer. (A) Kaplan–Meier curves of gastric cancer patients. (B) Kaplan–Meier curves of gastric cancer patients in tumour–nodes–metastasis (TNM) stages I–III. (C) Kaplan–Meier curves of gastric cancer patients in TNM stage IV.