Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

Abstract

Aim: To investigate the expression and methylation status of the secreted frizzled-related protein 2 (SFRP2) in esophageal squamous cell carcinoma (ESCC) and explore its role in ESCC carcinogenesis.

Methods: Seven ESCC cell lines (KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1) and one immortalized human esophageal epithelial cell line (Het-1A), 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2'-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.

Results: SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2'-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue (0.939 ± 0.398 vs 1.51 ± 0.399, P < 0.01). SFRP2 methylation was higher in tumor tissue than in paired normal tissue (95% vs 65%, P < 0.05). The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro (47.17% ± 15.61% vs 17% ± 3.6%, P = 0.031) and tumor growth in nude mice (917.86 ± 249.35 mm(3)vs 337.23 ± 124.43 mm(3), P < 0.05). Using flow cytometry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells (P = 0.025). The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts.

Conclusion: Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.

Keywords: Esophageal squamous cell carcinoma; Methylation; Secreted frizzled-related protein 2; Tumor suppressor gene; Wnt signaling pathway.

Figure 1

Expression and promoter methylation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma cell lines. A: Secreted frizzled-related protein 2 (SFRP2) mRNA expression as determined by reverse-transcription polymerase chain reaction (RT-PCR); B: The methylation status of the SFRP2 promoter as determined by methylation specific PCR; C: mRNA expression of SFRP2 (determined by RT-PCR) restored after treatment with the demethylation agent 5-Aza-dC in three esophageal squamous cell carcinoma cell lines. M: Methylated; U: Unmethylated.

Figure 2

A representative picture of bisulfite genomic sequencing in the secreted frizzled-related protein 2 gene promoter. A-293bp region with 34 CpG site was analyzed. B

Figure 3

Expression of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma and paired non-cancerous tissues. A: Reverse-transcription polymerase chain reaction (RT-PCR) analysis of secreted frizzled-related protein 2 (SFRP2) mRNA expression in 10 primary esophageal squamous cell carcinoma (ESCC) tissues and their adjacent non-cancerous tissues; B: The SFRP2 mRNA expression level normalized according to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. Data are expressed as mean ± SD. ESCC tissues vs adjacent non-tumor tissues (^aP < 0.01).

Figure 4

Effect of secreted frizzled-related protein 2 ectopic expression on cell proliferation. A: Strong secreted frizzled-related protein 2 (SFRP2) mRNA expression in KYSE30 cells transfected with pcDNA3.1/SFRP2, but not in KYSE30 and cells transfected with the empty vector; B: SFRP2-transfected KYSE30 cells growing significantly slower than control and parental cells (^aP < 0.05).

Figure 5

Ectopic expression of SFRP2 induces apoptosis in KYSE30 cells. A: The representative images of flow cytometry analysis; B: The data are the percentage of early apoptotic out of the total cell population of KYSE30 cells transfected with SFRP2 and cells with the empty vector.The early apoptotic cells in SFRP2-transfected cells were significantly higher than that in the control cells (^aP < 0.05).

Figure 6

Colony formation assays. A: The representative dishes of transfection with pcDNA3.1V5HisA/secreted frizzled-related protein 2 (SFRP2) or empty vector (pcDNA3.1 V5HisA). Left: colonies formed by control cells. Right: colonies formed by cell strains with stably transfected SFRP2; B: Quantitative analyses of colony formation efficiency. Values are the mean ± SD of at least 3 independent experiments. The relative colony formation efficiency of SFRP2-transfected esophageal squamous cell carcinoma cells were lower than those of mock-transfected cells (^aP < 0.05).

Figure 7

Secreted frizzled-related protein 2 inhibits growth of tumors derived from KYSE-30 in vivo. A: A representative picture of nude mice: at week 5 nude mice injected with KYSE-30, KYSE-30/vector, KYSE30/SFRP2 and phosphate buffered saline; B: The tumor growth curves of nude mice. The growth of tumors derived from the SFRP2-transfected esophageal squamous cell carcinoma cells cells were significantly slower than those from the mock-transfected cells (^aP < 0.05).