PCDH18 is frequently inactivated by promoter methylation in colorectal cancer

Abstract

Protocadherin18 (PCDH18) was found to be preferentially methylated and inactivated in colorectal cancer (CRC) using bioinformatics tools. However, its biologic role in tumorgenesis remains unclear. Herein, we aimed to elucidate its epigenetic regulation and biological functions in CRC. The methylation status of PCDH18 was significant higher in CRC tissues than in adjacent non-tumor tissues (median, 15.17% vs. median, 0.4438%). Expression level of PCDH18 was significantly lower in primary CRCs than in nonmalignant tissues. Importantly, methylation status of PCDH18 in cell-free DNA of CRC patients was also significantly higher than in healthy subjects. PCDH18 was readily expressed in NCM460 cells, but downregulated in 100% (4/4) of CRC cell lines by promoter methylation, despite its expression could be restored through demethylation treatment. Overexpression of PCDH18 suppressed CRC cell viability, colony formation and migration. Meanwhile, the depletion of PCDH18 by siRNA in NCM460 cells enhanced the colonogenicity and migration ability and promoted β-catenin nuclear accumulation, whereas it inhibited cell cycle arrest. These effects were associated with upregulation of phospho-GSK-3β and cyclin D1, and downregulation of caspase3 and p21. Our results suggested that PCDH18 was a putative tumor suppressor with epigenetic silencing in CRC and a potential biomarker for CRC diagnosis.

Figure 1

(A) Expression level of PCDH18 was significantly downregulated in colorectal cancer (CRC) tissues compared to normal controls in Hong Colorectal dataset and The Cancer Genome Altas expression array dataset from Oncomine. (B) A significant difference of PCDH18 methylation status was observed between 125 CRC tissues and 29 adjacent normal tissues in GSE25062 dataset. (C) Schematic structure of the PCDH18 promoter CpG islands, CpG sites (short vertical lines) and methylation specific PCR (MSP) region indicated (from forward primer to reverse primer). The transcription start site was showed by a curved arrow. (D) Methylation percentage of PCDH18 in 29 paired CRC tissues and normal tissues was detected by MSP. (E) Methylation percentage of PCDH18 in plasma from 20 CRC cases and 20 healthy subjects was examined by MSP. (F) Methylation percentage of PCDH18 in CRC cell lines (HT29, HCT116, SW480 and SW620) and normal colonic NCM460 cells were detected by MSP. (G) Receiver operating characteristic (ROC) curves showed the performance of PCDH18 methylation status from CRC tissues in predicting CRC. (H) ROC curves showed the performance of PCDH18 methylation status from CRC plasma in predicting CRC. Sensitivity means true-positive rate. Specificity means false-positive rate. Results were shown with means ± SD. *p < 0.05, ***p < 0.001.

Figure 2

PCDH18 was epigenetically silenced in colorectal cancer tissues and cell lines. (A) PCDH18 mRNA expression levels in 29 paired CRC tissues and paired normal tissues were detected using quantitative PCR (qPCR), relative to the value of GAPDH in each sample. (B) ROC curves showed the performance of PCDH18 expression levels from CRC tissues in predicting CRC. (C) Representative Western blot image for protein expression level of PCDH18 in CRC tissues and paired normal tissues. (D) Western blot image for protein expression level of PCDH18 in CRC cell lines (HT29, HCT116, SW480 and SW620) and normal colonic NCM460 cells. (E) PCDH18 mRNA expression levels in CRC cell lines (HT29, HCT116, SW480 and SW620) and normal colorectal epithelial NCM460 cells were detected using qPCR, relative to the value of GAPDH in each sample. (F) Representative images of PCDH18 staining in CRC tissues and paired normal tissues. Normal tissues showed strong positive staining, whereas CRC tissue displayed weak staining. The right panel (magnification x400) was magnification of where inset in the left tissue array (magnification x100). (G) A statistically significant difference was detected between the mean density of CRC tissues and that of paired normal tissues in tissue microarray. Results were shown with means ± SD. **p < 0.01***p < 0.001.

Figure 3

Demethylating agent 5-aza-2′-deoxycytidine (5-AZA) treatment induced PCDH18 expression and inhibited migration and growth in CRC cell lines. (A) PCDH18 methylation status after 5μm 5-AZA treatment in CRC cell lines (HT29, HCT116, SW480 and SW620) and normal colorectal NCM460 cells are measured by MSP. QPCR (B), Western blot (C) and Immunofluorescence staining (D) were used to examine mRNA and protein expression levels of PCDH18 after 5 μm 5-AZA treatment in CRC cell lines and NCM460 cells. (E) CRC cell lines treated with 5 μm 5-AZA were examined the ability of cell migration compared with NCM460 cells by Wound healing assays. (F) Growth curve of CRC cell lines and NCM460 cells treated with various concentrations of 5-AZA. A negative control treated with PBS (without 5-AZA) were included in each assay. Results were shown with means ± SD. *p < 0.05, **p < 0.01.

Figure 4

Overexpression of PCDH18 suppresses cell migration and proliferation in CRC cell lines. (A) Expression of PCDH18 in SW480 and SW620 cells after transfection with a PCDH18 expression vector was confirmed by Western blot. (B) PCDH18 expression significantly suppressed the number of viable cells of PCDH18-transfected SW480 and SW620 cells compared to that of control vector-transfected cells. (C) Representative images of colonies in PCDH18-transfecting as well as control vector-transfecting SW480 and SW620 cells. (D) Representative transwell assays in PCDH18-transfecting as well as control vector-transfecting SW480 and SW620 cells. Results were shown with means ± SD. *p < 0.05, **p < 0.01.

Figure 5

PCDH18 suppression promoted cell migration and proliferation in colonic NCM460 cells through the Wnt/β-catenin signaling pathway. (A) PCDH18 mRNA and protein expression was efficiently inhibited in NCM460 cells transfected with siPCDH18. GAPDH was used as an internal control. (B) Representative colony formation assays in NCM460 cells transfected with siRNA-PCDH18 (siPCDH18) and siRNA-negative control (siNC). (C) Representative transwell assays in NCM460 cells transfected with siPCDH18 and siNC. (D) Cell cycle distribution of NCM460 cells transfected with siPCDH18 and siNC was detected by flow cytometry analysis. (E) Molecular interaction networks involving PCDH18 were visualized by Cytoscape3.3.0 software from different species. Blue node represents gene from Homo species. Red node represents gene from Mus species. Green node represents gene from Bos taurus species. (F) Protein expression levels of nonphospho (active)-β-catenin, phospho-β-catenin, total-β-catenin, phospho-GSK-3β, GSK-3β, p21, cyclin A1, cyclin D1, cyclin E1 and caspase3 in NCM460 cells transfected with siPCDH18 and siNC. (G) Representative images of β-catenin staining in NCM460 cells transfected with siPCDH18 and siNC.