Hypermethylation of heparanase 2 promotes colorectal cancer proliferation and is associated with poor prognosis

Abstract

Background: The epigenetic abnormality of tumor-associated genes contributes to the pathogenesis of colorectal carcinoma (CRC). However, methylation in colorectal cancer is still poorly characterized.

Method: By integration of DNA methylation data from the GEO database and gene expression data from The Cancer Genome Atlas database, the aberrantly methylated genes involved in CRC tumorigenesis were identified. Subsequent in vitro experiments further validated their role in CRC.

Results: We performed integrative genomic analysis and identified HPSE2, a novel tumor suppressor gene that is frequently inactivated through promoter methylation in CRC. K-M survival analysis showed that hypermethylation-low expression of heparanase 2 (HPSE2) was related to poor patient prognosis. Overexpression of HPSE2 reduced cell proliferation in vivo and in vitro. HPSE2 could regulate the p53 signaling pathway to block the cell cycle in G1 phase.

Conclusion: HPSE2, a novel tumor suppressor gene that is frequently inactivated through promoter methylation in CRC. HPSE2 performs a tumor suppressive function by activating the p53/ p21 signaling cascade. The promoter hypermethylation of HPSE2 is a potential therapeutic target in patients with CRC, especially those with late-stage CRC.

Keywords: Biomarker; Colorectal cancer; Heparanase 2; Methylation; Proliferation.

Fig. 1

Identification of aberrantly methylation-regulated genes from GEO and TCGA databases. Aberrant DNA methylation between CRC tumors and adjacent normal tissues in GSE17648 (a) and GSE29490 (b); c Ectopically expressed genes in CRC tumor tissues from TCGA database; d Venn diagram of DNA methylation and expression in tumor tissue; e, f Heat map showing the differential expression and methylation of 14 genes between tumors and adjacent normal tissues from TCGA database

Fig. 2

Expression and methylation of HPSE2 was associated with 5-year overall survival in CRC patients. a Pearson correlation between HPSE2 expression and methylation; b Patients with hypermethylation–low expression of HPSE2 have a worse prognosis

Fig. 3

HPSE2, which was regulated by promoter DNA methylation, could be an independent prognostic factor. a Methylation levels of HPSE2 in 22 cases of CRC tissues in GSE17648; b Methylation levels of HPSE2 in 26 cases of matched CRC tumors and adjacent normal tissues in GSE29490; HPSE2 expression (c) and methylation (d) in TCGA database; e, f Kaplan–Meier curves showing that hypermethylation–low expression of HPSE2 was associated with the shortened survival of CRC patients at the late stages (f) but not at the early stages (e); g Univariate and multivariate COX regression model revealed that HPSE2 could be an independent prognostic risk factor. ***P < 0.001

Fig. 4

CpG islands methylation of the HPSE2 promoter. a Heat map of 49 methylated CpG islands located in the promoter region of HPSE2 from TCGA database; b Pearson correlation of methylation sites and HPSE2 expression with cor < − 0.3 were selected; c Differentially methylated CpG islands between normal and tumor tissues from TCGA database; d Differentially methylated CpG islands of the HPSE2 promoter among adenocarcinoma, adenoma, and normal tissues from the GSE68484 dataset. ***P < 0.001

Fig. 5

Changes in CpG island methylation in the HPSE2 promoter after DCA treatment. a CpG island methylation in 6 matched cancers and adjacent tissues; b Changes in methylation level in peripheral blood of mice treated with DCA in GSE77965 dataset; c, d Changes in methylation levels after adding DAC to HCT116 cells from the GSE51810 dataset

Fig. 6

HPSE2 was significantly downregulated in cancer tissues. We detected HPSE2 expression in CRC samples and peripheral blood. HPSE2 was aberrantly expressed in CRC tissues (a) but not in peripheral blood (b); HPSE2 protein showed differential expression in 10 pairs of CRC tumors and normal tissues (c)

Fig. 7

HPSE2 performs a tumor suppressive function by activating the p53/ p21 signaling cascade. a HPSE2 mRNA expression in 5 CRC cell lines and 1 intestinal epithelial cell; b HPSE2 overexpression in SW480 and HCT116 was confirmed through qRT-PCR; c, d HPSE2 overexpression significantly inhibited SW480 (c) and HCT116 (d) proliferation; e, f HPSE2 overexpression inhibited SW480 (e) and HCT116 (f) migration; g HPSE2 blocked the SW480 and HCT116 cell cycle in the G1–S stage; h, i Images of tumor specimens; j HPSE2 overexpression significantly suppressed tumorigenicity in vivo; k Immunohistochemistry results for mouse tumor tissues: ki67 expression was decreased in the HPSE2-overexperssing and control group; l GSEA plot showed pathways that were significantly related to HPSE2 (p < 0.05); m Western blot analysis showed that HPSE2 overexpression could upregulate p53/p21 levels in HCT116 and SW480. *P < 0.05, **P < 0.01, ***P < 0.001