Downregulation of ZNF365 by methylation predicts poor prognosis in patients with colorectal cancer by decreasing phospho-p53 (Ser15) expression

Abstract

ZNF365 is a transcription factor that plays important roles in different types of cancer, such as colorectal cancer, breast cancer and hepatocellular carcinoma. ZNF365 can promote stalled replication fork recovery to prevent genomic instability, which is a notable feature of sporadic and hereditary types of cancers. However, the function of ZNF365 in the tumor progression of colorectal cancer (CRC) remains unclear. Thus, immunohistochemical staining was used to investigate the association between ZNF365 expression and the clinicopathological characteristics of patients with colorectal cancer. The results demonstrated that ZNF365 protein was strongly expressed in the nucleus and cytoplasm of normal colorectal mucosa. Furthermore ZNF365, which is methylated and downregulated in most cancer cell lines and tissues, was significantly associated with lymph node metastasis (P=0.015), depth of invasion (P=0.031) and histopathological grading (P=0.042). A positive correlation was observed between ZNF365 expression and phosphorylated (P)-p53 (Ser15) protein expression (r=0.18; P=0.038). Survival analysis indicated that patients with high ZNF365 expression had a higher survival rate than those with low ZNF365 expression (P=0.009), suggesting that ZNF365 may be an independent prognostic factor for survival in colorectal cancer (P=0.046). Taken together, the results of the present study demonstrated that ZNF365 was frequently inactivated by promoter methylation and independently predicted poor prognosis in patients with colorectal cancer by downregulating P-p53 (Ser15) expression.

Keywords: DNA methylation; ZNF365; colorectal cancer; immunohistochemistry.

Figure 1.

Representative images of immunohistochemical staining of ZNF365 in colorectal cancer tissues and normal colorectal mucosa. (A) ZNF365 expression in normal colorectal mucosa. (B) ZNF365 weak staining, (C) ZNF365 moderate staining and (D) ZNF365 strong staining in colorectal cancer tissues (magnification, ×200).

Figure 2.

Kaplan-Meier survival curve of patients with colorectal cancer according to ZNF365 expression. The 3-year overall survival rate of patients with low ZNF365 expression was significantly lower than that of patients with high ZNF365 expression.

Figure 3.

Interaction network between ZNF365 and p53. ZNF365 can interact with p53 through RPRM and MAP4, as predicted by the GeneMANIA database.

Figure 4.

Images of immunohistochemical staining of ZNF365 and P-p53 (Ser15) in CRC tissues. ZNF365 protein expression was positively correlated with P-p53 (Ser15) expression in CRC (magnification, ×200). P, phosphorylated; CRC, colorectal cancer.

Figure 5.

Correlation between ZNF365 mRNA expression and promoter methylation analyzed using the cBioPortal database. There was a significantly negative correlation between ZNF365 gene expression and DNA methylation. Seq, sequence; RSEM, RNA-Seq by Expectation Maximization; CNA, copy number alteration.

Figure 6.

Expression and CGI methylation of ZNF365 in CRC cell lines and primary tumors. (A) ZNF365 expression was downregulated or silenced in several CRC cell lines due to its CGI methylation. (B) Pharmacological demethylation with 5-aza and TSA recovered ZNF365 expression in methylated and silenced cell lines. Typical results are presented. A+T, treatment with 5-aza and TSA. (C) Methylation of ZNF365 in primary T tissues and paired N tissues by MSP. CGI, CpG island; CRC, colorectal cancer; T, tumor; N, non-tumor; M, methylated; U, unmethylated; TSA, trichostatin A; MSP, methylation-specific PCR.

Figure 7.

ZNF365 CGI methylation in colorectal cancer cell lines and primary tumors. (A) Sequence of ZNF365 CGI with locations of the 49 CpG sites analyzed and primers used. MSP and BGS regions are also presented. (B) Each vertical line indicates individual CpG sites. Products of cloned BGS-PCR were sequenced and each clone is exhibited as an individual row, representing a single allele of the CGI. Open circles represent unmethylation, while filled circle represent methylation. CGI, CpG island; BGS, bisulfite genomic sequencing.