Identification of a novel hypermethylated tumor suppressor gene ZNF671 in human urothelial carcinoma and its implication in non-invasive cancer detection using voided urine

• Main Authors: Chia-Ming Yeh, 葉佳銘
• Other Authors: Michael Wing-Yan Chan
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/jyby3h

博士 === 國立中正大學 === 分子生物研究所 === 104 === Urothelial carcinoma (UC, also known as transitional cell carcinoma, TCC) is the second most common genitourinary malignancy and the ninth most common cancer in the world. UC is particularly common in Taiwan, where the highest incidence occurs in the southwestern coastal region. Although urothelial carcinoma patients have a low mortality rate, long term follow-up with repeated cystoscopy is required due to the high recurrence nature of the tumor. However the sensitivity of urine cytology, which is the current non-invasive diagnosis, is known to be low especially for low grade cancer patients. It’s therefore necessary to develop novel methods with high specificity and high sensitivity to improve the diagnosis of UC patients. The molecular mechanism underlying the lethal phenomenon of urothelial carcinoma (UC) tumor recurrence remains unresolved, while aberrant promoter hypermethylation which is considered as a hallmark of cancer for over a decade, plays an important role in controlling cancer progression. Nevertheless, detection of promoter hypermethylation in bodily fluid has been implicated as a non-invasive and sensitive tool for cancer diagnosis. In this regard, we aim to identify novel tumor suppressors that are epigenetically silenced by promoter methylation and can be used as a novel biomarker for non-invasive diagnosis of UC. By using illumina 27K CpG island methylation microarray, we identified promoter methylation of the zinc-finger protein gene, ZNF671 in UC tumor tissue samples, a finding that was independently validated by further assessments of DNA methylation in cell lines and tissue samples. In vitro promoter methylation assay and epigenetic treatment confirmed that the expression of ZNF671 is epigenetically controlled in UC cell lines. Re-expression of ZNF671 in UC cell lines inhibited tumor growth in soft agar colony formation assay and invasion assay. In vivo tumorigenicity assay, re-expression of ZNF671 suppressed tumor growth in nude mice, in possible conjunction with downregulation of cancer stem cell markers. Moreover, bisulphite pyrosequencing demonstrated high ZNF671 methylation in UC tumor tissues (n=96), associated with tumor grade and poor locoregional disease-free survival. To investigate the diagnostic potential of this novel epigenetic marker in non-invasive cancer detection, we performed quantitative methylation-specific PCR (qMSP) analysis in a training (n=97) and test (n=61) sets of voided urine samples from bladder UC patients revealed a sensitivity and specificity of 42%-48% and 89%-92.8%, respectively, for UC cancer detection. Moreover, combining DNA methylation of ZNF671 and 2 other genes (IRF8 and sFRP1) further increased the sensitivity to 96.2%, suggesting a possible three-gene UC biomarker. In summary, ZNF671, an epigenetically silenced novel tumor suppressor, represents a potential predictor for UC relapse and non-invasive biomarker that could assist in UC clinical diagnosis-making.