Epigenetic Identification of Paired Box Gene 5 as a Functional Tumor Suppressor Associated with Poor Prognosis in Patients with Gastric Cancer.

摘要

Background & aims. DNA methylation induced tumor suppressor gene silencing plays an important role in carcinogenesis. By using methylation-sensitive representational difference analysis, we identified paired box gene 5 (PAX5) being methylated in human cancer. PAX5 locates at human chromosome 9p13.2 and encodes a 391 amino acids transcription factor. However, the role of PAX5 in gastric cancer is still unclear. Hence, we analyzed its epigenetic inactivation, biological functions, and clinical implications in gastric cancer.;Methods. Methylation status of PAX5 promoter in gastric cancer cell lines and clinical samples was evaluated by methylation specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS). The effects of PAX5 re-expression in cancer cell lines were determined in proliferation, cell cycle, apoptosis, migration and invasion assays. Its in vivo tumorigenicity was investigated by injecting cancer cells with PAX5 expression vector subcutaneously into the dorsal flank of nude mice. Chromosome Immunoprecipitation (ChIP) and cDNA expression array were performed to reveal the molecular mechanism of the biological function of PAX5.;Results. PAX5 was silenced or down-regulated in seven out of eight of gastric cancer cell lines examined. A significant down-regulation was also detected in paired gastric tumors compared with their adjacent non-cancer tissues (n = 18, P = 0.0196). In contrast, PAX5 is broadly expressed in all kinds of normal adult and fetal tissues. The gene expression of PAX5 in the gastric cancer cell line is closely linked to the promoter hypermethylation status. In addition, the expression levels could be restored by exposure to demethylating agents 5-aza-21-deoxycytidine. Re-expression of PAX5 in AGS, BGC823 and HCT116 cancer cells reduced colony formation (P < 0.01) and cell viability (P < 0.05), arrested cell cycle in G0/G1 phase (P = 0.0055), induced cell apoptosis (P < 0.05), repressed cell migration and invasion (P = 0.0218) in vitro. It also inhibited tumor growth in nude mice (P < 0.05). The molecular basis of its function were investigated by cDNA expression array and demonstrated that ectopic expression of PAX5 up-regulated tumor suppressor gene P53, anti-proliferation gene P21, pro-apoptosis gene BAX, anti-invasion gene MTSS1 and TIMP1; and down-regulated anti-apoptosis gene BCL2, cell cycle regulator cyclinD1, migration related gene MET and MMP1. ChIP assay indicated that P53 and MET are direct transcriptional target of PAX5. Moreover, PAX5 hypermethylation was detected in 90% (145 of 161) of primary gastric cancers compared with 16% (3 of 19) of non-cancer tissues (P < 0.0001). After a median follow-up period of 15.4 months, multivariate analysis revealed that gastric cancer patients with PAX5 methylation had a significant poor overall survival compared with the unmethylated cases (P = 0.0201).;Conclusions. Our results demonstrated that PAX5 promoter methylation directly mediates its transcriptional silence and commonly occurs in gastric cancer. PAX5 gene can act as a functional tumor suppressor in gastric carcinogenesis by playing an important role in suppression of cell proliferation, migration, invasion, and induction of cell apoptosis. Detection of methylated PAX5 may be utilized as a biomarker for the prognosis of gastric cancer patients.