Aberrant Methylation And Expression Of DAPK1in Human Hypopharyngeal Squamous Cell Carcinoma

BackgroundHypopharyngeal malignant tumor is one of the most aggressive cancers in the head and neck area, and squamous cell carcinoma is the most common pathological types, hypopharyngeal squamous cell carcinoma (HSCC) accounts for5%of head neck cancers. The initiation and progression of HSCC is a multi-stage process involving a variety of changes at the gene level. Due to the particularity of its anatomical site, early symptoms of HSCC are not obvious. Most of HSCC presents poor differentiation, aggressive growth by submucosal infiltration and diffusion and early regional lymph node involvement. At present, the multidisciplinary plan, including surgery, ionizing radiation and chemotherapy, is the mainstay of the treatments of HSCC. Despite the improvements made in recent years, clinical results of HSCC are still dissatisfying with the5-year survival rate at only approximately40-50%.The Death-Associated Protein kinase-1(DAPkl or simply, DAPk) is a tumor suppressor gene found in recent years, which is one of five kinases of DAPk protein family, including DAPkl, DAPk2/DRP-1, DAPk3/ZIPk/Dlk, DRAK1and DRAK2. Mapped to chromosome9q34.1, DAPkl is a160-kD protein consisting of1430amino acids, which contains kinase domain, Ca2+/CaM autoregulatory domain, eight ankyrin repeats, ROC-COR domains, a death domain and a serine-rich C-terminal tail. DAPkl was a positive mediator of apoptosis induced. As a multi-domain serine/threonine kinase, DAPk1participates into multiple signaling pathways that regulate apoptosis, autophagy, caspase-dependent cell death, cell adhesion and migration. Therefore, it functions not only in inflammation but also in antitumor and anti-metastasis. At present, more and more anti-tumor effects of DAPkl were discovered, including disruption of integrin signaling, promotion of anoikis, suppression of cell motility, regulation of tumor metabolism and so on.As a tumor suppressor gene, expression of DAPkl is frequently down-regulated in many kinds of tumors, and the methylation of a CpG island in the promoter region appears to be one major mechanism, which will modify the structure but not the sequence of DNA. Because DNA methylation is one of the essential epigenetic mechanisms that are closely correlated with cell growth, differentiation and transformation, it should play significant roles in the initiation and progression of cancers. Meanwhile, aberrant CpG island methylation of the suppressor genes is a frequent event in many cancers. Such DNA promoters’ hypermethylation status is to silence gene expression by providing an alternative pathway to gene’s inactivation, deletions or mutations. Aberrant CpG island methylation of the DAPkl gene is common in the tumors, including laryngeal cancer, colorectal cancer, breast cancer and so on. In our study, we determined the extent of aberrant DNA methylation and expression of DAPk1in HSCC, and assessed its clinicopathological features and potential prognostic values. ObjectiveFifty-three pairs of primary tumor tissues and adjacent non-tumor tissues samples were collected from HSCC patients. This study was aimed to investigate extent of aberrant DNA methylation and expression of DAPkl in HSCC, and assessed its clinicopathological features and potential prognostic values.Methods1. Fifty-three pairs of primary tumor tissues and adjacent non-tumor tissues samples were collected from HSCC patients hospitalized in2010. Tumor tissues were routinely harvested from the center of the lesions, and corresponding non-tumor tissues were procured at least2cm distal to tumor margins.2. Methylation status of DAPk1was detected by methylation-specific polymerase chain reaction (MSP), and expression of DAPk1was determined with real-time reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry and western blot at mRNA or protein levels. Correlations between the findings and patients’clinicopathological parameters were further evaluated.Results1. Methylation ratio of tumor tissues was60.38%(32/53), while the ratio of adjacent non-tumor tissues was26.42%(14/53) in HSCC. Continuous correction Chi-square test showed that methylation ratio in tumor group was significantly higher than non-tumor tissue group (P=0.001).2. With real-time RT-PCR, the mRNA level of DAPkl was considerably lower in tumor tissues than in adjacent non-tumor tissues (0.863±0.095v.s.1.000)(P=0.002). With western blot, the protein level of DAPkl was also much lower in tumor tissues than in adjacent non-tumor tissues (0.459±0.036v.s.0.666±0.037)(P<0.001). With immunohistochemistry, DAPk1located mainly in the cytoplasm. Expressions of DAPk1were identified in32.08%HSCC tissues (17/53), which was markedly lower than adjacent non-tumor tissues (58.49%,31/53)(P=0.006). The results demonstrated that DAPkl expression was down-regulated notably in the tumor group at mRNA or protein levels, and the difference is statistically significant.3. The mRNA level of DAPkl was significantly different between the former (0.633±0.095) and the latter group (1.212±0.167)(P=0.002, r=-0.521).DAPkl methylation was more frequently found in the tumors with lymph node metastasis than those without (P=0.001), and in the tumors with lymph node metastasis (N1+N2), mRNA expression of DAPk1was significantly down-regulated (0.516±0.069v.s.1.128±0.142, P=0.001). And significant correlation was also found between hypermethylation, down-regulation of DAPkl and clinical TNM stage (P=0.009and0.019, respectively). But there was no other significant association with clinicopathological parameters.4. Survival analyses among53HSCC patients showed that the overall survival was better in the patients with unmethylation of DAPkl (P=0.031) and up-regulated DAPk1expression (P=0.045). In the multivariate Cox’s proportional hazards regression analyses, invasion of regional lymph nodes was an independent prognostic factor (P=0.043, HR5.387,95%CI1.052-27.600), while methylation and expression of DAPk1, tumor stage and clinical TNM stage were not, rather than an independent factor for HSCC, DAPk1might work in a cooperative way with other genes to exert its influence on the prognosis.ConclusionsOur findings indicates that in HSCC, hypermethylation and down-regulation of Death-Associated Protein kinase-1(DAPkl) are common events, and there is a negative correlation between hypermethylation and down-regulation. Hypermethylation and down-regulation is associated with lymph node metastasis, advanced TNM stage and poor prognosis, and invasion of regional lymph nodes was an independent prognostic factor.