Esophageal squamous cell carcinoma (ESCC) is usually a common malignant disease

Esophageal squamous cell carcinoma (ESCC) is usually a common malignant disease characterized by poor prognosis. metastasis [17]. However, the exact clinical and biological role of VRK1 in the initiation and progression of ESCC remains unknown. The oncogene c-Jun was initially identified as Fos-associated protein p39, which acted as a transcription factor by forming dimers with Jun-related protein, Fos-related protein or ATF/CREB protein family members to activate AP-1 [18, 19]. In cells, c-Jun phosphorylation is usually rapidly and transiently induced by numerous SC35 extracellular stress stimuli, which are mediated and relayed by mitogen-activated protein kinases (MAPKs), such as ERK1/2, p38K and c-Jun N-terminal kinase (JNK) [20C22]. Phosphorylated c-Jun becomes transcriptionally active and participates in cell proliferation, differentiation and apoptosis by controlling the manifestation of relevant genes [23]. A previous study discovered that VRK1 can phosphorylate c-Jun at serine 63 and serine 73 and interact with JNK [24]. Thus, aberrant VRK1 manifestation might induce constitutive c-Jun activity impartial of extracellular stress or upstream signals and eventually contribute to oncogenesis. Although previous research has reported that VRK1 manifestation levels are elevated in ESCC [25], the biological functions TAK-733 of VRK1 in ESCC are still unclear. In this study, we investigate the manifestation profile of VRK1 in ESCC TAK-733 and its correlation with clinicopathological characteristics. VRK1 was found to promote cell proliferation and migration as well as cisplatin (CDDP) resistance in ESCC. Furthermore, our results indicated that VRK1 up-regulates c-MYC through c-Jun activation and that this axis is usually responsible for VRK1-mediated CDDP resistance. We also examined the inhibitory effect of VRK1 using luteolin, a VRK1 inhibitor, in ESCC cells both and < 0.001) and TNM stage (< 0.001) (Physique 1B, 1C). Subsequently, we examined the VRK1 mRNA and protein levels in normal esophageal epithelial cells and in a panel of ESCC cell lines and found that VRK1 was differentially up-regulated in the ESCC cell lines compared with that in the TAK-733 normal Het-1a and HECC cells (Physique 1D, 1E). Physique 1 VRK1 is usually overexpressed in ESCC and correlated with the clinical outcome of ESCC patients The VRK1 protein level was further examined in 132 paraffin-embedded, archived ESCC tissues using immunohistochemistry (IHC). Positive staining was reflected by yellow or brown particles, which were predominantly aggregated in the nucleus. The semi-quantitative IHC analysis score for staining intensity and the percentage of positively stained cells showed that high VRK1 manifestation (staining index (SI) 8) was observed in 59.84% (79/132) of tumor tissues (Figure ?(Figure1F).1F). Consistent with this observation, statistical analysis revealed that aberrant VRK1 protein levels were significantly correlated with the depth of invasion, lymphatic involvement and TNM stage (Table ?(Table1).1). Furthermore, Kaplan-Meier and log-rank analyses indicated that patients with high levels of VRK1 manifestation (SI 8) had worse overall survival (OS) or disease-free survival (DFS) than patients with low levels of VRK1 (SI < 8) manifestation (Physique 1G, 1H). Ultimately, the univariate and multivariate Cox regression analyses exhibited that VRK1 manifestation, along with T classification and N classification, were impartial prognostic factors in ESCC (Table ?(Table22). Table 1 Association between VRK1 protein level and several clinical characteristics in 132 ESCC cases Table 2 Univariate and multivariate Cox regression analysis for overall survival and disease-free survival in 132 ESCC patients VRK1 mediates invasion and proliferation of ESCC cell lines Given that VRK1 manifestation was correlated with malignant clinical parameters (depth of invasion, lymphatic involvement and TNM stage) in ESCC patients, we therefore investigated the biological role of VRK1 in initiation and progression of ESCC. To explore the effect of VRK1 on the malignant ESCC phenotype, Ec9706 cells stably overexpressing full-length VRK1 DNA (OEVRK1-Ec9706) as well TAK-733 as Eca109 (shVRK1-Eca109) and Kyse150 (shVRK1 -Kyse150) cells transfected with VRK1-shRNA lentivirus were constructed. The efficiency of VRK1 knockdown or overexpression in ESCC cells was decided by western blotting (Physique ?(Figure2A2A). Physique 2 VRK1 is usually essential for the proliferation, survival, migration and invasion of ESCC cells As expected, the proliferative capacity and colony-forming ability of Kyse150 and Eca109 cells transfected with shRNA targeting VRK1 were impaired compared with that of control cells, as decided with CCK-8 (Physique ?(Physique2W2W and Supplementary Physique 1) and colony formation (Physique ?(Figure2C)2C) assays, respectively. In contrast, ectopic manifestation of VRK1 in Ec9706 cells led to a significant increase in cell proliferation (Physique ?(Physique2W,2B, Supplementary Physique 1) and colony formation (Physique ?(Figure2C).2C). Additionally, an apoptosis assay (Physique ?(Figure2D)2D) revealed significantly decreased apoptosis in VRK1-overexpressing cells (OEVRK1-Ec9706) and increased apoptosis in VRK1-knockdown cells (shVRK1 -Eca109 and shVRK1 -Eca109) compared with that in control.