Characterization of Pontin, Reptin and CD82 in Esophageal Adenocarcinoma Cells

Background: Barrett’s esophagus (BE), the result of chronic exposure of esophageal mucosa to acid exposure, predisposes to the development of esophageal adenocarcinoma (EAC). While the incidence of EAC is rapidly increasing, its prognosis remains poor, with a five-year survival rate of 20%. For advanced cancer stages, chemotherapy together with surgery is considered to be the standard in therapy. Consequently, the discovery of new molecular targets for an efficient characterization of EAC patients is necessary. CKAP4 has been attributed to be increased in various cancer entities and has a role in tumorigenesis. However, its role in EAC development and progression is widely unknown. Materials and Methods: In a Barrett’s esophagus, in vitro cell culture model of BE and EAC, CKAP4 expression levels were verified with qPCR and western blot. In OE33 and OE19 cells a knockdown of CKAP4-expression was done by siRNA for 48h and 72h. In siCKAP4 transfected cells FACS (apoptosis), western blot analysis, colony forming assays, spheroid formation and proliferation assays were performed. Using western blot, the impact of CKAP4-knockdown on the phosphorylation of Akt, MAP-Kinase, GSK3ß and ß-Catenin was investigated. Furthermore, Ki67and p21-mRNA-expression was analyzed in siCKAP4 cells using qPCR. Results: CKAP4 was present in all cell lines of the Barrett’s sequence (squamous epithelium, metaplasia, dysplasia and EAC). CKAP4 expression was lower in OE19 cells than in OE33 cells. Western blot and qPCR analyses confirmed an efficient siRNArelated knockdown of CKAP4 in EAC cells after transfection for 48h and 72h. The phosphorylation of Akt and MAP-Kinase was decreased in OE33 cells only, suggesting a role of CKAP4 in tumorassociated proliferation, which was confirmed by a decreased number of colonies in a colony forming assay and smaller spheroids in siCKAP4 cells. There were no significant changes in Ki67and p21-mRNA-expression in siCKAP4 cells. Conclusion: The role of CKAP4 is barely investigated in EAC at this time. Acting as a Dickkopf-1 (DKK1) receptor, the DKK1CKAP4-axis might promote cell proliferation independently to the Wnt-pathway. Based on our current results, it can be assumed, that CKAP4 is important for EAC cell growth and proliferation. Nevertheless, further characterization of CKAP4 is necessary, particularly focusing on the CKAP4-DKK1-axis to provide more evidence for its contribution in EAC development.