Regulation of HDM2 E3-ubiquitin ligase in esophageal adenocarcinoma cells by AURKA.

Abstract

Listen

Translate article

35 Background: Esophageal adenocarcinomas (EAC) exhibit intrinsic resistance against chemotherapy. AURKA regulates cell cycle progression and its overexpression is associated with oncogenic transformation. We have recently reported that AURKA is significantly overexpressed in about 70% of human EAC tissue samples and EAC cell lines. We have previously shown that AURKA inhibits p53- and p73-mediated apoptotic pathways in GI adenocarcinomas. HDM2 is an E3-ubiquitin ligase which is closely involved in regulating p53 and p73 protein stability and activity. In this study we demonstrate that AURKA directly interacts with HDM2 and regulates HDM2 protein expression and phosphorylation in both FLO-1 and OE33 EAC cells. Methods and Results: Western blot analyses were done following AURKA overexpression with adenovirus, knockdown with si-RNA or inhibition with MLN 8237 (0.5µM) in FLO-1 and OE33 EAC cell lines. The data indicated that overexpression of AURKA induced both total and phospho-HDM2-(Ser166) protein levels. Knockdown or inhibition of AURKA significantly decreased expression of both total and phospho-HDM2-(Ser166) protein levels in FLO-1 and OE33 EAC cells. Additionally, following adenovirus mediated overexpression of AURKA, co-immunoprecipitaion (Co-IP) was done for AURKA and HDM2 in FLO-1 and OE33 EAC cells. The two-way Co-IP data indicated the presence of HDM2 in a complex associated with AURKA and vice-versa. The data from in vitro protein kinase assay indicated that recombinant AURKA directly phosphorylates recombinant HDM2 at Ser166 site. To confirm direct interaction between recombinant AURKA and HDM2 proteins we performed IP following the in vitro kinase assay. The in vitro kinase IP data indicates that kinase intact recombinant AURKA directly interacts and phosphorylates recombinant HDM2 protein. Conclusions: Our data indicate that AURKA regulates HDM2 expression and phosphorylation in both FLO-1 and OE33 EAC cells. Additionally, we also report for the first time that AURKA directly interacts with HDM2 and phosphorylates it at Ser166 site. Therefore, our study suggests that AURKA-mediated regulation of HDM2 could be the major underlying mechanism for induction of apoptosis in p53-negative EAC.