Akt1 enhances the chemoradiotherapy resistance of gliomas through phosphorylation of ubiquitin-conjugating enzyme 2S: An experimental study

Abstract

Objective To explore the mechanism of Akt1-associated enhancement of the chemoradiotherapy resistance of gliomas. Methods The immunofluorescence (IF) experiment was conducted to test the expression of ubiquitin-conjugating enzyme 2S (UBE2S) in the normal astrocytes and glioma cells (U87, U251, SF767 and U373). The Western blot was used to assess the differences of UBE2S expression in PTEN mutant and wild type glioma cell lines. To explore the role of PTEN/Akt signal pathway in UBE2S functions and the effect of UBE2S on the non-homologous end joining (NHEJ) repair, a constitutively activated Akt1 (Myr-HA-Akt1) and the UBE2S wild type or UBE2S T152A mutant were coexpressed. The flow cytometry (FCM) was used to test in the chemoradiotherapy sensitivity of the stable UBE2S knockdown glioma cells. Results Compared with the normal astrocytes, UBE2S was overexpressed in the malignant glioma cells. The expression of UBE2S was more stable in the PTEN mutant glioma cell line. Akt1 phosphorylated UBE2S at T152, which promoted the stabilization of UBE2S. UBE2S interacted with Ku70 and Ku80, which increased the expression of Ku70 (P=0.009). The results of Western blot showed that the expression of Ku70, Ku80 and DNA-PKcs decreased significantly after knockdown of UBE2S expression (P<0.001). The results of FCM showed that the knockdown of UBE2S could enhance the chemoradiotherapy sensitivity of gliomas(P<0.001). Conclusions UBE2S is highly expressed in the malignant glioma. Akt1 physically interacts with UBE2S and phosphorylates UBE2S at T152, which is critical for the stabilization of UBE2S. The knockdown of UBE2S expression could retard NHEJ-mediated DNA repair and thus increase the sensitivity of chemoradiotherapy. Key words: Glioma; Chemoradiotherapy; Ubiquitin-conjugating enzyme 2S; PTEN/Akt signal pathway