Abstract 4549: Proteomic analysis of ubiquitination identifies the interplay between HSP90 inhibition and CUL5 in the control of autophagy

Abstract

Abstract HSP90 has emerged as an important target in cancer therapy as inhibition of HSP90 can result in the degradation of many oncogenic client proteins. The two major cellular protein degradation pathways are the ubiquitin-proteasome system and autophagy. The role of the ubiquitin-proteasome pathway in client protein degradation following HSP90 inhibition is well established. We recently described how this process is controlled by an E3-ubiquitin ligase, cullin 5 (CUL5). We sought to further understand how both major protein degradation pathways are regulated in response to HSP90 inhibition. To do this, we performed a proteomic screen on the ubiquitination status of proteins extracted from HT29 cells following HSP90 inhibition by 17-AAG and / or siRNA silencing of CUL5 expression. We found the level of ubiquitination of 125 proteins increased by more than 2 fold in response to 17-AAG treatment. For 66 of these proteins, CUL5 silencing eliminated the 17-AAG-induced increase in ubiquitination; for 30 proteins CUL5 silencing partially reduced the 17-AAG-induced increase in ubiquitination; and for 29 proteins CUL5 silencing had no effect. Importantly, we found some known regulators of autophagy to be among those proteins ubiquitinated in response to 17-AAG treatment and rescued by CUL5 silencing. There was a correlation between levels of ubiquitination and levels of protein degradation. We measured markers of autophagic flux, LC3B and p62, and found clear evidence for altered pathway flux in response to HSP90 inhibition and CUL5 silencing. This study indicates that the interplay between HSP90 inhibition and CUL5 can control both proteasomal degradation and autophagy. Citation Format: Silvia A a Batista, Rahul Samant, Paul A. Clarke, Paul Workman. Proteomic analysis of ubiquitination identifies the interplay between HSP90 inhibition and CUL5 in the control of autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4549.