Abstract 2098 Characterization of the effects of the N-terminal Hsp90 Inhibitor 17-DMAG to the effects of the C-terminal inhibitor KU174 on the proteomic profiles of HCT116 colon cancer cells

Hsp90 functions to chaperone proteins that are involved in tumor development, progression, metastasis and evasion of cell death. Because Hsp90 is required for oncogenic proteins to acquire and maintain their activated state, Hsp90 inhibitors have been developed with the potential of being effective anti-cancer drugs. Several inhibitors which target its N-terminal ATPase domain of Hsp90 have made it to clinical trials. However, these inhibitors have been found to have limited efficacy due to dose limiting toxicities and the concomitant induction of the anti-apoptotic heat shock response (HSR). The development in inhibitors that interact with C-terminal domain of Hsp90 have been pursued based on the scaffold of the natural product novobiocin, as they do not similarly induce the HSR. Using SILAC and MudPIT, followed by LC/MS-MS analysis utilizing an Orbitrap Tribrid Fusion mass spectrometer, we identified 4,235 proteins from extracts from HCT116 cells treated with the second-generation C-terminal inhibitor KU174 and 5,520 proteins from cells treated with 17-DMAG, a traditional N-terminal inhibitor. Significant protein expression changes were determined by a one-sample two-sided t-test, using Benjamini-Hochberg FDR set to 0.05. Differentially expressed proteins (DEP) were analyzed for Gene Ontology and Pathways enrichment using an arbitrary cut-off of ± 30% change in expression levels. Analysis of DEPs that were increased by the drugs treatments, indicated that there was little commonality in their effects, and as expected no induction of the HSR by KU174. Analysis of DEPs that were decreased by the drugs treatments indicated that KU174 did not deplete cells of Hsp90-dependent kinases and did not cause a generalized depletion of Hsp90-dependent proteins, suggesting that KU174 does not target the canonical Hsp90/Hsp70/HOP/Cdc37 folding pathway. Interestingly, KU174 treatment of HCT116 cells deplete proteins involved RNA processing and splicing, and RNA polymerase complexes, particularly RNA Polymerase II. These are pathways that require the function of the Hsp90-R2TP/PAQosome complex, which suggests a new mechanism through which Hsp90-dependent processes can be inhibited. This work was support by a Stephenson Cancer Center seed grant and the Oklahoma Agricultural Experiment Station Project OKL03159.