Proteomic Analysis of Hsp90β-Selective Inhibitors Against Triple-Negative Breast Cancer to Gain a Mechanistic Insight.

Abstract

Hsp90 (90 kDa heat shock protein) is a central molecular chaperone responsible for the folding and activation of >400 client proteins, causing it to be a highly sought after drug target for the treatment of cancer. Hsp90 pan-inhibitors have been evaluated in the clinic, but on- and off-target toxicities have limited their development. The emergence of Hsp90β-selective inhibitors has been proposed as a safer therapeutic alternative. However, since they in theory only target a portion of the Hsp90-regulated proteome, a deeper understanding of their mechanism of action and whether they exhibit selectivity for cancer over normal cells has remained uninvestigated. Herein, we show that Hsp90β-selective inhibitors, NDNB1 and NDNB1182, exhibit a moderate selectivity for triple-negative breast cancer (TNBC) over normalized MCF-10A cells in contrast to pan-inhibitors, which do not exhibit a selectivity. This article contains the first proteomic analysis of Hsp90β-selective inhibitors. We have employed a traditional bottom-up LC-MS/MS proteomics approach to explore the potential mechanisms of action underlying the anticancer effects of NDNB1 and NDNB1182 against TNBC. Primarily, inhibition of kinases and associated cell signaling pathways, cell cycle proteins, and DNA repair were notable processes affected by Hsp90β inhibition, to name a few. Further investigation of the impact on Hsp90β interactors allowed a fuller understanding of Hsp90β-dependent processes. We also identified RAD9A, cyclin-dependent kinase 1 (CDK1), and ribosomal protein S9 (RPS9) as potential Hsp90β client substrates. The three example proteins selected exemplify a mechanistic explanation for inhibition of DNA repair (RAD9A), cell cycle (CDK1), and translation (RPS9), shedding some light on some of the implications of Hsp90β inhibition as it pertains to TNBC. Therefore, previously unknown clients, Hsp90β interactors, or Hsp90β-regulated proteins could be determined using the results from this study.