Proteomics analysis of Hsp90 modulation

B160

Background: Hsp90 is a pleiotropic cytosolic chaperone protein that is involved in stabilization and maturation of over 100 ‘client’ proteins, many of which are upregulated or mutated in human cancers. Preclinical data indicates that not all client proteins are affected by Hsp90 inhibition equally and some proteins such as HER-2 have been identified as more sensitive client proteins than others. The wealth of potential cellular functions in which Hsp90 plays a role potentially offers a plethora of biomarkers for drug sensitivity, and activity. Proteomic analysis represents a comprehensive and efficient method to improve our understanding of the cellular consequences of Hsp90 modulation. Hsp90 and other cellular chaperones function primarily by regulating protein stability, sub-cellular protein location, composition of the secreted proteome, and affects protein phosphorylation, all of which can be monitored globally and simultaneously by proteomic analysis.
 Methods: To explore the astounding complexity of proteome, we have developed a robust and fully automated non-labeled tandem multidimensional separation system on-line coupled with ESI-MS (LC/LC/LC-MS/MS/MS). This platform provides reliable and sensitive analysis and gives over 10,000 high-confidence protein identifications from a typical sample load (100 ug). Multiplex iTraq labeling has been applied to provide accurate quantification. This mass spectrometry based platform was utilized to investigate the consequences of small-molecule inhibition of Hsp90. Both in vitro and ex vivo experiments were designed to focus on the changes occurring over 1 to 48 hours by both marginally and strongly active concentrations of the Hsp90 inhibitor BIIB021 in either HER-2⁺ breast cancer cells or normal human PBMCs. Additionally, tumor interstitial fluid (TIF) was explored as a source of secreted biomarker candidates in an in vivo setting using a xenograft model of tumor regression.
 Results: Consistent changes in protein expression level were observed in various metabolic and signaling pathways after BIIB021 exposure, including ribosomal proteins and purine metabolism. In agreement with results from antibody based techniques, BIIB021 induced HER-2 degradation in both a dose and time dependant manner. Both the intracellular and extracellular domains of HER-2 peptides were detected. Additionally, we found other proteins that also decreased in a similar dose and/or time dependant manner. Conversely, antibody based techniques have revealed the upregulation of heat shock protein Hsp70 as a consequence of Hsp90 inhibition. Our results confirmed Hsp70 upregulation but it does not show a dose dependant response. In addition, more proteins were identified that demonstrated a dose dependant upregulation in response to Hsp90 inhibition.
 Conclusions: Proteomics analysis of Hsp90 modulation has confirmed and extended our knowledge of the dose dependent changes in abundance for existing biomarkers such as HER-2 and HSP70.