Modulation of the Co-Chaperone AHA1 Affects HSP90 Function in Cancer Cells

• Main Author: Holmes, Joanna Louise
• Published: Institute of Cancer Research (University Of London) 2008
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504791

AHAl (Activator ofHSP90 ATPase) is a co-chaperone of the ATP-dependent molecular chaperone Heat shock protein 90 (HSP90) which is involved in the maturation, stabilization/degradation and function of oncogenic proteins. HSP90 operates in a multimeric complex driven by the binding and hydrolysis of ATP. Treatment of human cancer cells with the clinically evaluated HSP90 inhibitor 17-allylamino-l7- . demethoxygeldanamycin (l7-AAG) results in degradation of oncogenic client proteins via the ubiquitin-proteasome pathway. As AHAI increases the ATPase activity of HSP90, it was hypothesized that modulation of AHAI expression could influence the activity of client proteins and/or the cellular response to 17-AAG. Initial. studies were carried out to characterise the expression of AHAl in a panel of human cancer cell lines and the co-chaperone complexes in which it is found. Published data have suggested that AHAl was present only in the mature complexes with HSP90 once ATP had bound. Here, however, more detailed studies have shown that AHAI is found in both early and intermediate complexes and may have a role in driving the transition between the intermediate and mature complexes. The role of AHA 1 in the .cellular sensitivity to 17- AAG was determined by modulating AHAl expression. Overexpression of AHAl did not affect cellular sensitivity to 17-AAG, but did increase client protein activation without affecting their total levels. When AHAl levels were selectively knocked down (>80%) using an RNAi approach, client protein activation was decreased. The AHAl knockdown also resulted in a significant increase in sensitivity to 17-AAG, due in part to an increase in apoptosis. Inducible and constitutively stable RNAi models were also developed to further confirm these findings ill vitro and ill vivo. Overall, the results demonstrate that reduction of AHAl levels decreased activation of key signal transduction proteins and sensitized cancer cells to 17-AAG. Gene expression microarray analysis of AHAl knockdown revealed that the mechanism by which AHAI depletion sensitized cells to l7-AAG involved NAD(P)H dehydrogenase quinone 1 (NQOl), the expression of which was significantly increased with AHAl knockdown. Thus modulation of AHAI could be a therapeutic strategy to increase sensitivity to HSP90 inhibitors. The cDNA microarray analysis was also used to identify potential pharmacodynamic markers for future AHAl inhibitors.