Silencing of the heat shock protein 90 (HSP90) cochaperone CDC37 by RNA interference sensitises cancer cells to HSP90 inhibitors

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The molecular chaperone HSP90 maintains the stability and activity of an array of proteins involved in cellular signalling pathways. A large subset of these HSP90 client proteins are protein kinases, which are frequently mutated or overexpressed in cancers. Kinase clients specifically require association with the cochaperone CDC37 to facilitate their recruitment to HSP90. CDC37 itself is known to exhibit oncogenic characteristics, with the ability to promote proliferation and induce transformation. In view of the important role of CDC37 in cancer cells, we set out to validate CDC37 as a potential target for therapeutic intervention and to determine whether modulation of CDC37 affects the sensitivity to HSP90 inhibitors. Using the colon carcinoma cell lines, HCT116 and HT29, CDC37 expression was silenced using siRNAs. Silencing of CDC37 expression partially reduced the expression of the protein kinase clients CDK4, CDK6, AKT, ERBB2 and CRAF. This was consistent with the observed decrease in association between kinase clients and HSP90. Decreased activation of pathways regulated by these kinase clients was also determined by a reduction in the levels of phosphorylated substrates. Subsequently, prolonged knockdown of CDC37 expression resulted in modest inhibition of cellular growth. Furthermore, silencing of CDC37 in HCT116 cells caused sensitisation to the HSP90 inhibitor 17-AAG, as determined by a significant 3-fold decrease in the GI₅₀. A similar sensitisation was also observed in a prostate and breast cancer cell line in which CDC37 expression was silenced, suggesting that the depletion of kinase clients by CDC37 knockdown significantly contributes to the antiproliferative activity of 17-AAG. Combining the knockdown of CDC37 expression with 17-AAG treatment for 96hrs resulted in less recovery of kinase client proteins compared with controls treated at equitoxic doses. Kinase clients could also be significantly depleted using lower doses of 17-AAG compared with controls when CDC37 was silenced. A G1/S cell cycle block was observed following CDC37 knockdown combined with 17-AAG, again at doses that were sub-effective in controls. As well as enhancing 17-AAG-induced growth inhibition, silencing of CDC37 expression also promoted apoptosis following 17-AAG treatment, as shown by the level of cleaved PARP. In conclusion, we have demonstrated that silencing of CDC37 expression compromises the stabilisation of protein kinase clients and acts synergistically with HSP90 inhibitors by potentiating growth inhibition and apoptosis. This is further supportive of the potential that CDC37 inhibition could have as a more selective therapeutic approach for treating kinase-driven cancers