Hsp90 has emerged as an exciting anticancer drug target due to its role in maintaining the conformational stability of a plethora of oncogenic client proteins. The molecular chaperone function of Hsp90 is dependent on ATP hydrolysis which is orchestrated by interactions with a number of co-chaperones including Hsp70. 17-allylamino-17-demethoxygeldanamycin (17AAG) competitively inhibits the intrinsic ATPase of Hsp90 which we and others have previously demonstrated to cause cytostasis and cell line-dependent cell death in vitro, the extent of which is relatively small when compared to that induced by other clinically relevant agents such as 5-FU and oxaliplatin (Clarke et al unpublished observations). This may be due to induction of the major constitutive (Hsc70) and inducible (Hsp72) isoforms of the Hsp70 family, which we and others have previously shown to occur in response to Hsp90 inhibition. Since the Hsp70 family can have anti-apoptotic activity, we used an siRNA approach to reduce the expression of Hsp72 and/or Hsc70 in the human colon cancer cell line HCT116. Transient knockdown of Hsp72, but not Hsc70, prior to treatment with 17AAG caused an increase in the cell death response to this Hsp90 inhibitor. We hypothesized that the absence of increased cell death following Hsc70 reduction may be due to concurrent induction of Hsp72. To investigate this, the expression of the two isoforms were simultaneously reduced which resulted in increased cell death from 13.1% (± 2.18 s.e) to 40.3% (± 13.79 s.e) of the total cell population following only 24 hours exposure to 17AAG. In addition, we observed a time-dependent increase in cell death following reduction of Hsp72 and Hsc70 expression, in the absence of 17AAG exposure. This increase in cell death may be in part due to inhibition of Hsp90 since knockdown of Hsp72 and Hsc70 resulted in reduced expression of Hsp90 client proteins c-RAF, CDK4 and ErbB2. The observed reduction in Hsp90 client proteins was shown to involve ubiquitin-dependent proteasome degradation, as co-treatment with proteasome inhibitors bortezomib and MG-132 resulted in reduced degradation of these clients. In conclusion, we have demonstrated that induction of Hsp72 provides a cytoprotective effect against the cell death activity of 17AAG. In addition, reducing the expression of Hsc70 with Hsp72 produces a molecular phenotype similar to that of Hsp90 inhibition but with a degree of cell death far greater than is associated with existing Hsp90 inhibitors. This is consistent with an anti-apoptotic effect of Hsp72 and Hsc70, which makes the development of Hsp70 inhibitors an exciting prospect, both in terms of enhancing the cell death activity of existing Hsp90 inhibitors and also as anticancer therapeutics in their own right.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]