Abstract
Heat shock protein 70 (Hsp70) and Hsp90 are molecular chaperones that play essential roles in tumor growth by stabilizing pro-survival client proteins. However, although the development of Hsp90 inhibitors has benefited from the identification of clients, such as Raf-1 proto-oncogene, Ser/Thr kinase (RAF1), that are particularly dependent on this chaperone, no equivalent clients for Hsp70 have been reported. Using chemical probes and MDA-MB-231 breast cancer cells, we found here that the inhibitors of apoptosis proteins, including c-IAP1 and X-linked inhibitor of apoptosis protein (XIAP), are obligate Hsp70 clients that are rapidly (within ∼3-12 h) lost after inhibition of Hsp70 but not of Hsp90. Mutagenesis and pulldown experiments revealed multiple Hsp70-binding sites on XIAP, suggesting that it is a direct, physical Hsp70 client. Interestingly, this interaction was unusually tight (∼260 nm) for an Hsp70-client interaction and involved non-canonical regions of the chaperone. Finally, we also found that Hsp70 inhibitor treatments caused loss of c-IAP1 and XIAP in multiple cancer cell lines and in tumor xenografts, but not in healthy cells. These results are expected to significantly accelerate Hsp70 drug discovery by providing XIAP as a pharmacodynamic biomarker. More broadly, our findings further suggest that Hsp70 and Hsp90 have partially non-overlapping sets of obligate protein clients in cancer cells.
Highlights
Heat shock protein 70 (Hsp70) and Hsp90 are molecular chaperones that play essential roles in tumor growth by stabilizing pro-survival client proteins
We show that treatment with Hsp70 inhibitors leads to rapid and dramatic loss of the inhibitor of apoptosis proteins (IAPs) in MDA-MB-231 breast cancer cells. To understand this relationship in more detail, we explored the interaction between X-linked inhibitor of apoptosis protein (XIAP) and Hsp70 in vitro and found that the chaperone binds to multiple sites within the BIR2 and BIR3 domains
We first examined the levels of XIAP, c-IAP1, and Raf-1 after treatment with either Hsp70 or Hsp90 inhibitors
Summary
Chemical inhibitors of Hsp have been reported to enhance turnover of a number of proteins, including IAP-1, XIAP, Raf-1, tau, androgen receptor and others [35, 40, 46]. Using co-immunoprecipitations, we found that endogenous XIAP was bound to Hsp in MDA-MB-231 cell lysate (Fig. 2B) To understand whether this interaction might be direct, we first used a computational algorithm to predict possible Hsp70-binding sites in full-length human XIAP [18]. In the ELISA platform, we found that Y190E, L207S, L307S, and L331S all had weaker affinity for Hsp, with dissociation constants ranging from 2-fold to greater than 10-fold higher than the wildtype protein (Fig. 3C) To test whether this difference in affinity would translate into resistance to Hsp inhibitors, we expressed full-length XIAP (XIAPWT, XIAPY190E, and XIAPL207S) in HeLa cells and measured protein stability after treatment with JG-98. Tant because clients often depend on chaperone only in a cancer cell-specific context [4] Consistent with this idea, JG-98 did not reduce levels of XIAP or c-IAP1 in IMR90 normal human lung fibroblasts (Fig. 5B). These results suggest that XIAP and c-IAP1 are candidate biomarkers of Hsp in cells and animals
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