Anticancer Drugs Up-regulate HspBP1 and Thereby Antagonize the Prosurvival Function of Hsp70 in Tumor Cells

Abstract

The 70-kDa heat shock protein (Hsp70) is up-regulated in a wide variety of tumor cell types and contributes to the resistance of these cells to the induction of cell death by anticancer drugs. Hsp70 binding protein 1 (HspBP1) modulates the activity of Hsp70 but its biological significance has remained unclear. We have now examined whether HspBP1 might interfere with the prosurvival function of Hsp70, which is mediated, at least in part, by inhibition of the death-associated permeabilization of lysosomal membranes. HspBP1 was found to be expressed at a higher level than Hsp70 in all normal and tumor cell types examined. Tumor cells with a high HspBP1/Hsp70 molar ratio were more susceptible to anticancer drugs than were those with a low ratio. Ectopic expression of HspBP1 enhanced this effect of anticancer drugs in a manner that was both dependent on the ability of HspBP1 to bind to Hsp70 and sensitive to the induction of Hsp70 by mild heat shock. Furthermore, anticancer drugs upregulated HspBP1 expression, whereas prevention of such upregulation by RNA interference reduced the susceptibility of tumor cells to anticancer drugs. Overexpression of HspBP1 promoted the permeabilization of lysosomal membranes, the release of cathepsins from lysosomes into the cytosol, and the activation of caspase-3 induced by anticancer drugs. These results suggest that HspBP1, by antagonizing the prosurvival activity of Hsp70, sensitizes tumor cells to cathepsin-mediated cell death.Members of the 70-kDa heat shock protein (Hsp70) 2 family play an essential role in quality control of cellular proteins (1-3) and include stress-inducible Hsp70, constitutively expressed Hsc70, mitochondrial Hsp75, and endoplasmic reticulum GRP78 (4, 5). Under normal conditions, Hsp70 proteins function as ATP-dependent molecular chaperones by facilitating the folding of newly synthesized polypeptides, the assembly of multiprotein complexes, and the transport of proteins across cellular membranes. Under stressful conditions, the synthesis of inducible Hsp70 enhances the ability of cells to cope with increased concentrations of unfolded or denatured proteins (1-6).The Hsp70 proteins undergo a cycle of substrate binding and release that is accelerated by ATP hydrolysis (1-3). The substrate binding domain of Hsp70 is localized to a 25-kDa COOH-terminal region, with substrate access to this domain being controlled by a COOH-terminal "lid" that exposes the domain in the ATP-bound form and allows substrate binding to occur when Hsp70 is in the ADP-bound form. Opening and closing of the lid are governed by conformational changes associated with ATP binding and hydrolysis, which occur within the cleft of the 45-kDa NH 2 -terminal ATPase domain. The exchange of bound ADP for ATP results in substrate release, thus allowing Hsp70 to enter a new round of substrate binding and release.The chaperone activity of Hsp70 proteins is regulated by various accessory proteins, known as cochaperones (1-3). For example, Hsp40 binds to the COOH terminus of Hsp70, stimulates its AT...

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