Increasing evidence points to the multi-functional HSP70 molecular chaperone as a key factor in the ability of cancer cells to survive the potentially toxic effects of enhanced proteotoxic stress. This stress-induced factor helps to maintain the function and/or stability of a large number of client proteins, including many oncogenes and signaling factors that directly contribute to a tumorigenic phenotype. The close functional interaction among various molecular chaperones and with systems that maintain protein homeostasis helps to explain how disrupting the function of HSP70 simultaneously impacts multiple signaling pathways that sustain cancer cells. Thus, targeting the actions of HSP70 offers a promising therapeutic approach for cancer treatment. PES is a recently identified small molecule that selectively interacts with the closely related HSP70 and HSC70 proteins and impairs their actions.
The autophagy-lysosome system and the proteasome machinery represent the two major routes used by mammalian cells for protein degradation and organelle clearance. One important consequence of PES treatment in solid tumors is a disruption of both of these degradative systems, as shown here and in our previous work (24
). As a consequence, potentially toxic misfolded, aggregated, or otherwise altered proteins accumulate in the cell. Among the proteins affected by PES treatment is p62/SQSTM1. The p62/SQSTM1 adapter scaffold protein participates in, and is degraded by, autophagy; however, when autophagy is inhibited this protein accumulates and oligomerizes. Interestingly, overexpression of p62 also can have an inhibitory effect on proteasome function (34
), which would be expected to contribute to the cytotoxic effects of PES. A disruption of both the autophagy-lysosome and proteasome systems likely impacts a range of cell signaling pathways that depend on the appropriate expression and activity of both long-lived and short-lived regulatory proteins (39
). PES promotes an accumulation and oligomerization of p62/SQSTM1 in a time-dependent and dosage-dependent manner (see for example and ref. 24
). Indeed, our investigations suggest that an accumulation of p62/SQSTM1 represents a sensitive molecular signature for PES-mediated alterations in protein quality control pathways.
Like other members of the 70-kDa family of molecular chaperones, HSP70 and HSC70 are ATPases comprised of two major domains, an N-terminal adenine-nucleotide binding domain (NBD or ATPase domain) and a C-terminal substrate/client binding domain (SBD) (5
). A conformational change in structure accompanies cycles of substrate binding and release. The HSP70 and HSC70 proteins dynamically interact with a large and diverse array of substrate proteins via the SBD, and with co-chaperones via both the N-terminal and C-terminal regions. Co-chaperone partners, such as HSP90, CHIP and HSP40 are essential in carrying out HSP70/HSC70 activities, such as client protein selection, allosteric cycling and client processing (5
). Although the broad outlines of HSP70/HSC70 actions have been determined, key questions remain about the molecular mechanisms involved in these activities. These include how critical conformational changes in HSP70 are modulated and how specific client protein selection, binding, and fate are determined. Given that HSP70/HSC70 participate in many cellular pathways, selective binding of PES to the C-terminal domain of the proteins may alter cellular homeostasis in many ways. PES may promote a dissociation of chaperone complexes and/or prevent the release of client proteins from the complex, thereby preventing the proteins from achieving stable conformation and participating in cellular processes. It is likely that more than one pathway of PES-mediated HSP70/HSC70 inhibition may be operative in the cell, and future studies will be important in clarifying the way in which PES alters chaperone activities.
A critical function of HSP70 and HSC70 involves delivery of clients to the HSP90 chaperone machinery. As assayed by immunoprecipitation-Western blotting, the interactions of HSP70 with HSC70 and with HSP90 are not detectably impaired by PES. Following treatment of cells with different PES concentrations, we noted that the abundance of protein complexes containing these molecular chaperones with certain client proteins is not significantly changed until higher concentrations of PES are employed (). However, some cellular proteins may be more sensitive to HSP70 inhibition than others, and this may also vary among tumor cell types. In either case, a reduction in the overall abundance of chaperone complexes tends to correlate with the accumulation of various cellular proteins in the detergent insoluble cellular fraction, indicative of aggregation ( and ). These data suggest that when protein turnover is sufficiently impaired, a significant fraction of proteins become denatured or aggregated and subsequently are relocated, together with accompanying molecular chaperones, into the detergent-insoluble fraction perhaps as a means of disposal. If not resolved, the continued accumulation of aggregated macromolecules in the detergent-soluble and detergent-insoluble cellular compartments would result in cell death. Accordingly, PES-mediated inhibition of HSP70/HSC70 results in impaired autophagy-lysosome and proteasome systems, thereby promoting the simultaneous disruption of many tumor-promoting signaling pathways.
In a recent seminal study, Powers and colleagues (18
), investigated the effects on HSP90 resulting from the expression knockdown of HSP70, HSC70 or both. Using an siRNA gene silencing approach, they determined that simultaneous silencing of both HSP70 and HSC70 was needed to inhibit proliferation and to induce apoptosis in a consistent manner (18
). Although they are highly homologous structurally and functionally, the activities of HSP70 and HSC70 are not altogether equivalent. Some of the observed functional differences between these proteins relate to different interactions with some co-chaperones or substrates (47
). Moreover, in certain cancer cell lines, sensitivity to HSP90 inhibitors can be enhanced by the silencing of HSP70, but not of HSC70 (46
). The knockdown of expression of HSP70, HSC70, or both does not impair cellular proteasome activity (46
). Indeed, studies have shown that the dual knockdown of both HSP70 and HSC70 expression, or the use of an adenosine-derived inhibitor that targets the ATPase domain of these proteins, promotes HSP70/HSC70/HSP90 client proteins for proteasomal degradation (22
). This outcome is similar to that achieved by targeting HSP90 with pharmacologic inhibitors like 17-AAG. However, previous work has shown that the simultaneous silencing of HSP70 and HSC70 in non-tumorigenic cell lines does not produce the anti-proliferative or apoptotic effect noted in tumor cells (46
). This observation is consistent with the idea that tumor cells seem to be much more dependent on the actions of molecular chaperones in order to cope with constitutively enhanced proteotoxic and physiologic stress associated with tumorigenesis. While certain levels of HSP70/HSC70 inhibitors may be toxic to normal cells, studies on cultured cells and an in vivo Eµ-myc
animal tumor model indicate that differential responses to PES between normal and tumor cells can be achieved (24
). It should also be noted that, taken together, the data obtained to date indicate that PES-mediated HSP70/HSC70 inhibition may promote a "gain-of-function" phenotype distinct from that achieved by expression silencing of these proteins. Future biophysical studies should provide needed insight regarding the molecular mechanisms by which PES interferes with HSP70/HSC70 actions.
The development of new agents and new approaches to therapy are needed to improve the survival of cancer patients. An inhibition of autophagy and/or of the proteasome is being considered for the treatment of some tumors, including those that are resistant to conventional therapies (49
). Also, recent studies show that a combination treatment of an HSP90 inhibitor together with a proteasome inhibitor is more effective at killing cultured tumor cells when compared to using either alone (41
). The small molecule PES represents a novel selective inhibitor of the HSP70 family proteins that leads to a dysfunctional molecular chaperone machinery and impairs protein homeostasis. These findings support the further development of compounds for targeting HSP70 in human malignancies. The small-molecule PES also represents a valuable new tool to interrogate the critical functions of HSP70 proteins in normal biology and in various pathologies.