The stress-inducible molecular chaperone HSP70 participates in numerous cellular pathways and interacts with a varied group of proteins, including key factors in signal transduction, transcription, cell cycle control and stress response (Mayer and Bukau, 2005
; Brodsky and Chiosis, 2006
; Garrido et al., 2006
; Schmitt et al., 2006
; Powers and Workman, 2007
). HSP70 activities also have been implicated in the pathogenesis of several human diseases, including cancer. Thus, there is growing interest in the identification of HSP70 modulators to better understand the many cellular activities of this protein. In this study we provide evidence that the small molecule PES interacts with HSP70, alters its functions, and is cytotoxic to tumor cells. PES-induced tumor cell death is not dependent on caspase activation or p53 function, and is not inhibited by overexpression of the anti-apoptotic BCL-xL protein. Rather, loss of cell viability is associated with protein aggregation and an impairment of lysosomal functions resulting in a disruption of autophagic processes.
Autophagy is a catabolic process characterized by the self-digestion of cellular constituents in lysosomal compartments. This degradative process is an important mechanism for the disposal of altered cytoplasmic constituents, including aggregated proteins, and it can be activated in tumor cells by various stressors, including as a response to therapies, nutrient deprivation, or following an inhibition of apoptosis. The process generally serves to promote survival under adverse conditions, in part by helping to prevent the accumulation of damaged proteins and organelles, and by supporting the metabolic needs of the cell (Debnath et al., 2005
; Eskelinen, 2005
; Levine and Kroemer, 2008
; Mizushima et al., 2008
). Several lines of investigation point to HSP70 as a regulator of lysosomal activities and, thereby, of autophagy. For example, recent studies demonstrate that the stress-inducible HSP70 protein exhibits tumor-specific localization at membranes of the endosomal/lysosomal compartment, and that it contributes to tumor cell survival by inhibiting lysosomal permeabilization induced by diverse stimuli (Nylandsted et al., 2004
; Daugard et al., 2007; Ryhänen et al., 2008
). In addition, the interaction of HSP70 and the lysosomal marker LAMP2, which is disrupted by PES, has been implicated in the formation of complexes at lysosomes that are important for lysosomal activities such as the translocation of soluble substrates during chaperone-mediated autophagy. Previous work indicates that inhibiting lysosomal functions following activation of autophagy can result in cancer cell death (Amaravadi et al., 2007
; Degtyarev et al., 2008
; Maclean et al., 2008
). As presented here, PES-treated cells exhibit a significant reduction in the degradation of long-lived proteins and an obvious defect in the processing of the precursor form of cathepsin L to the mature lysosomal form of this cysteine protease. Such observations support the conclusion that PES impairs autophagy in part by its inhibitory effects on lysosomal functions.
HSP70 also is a regulator of apoptosis that has been reported to associate with APAF1 and to either block, or promote, apoptosome formation, depending on experimental conditions (Beere et al., 2000
; Saleh et al., 2000
; Kim et al., 2008
). Recent studies indicate that HSP70 interacts with the tumor suppressor protein PHAPI and the cellular apoptosis susceptibility protein CAS; together these three proteins play an important role in helping with the proper folding of APAF1 to prevent its aggregation and to stimulate apoptosome assembly and caspase activation (Kim et al., 2008
). Consistent with such a model, our studies reveal that in the presence of PES, the interaction of HSP70 with its substrate APAF1 is diminished, and this correlates with a significant reduction in caspase activation following cisplatin treatment of tumor cell lines. PES also inhibits the appearance of the HSP70/p53 complex and the stress-induced localization of p53 to mitochondria, thus interfering with p53-mediated apoptosis.
HSP70 regulation and function is mediated by its interactions with co-factors or co-chaperones. PES disrupts several of these interactions, including the association of HSP70 with CHIP, BAG-1M, and HSP40. Both CHIP and BAG-1 help regulate the ATPase activity of HSP70. It has been suggested that, in binding to both HSP70 and substrates, co-factors like CHIP and BAG-1 also may serve as a direct physical link between the chaperone and the proteasome, perhaps aiding in the targeting or selection of substrates for degradation (Mayer and Bukau, 2005
; Townsend et al., 2005
; Kabbage and Dickman, 2008
). The HSP40 co-factor also works with HSP70 to induce conformational changes of certain substrates, in part by promoting ATP hydrolysis and preventing protein aggregation (Fan et al., 2003
; Vos et al., 2008
). HSP-interacting proteins, including CHIP, HSP40, BAG-1 and the scaffold-adapter protein p62/SQSTM1, also associate with many other cellular proteins to mediate diverse biological processes and signaling pathways. Altering HSP70 function therefore also has consequences for the activities of these other regulatory proteins. For example, recent data show that an inability to eliminate p62 through autophagy can lead to a toxic increase in oxidative stress and DNA damage in some tissues, and that autophagy inhibition also can compromise the ubiquitin-proteasome system, leading to a potentially lethal accumulation of aggregation-prone or misfolded proteins (Korolchuk et al., 2008; Matthew et al., 2009). Considered together, these data are consistent with the idea that PES alters the activities of HSP70 in multiple cellular processes, disabling the normally cytoprotective role of this molecular chaperone. Interestingly, since PES selectively interacts with HSP70, the deleterious consequences of this compound may depend on the presence of this protein. Our investigations on the cell-death inducing properties of PES are supportive of this idea, in that cultured tumor cells which generally have greater levels of HSP70 are much more sensitive to the cell death effects of this small molecule than are non-transformed fibroblasts. Additionally, reducing HSP70 levels in tumor cells reduces the cytotoxic effects of PES exposure. In this regard, PES may cause HSP70 to adopt a potentially lethal “gain-of-function” activity along with a loss of its prosurvival role. Future studies, including structural analysis of the HSP70-PES interaction, should provide needed insight about this issue.
HSP70 has a key cytoprotective role in a broad range of activities that promote protein homeostasis, including the targeting of potentially toxic proteins for proteolysis. Cancer cells experience high levels of protein-modifying- and metabolic-stresses and seem to be particularly dependent on the various actions of HSP70 for survival. This phenomenon, referred to as “non-oncogene addiction”, suggests that it may be possible to target such critical survival proteins for the development of therapies aimed at the selective killing of neoplastic cells (Solomini et al., 2007). In support of this idea, our in vivo analysis indicate that administration of PES inhibits spontaneous tumor development and enhances survival in the Eμ-Myc model of lymphomagenesis. Thus PES represents a valuable new tool to advance basic investigations on the varied activities of the HSP70 protein, and also should have application in the development of effective therapies aimed at simultaneously disabling multiple cancer-critical biological processes.