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The protein chaperone HSP70 is overexpressed in many cancers including colorectal cancer, where overexpression is associated with poor survival. We report here the creation of a uniquely acting HSP70 inhibitor (HSP70i) that targets multiple compartments in the cancer cell, including mitochondria. This inhibitor was mitochondria toxic and cytotoxic to colorectal cancer cells, but not to normal colon epithelial cells. Inhibition of HSP70 was efficacious as a single agent in primary and metastatic models of colorectal cancer and enabled identification of novel mitochondrial client proteins for HSP70. In a syngeneic colorectal cancer model, the inhibitor increased immune cell recruitment into tumors. Cells treated with the inhibitor secreted danger-associated molecular patterns (DAMP), including ATP and HMGB1, and functioned effectively as a tumor vaccine. Interestingly, the unique properties of this HSP70i in the disruption of mitochondrial function and the inhibition of proteostasis both contributed to DAMP release. This HSP70i constitutes a promising therapeutic opportunity in colorectal cancer and may exhibit antitumor activity against other tumor types. SIGNIFICANCE: These findings describe a novel HSP70i that disrupts mitochondrial proteostasis, demonstrating single-agent efficacy that induces immunogenic cell death in treated tumors., (©2020 American Association for Cancer Research.)

Protein quality control is an important component of survival for all cells. The use of proteasome inhibitors for cancer therapy derives from the fact that tumor cells generally exhibit greater levels of proteotoxic stress than do normal cells, and thus cancer cells tend to be more sensitive to proteasome inhibition. However, this approach has been limited in some cases by toxicity to normal cells. Recently, the concept of inhibiting proteostasis in organelles for cancer therapy has been advanced, in part because it is predicted to have reduced toxicity for normal cells. Here we demonstrate that a fraction of the major stress-induced chaperone HSP70 (also called HSPA1A or HSP72, but hereafter HSP70) is abundantly present in mitochondria of tumor cells, but is expressed at quite low or undetectable levels in mitochondria of most normal tissues and non-tumor cell lines. We show that treatment of tumor cells with HSP70 inhibitors causes a marked change in mitochondrial protein quality control, loss of mitochondrial membrane potential, reduced oxygen consumption rate, and loss of ATP production. We identify several nuclear-encoded mitochondrial proteins, including polyadenylate binding protein-1 (PABPC1), which exhibit decreased abundance in mitochondria following treatment with HSP70 inhibitors. We also show that targeting HSP70 function leads to reduced levels of several mitochondrial-encoded RNA species that encode components of the electron transport chain. Our data indicate that small molecule inhibitors of HSP70 represent a new class of organelle proteostasis inhibitors that impair mitochondrial function in cancer cells, and therefore constitute novel therapeutics.

The stress-inducible chaperone protein HSP70 (HSPA1) is implicated in melanoma development, and HSP70 inhibitors exert tumor-specific cytotoxic activity in cancer. In this study, we documented that a significant proportion of melanoma tumors express high levels of HSP70, particularly at advanced stages, and that phospho-FAK (PTK2) and BRAF are HSP70 client proteins. Treatment of melanoma cells with HSP70 inhibitors decreased levels of phospho-FAK along with impaired migration, invasion, and metastasis in vitro and in vivo Moreover, the HSP70 inhibitor PET-16 reduced levels of mutant BRAF, synergized with the BRAF inhibitor PLX4032 in vitro, and enhanced the durability of response to BRAF inhibition in vivo Collectively, these findings provide strong support for HSP70 inhibition as a therapeutic strategy in melanoma, especially as an adjuvant approach for overcoming the resistance to BRAF inhibitors frequently observed in melanoma patients. Cancer Res; 76(9); 2720-30. ©2016 AACR., (©2016 American Association for Cancer Research.)

Multiple myeloma (MM) is a common and largely incurable blood cancer for which new treatment options are needed, as resistance to current modalities is an issue. Additionally, because this tumor type often resides in a hypoxic niche of the bone marrow, new therapeutics that remain effective even under hypoxic conditions are sought. Because of the secretory nature of MM cells they are uniquely under proteotoxic stress, and we hypothesized that these tumor cells may alleviate this stress by upregulating the major stress-induced cytosolic form of the chaperone HSP70. In this work we test the efficacy of the HSP70 inhibitor PET-16 for MM. We show that MM cell lines express significant levels of HSP70, and further that inhibition of HSP70 causes decreased viability and apoptosis, along with proteotoxic stress, as assessed by the accumulation of poly-ubiquitylated proteins. Importantly, we show that growth of these tumor cells under hypoxic conditions has no effect on the ability of PET-16 to be cytotoxic. The HSP70 inhibitor PET-16 should thus be considered further for pre-clinical analyses of efficacy in MM.

The stress-inducible mammalian heat shock protein 70 (HSP70) and its bacterial orthologue DnaK are highly conserved nucleotide binding molecular chaperones. They represent critical regulators of cellular proteostasis, especially during conditions of enhanced stress. Cancer cells rely on HSP70 for survival, and this chaperone represents an attractive new therapeutic target. We have used a structure-activity approach and biophysical methods to characterize a class of inhibitors that bind to a unique allosteric site within the C-terminus of HSP70 and DnaK. Data from X-ray crystallography together with isothermal titration calorimetry, mutagenesis, and cell-based assays indicate that these inhibitors bind to a previously unappreciated allosteric pocket formed within the non-ATP-bound protein state. Moreover, binding of inhibitor alters the local protein conformation, resulting in reduced chaperone-client interactions and impairment of proteostasis. Our findings thereby provide a new chemical scaffold and target platform for both HSP70 and DnaK; these will be important tools with which to interrogate chaperone function and to aid ongoing efforts to optimize potency and efficacy in developing modulators of these chaperones for therapeutic use.

The HSP70 family of molecular chaperones function to maintain protein quality control and homeostasis. The major stress-induced form, HSP70 (also called HSP72 or HSPA1A) is considered an important anti-cancer drug target because it is constitutively overexpressed in a number of human cancers and promotes cancer cell survival. All HSP70 family members contain two functional domains: an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate-binding domain (SBD); the latter is subdivided into SBDα and SBDβ subdomains. The NBD and SBD structures of the bacterial ortholog, DnaK, have been characterized, but only the isolated NBD and SBDα segments of eukaryotic HSP70 proteins have been determined. Here we report the crystal structure of the substrate-bound human HSP70-SBD to 2 angstrom resolution. The overall fold of this SBD is similar to the corresponding domain in the substrate-bound DnaK structures, confirming a similar overall architecture of the orthologous bacterial and human HSP70 proteins. However, conformational differences are observed in the peptide-HSP70-SBD complex, particularly in the loop L(α, β) that bridges SBDα to SBDβ, and the loop L(L,1) that connects the SBD and NBD. The interaction between the SBDα and SBDβ subdomains and the mode of substrate recognition is also different between DnaK and HSP70. This suggests that differences may exist in how different HSP70 proteins recognize their respective substrates. The high-resolution structure of the substrate-bound-HSP70-SBD complex provides a molecular platform for the rational design of small molecule compounds that preferentially target this C-terminal domain, in order to modulate human HSP70 function.

The chaperone HSP70 promotes the survival of cells exposed to many different types of stresses, and is also potently anti-apoptotic. The major stress-induced form of this protein, HSP70-1, is overexpressed in a number of human cancers, yet is negligibly expressed in normal cells. Silencing of the gene encoding HSP70-1 (HSPA1A) is cytotoxic to transformed but not normal cells. Therefore, HSP70 is considered to be a promising cancer drug target, and there has been active interest in the identification and characterization of HSP70 inhibitors for cancer therapy. Because HSP70 behaves in a relatively non-specific manner in the control of protein folding, to date there are no reliably-identified "clients" of this protein, nor is there consensus as to what the phenotypic effects of HSP70 inhibitors are on a cancer cell. Here for the first time we compare three recently-identified HSP70 inhibitors, PES-Cl, MKT-077, and Ver-155008, for their ability to impact some of the known and reported functions of this chaperone; specifically, the ability to inhibit autophagy, to influence the level of HSP90 client proteins, to induce cell cycle arrest, and to inhibit the enzymatic activity of the anaphase-promoting complex/cyclosome (APC/C). We report that all three of these compounds can inhibit autophagy and cause reduced levels of HSP90 client proteins; however, only PES-Cl can inhibit the APC/C and induce G 2/M arrest. Possible reasons for these differences, and the implications for the further development of these prototype compounds as anti-cancer agents, are discussed.

The HSP70 family of heat shock proteins consists of molecular chaperones of approximately 70kDa in size that serve critical roles in protein homeostasis. These adenosine triphosphatases unfold misfolded or denatured proteins and can keep these proteins in an unfolded, folding-competent state. They also protect nascently translating proteins, promote the cellular or organellar transport of proteins, reduce proteotoxic protein aggregates and serve general housekeeping roles in maintaining protein homeostasis. The HSP70 family is the most conserved in evolution, and all eukaryotes contain multiple members. Some members of this family serve specific organellar- or tissue-specific functions; however, in many cases, these members can function redundantly. Overall, the HSP70 family of proteins can be thought of as a potent buffering system for cellular stress, either from extrinsic (physiological, viral and environmental) or intrinsic (replicative or oncogenic) stimuli. As such, this family serves a critical survival function in the cell. Not surprisingly, cancer cells rely heavily on this buffering system for survival. The overwhelming majority of human tumors overexpress HSP70 family members, and expression of these proteins is typically a marker for poor prognosis. With the proof of principle that inhibitors of the HSP90 chaperone have emerged as important anticancer agents, intense focus has now been placed on the potential for HSP70 inhibitors to assume a role as a significant chemotherapeutic avenue. In this review, the history, regulation, mechanism of action and role in cancer of the HSP70 family are reviewed. Additionally, the promise of pharmacologically targeting this protein for cancer therapy is addressed.

The p14/p19 (ARF) (ARF) tumor suppressor gene is frequently mutated in human cancer. Recently ARF has been shown to localize to mitochondria and to induce autophagy. However the controls that regulate the trafficking of ARF to mitochondria remain unknown. We recently reported that 2-phenylethynesulfonamide (PES) selectively interacts with cytosolic heat shock protein 70 (HSP70) and inhibits its function; we further showed that PES promotes the death of tumor cells, and that this is associated with an impairment of lysosome function and an inhibition of autophagy. In the present work we used a mass spectrometry-based approach to identify mitochondrial ARF-binding proteins. We report that mitochondrial ARF interacts with HSP70. We show that treatment of cells with PES blocks the trafficking of ARF to mitochondria, indicating that interaction with HSP70 mediates the mitochondrial localization of ARF. We also show that PES inhibits the ability of ARF to induce autophagy, supporting the premise that localization to this organelle is critical for ARF-induced autophagy. Finally, we report that cells expressing high levels of ARF are more sensitive to PES than counterparts with ARF silenced. High levels of ARF are characteristic of tumor cells with enhanced MAPK signaling and advanced stage; therefore, these data support the premise that PES may show preferential cytotoxicity to advanced stage cancers.

The evolutionarily conserved stress-inducible HSP70 molecular chaperone plays a central role in maintaining protein quality control in response to various forms of stress. Constitutively elevated HSP70 expression is a characteristic of many tumor cells and contributes to their survival. We recently identified the small-molecule 2-phenylethyenesulfonamide (PES) as a novel HSP70 inhibitor. Here, we present evidence that PES-mediated inhibition of HSP70 family proteins in tumor cells results in an impairment of the two major protein degradation systems, namely, the autophagy-lysosome system and the proteasome pathway. HSP70 family proteins work closely with the HSP90 molecular chaperone to maintain the stability and activities of their many client proteins, and PES causes a disruption in the HSP70/HSP90 chaperone system. As a consequence, many cellular proteins, including known HSP70/HSP90 substrates, accumulate in detergent-insoluble cell fractions, indicative of aggregation and functional inactivation. Overall, PES simultaneously disrupts several cancer critical survival pathways, supporting the idea of targeting HSP70 as a potential approach for cancer therapeutics., (©2011 AACR.)

The multifunctional, stress-inducible molecular chaperone HSP70 has important roles in aiding protein folding and maintaining protein homeostasis. HSP70 expression is elevated in many cancers, contributing to tumor cell survival and resistance to therapy. We have determined that a small molecule called 2-phenylethynesulfonamide (PES) interacts selectively with HSP70 and leads to a disruption of the association between HSP70 and several of its cochaperones and substrate proteins. Treatment of cultured tumor cells with PES promotes cell death that is associated with protein aggregation, impaired autophagy, and inhibition of lysosomal function. Moreover, this small molecule is able to suppress tumor development and enhance survival in a mouse model of Myc-induced lymphomagenesis. The data demonstrate that PES disrupts actions of HSP70 in multiple cell signaling pathways, offering an opportunity to better understand the diverse functions of this molecular chaperone and also to aid in the development of new cancer therapies.