HSP70 Inhibition by the Small-Molecule 2-Phenylethynesulfonamide Impairs Protein Clearance Pathways in Tumor Cells

Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Molecular Cancer Research (Impact Factor: 4.38). 06/2011; 9(7):936-47. DOI: 10.1158/1541-7786.MCR-11-0019
Source: PubMed


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.

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    • "PES is a small molecule with chemical traits for being a highly reactive and oxidant agent (Fig. 3A). A characterization of its mode of action revealed its ability to block HSP70 function in vivo and as a consequence: i) the secondary inhibition of HSP90 and the proteasome; ii) the blockage of the chaperone-mediated type of autophagy, which results from HSC70 inhibition; iii) the impairment of the completion of macroautophagy (named autophagy hereafter) [12] [34]. "
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    ABSTRACT: Aerobic metabolism of mammalian cells leads to the generation of reactive oxygen species (ROS). To cope with this toxicity, evolution provided cells with effective antioxidant systems like glutathione. Current anticancer therapies focus on the cancer dependence on oncogenes and non-oncogenes. Tumours trigger mechanisms to circumvent the oncogenic stress and to escape cell death. In this context we have studied 2-phenylethinesulfoxamine (PES), which disables the cell protective mechanisms to confront the proteotoxicity of damaged and unfolded proteins. Proteotoxic stress is increased in tumour cells, thus providing an explanation for the anticancer selectivity of PES. In addition, we have found that PES induces a severe oxidative stress and the activation of p53. The cell content reduction of glutathione by means of L-buthionine-sulfoximine (BSO) synergizes with PES. In conclusion, we have found that ROS constitutes a central element in a series of positive feed-backs in the cell. ROS, p53, proteotoxicity, autophagy, mitochondrial dynamics are interconnected with the mechanisms leading to cell death, either apoptotic or necrotic. This network of interactions provides multiple targets for drug discovery and development in cancer.
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    • "Importantly, it is constitutively overexpressed in most human cancer cells, and its elevated expression correlates with resistance to therapy and poor prognosis [6], [13], [15], [16]. Evidence indicates that, among its cancer-supporting activities, HSP70 protects cells from apoptosis and the proteotoxic stress associated with oncoproteins and aberrant metabolism [20]–[22]. Accordingly, this molecular chaperone has emerged as an attractive therapeutic target, and several groups have focused efforts on the identification of HSP70 inhibitors for use in cancer therapy. To date, however, relatively few effective, well-characterized modulators of HSP70 activities have been reported [7], [8], [22]–[30]. "
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    ABSTRACT: 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 Lα, β that bridges SBDα to SBDβ, and the loop LL,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.
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    • "Therefore, we examined the effect of knockdown of HSP70 or PFT-μ on the expression of HSP90 in cancer cells, while no change in the HSP90 expression was observed (Fig. 2B). However, this result cannot exclude the possibility that inhibition of HSP70 or PFT-μ influenced the sequestration of HSP90 client proteins, including epidermal growth factor receptor, HER2/ErbB2 and AKT, into an insoluble fraction and promoted their aggregation and inactivation, as reported [40]. "
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    ABSTRACT: Hyperthermia (HT) improves the efficacy of anti-cancer radiotherapy and chemotherapy. However, HT also inevitably evokes stress responses and increases the expression of heat-shock proteins (HSPs) in cancer cells. Among the HSPs, HSP70 is known as a pro-survival protein. In this study, we investigated the sensitizing effect of pifithrin (PFT)-μ, a small molecule inhibitor of HSP70, when three human prostate cancer cell lines (LNCaP, PC-3, and DU-145) were treated with HT (43°C for 2 h). All cell lines constitutively expressed HSP70, and HT further increased its expression in LNCaP and DU-145. Knockdown of HSP70 with RNA interference decreased the viability and colony-forming ability of cancer cells. PFT-μ decreased the viabilities of all cell lines at one-tenth the dose of Quercetin, a well-known HSP inhibitor. The combination therapy with suboptimal doses of PFT-μ and HT decreased the viability of cancer cells most effectively when PFT-μ was added immediately before HT, and this combination effect was abolished by pre-knockdown of HSP70, suggesting that the effect was mediated via HSP70 inhibition. The combination therapy induced cell death, partially caspase-dependent, and decreased proliferating cancer cells, with decreased expression of c-Myc and cyclin D1 and increased expression of p21(WAF1/Cip), indicating arrest of cell growth. Additionally, the combination therapy significantly decreased the colony-forming ability of cancer cells compared to therapy with either alone. Furthermore, in a xenograft mouse model, the combination therapy significantly inhibited PC-3 tumor growth. These findings suggest that PFT-μ can effectively enhance HT-induced antitumor effects via HSP70 inhibition by inducing cell death and arrest of cell growth, and that PFT-μ is a promising agent for use in combination with HT to treat prostate cancer.
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