Triple-negative breast cancers (TNBCs) are defined by a lack of expression of estrogen, progesterone, and HER2 receptors. Because of the absence of identified targets and targeted therapies, and due to a heterogeneous molecular presentation, treatment guidelines for patients with TNBC include only conventional chemotherapy. Such treatment, while effective for some, leaves others with high rates of early relapse and is not curative for any patient with metastatic disease. Here, we demonstrate that these tumors are sensitive to the heat shock protein 90 (Hsp90) inhibitor PU-H71. Potent and durable anti-tumor effects in TNBC xenografts, including complete response and tumor regression, without toxicity to the host are achieved with this agent. Notably, TNBC tumors respond to retreatment with PU-H71 for several cycles extending for over 5 months without evidence of resistance or toxicity. Through a proteomics approach, we show that multiple oncoproteins involved in tumor proliferation, survival, and invasive potential are in complex with PU-H71-bound Hsp90 in TNBC. PU-H71 induces efficient and sustained downregulation and inactivation, both in vitro and in vivo, of these proteins. Among them, we identify downregulation of components of the Ras/Raf/MAPK pathway and G(2)-M phase to contribute to its anti-proliferative effect, degradation of activated Akt and Bcl-xL to induce apoptosis, and inhibition of activated NF-kappaB, Akt, ERK2, Tyk2, and PKC to reduce TNBC invasive potential. The results identify Hsp90 as a critical and multimodal target in this most difficult to treat breast cancer subtype and support the use of the Hsp90 inhibitor PU-H71 for clinical trials involving patients with TNBC.
Hsp90 is a molecular chaperone with important roles in regulating pathogenic transformation. In addition to its well-characterized functions in malignancy, recent evidence from several laboratories suggests a role for Hsp90 in maintaining the functional stability of neuronal proteins of aberrant capacity, whether mutated or over-activated, allowing and sustaining the accumulation of toxic aggregates. In addition, Hsp90 regulates the activity of the transcription factor heat shock factor-1 (HSF-1), the master regulator of the heat shock response, mechanism that cells use for protection when exposed to conditions of stress. These biological functions therefore propose Hsp90 inhibition as a dual therapeutic modality in neurodegenerative diseases. First, by suppressing aberrant neuronal activity, Hsp90 inhibitors may ameliorate protein aggregation and its associated toxicity. Second, by activation of HSF-1 and the subsequent induction of heat shock proteins, such as Hsp70, Hsp90 inhibitors may redirect neuronal aggregate formation, and protect against protein toxicity. This mini-review will summarize our current knowledge on Hsp90 in neurodegeneration and will focus on the potential beneficial application of Hsp90 inhibitors in neurodegenerative diseases.
Heat shock protein 70 (Hsp70) is a family of proteins with key roles in regulating malignancy. Cancer cells rely on Hsp70 to inhibit apoptosis, regulate senescence and autophagy, and maintain the stability of numerous onco-proteins. Despite these important biological functions in cancer, robust chemical tools that enable the analysis of the Hsp70-regulated proteome in a tumor-by-tumor manner are yet unavailable. Here we take advantage of a recently reported Hsp70 ligand to design and develop an affinity purification chemical toolset for potential use in the investigation of the endogenous Hsp70-interacting proteome in cancer. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins.
Abstract As molecularly targeted agents assume a more prominent role in anticancer therapy there is a growing need to determine in a noninvasive manner whether the target is being engaged and to what extent such drug-target binding results in desirable effects. We address this need in the context of Hsp90, a target of significant value and one in critical need for such assessment tools, by combining a novel chemical tool selective for tumor Hsp90 with PET imaging and mathematical modeling. The chemical tool is [124I]-PU-H71, the iodine-124 radiolabeled analog of the potent Hsp90 inhibitor PU-H71, which can be administered in tracer quantities for PET imaging. The resulting diagnostic, PU-PET, has been optimized and validated preclinically in mouse models of cancer and then translated to the clinic. The exquisite design of this assay is based on three essential concepts as it relates to the target (Hsp90) as well as to the PET tracer (124I-PU-H71). First, the target is “oncogenic” Hsp90 and has been shown by numerous biochemical and pharmacokinetic studies to have a strong affinity for inhibitors and a very low koff resulting in selective and prolonged retention in tumor. Secondly, the tracer incorporates a 124I in place of the naturally occurring 127I in the structure of PU-H71 and therefore there is no change in the chemical structure. This feature in a PET tracer intended as a companion diagnostic is unprecedented and ensures that the PK properties are identical to the therapeutic agent (PU-H71). Finally, the radionuclide 124I has a four-day half-life and thus is well-suited to monitor the extended tumor retention profile observed for Hsp90 inhibitors. We here demonstrate that this PET assay informs on Hsp90 targeting in individual tumors in real time and provides accurate tumor drug concentrations for at least four chemically distinct Hsp90 drugs. In contrast, we find that plasma pharmacokinetics is not predictive of intratumor parameters and therefore provides limited value in estimating target engagement. Using PU-PET we demonstrate that at least one Hsp90 inhibitor exhibits tumor targeting and retention in humans, delivering and retaining therapeutic, micromolar, concentrations at safe doses. PU-PET is currently being evaluated in Phase 0/1 (NCT01269593) clinical trials as a noninvasive companion diagnostic to determine intratumoral concentration as well as to identify those patients who would best benefit from Hsp90 inhibitor therapy. This diagnostic assay is intended to be incorporated into future Phase 2 clinical trials in order to preselect those patients who would most likely benefit from Hsp90 inhibitor treatment. Citation Format: Tony Taldone, Nagavarakishore Pillarsetty, Mark PS Dunphy, John F. Gerecitano, Eloisi Caldas-Lopes, Brad Beattie, Radu I. Peter, Yanlong Kang, Anna Rodina, Pengrong Yan, Erica M. DaGama Gomes, Alexander Bolaender, Christina Pressl, Blesida Punzalan, Anson Ku, Thomas Ku, Smit Shah, Mohammad Uddin, Mei H. Chen, Elmer Santos, Jacek Koziorowski, Adriana Corben, Shanu Modi, Komal Jhaveri, Oscar Lin, Efsevia Vakiani, Yelena Janjigian, Pat Zanzonico, Clifford Hudis, Steven M. Larson, Jason S. Lewis, Gabriela Chiosis. Development of a noninvasive assay to determine drug concentration in tumor during hsp90 inhibitor therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5444. doi:10.1158/1538-7445.AM2015-5444
Abstract Background. The cancerous phenotype evolves through accumulation of a large repertoire of elements that allow for increased proliferative potential, self-sufficiency in growth signals and resistance to apoptotic and antiproliferative signals. While much is now gained in understanding the many factors that lead to a malignant phenotype, little remains known on how cancer cells are able to accumulate and to survive with such complexity and high load of aberrant processes. Study design. Here we use the novel dual Hsp70 and Hsc70 inhibitor, YK5, and its biotinylated derivative, YK55, to investigate the interactome of cytosolic Hsp70s. Results. We show that heat shock protein 70 family members (Hsp70s) keep active conformations of key oncoproteins and inhibit the function of tumor suppressors. Hsp70s also buffer levels of activated molecules by maintaining them in a conformation that alters their dephosphorylation by phosphatases. Pharmacologic inhibition of Hsp70s by YK5 impairs oncoprotein stability and function, restores tumor suppressor activity, affects major cancer hallmarks and is selectively lethal to cancer cells. We show that enhanced affinity of YK5 for cancer cell-Hsp70s species is an essential mechanism for providing selective tumor lethality. Conclusion. By demonstrating that pharmacologic dual inhibitors of Hsp70 and Hsc70, such as YK5, confer specific and broad tumor lethality, and interact selectively with tumor Hsp70s species, this work proposes Hsp70s inhibitors as potential anti-cancer therapeutics with a large spectrum of activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2011-2579
Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.
Supplementary Data 1. Input datasets, protein quality control assessments and Reactome pathway analyses. Supplementary Data 2. epiHSP70s and epiHSP90s interactor datasets and network topology analyses. Supplementary Data 3. Input datasets and Reactome pathway analyses.
