Hsp90 is a cellular chaperone that stabilizes several signal transduction networks important to cancer cells. In FY14, our research accomplishments include further development and evaluation of a novel lead compound that interferes with Hsp90/FKBP52-dependent chaperoning of the androgen receptor. This compound acts via a mechanism that is unique compared to other androgen receptor antagonists. Also, we found that the HSP90 inhibitor ganetespib synergizes with the MET kinase inhibitor crizotinib in both crizotinib-sensitive and -resistant MET-driven tumor models. The clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. MET is an HSP90-dependent kinase, and in this report we show that HSP90 preferentially interacts with and stabilizes activated MET, regardless of whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET-TKI show a preference for the inactive form of the kinase, and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET-TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in both TKI-sensitive and -resistant MET-driven tumor models. These data suggest that inclusion of an HSP90 inhibitor can partially restore TKI sensitivity to previously resistant MET mutants, and they provide the foundation for clinical evaluation of this therapeutic combination in patients with MET-driven cancers. In addition, we showed that the Hsp90-related and mitochondrially localized molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis. TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor. We collaborated on a study targeting heat shock protein 90 for the treatment of malignant pheochromocytoma. Metastatic pheochromocytoma represents one of the major clinical challenges in the field of neuroendocrine oncology. Recent molecular characterization of pheochromocytoma suggests new treatment options with targeted therapies. In this study, we investigated Hsp90 as a potential therapeutic target for advanced pheochromocytoma. Both the first generation, natural product Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin), and the second-generation synthetic Hsp90 inhibitor STA-9090 (ganetespib) demonstrated potent inhibition of proliferation and migration of pheochromocytoma cell lines and induced degradation of key Hsp90 clients. Furthermore, ganetespib induced dose-dependent cytotoxicity in primary pheochromocytoma cells. Using metastatic models of pheochromocytoma, we demonstrated the efficacy of 17-AAG and ganetespib in reducing metastatic burden and increasing survival. These findings suggest that targeting Hsp90 may benefit patients with advanced pheochromocytoma. We collaborating on a study showing that optical and radioiodinated tethered Hsp90 inhibitors reveal selective internalization of ectopic Hsp90 in malignant breast tumor cells. These tethered inhibitors selectively recognize cells expressing ectopic Hsp90 and become internalized. The internalization process is blocked by Hsp90 antibodies, suggesting that active cycling of the protein occurs at the plasma membrane. In mice, we observed exquisite accumulation of the fluor-tethered versions within breast tumors at very sensitive levels. Cell-based assays with the radiolabeled version showed picomolar detection in cells that express ectopic Hsp90. Our findings show that fluor-tethered or radiolabeled inhibitors that target ectopic Hsp90 can be used to detect breast cancer malignancies through noninvasive imaging. Finally, we identified coordinated regulation of serum- and glucocorticoid-inducible kinase 3 (SGK3) by a C-terminal hydrophobic motif and Hsp90-Cdc37 chaperone complex. SGK3 mediates a variety of cellular processes including membrane transport, cell proliferation, and survival, and it has been implicated in Akt-independent signaling downstream of oncogenic PIK3CA mutations (activating mutations in the catalytic subunit of PI3K) in human cancers. However, the regulation of SGK3 is poorly understood. Here we report that SGK3 stability and kinase activation are regulated by the Hsp90-Cdc37 chaperone complex. Hsp90-Cdc37 associates with the kinase domain of SGK3 and acts in concert with a C-terminal hydrophobic motif of SGK3 to prevent Hsp70 association and ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein)-mediated degradation. Phosphorylation of hydrophobic motif triggers release of Cdc37 and concomitant association of 3-phosphoinositide dependent kinase 1 (PDK1) to activate SGK3. Our study provides new insights into regulation of SGK3 stability and activation and the rationale for application of Hsp90 inhibitors in treating SGK3-dependent cancers.
| Kijima, Toshiki; Eguchi, Takanori; Neckers, Len et al. (2018) Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter. Methods Mol Biol 1709:35-45 |
| Zuehlke, Abbey D; Moses, Michael A; Neckers, Len (2018) Heat shock protein 90: its inhibition and function. Philos Trans R Soc Lond B Biol Sci 373: |
| Zuehlke, Abbey D; Neckers, Leonard (2017) Out with the old: Hsp90 finds amino acid residue more useful than co-chaperone protein. Microb Cell 4:273-274 |
| Xu, Wanping; Neckers, Len (2017) A USE1ful Biomarker and Molecular Target in Lung Cancer? J Natl Cancer Inst 109: |
| Woodford, Mark R; Dunn, Diana; Miller, Jonelle B et al. (2016) Impact of Posttranslational Modifications on the Anticancer Activity of Hsp90 Inhibitors. Adv Cancer Res 129:31-50 |
| Yim, Kendrick H; Prince, Thomas L; Qu, Shiwei et al. (2016) Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90?. Proc Natl Acad Sci U S A 113:E4801-9 |
| Zuehlke, Abbey D; Neckers, Len (2016) Clients Place Unique Functional Constraints on Hsp90. Trends Biochem Sci 41:562-564 |
| Xu, Wanping; Neckers, Len (2016) Gr(i)p the ER to Stress Out Melanoma. Cancer Cell 29:769-71 |
| Calderwood, Stuart K; Neckers, Len (2016) Hsp90 in Cancer: Transcriptional Roles in the Nucleus. Adv Cancer Res 129:89-106 |
| Woodford, Mark R; Dunn, Diana M; Ciciarelli, Joseph G et al. (2016) Targeting Hsp90 in Non-Cancerous Maladies. Curr Top Med Chem 16:2792-804 |
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