HSP90 and GRP94 are homologous cellular chaperones found in cytosol and endoplasmic reticulum, respectively. Several years ago, we discovered that members of the benzoquinone and ansamycin class of antibiotic, including herbimycin A and geldanamycin (GA) bound to HSP90 and GRP94 and disrupted certain multi-molecular complexes of which these proteins were a part. We have utilized pharmacologic disruption of HSP90 and GRP94 activity to study the function of these chaperones in cellular signal transduction. Multiple signal transduction proteins interact with these charperones, including the kinasesz src, erbB2 and c-raf-1, and mutated (but not wild type) p53. A general consequence of pharmacologic disruption of the chaperone/signal protein complex is the resultant marked instability and incorrect subcellular localization of the signalling protein. The instability is due to stimulation of targeted degradation of the signalling protein by the 26S proteasome proteolytic complex following chaperone dissociation. Within the last year, we have made the novel observation that HSP90 associates with the cytosolic kinase RIP, a key component of the TNF signalling pathway which leads to NFkB activation. We have determined that disruption of RIP stability by geldanamycin prevents NFkB induction by TNF, but not TNF signalling to Jnk, thus sensitizing cells to the apoptotic properties of TNF. We have additionally observed that another kinase associated with cell survival, Akt, is sensitive to geldanamycin. Geldanamycin blocks NFkB induction by a wide variety of stimuli other than TNF, including chemotherapeutic drugs and IL-1. Its ability to do this may relate to its destabilization of Akt. Experiments to test this hypothesis are currently underway. Benzoquinone ansamycins (geldanamycin) had been the only agents capable of specifically interfering in HSP90/GRP94 function. Recently, we identified radicicol as representing a novel class of natural product capable of binding to HSP90. Both radicicol and the ansamycins bind to HSP90 at an amino terminal nucleotide pocket. Most recently, we have identified a third class of natural product, novobiocin, which also binds to HSP90, although at a lower affinity than either benzoquinone ansamycins or radicicol. Nonetheless, novobiocin appears to cause the same biologic effects on """"""""client proteins"""""""" as ansamycins and radicicol. Surprisingly, novobiocin appears to interact with a carboxyl terminal region on HSP90, which may be a previously unrecognized second nucleotide binding site. Preliminary animal testing has revealed no toxicity after twice daily administration of novobiocin for more than one month. This regimen demonstrates significant anti-tumor activity in a transgenic murine model of erbB2-driven breast cancer. Finally, we have observed that geldanamycin reverses beta-catenin tyrosine phosphorylation in melanoma cells, probably due to the rapid loss of erbB2 from these cells. In untreated cells, erbB2 and beta-catenin can be readily co-precipitated. Loss of beta-catenin tyrosine phosphorylation leads to an increased association with E-cadherin and decreased cell motility in vitro. This is the first indication that modulation of the tyrosine phosphorylation status of beta catenin in melanoma cells is associated with decreased motility. The fact that beta-catenin tyrosine phosphorylation seems to be mediated, in 3/3 melanoma cell lines examined, by erbB2 - a geldanamycin-sensitive tyrosine kinase - suggests that geldanamycin treatment may be anti-metastatic. This hypothesis is currently being tested in an in vivo metastasis model. The ErbB family of receptor tyrosine kinases contains four members. We have found that ErbB2, the only ligandless member of the family, is one of the most sensitive geldanamycin substrates. Since the ErbBs are transmembrane proteins, they are likely to come in contact with both Hsp90 and Grp94, and one or both chaperones may be responsible for ErbB2's geldanamycin sensitivity. Our current data demonstrate that the kinase domain of ErbB2 mediates its geldanamycin responsiveness and that Hsp90 binds to this domain in the mature protein. In contrast, mature ErbB1, much less sensitive to geldanamycin than ErbB2, does not associate with Hsp90. Geldanamycin-induced instability of nascent ErbB2 is also mediated by its kinase domain, and we can find little evidence to support a role for Grp94 in ErbB2 maturation.

Agency
National Institute of Health (NIH)
Institute
Division of Clinical Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01SC010074-07
Application #
6757105
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Clinical Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Bachman, Ashleigh B; Keramisanou, Dimitra; Xu, Wanping et al. (2018) Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation. Nat Commun 9:265
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
Calderwood, Stuart K; Neckers, Len (2016) Hsp90 in Cancer: Transcriptional Roles in the Nucleus. Adv Cancer Res 129:89-106
Prince, Thomas L; Kijima, Toshiki; Tatokoro, Manabu et al. (2015) Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants. PLoS One 10:e0141786
Sourbier, Carole; Scroggins, Bradley T; Ratnayake, Ranjala et al. (2013) Englerin A stimulates PKC? to inhibit insulin signaling and to simultaneously activate HSF1: pharmacologically induced synthetic lethality. Cancer Cell 23:228-37
Alarcon, S V; Mollapour, M; Lee, M-J et al. (2012) Tumor-intrinsic and tumor-extrinsic factors impacting hsp90- targeted therapy. Curr Mol Med 12:1125-41
Xu, Wanping; Neckers, Len (2012) The double edge of the HSP90-CDC37 chaperone machinery: opposing determinants of kinase stability and activity. Future Oncol 8:939-42
Mollapour, Mehdi; Neckers, Len (2012) Post-translational modifications of Hsp90 and their contributions to chaperone regulation. Biochim Biophys Acta 1823:648-55
Mollapour, Mehdi; Tsutsumi, Shinji; Truman, Andrew W et al. (2011) Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity. Mol Cell 41:672-81
Wang, Suiquan; Pashtan, Itai; Tsutsumi, Shinji et al. (2009) Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors. Cell Cycle 8:2050-6

Showing the most recent 10 out of 62 publications