title: Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activity.
abstract: The ansamycin antibiotic, geldanamycin, targets the hsp 90 protein chaperone and promotes ubiquitin-dependent proteasomal degradation of its numerous client proteins. Bortezomib is a specific and potent proteasome inhibitor. Both bortezomib and the geldanamycin analogue, 17-N-allylamino-17-demethoxy geldanamycin, are in separate clinical trials as new anticancer drugs. We hypothesized that destabilization of hsp 90 client proteins with geldanamycin, while blocking their degradation with bortezomib, would promote the accumulation of aggregated, ubiquitinated, and potentially cytotoxic proteins. Indeed, geldanamycin plus bortezomib inhibited MCF-7 tumor cell proliferation significantly more than either drug alone. Importantly, while control cells were unaffected, human papillomavirus E6 and E7 transformed fibroblasts were selectively sensitive to geldanamycin plus bortezomib. Geldanamycin alone slightly increased protein ubiquitination, but when geldanamycin was combined with bortezomib, protein ubiquitination was massively increased, beyond the amount stabilized by bortezomib alone. In geldanamycin plus bortezomib-treated cells, ubiquitinated proteins were mostly detergent insoluble, indicating that they were aggregated. Individually, both geldanamycin and bortezomib induced hsp 90, hsp 70, and GRP78 stress proteins, but the drug combination superinduced these chaperones and caused them to become detergent insoluble. Geldanamycin plus bortezomib also induced the formation of abundant, perinuclear vacuoles, which were neither lysosomes nor autophagosomes and did not contain engulfed cytosolic ubiquitin or hsp 70. Fluorescence marker experiments indicated that these vacuoles were endoplasmic reticulum derived and that their formation was prevented by cycloheximide, suggesting a role for protein synthesis in their genesis. These observations support a mechanism whereby the geldanamycin plus bortezomib combination simultaneously disrupts hsp 90 and proteasome function, promotes the accumulation of aggregated, ubiquitinated proteins, and results in enhanced antitumor activity.
authors.affiliation: Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Rockville, Maryland 20850, USA. edmim@mail.nih.gov
authors.forename: Edward G
authors.initials: EG
authors.lastname: Mimnaugh
authors.forename: Wanping
authors.initials: W
authors.lastname: Xu
authors.forename: Michele
authors.initials: M
authors.lastname: Vos
authors.forename: Xitong
authors.initials: X
authors.lastname: Yuan
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Isaacs
authors.forename: Kheem S
authors.initials: KS
authors.lastname: Bisht
authors.forename: David
authors.initials: D
authors.lastname: Gius
authors.forename: Len
authors.initials: L
authors.lastname: Neckers
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D016227:Benzoquinones; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002472:Cell Transformation, Viral; D003513:Cycloheximide / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects* / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D047029:Lactams, Macrocyclic; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011506:Proteins / Q000378:metabolism*; D011719:Pyrazines / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives* / Q000494:pharmacology; D012995:Solubility; D014452:Ubiquitins / Q000378:metabolism*; D014617:Vacuoles / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D016227:Benzoquinones; D001897:Boronic Acids; D003902:Detergents; C068651:E6 protein, Human papillomavirus type 16; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D047029:Lactams, Macrocyclic; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D061988:Proteasome Inhibitors; D011506:Proteins; D011719:Pyrazines; D011809:Quinones; D012097:Repressor Proteins; D014452:Ubiquitins; C059731:oncogene protein E7, Human papillomavirus type 16; D017828:Rifabutin; C112765:tanespimycin; D000069286:Bortezomib; D003513:Cycloheximide; D046988:Proteasome Endopeptidase Complex; C001277:geldanamycin