Edward G. Mimnaugh

Hsp90 Regulates a von Hippel Lindau-independent Hypoxia-inducible Factor-1α-degradative Pathway

HIF-1α is a normally labile proangiogenic transcription factor that is stabilized and activated in hypoxia. Although the von Hippel Lindau (VHL) gene product, the ubiquitin ligase responsible for regulating HIF-1α protein levels, efficiently targets HIF-1α for rapid proteasome-dependent degradation under normoxia, HIF-1α is resistant to the destabilizing effects of VHL under hypoxia. HIF-1α also associates with the molecular chaperone Hsp90. To examine the role of Hsp90 in HIF-1α function, we used renal carcinoma cell (RCC) lines that lack functional VHL and express stable HIF-1α protein under normoxia. Geldanamycin (GA), an Hsp90 antagonist, promoted efficient ubiquitination and proteasome-mediated degradation of HIF-1α in RCC in both normoxia and hypoxia. Furthermore, HIF-1α point mutations that block VHL association did not protect HIF-1α from GA-induced destabilization. Hsp90 antagonists also inhibited HIF-1α transcriptional activity and dramatically reduced both hypoxia-induced accumulation of VEGF mRNA and hypoxia-dependent angiogenic activity. These findings demonstrate that disruption of Hsp90 function 1) promotes HIF-1α degradation via a novel, oxygen-independent E3 ubiquitin ligase and 2) diminishes HIF-1α transcriptional activity. Existence of an Hsp90-dependent pathway for elimination of HIF-1α predicts that Hsp90 antagonists may be hypoxic cell sensitizers and possess antiangiogenic activityin vivo, thus extending the utility of these drugs as therapeutic anticancer agents.

Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu

Overexpression of the transmembrane receptor tyrosine kinase ErbB2 is common in multiple malignancies, including breast and ovarian cancer. ErbB2 is resistant to degradation mediated by c-Cbl, the E3 ubiquitin ligase responsible for ligand-induced ubiquitination of ErbB1 (epidermal growth factor receptor). Because of its resistance to degradation, ErbB2 is the preferred dimerization partner for other members of the ErbB family, and its overexpression in vivo is associated with poor prognosis. We now show that the chaperone-binding ubiquitin ligase CHIP efficiently ubiquitinates and down-regulates ErbB2. CHIP expression shortens the half-life of both nascent and mature ErbB2 protein. In vitro ubiquitination assay shows that CHIP serves as a ubiquitin ligase for ErbB2, and both exogenously expressed and endogenous CHIP coprecipitate with the kinase. Furthermore, CHIP association with ErbB2 requires a chaperone intermediate and is increased by the chaperone-binding drug geldanamycin, a potent stimulator of ErbB2 ubiquitination and degradation. These data describe a previously unrecognized pathway, amenable to pharmacologic manipulation, that mediates ErbB2 stability.

Geldanamycin as a Potential Anti-Cancer Agent: Its Molecular Target and Biochemical Activity

Heat shock protein 90 is one of the most abundantcellularproteins. Although its functions are still being characterized,itappears to serve as a chaperone for a growing list of cellsignaling proteins, including many tyrosine and serine/threoninekinases, involved in proliferation and/or survival. Thebenzoquinone ansamycin geldanamycin has been shown to bind toHsp90and to specifically inhibit this chaperones function, resultinginclient protein destabilization. Its ability to simultaneouslystimulate depletion of multiple oncogenic proteins suggests thatgeldanamycin, or other molecules capable of targeting Hsp90 incancer cells, may be of clinical benefit.

P185ERBB2 BINDS TO GRP94 IN VIVO : DISSOCIATION OF THE P185ERBB2/GRP94 HETEROCOMPLEX BY BENZOQUINONE ANSAMYCINS PRECEDES DEPLETION OF P185ERBB2

Treatment of SKBr3 cells with benzoquinone ansamycins, such as geldanamycin (GA), depletes p185, the receptor tyrosine kinase encoded by the erbB2 gene. In the same cells, a biologically active benzoquinone photoaffinity label specifically binds a protein of about 100 kDa, and the ability of various GA derivatives to reduce the intracellular level of p185 correlates with their ability to compete with the photoaffinity label for binding to this protein. In this report, we present evidence that the 100-kDa ansamycin-binding protein is GRP94. Membrane-associated p185 exists in a stable complex with GRP94. GA binding to GRP94 disrupts this complex, leading to degradation of pre-existing p185 protein, and resulting in an altered subcellular distribution of newly synthesized p185.

Stimulation by adriamycin of rat heart and liver microsomal NADPH-dependent lipid peroxidation

Abstract Rat liver and heart microsomes catalyze the transfer of single electrons from NADPH to adriamycin forming semiquinone radicals which, in turn, activate molecular oxygen. This process stimulated lipid peroxidation 5- to 7-fold as measured by malonaldehyde formation. Adriamycinaugmented lipid peroxidation was linear with time to 60 min, optimal at 1.0 mg of microsomal protein/ml and pH 7.5, and was proportional to the adriamycin concentration up to 100 μM. An NADPH-generating system was superior to NADPH, and an oxygen atmosphere tripled the rate of peroxidation as compared to air. Nitrogen abolished adriamycin-stimulated peroxidation. Superoxide dismutase, reduced glutathione, α-tocopherol, EDTA, dioxopiperazinylpropane (ICRF-187), and dimethylurea were effective inhibitors of lipid peroxidation. This suggests that Superoxide anion and possibly hydroxyl radical may be formed by the oxidation of the adriamycin semiquinone radical and thus stimulate the peroxidation of microsomal unsaturated fatty acids. Although adriamycin failed to stimulate lipid peroxidation in heart microsomes from control animals, peroxidation was dramatically increased when adriamycin was added to cardiac microsomes from α-tocopherol-deficient rats. Lipid peroxidation in α-tocopheroldeficient liver microsomes was four times greater than in control microsomes with the NADPH-generating system, and adriamycin did not further increase that high rate of peroxidation; however, when NADPH was used as the source of electrons in place of the NADPH-generating system, adriamycin stimulated peroxidation more than 2-fold. These results suggest that microsomal lipid peroxidation may play a role in the cytotoxicity and cardiotoxicity of adriamycin.

Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin

Mitochondrial degeneration is a consistently prominent morphological alteration associated with adriamycin toxicity which may be the consequence of adriamycin-enhanced peroxidative damage to unsaturated mitochondrial membrane lipids. Using isolated rat liver mitochondria as an in vitro model system to study the effects of the anticancer drug adriamycin on lipid peroxidation, we found that NADH-dependent mitochondrial peroxidation--measured by the 2-thiobarbituric acid method--was stimulated by adriamycin as much as 4-fold. Marker enzyme analysis indicated that the mitochondria were substantially free of contaminating microsomes (less than 5%). Lipid peroxidation in mitochondria incubated in KCl-Tris-HCl buffer (pH 7.4) under an oxygen atmosphere was optimal at 1-2 mg of mitochondrial protein/ml and with NADH at 2.5 mM. Malonaldehyde production was linear with time to beyond 60 min, and the maximum enhancement of peroxidation was observed with adriamycin at 50-100 microM. Interestingly, in contrast to its stimulatory effect on NADH-supported mitochondrial peroxidation, adriamycin markedly diminished ascorbate-promoted lipid peroxidation in mitochondria. Superoxide dismutase, catalase, 1,3-dimethylurea, reduced glutathione, alpha-tocopherol and EDTA added to incubation mixtures inhibited endogenous and adriamycin-augmented NADH-dependent peroxidation of mitochondrial lipids, indicating that multiple species of reactive oxygen (superoxide anion radical, hydrogen peroxide and hydroxyl radical) and possibly trace amounts of endogenous ferric iron participated in the peroxidation reactions. In submitochondrial particles freed of endogenous defenses against oxyradicals, lipid peroxidation was increased 7-fold by adriamycin. These observations suggest that some of the effects of adriamycin on mitochondrial morphology and biochemical function may be mediated by adriamycin-enhanced reactive oxygen-dependent mitochondrial lipid peroxidation.

Free radicals and anticancer drug resistance : oxygen free radicals in the mechanisms of drug cytotoxicity and resistance by certain tumors

Certain anticancer agents form free radical intermediates during enzymatic activation. Recent studies have indicated that free radicals generated from adriamycin and mitomycin C may play a critical role in their toxicity to human tumor cells. Furthermore, it is becoming increasingly apparent that reduced drug activation and or enhanced detoxification of reactive oxygen species may be related to the resistance to these anticancer agents by certain tumor cell lines. The purposes of this review are to summarize the evidence pointing toward the significance of free radicals formation in drug toxicity and to evaluate the role of decreased free radical formation and enhanced free radical scavenging and detoxification in the development of anticancer drug resistance by a spectrum of tumor cell types. Studies failing to support the participation of oxyradicals in the cytotoxicity and resistance of adriamycin are also discussed.

Surface charge and hydrophobicity determine ErbB2 binding to the Hsp90 chaperone complex

The molecular chaperone Hsp90 modulates the function of specific cell signaling proteins. Although targeting Hsp90 with the antibiotic inhibitor geldanamycin (GA) may be a promising approach for cancer treatment, little is known about the determinants of Hsp90 interaction with its client proteins. Here we identify a loop within the N lobe of the kinase domain of ErbB2 that determines Hsp90 binding. The amino acid sequence of the loop determines the electrostatic and hydrophobic character of the proteins surface, which in turn govern interaction with Hsp90. A point mutation within the loop that alters ErbB2 surface properties disrupts Hsp90 association and confers GA resistance. Notably, the immature ErbB2 point mutant remains sensitive to GA, suggesting that mature and nascent client kinases may use distinct motifs to interact with the Hsp90 chaperone complex.

In vitro stimulation by paraquat of reactive oxygen-mediated lipid peroxidation in rat lung microsomes

Abstract Paraquat significantly stimulated lipid peroxidation in rat lung microsomes without the addition of exogenous iron. The ability of paraquat to stimulate this lipid peroxidation was dependent on the presence of adequate reducing equivalents (NADPH), aerobic conditions, and the duration of incubation, viz. optimal in vitro conditions. Even greater paraquat-mediated lipid peroxidation was observed if incubations were conducted under O2 or if vitamin E-deficient microsomes were utilized, factors which have previously been reported to increase the in vivo pulnonary toxicity of paraquat. Superoxide dismutase (3 μg/ml) significantly inhibited paraquat-stimulated lipid peroxidation in rat lung microsomes (73%), demonstrating a pivotal role for superoxide in this process. Thus, the redox cycling of paraquat and accompanying reactive oxygen generation was capable of mediating lipid peroxidation not only in mouse lung and rat liver microsomes but also in rat lung microsomes.

Possible Role for Serine/Threonine Phosphorylation in the Regulation of the Heteroprotein Complex between the hsp90 Stress Protein and the pp60v-src Tyrosine Kinase

The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-srcchaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90. Although phosphorylation of the total pool of immunoprecipitable pp60v-src was unchanged, okadaic acid slightly increased phosphoserine and phosphothreonine levels specifically in pp60v-src bound to heat-shock protein 90. The low level of tyrosine phosphorylation in the pp60v-src complexed with heat-shock protein 90 was further decreased by okadaic acid. Interestingly, okadaic acid-stabilized hyperphosphorylation of the heat-shock protein 90-pp60v-src complex lowered the level of pp60v-src in cell membranes, the functional location for pp60v-src. We suggest that serine/threonine phosphorylation of heat-shock protein 90 and/or pp60v-src functions as a regulatory molecular trigger to release pp60v-src from the chaperone complex at the inner surface of cell membranes.