The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis.

Mitochondrial topoisomerase IB (TOP1MT) is a nuclear-encoded topoisomerase, exclusively localized to mitochondria, which resolves topological stress generated during mtDNA replication and transcription. Here, we report that TOP1MT is overexpressed in cancer tissues and demonstrate that TOP1MT deficiency attenuates tumor growth in human and mouse models of colon and liver cancer. Due to their mitochondrial dysfunction, TOP1MT-KO cells become addicted to glycolysis, which limits synthetic building blocks and energy supply required for the proliferation of cancer cells in a nutrient-deprived tumor microenvironment. Mechanistically, we show that TOP1MT associates with mitoribosomal subunits, ensuring optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The TOP1MT genomic signature profile, based on Top1mt-KO liver cancers, is correlated with enhanced survival of hepatocellular carcinoma patients. Our results highlight the importance of TOP1MT for tumor development, providing a potential rationale to develop TOP1MT-targeted drugs as anticancer therapies.

Tumor-intrinsic and tumor-extrinsic factors impacting hsp90- targeted therapy.

In 1994 the first heat shock protein 90 (Hsp90) inhibitor was identified and Hsp90 was reported to be a target for anticancer therapeutics. In the past 18 years there have been 17 distinct Hsp90 inhibitors entered into clinical trial, and the small molecule Hsp90 inhibitors have been highly valuable as probes of the role of Hsp90 and its client proteins in cancer. Although no Hsp90 inhibitor has achieved regulatory approval, recently there has been significant progress in Hsp90 inhibitor clinical development, and in the past year RECIST responses have been documented in HER2-positive breast cancer and EML4-ALK-positive non-small cell lung cancer. All of the clinical Hsp90 inhibitors studied to date are specific in their target, i.e. they bind exclusively to Hsp90 and two related heat shock proteins. However, Hsp90 inhibitors are markedly pleiotropic, causing degradation of over 200 client proteins and impacting critical multiprotein complexes. Furthermore, it has only recently been appreciated that Hsp90 inhibitors can, paradoxically, cause transient activation of the protein kinase clients they are chaperoning, resulting in initiation of signal transduction and significant physiological events in both tumor and tumor microenvironment. An additional area of recent progress in Hsp90 research is in studies of the posttranslational modifications of Hsp90 itself and Hsp90 co-chaperone proteins. Together, a picture is emerging in which the impact of Hsp90 inhibitors is shaped by the tumor intracellular and extracellular milieu, and in which Hsp90 inhibitors impact tumor and host on a microenvironmental and systems level. Here we review the tumor intrinsic and extrinsic factors that impact the efficacy of small molecules engaging the Hsp90 chaperone machine.

Update on Hsp90 inhibitors in clinical trial.

Twenty-five years ago the first small molecule inhibitors of Hsp90 were identified. In the intervening years there has been dramatic progress in basic scientific understanding of the Hsp90 chaperone machinery and in the role of Hsp90 in malignancy. The first-in-class Hsp90 inhibitor 17-AAG entered into Phase I clinical trials in 1999. There are now 13 Hsp90 inhibitors in clinical trial, representing multiple drug classes, and hundreds of patients have been treated in adult oncology and pediatric oncology trials. This review will provide an overview of the clinical trial results thus far. In addition, pivotal issues in further development of Hsp90 inhibitors as anticancer drugs will be discussed.

A small molecule cell-impermeant Hsp90 antagonist inhibits tumor cell motility and invasion.

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains function of numerous intracellular signaling nodes utilized by cancer cells for proliferation and survival. Hsp90 is also detected on the plasma membrane of tumor cells and its expression has been suggested to correlate with metastatic potential. Given the abundance and diverse functions of the intracellular pool of this protein, the precise contribution of cell surface Hsp90 to cell motility and tumor metastasis remains to be determined. In this study we utilized the small molecule DMAG-N-oxide, a novel cell-impermeable Hsp90 inhibitor, to specifically examine the role of cell surface Hsp90 in cell motility. We observed that, while not affecting intracellular Hsp90 function, DMAG-N-oxide significantly retarded tumor cell migration and integrin/extracellular matrix-dependent cytoskeletal reorganization. Concomitant with these findings, targeting cell surface Hsp90 significantly inhibited tumor cell motility and invasion in vitro, and had a dramatic impact on melanoma cell lung colonization in vivo. These data indicate that cell surface Hsp90 plays an important role in modulating cancer cell migration that is independent of the function of the intracellular Hsp90 pool, and that small molecule inhibitors of surface Hsp90 may provide a new approach to targeting the metastatic phenotype.

FLT3 regulates beta-catenin tyrosine phosphorylation, nuclear localization, and transcriptional activity in acute myeloid leukemia cells.

Deregulated accumulation of nuclear beta-catenin enhances transcription of beta-catenin target genes and promotes malignant transformation. Recently, acute myeloid leukemia (AML) cells with activating mutations of FMS-like tyrosine kinase-3 (FLT3) were reported to display elevated beta-catenin-dependent nuclear signaling. Tyrosine phosphorylation of beta-catenin has been shown to promote its nuclear localization. Here, we examined the causal relationship between FLT3 activity and beta-catenin nuclear localization. Compared to cells with wild-type FLT3 (FLT3-WT), cells with the FLT3 internal tandem duplication (FLT3-ITD) and tyrosine kinase domain mutation (FLT3-TKD) had elevated levels of tyrosine-phosphorylated beta-catenin. Although beta-catenin was localized mainly in the cytoplasm in FLT3-WT cells, it was primarily nuclear in FLT3-ITD cells. Treatment with FLT3 kinase inhibitors or FLT3 silencing with RNAi decreased beta-catenin tyrosine phosphorylation and nuclear localization. Conversely, treatment of FLT3-WT cells with FLT3 ligand increased tyrosine phosphorylation and nuclear accumulation of beta-catenin. Endogenous beta-catenin co-immunoprecipitated with endogenous activated FLT3, and recombinant activated FLT3 directly phosphorylated recombinant beta-catenin. Finally, FLT3 inhibitor decreased tyrosine phosphorylation of beta-catenin in leukemia cells obtained from FLT3-ITD-positive AML patients. These data demonstrate that FLT3 activation induces beta-catenin tyrosine phosphorylation and nuclear localization, and thus suggest a mechanism for the association of FLT3 activation and beta-catenin oncogeneic signaling in AML.

Sensitivity of epidermal growth factor receptor and ErbB2 exon 20 insertion mutants to Hsp90 inhibition.

The mature epidermal growth factor receptor (EGFR) neither associates with nor requires the molecular chaperone heat-shock protein 90 (Hsp90). Mutations in EGFR exons 18, 19, and 21 confer Hsp90 chaperone dependence. In non-small cell lung cancer (NSCLC), these mutations are associated with enhanced sensitivity to EGFR inhibitors in vitro and with clinical response in vivo. Although less prevalent, insertions in EGFR exon 20 have also been described in NSCLC. These mutations, however, confer resistance to EGFR inhibitors. In NSCLC, exon 20 insertions have also been identified in the EGFR family member ErbB2. Here, we examined the sensitivity of exon 20 insertion mutants to an Hsp90 inhibitor currently in the clinic. Our data demonstrate that both EGFR and ErbB2 exon 20 insertion mutants retain dependence on Hsp90 for stability and downstream-signalling capability, and remain highly sensitive to Hsp90 inhibition. Use of Hsp90 inhibitors should be considered in NSCLC harbouring exon 20 insertions in either EGFR or ErbB2.

HIF and fumarate hydratase in renal cancer.

Hereditary leiomyomatosis and renal cell cancer is a recently described hereditary cancer syndrome in which affected individuals are predisposed to the development of leiomyomas of the skin and uterus. In addition, this clinical entity also can result in the development of biologically aggressive kidney cancer. Affected individuals harbour a germline mutation of the fumarate hydratase (FH) gene, which encodes an enzyme that catalyses conversion of fumarate to malate in the Kreb's cycle. Thus far, proposed mechanisms for carcinogeneis associated with this syndrome include aberrant apoptosis, oxidative stress, and pseudohypoxic drive. At this time, the majority of accumulating data support a role for pseudohypoxic drive in tumour development. The link between FH mutation and pseudohypoxic drive may reside in the biochemical alterations resulting from diminished/absent FH activity. These biochemical derangements may interfere with oxygen homeostasis and result in a cellular environment conducive to tumour formation.

Chaperoning oncogenes: Hsp90 as a target of geldanamycin.

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signaling proteins, as well as multiple mutated, chimeric, and/or over-expressed signaling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors, by interacting specifically with a single molecular target, cause the inactivation, destabilization, and eventual degradation of Hsp90 client proteins, and they have shown promising anti-tumor activity in preclinical model systems. One Hsp90 inhibitor, 17-AAG, has completed Phase I clinical trial and several Phase II trials of this agent are in progress. Hsp90 inhibitors are unique in that, although they are directed toward a specific molecular target, they simultaneously inhibit multiple signaling pathways that frequently interact to promote cancer cell survival. Further, by inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, a combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer.

Novel mutations in FH and expansion of the spectrum of phenotypes expressed in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC; OMIM 605839) is the predisposition to develop smooth muscle tumours of the skin and uterus and/or renal cancer and is associated with mutations in the fumarate hydratase gene (FH). Here we characterise the clinical and genetic features of 21 new families and present the first report of two African-American families with HLRCC. METHODS: Using direct sequencing analysis we identified FH germline mutations in 100% (21/21) of new families with HLRCC. RESULTS: We identified 14 germline FH mutations (10 missense, one insertion, two nonsense, and one splice site) located along the entire length of the coding region. Nine of these were novel, with six missense (L89S, R117G, R190C, A342D, S376P, Q396P), one nonsense (S102X), one insertion (111insA), and one splice site (138+1G>C) mutation. Four unrelated families had the R58X mutation and five unrelated families the R190H mutation. Of families with HLRCC, 62% (13/21) had renal cancer and 76% (16/21) cutaneous leiomyomas. Of women FH mutation carriers from 16 families, 100% (22/22) had uterine fibroids. Our study shows that expression of cutaneous manifestations in HLRCC ranges from absent to mild to severe cutaneous leiomyomas. FH mutations were associated with a spectrum of renal tumours. No genotype-phenotype correlations were identified. CONCLUSIONS: In combination with our previous report, we identify 31 different germline FH mutations in 56 families with HLRCC (20 missense, eight frameshifts, two nonsense, and one splice site). Our FH mutation detection rate is 93% (52/56) in families suspected of HLRCC.

Modulation of prion protein structural integrity by geldanamycin.

The cellular prion protein PrPc is of crucial importance for the development of neurodegenerative diseases called transmissible spongiform encephalopathies. We investigated if the function of members of the HSP90 family is required for the integrity of the normal, nonpathogenic prion protein called PrPc. Eukaryotic cells were treated with the structurally unrelated HSP90-inhibitors geldanamycin (GA) or radicicol (RC). In either case the cellular prion protein was induced and exhibited faster migrating bands on western blot analysis, whereas geldampicin (GE), an analog of GA known not to bind to HSP90, had no effect. Ongoing protein and messenger RNA synthesis during treatment were found to be necessary for the appearance of these bands. Cotreatment with tunicamycin abrogated any effect of HSP90 inhibitors on the cellular prion protein. Finally, enzymatic deglycosylation with peptide:N-glycosidase F of the normal prion protein as well as the variant induced by benzoquinone ansamycins resulted in very similar band patterns. These experiments indicate that either altered glycosylation, or a change in conformation, or both are involved in the induction of faster migrating bands by HSP90 inhibitors. Thus the inhibition of the function of members of the HSP90 family of molecular chaperones results in profound changes in the physicochemical properties of PrPc.

Retinoic acid decreases targeting of p27 for degradation via an N-myc-dependent decrease in p27 phosphorylation and an N-myc-independent decrease in Skp2.

Poor prognosis neuroblastoma (NB) tumors are marked by amplification and overexpression of N-myc. Retinoic acid (RA) decreases N-myc levels and induces cell cycle arrest in vitro and increases event-free survival in advanced stage NB patients. In this study, we investigated the mechanism(s) by which RA regulates cell cycle and how N-myc affects NB cell cycle progression. Constitutive N-myc overexpression stimulates increases in cyclin E-dependent kinase activity and decreases in p27 resulting in increased DNA synthesis. N-myc regulates p27 levels through an increase in targeting of p27 to the proteasome via cyclin E kinase-dependent phosphorylation of p27 and its ubiquitination. N-myc also stimulates an increase in proteasome activity. In RA-treated cells in which N-myc levels decline as p27 levels increase, degradation of p27 is also decreased. However, RA does not affect the activity of proteasome. The decrease in the degradation of p27 in RA-treated cells is due in part to a decrease in the N-myc stimulated phosphorylation of p27. However, RA also decreases Skp2 levels thus impairing the ability of p27 to be ubiquitinated. Thus, RA induces both N-myc-dependent and -independent mechanisms to minimize the degradation of p27 and arrest NB cell growth.

Caspase-dependent deubiquitination of monoubiquitinated nucleosomal histone H2A induced by diverse apoptogenic stimuli.

Enzymatic deubiquitination of mono-ubiquitinated nucleosomal histone H2A (uH2A) and H2B (uH2B) is closely associated with mitotic chromatin condensation, although the function of this histone modification in cell division remains ambiguous. Here we show that rapid and extensive deubiquitination of nucleosomal uH2A occurs in Jurkat cells undergoing apoptosis initiated by anti-Fas activating antibody, staurosporine, etoposide, doxorubicin and the proteasome inhibitor, N-acetyl-leucyl-leucyl-norlucinal. These diverse apoptosis inducers also promoted the accumulation of slowly migrating, high molecular weight ubiquitinated proteins and depleted the cellular pool of unconjugated ubiquitin. In apoptotic cells, ubiquitin was cleaved from uH2A subsequent to the appearance of plasma membrane blebbing, and deubiquitination of uH2A closely coincided with the onset of nuclear pyknosis and chromatin condensation. Nucleosomal uH2A deubiquitination, poly (ADP-ribose)polymerase (PARP) cleavage and chromatin condensation were prevented in cells challenged with apoptosis inducers by pretreatment with the pan-caspase inhibitor, zVAD-fmk, or by over-expressing anti-apoptotic Bcl-xL protein. These results implicate a connection between caspase cascade activation and nucleosomal uH2A deubiquitination. Transient transfection of 293 cells with the gene encoding Ubp-M, a human deubiquitinating enzyme, promoted uH2A deubiquitination, while an inactive mutated Ubp-M enzyme did not. However, Ubp-M-promoted deubiquitination of uH2A was insufficient to initiate apoptosis in these cells. We conclude that uH2A deubiquitination is a down-stream consequence of procaspase activation and that unscheduled cleavage of ubiquitin from uH2A is a consistent feature of the execution phase of apoptosis rather than a determining or initiating apoptogenic event. Nucleosomal uH2A deubiquitination may function as a cellular sensor of stress in situations like apoptosis through which cells attempt to preserve genomic integrity.

The Hsp90 inhibitor geldanamycin selectively sensitizes Bcr-Abl-expressing leukemia cells to cytotoxic chemotherapy.

The Bcr-Abl fusion protein drives leukemogenesis and can render leukemia cells resistant to conventional chemotherapy. Geldanamycin (GA), a drug which destabilizes Hsp90-associated proteins, depletes cells of Bcr-Abl, an Hsp90 client, but not of Abl. Both HL60 cells transfected with Bcr-Abl and naturally Ph1-positive K562 leukemia cells are resistant to most cytotoxic drugs, but were found to be sensitive to GA. Furthermore, GA sensitized Bcr-Abl-expressing cells to doxorubicin (DOX) and paclitaxel (PTX). In contrast, in parental HL60 cells, 90 nM GA inhibited PARP cleavage, nuclear fragmentation, and cell death caused by 500 ng/ml DOX. Like GA, STI 571 (an inhibitor of the Abl kinase) sensitized Bcr-Abl-expressing cells to DOX. Unlike GA, STI 571 did not antagonize the cytotoxic effects of DOX in parental HL60 cells. These results indicate that sensitization of Bcr-Abl-expressing cells, but not desensitization of HL60 cells, depends on inhibition of Bcr-Abl. Thus, GA differentially affects leukemia cells depending on their Bcr-Abl expression and selectively increases apoptosis in Bcr-Abl-expressing cells.

Effect of the proteasome inhibitor ALLnL on cisplatin sensitivity in human ovarian tumor cells.

Small molecules suppressing proteasome function inhibit the post-translational ubiquitination of selected proteins. Ubiquitin H2A is an example of an abundant chromatin-associated protein that is known to be ubiquitinated, which is important for several proteins involved in the repair of DNA damage. We therefore studied the effect of the proteasome inhibitor, N-acetyl leucyl-leucyl norlucinal (ALLnL), on cisplatin sensitivity in three human ovarian tumor cell lines. The proteasome inhibitor ALLnL was administered for 4 h before cells were subsequently exposed to cisplatin for 1 h. Our results showed that ALLnL, at its respective IC20 concentration, increased cellular sensitivity to cisplatin in an additive manner in human ovarian cancer A2780, A2780/CP70, and OVCAR3 cells. We also demonstrated that ALLnL caused a 50% increase in total cellular accumulation of cisplatin, and reduced the rate of cisplatin efflux by about 50%. In addition, DNA damage levels were increased after ALLnL treatment. By contrast, DNA repair was inhibited 2 to 3-fold in ALLnL-pretreated cells, as compared to the controls. Furthermore, our study showed that ALLnL deubiquitinated nucleosomal histone H2A in these cells in a concentration-dependent fashion, as assessed by Western blot analysis. These data suggest that sublethal levels of exposure to agents that inhibit proteasome function may alter the subcellular pharmacology of platinum in human ovarian carcinoma cells.

Measurement of the novel antitumor agent 17-(allylamino)-17-demethoxygeldanamycin in human plasma by high-performance liquid chromatography.

A sensitive HPLC assay has been developed to determine the concentration of 17-(allylamino)-17-demethoxygeldanamycin (AAG) in human plasma over the concentration range of 12.5 to 2,500 nM (7.33 to 1,465 ng/mL). After the addition of 1,000 nM geldanamycin as the internal standard, 1 mL samples of human plasma were subjected to solid-phase extraction, via Bond-Elut C18 cartridges, followed by analysis using an isocratic reversed-phase HPLC assay with UV detection. A Phenomenex Kingsorb, 3 micron, C18, 150x4.60 mm column and a Phenomenex Security Guard pre-column, C18 (ODS, Octadecyl), were used to achieve separation. AAG and GM were monitored at 334 and 308 nm, respectively, on a Hewlett-Packard 1050 Diode-Array Detector. The mobile phase, run at a flow-rate of 1 mL/min, was composed of 50% (v/v) 25 mM sodium phosphate (pH 3.00) with 10 mM triethylamine and 50% acetonitrile. HPLC effectively resolved AAG with retention times of 14.60 +/- 0.54 min and the internal standard geldanamycin at 10.72+/-0.38 min (n = 15). This assay was able to measure plasma concentrations of AAG, the lower limit of quantitation being 12.5 nM, at a starting dose of 10 mg/m2 infused intravenously over 1 h in a Phase I clinical trial in adult patients with solid tumors.

Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription.

Beta-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets beta-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of beta-catenin in melanoma cells expressing proteasome-resistant beta-catenin, as well as the effects that perturbation of beta-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity. Beta-catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with beta-catenin without perturbing either LAR or beta-catenin levels or LAR/beta-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of beta-catenin and increased beta-catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclear beta-catenin level and inhibited beta-catenin-driven transcription, as assessed using two different beta-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two beta-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the beta-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant beta-catenin is regulated further by geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.

Requirement for HDM2 activity in the rapid degradation of p53 in neuroblastoma.

The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.

Prostaglandin E2 and vasoactive intestinal peptide increase vascular endothelial cell growth factor mRNAs in lung cancer cells.

The effects of prostaglandin E2 (PGE2) and vasoactive intestinal peptide (VIP) on vascular endothelial cell growth factor (VEGF) mRNAs were investigated using lung cancer cells. By RT-PCR, VEGF(121), VEGF(165), and VEGF(189), but not VEGF(206) isoforms were detected in all lung cancer cell lines and biopsy specimens examined. By Northern blot, VEGF mRNA was detected in all small cell lung cancer (SCLC) and non-SCLC (NSCLC) cell lines examined. PGE2, VIP and forskolin caused increased VEGF expression in a time- and concentration-dependent manner using NSCLC cell line NCI-H157. Approximately 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin caused cAMP elevation, 64-, 33- and 128-fold, respectively, using NCI-H157 cells after 5 min. The increase in cAMP caused by PGE(2) and VIP was reversed by somatostatin (SST). Also 1 microM PGE2, 0.1 microM VIP and 50 microM forskolin increased the VEGF mRNA 2.0-, 1.5- and 2.3-fold, respectively, after 4 h. The increase in VEGF mRNA caused by PGE2, VIP and forskolin was inhibited by H-89, a protein kinase A inhibitor. A VIP receptor antagonist, VIPhybrid, inhibited the increase in cAMP and VEGF mRNA caused by VIP. By ELISA, VEGF was detected in the conditioned media exposed to the lung cancer cell lines. These results suggest that VEGF synthesis in and secretion from lung cancer cells can be regulated by agents, which cause adenylyl cyclase activation.

Stereospecific antitumor activity of radicicol oxime derivatives.

PURPOSE: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. METHODS: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. RESULTS: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. CONCLUSIONS: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

Lactacystin enhances cisplatin sensitivity in resistant human ovarian cancer cell lines via inhibition of DNA repair and ERCC-1 expression.

Cisplatin is among the most effective chemotherapeutic agents in the treatment of human ovarian cancer. The cytotoxicity of cisplatin results primarily from its ability to bind covalently to DNA and prevent DNA replication and transcription. The ubiquitin-proteasome pathway plays important roles in a broad array of basic cellular processes. Lactacystin is a selective inhibitor of the proteasome that can inhibit the ubiquitin pathway. However, the effect of lactacystin on DNA repair and the antitumor activity of cisplatin in ovarian cancer have not been evaluated. We report in this work that lactacystin, at concentrations that do not appear harmful, increased cisplatin toxicity in three resistant human ovarian carcinoma cell lines. In addition, lactacystin significantly enhanced DNA platination and decreased DNA repair of cisplatin-DNA adducts in these cell lines, as measured by atomic absorption spectrometry. Furthermore, Northem blot analysis and in vitro nuclear transcript elongation assay demonstrated that lactacystin dramatically reduced the steady-state mRNA expression and the rate of transcription of the DNA repair gene ERCC-1 in these cells. These observations indicate that proteasome inhibition has impact on nucleotide excision repair in several ways: i/ the normal ERCC-1 message upregulation is suppressed; ii/ cisplatin-DNA adduct repair is inhibited, and iii/ DNA platination, as well as cisplatin cytotoxicity, is enhanced.