Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.

The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis.

Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.

BACKGROUND: KRAS mutations in NSCLC are associated with a lack of response to epidermal growth factor receptor inhibitors. Selumetinib (AZD6244; ARRY-142886) is an oral selective MEK kinase inhibitor of the Ras/Raf/MEK/ERK pathway. PATIENTS AND METHODS: Advanced nonsmall-cell lung cancer (NSCLC) patients failing one to two prior regimens underwent KRAS profiling. KRAS wild-type patients were randomized to erlotinib (150 mg daily) or a combination of selumetinib (150 mg daily) with erlotinib (100 mg daily). KRAS mutant patients were randomized to selumetinib (75 mg b.i.d.) or the combination. The primary end points were progression-free survival (PFS) for the KRAS wild-type cohort and objective response rate (ORR) for the KRAS mutant cohort. Biomarker studies of ERK phosphorylation and immune subsets were carried out. RESULTS: From March 2010 to May 2013, 89 patients were screened; 41 KRAS mutant and 38 KRAS wild-type patients were enrolled. Median PFS in the KRAS wild-type arm was 2.4 months [95% confidence interval (CI) 1.3-3.7] for erlotinib alone and 2.1 months (95% CI 1.8-5.1) for the combination. The ORR in the KRAS mutant group was 0% (95% CI 0.0% to 33.6%) for selumetinib alone and 10% (95% CI 2.1% to 26.3%) for the combination. Combination therapy resulted in increased toxicities, requiring dose reductions (56%) and discontinuation (8%). Programmed cell death-1 expression on regulatory T cells (Tregs), Tim-3 on CD8+ T cells and Th17 levels were associated with PFS and overall survival in patients receiving selumetinib. CONCLUSIONS: This study failed to show improvement in ORR or PFS with combination therapy of selumetinib and erlotinib over monotherapy in KRAS mutant and KRAS wild-type advanced NSCLC. The association of immune subsets and immune checkpoint receptor expression with selumetinib may warrant further studies.

A phase II study of TRC105 in patients with hepatocellular carcinoma who have progressed on sorafenib.

BACKGROUND: Endoglin is an endothelial cell membrane receptor essential for angiogenesis and highly expressed on the vasculature of many tumor types, including hepatocellular carcinoma (HCC). TRC105 is a chimeric IgG1 anti-CD105 monoclonal antibody that inhibits angiogenesis and tumor growth by endothelial cell growth inhibition, ADCC and apoptosis, and complements VEGF inhibitors. OBJECTIVE: The aim of this phase II study was to evaluate the efficacy of anti-endoglin therapy with TRC105 in patients with advanced HCC, post-sorafenib. METHODS: Patients with HCC and compensated liver function (Childs-Pugh A/B7), ECOG 0/1, were enrolled to a single-arm, phase II study of TRC105 15 mg/kg IV every two weeks. Patients must have progressed on or been intolerant of prior sorafenib. A Simon optimal two-stage design was employed with a 50% four-month PFS target for progression to the second stage. Correlative biomarkers evaluated included DCE-MRI as well as plasma levels of angiogenic biomarkers and soluble CD105. RESULTS: A total accrual of 27 patients was planned. However, because of lack of efficacy and in accordance with the Simon two-stage design, 11 patients were enrolled. There were no grade 3/4 treatment-related toxicities. Most frequent toxicities were headache (G2; N = 3) and epistaxis (G1; N = 4). One patient had a confirmed partial response by standard RECIST criteria and biologic response on DCE-MRI but the four-month PFS was insufficient to proceed to the second stage of the study. CONCLUSIONS: TRC105 was well tolerated in this HCC population following sorafenib. Although there was evidence of clinical activity, this did not meet prespecified criteria to proceed to the second stage. TRC105 development in HCC continues as combination therapy with sorafenib.

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.

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.

DNA damage and cell cycle arrest induced by 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

Failure of hormone therapy in prostate cancer involves systematic restoration of androgen responsive genes and activation of rapamycin sensitive signaling.

Androgen deprivation therapy for advanced prostate cancer is often effective, but not curative. Molecular pathways mediating the therapeutic response and those contributing to the subsequent hormone-refractory cell growth remain poorly understood. Here, cDNA microarray analysis of human CWR22 prostate cancer xenografts during the course of androgen deprivation therapy revealed distinct global gene expression profiles in primary, regressing and recurrent tumors. Elucidation of the genes involved in the transition between these states implicated specific molecular mechanisms in therapy failure and tumor progression. First, we identified a set of androgen-responsive genes whose expression decreased during the therapy response, but was then systematically restored in the recurrent tumors. In addition, altered expression of genes that encode known targets of rapamycin or that converge on the PI3K/AKT/FRAP pathway was observed in the recurrent tumors. Further suggestion for the involvement of these genes in hormone-refractory prostate cancer came from the observation that cells established from the recurrent xenografts were strongly inhibited in vitro by rapamycin. The results of this functional genomic analysis suggest that the combined effect of re-expression of androgen-responsive genes as well as the activation of rapamycin-sensitive signaling may drive prostate cancer progression, and contribute to the failure of androgen-deprivation therapy.

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.

Impact of extracellular folate levels on global gene expression.

Methylation of DNA is associated with gene silencing. DNA methylation uses S-adenosylmethionine (SAM) as the methyl donor and the formation of SAM requires a continuous supply of folate from the extracellular milieu. Low extracellular folate levels are known to result in induction of expression of the human alpha folate receptor in nasopharyngeal epidermoid carcinoma cells. Low folate levels have been implicated in global activation of gene expression. We have investigated the impact of lowering the level of extracellular folate by performing cDNA microarray analysis of global gene expression in human nasopharyngeal carcinoma KB cells grown in folate-deplete and folate-replete medium. We found that expression of only eight genes reproducibly responded to variation of folate levels. Among those, three were up-regulated and five were down-regulated. Examination of one gene, H-cadherin, demonstrated down-regulation in response to folate depletion. Despite the low level of extracellular folate, there was hypermethylation of H-cadherin 5' sequences. These data indicate that low extracellular folate positively and negatively influences the expression levels of a small cohort of genes. The data suggest that folate deficiency is associated with gene-specific methylation/demethylation, rather than global DNA demethylation and transcriptional activation.

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.

Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions.

Many arachidonic acid metabolites function in growth signaling for epithelial cells, and we previously reported the expression of the major arachidonic acid enzymes in human breast cancer cell lines. To evaluate the role of the 5-lipoxygenase (5-LO) pathway on breast cancer growth regulation, we exposed cells to insulinlike growth factor-1 or transferrin, which increased the levels of the 5-LO metabolite, 5(S)-hydrooxyeicosa-6E,8C,11Z,14Z-tetraenoic acid (5-HETE), by radioimmunoassay and high-performance liquid chromatography. Addition of 5-HETE to breast cancer cells resulted in growth stimulation, whereas selective biochemical inhibitors of 5-LO reduced the levels of 5-HETE and related metabolites. Application of 5-LO or 5-LO activating protein-directed inhibitors, but not a cyclooxygenase inhibitor, reduced growth, increased apoptosis, down-regulated bcl-2, up-regulated bax, and increased G1 arrest. Exposure of breast cancer cells to a 5-LO inhibitor up-regulated peroxisome proliferator-activated receptor (PPAR)a and PPARg expression, and these same cells were growth inhibited when exposed to relevant PPAR agonists. These results suggest that disruption of the 5-LO signaling pathway mediates growth arrest and apoptosis in breast cancer cells. Additional experiments suggest that this involves the interplay of several factors, including the loss of growth stimulation by 5-LO products, the induction of PPARg, and the potential activation of PPARg by interactions with shunted endoperoxides.

Metastasis-associated differences in gene expression in a murine model of osteosarcoma.

Despite advances in the management of osteosarcoma (OSA) and other solid tumors, the development of metastasis continues to be the most significant problem and cause of death for cancer patients. To define genetic determinants of pulmonary metastasis, we have applied cDNA microarrays to a recently described murine model of OSA that is characterized by orthotopic tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and cell line variants that differ in metastatic potential. Microarray analysis defined 53 genes (of 3166 unique cDNAs) that were differentially expressed between the primary tumors of the more aggressive (K7M2) and less aggressive (K12) OSA models. By review of the literature, these differentially expressed genes were assigned to six nonmutually exclusive metastasis-associated categories (proliferation and apoptosis, motility and cytoskeleton, invasion, immune surveillance, adherence, and angiogenesis). Functional studies to evaluate K7M2 and K12 for differences in each of these metastasis-associated processes revealed enhanced motility, adherence, and angiogenesis in the more aggressive K7M2 model. For this reason, 10 of the 53 differentially expressed genes that were assigned to the motility and cytoskeleton, adherence, and angiogenesis categories were considered as most likely to define differences in the metastatic behavior of the two models. Ezrin, a gene not described previously in OSA, with functions in motility, invasion, and adherence, was 3-fold overexpressed in K7M2 compared with K12 by microarray. Differential expression for RNA was confirmed by Northern analysis and for protein by immunostaining. Alterations in ezrin protein levels and concomitant cytoskeletal changes in our model confirmed predictions from the arrays. The potential relevance of ezrin in OSA was suggested by its expression in five of five human OSA cell lines. This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis.

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.

Transcriptional activation of p21(WAF1/CIP1) by apicidin, a novel histone deacetylase inhibitor.

Apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-l-2-amino-8-oxodecanoyl)], a novel histone deacetylase inhibitor, has been identified as an antiprotozoal and antiproliferative agent. In this study, we show apicidin induces transcriptional activation of p21(WAF1/CIP1) (p21) in human prostate carcinoma cells. Apicidin induces expression of p21 protein and mRNA and activation of p21 promoter-luciferase reporter constructs. Apicidin causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin. Chromatin immunoprecipitation shows p21 promoter DNA is associated with hyperacetylated histones H3 and H4 after treatment with apicidin. Therefore, the data here demonstrate that apicidin activates p21 transcription associated with the acetylation of histones H3 and H4.

MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells.

Transcriptional repression of the transforming growth factor (TGF)-1P type II receptor (TPRII) gene appears to be a major mechanism to inactivate TGF-beta responsiveness in many human cancers. Because histone acetylation/deacetylation plays a role in transcriptional regulation, we have examined the effect of MS-275, a synthetic inhibitor of histone deacetylase, in human breast cancer cell lines. MS-275 showed antiproliferative activity against all human breast cancer cell lines examined and induced TbetaRII mRNA, but not TGF-beta type I receptor mRNA. MS-275 caused an accumulation of acetylated histones H3 and H4 in total cellular chromatin. An increase in the accumulation of acetylated histones H3 and H4 was detected in the TbetaRII promoter after treatment with MS-275. However, the level of histone acetylation did not change in chromatin associated with the TGF-beta type I receptor gene. MS-275 treatment enhanced TGF-beta1-induced plasminogen activator inhibitor 1 expression. Thus, antitumor activity of MS-275 may be mediated in part through the induction of TbetaRII expression and consequent potentiation of TGF-beta signaling.

Prevention of cisplatin-DNA adduct repair and potentiation of cisplatin-induced apoptosis in ovarian carcinoma cells by proteasome inhibitors.

Histones H2A and H2B are known to be reversibly post-translationally modified by ubiquitination. We previously observed in cultured tumor cells that proteasome inhibition stabilizes polyubiquitinated proteins, depletes unconjugated ubiquitin, and thereby promotes the deubiquitination of nucleosomal histones in chromatin. Provocative indirect evidence suggests that histone ubiquitination/deubiquitination cycles alter chromatin structure, which may limit accessibility of DNA repair proteins to damaged sites. In the present study, we focused on the relationship between the ubiquitination status of histone H2A, the structure of chromatin, and the efficiency of nucleotide excision repair (NER) of cisplatin-DNA adducts in human ovarian carcinoma cells exposed to the antitumor drug cisplatin. Pretreating cells with the proteasome inhibitor lactacystin (LC) or N-acetyl-leucyl-leucyl-norleucinal (ALLnL) induced deubiquitination of ubiquitinated histone H2A (uH2A) and concomitantly promoted chromatin condensation, increased the extent of cisplatin-DNA adducts, and diminished NER-dependent repair of cisplatin-DNA lesions, compared with control cells treated with cisplatin alone. Both proteasome inhibitors also prevented the increase in ERCC-1 mRNA expression that occurs in cells exposed to cisplatin. Cells treated with the combination of ALLnL and cisplatin underwent apoptosis, as indicated by caspase-dependent poly(ADP-ribose) polymerase (PARP) cleavage, more quickly than cells treated with either agent alone. Additionally, the combination of ALLnL and cisplatin potently increased p53 levels in cell lysates and stimulated the binding of p53 to chromatin. Together, these observations suggest that proteasome inhibition may be exploited therapeutically for its potential to sensitize ovarian tumor cells to cisplatin.