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Biomedical subjects

Neal Rosen

Publications and source records attributed to Neal Rosen.

At least 37 records · Page 2Linked to original sources
PubMed2003

Combination treatment with 17-N-allylamino-17-demethoxy geldanamycin and acute irradiation produces supra-additive growth suppression in human prostate carcinoma spheroids.

Failure to control localized prostate cancer can result not only in localized disease progression but also distant metastatic spread. Whereas significant advances in both surgical technique and radiation therapy have improved local control rates with decreased morbidity, consistent long-term control remains elusive. This study investigates the potential of 17-N-allylamino-17-demethoxy geldanamycin (17AAG), a geldanamycin derivative, to sensitize tumor cells to ionizing radiation, permitting a significant improvement to targeted radiotherapies of prostate carcinoma. As a monotherapeutic, 17AAG functions to modulate the action of heat shock protein 90, ultimately affecting a multitude of cellular signaling pathways. It is in Phase I trial and has shown promise in controlling prostate cancer progression. Human prostate tumor cells (LNCaP and CWR22Rv1) were grown as spheroids and incubated for 96 h with increasing doses of 17AAG immediately before and after 2 or 6 Gy low linear energy transfer (LET), high dose-rate irradiation (Cs-137 irradiator). Twelve or 24 spheroids (initial diameter, 150-200 microm) were used per experiment. Response was determined by spheroid volume measurements taken over at least 40 days, after treatment. Incubation of either cell line with 17AAG (<or=1000 nM) or irradiation (<or=6 Gy) alone resulted in transient median growth delays ranging from 2 to 9 days (relative to controls). Combining treatments produced dose- and cell line-dependent supra-additive responses. For LNCaP spheroids, the combination of 2 Gy and 100 nM 17AAG resulted in growth delays additive of the treatments individually; however, increasing either the radiation to 6 Gy or the 17AAG concentration to 1000 nM led to synergistic interactions. Similarly, synergy was noted in CWR22Rv1 studies at only 6 Gy and 1000 nM 17AAG. Terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and Ki67 staining of spheroid sections revealed the increased growth control to be a function of spheroids failing to re-enter the cell cycle. For all 6 Gy experiments, cells remaining from each of the spheroids that failed to regrow were transferred to adherent dishes to evaluate clonogenicity; growth-controlled spheroids also failed to form colonies within 2 weeks of being plated. These results suggest that significant gains in treatment effectiveness may be obtained by combining these treatment modalities, warranting additional preclinical investigation.

Benzoquinones↗
PubMed2003

Synthesis of novel fluorescent probes for the molecular chaperone Hsp90.

Heat shock protein 90 (Hsp90) is a molecular chaperone necessary for maintaining oncogenic transformation. There is substantial interest in developing novel agents that bind to the N-terminal of the chaperone. Here we report the synthesis and characterization of two fluorescent Hsp90 inhibitors and probe their use in an Hsp90 fluorescent polarization assay.

Binding, Competitive↗
PubMed2003

The heat shock protein 90 inhibitor geldanamycin and the ErbB inhibitor ZD1839 promote rapid PP1 phosphatase-dependent inactivation of AKT in ErbB2 overexpressing breast cancer cells.

AKT, a serine/threonine kinase that promotes cell survival, can be activated by overexpression of the receptor tyrosine kinase ErbB2. Conversely, down-regulation of ErbB2 inhibits AKT activation. Here, we identify PP1 as a serine/threonine phosphatase that associates with and dephosphorylates AKT in breast cancer cells, and we show that ErbB2 inhibits PP1-dependent dephosphorylation of AKT. Inhibition of ErbB2 by either the HSP (heat shock protein) 90 inhibitor geldanamycin or the ErbB inhibitor ZD1839 in SKBR3 cells, a human breast cancer cell line overexpressing ErbB2 protein, induces a rapid and dramatic decrease in AKT activity. Decreased AKT activity occurs many hours before the HSP90-dependent decline of AKT protein but is correlated with loss of AKT phosphorylation. Decreased AKT phosphorylation is not due to blockade of AKT activation or to preferential HSP90-mediated degradation of phosphorylated AKT. Instead, it is caused by increased AKT dephosphorylation. Sensitivity to a panel of phosphatase inhibitors suggests involvement of the phosphatase PP1 in this process. In vitro phosphatase assay (using PP1 immunoprecipitated from COS7 cells transiently transfected with the wild-type protein, as well as purified PP1) confirmed that AKT is a substrate of PP1. Furthermore, endogenous PP1 and AKT associate with each other in SKBR3. However, the phosphatase is phosphorylated and its activity is suppressed (determined by in vitro assay). In contrast, ErbB2 inhibition abrogates PP1 phosphorylation and restores its activity (measured by its ability to dephosphorylate AKT in vitro). Finally, transient overexpression of constitutively active PP1 in SKBR3 cells promotes marked dephosphorylation of endogenous AKT protein. These data indicate that ErbB2 acts to preserve the phosphorylation, and hence to prolong the activation, of AKT kinase by repressing the activity of the phosphatase PP1. ErbB2 thus functions to regulate AKT kinase by simultaneously promoting its activation while inhibiting its inactivation.

Animals↗
PubMed2003

Angiogenesis impairment in Id-deficient mice cooperates with an Hsp90 inhibitor to completely suppress HER2/neu-dependent breast tumors.

Id proteins bind basic helix-loop-helix transcription factors and function as dominant negative inhibitors of gene expression. Id1 and Id3 are required for the recruitment of bone marrow-derived endothelial cell precursors and tumors transplanted into Id-deficient mice demonstrate impaired angiogenesis. Mouse mammary tumor virus-neu mice were bred with Id1-/-Id3+/- mice to ascertain the role of Id1 and Id3 in mammary tumorigenesis in a more physiologically relevant model. In mammary tumors from these mice, Id1 and Id3 expression was restricted to the vascular endothelium. Id1 and Id3 deficiency did not prevent or delay tumor formation but did alter tumor phenotype. The tumors that developed in the Id-deficient mice were larger and cystic with a viable rim of tumor cells surrounding a nonviable core of cellular debris. The Hsp90 chaperone protein is required for cellular survival under condition of environmental stress and for the stability of the neu oncogene. 17-Allylamino-17-demethoxygeldanamycin, an Hsp90 inhibitor, was used to treat these mice. Whereas 17-allylamino-17-demethoxygeldanamycin only modestly delayed the growth of established mammary tumors in WT mice for Id, tumor suppression was dramatically more effective in an Id1- or Id3-deficient background. These data suggest that tumorigenesis can occur in a background of defective angiogenesis but that tumors developing in such an environment may be especially sensitive to inhibitors of neu and stress-activated survival pathways. Thus angiogenesis inhibitors in combination with inhibitors of Hsp90 function should be evaluated for the treatment of advanced breast cancer.

Animals↗
PubMed2003

Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors.

We have examined the possible mechanisms of resistance to the epidermal growth factor receptor (EGFR) inhibitors in tumor cells with variable levels of EGFR. ZD1839 (Iressa) is a small-molecular-weight, ATP-mimetic that specifically inhibits the EGFR tyrosine kinase. A431 cell growth was markedly inhibited by ZD1839 (IC(50)< or =0.1 microM) whereas the MDA-468 cells were relatively resistant (IC(50)2 microM). Low doses of ZD1839 delayed cell cycle progression and induced apoptosis in A431 cells but not in MDA-468 cells. In both cell lines, 0.1 microM ZD1839 eliminated EGFR phosphorylation. However, the basal activity of the phosphatidylinositol-3 kinase (PI3 K) target Akt was eliminated in A431 but not in MDA-468 cells, implying that their Akt activity is independent of EGFR signals. A431 cells express PTEN/MMAC1/TEP, a phosphatase that can dephosphorylate position D3 of phosphatidylinositol-3,4,5 trisphosphate, the site that recruits the plecstrin-homology domain of Akt to the cell membrane. On the contrary, MDA-468 cells lack the phosphatase and tensin homolog (PTEN), potentially setting Akt activity at a high threshold that is unresponsive to EGFR inhibition alone. Therefore, we reintroduced (PTEN) by retroviral infection in MDA-468 cells. In MDA-468/PTEN but not in vector controls, treatment with ZD1839 inhibited P-Akt levels, induced relocalization of the Forkhead factor FKHRL1 to the cell nucleus, and increased FKHRL1-dependent transcriptional activity. ZD1839 induced a greater degree of apoptosis and cell cycle delay in PTEN-reconstituted than in control cells. These data suggest that loss of PTEN, by permitting a high level of Akt activity independent of receptor tyrosine kinase inputs, can temporally dissociate the inhibition of the EGFR with that of Akt induced by EGFR inhibitors. Thus, in EGFR-expressing tumor cells with concomitant amplification(s) of PI3K-Akt signaling, combined blockade of the EGFR tyrosine kinase and Akt should be considered as a therapeutic approach.

Antineoplastic Agents↗
PubMed2003

Inhibition of heat shock protein 90 function down-regulates Akt kinase and sensitizes tumors to Taxol.

The phosphatidylinositol 3'-kinase/Akt pathway is activated frequently in human cancer, and has been implicated in tumor proliferation, cell survival, and resistance to apoptotic stimuli. Akt forms a complex with heat shock protein (Hsp) 90 and Cdc37, and inhibitors of Hsp90 cause Akt degradation. 17-allylamino-17-demethoxygeldanamycin (17-AGG) is an Hsp90 inhibitor currently in Phase I clinical trial. 17-AAG inhibits Akt activation and expression in tumors, and has antitumor activity in breast cancer xenografts. The combination of 17-AAG and Taxol is synergistic, and 17-AAG sensitizes tumor cells to Taxol-induced apoptosis in a schedule-dependent manner. Transfection of membrane-bound p110 PI3k prevented 17-AAG inactivation of Akt and abrogated the enhancement of Taxol-induced apoptosis caused by the drug. 17-AAG and Taxol could be administered together at their maximally tolerated doses to tumor-bearing mice. Doses of 17-AAG that induce HER2 degradation and cause Akt inactivation but have no single agent activity were effective in sensitizing tumors to Taxol. Enhancement was schedule-dependent and maximal when Taxol and 17-AAG were administered on the same day. These results suggest that Hsp90 inhibitors can effectively suppress Akt activity in animal models of human cancer at nontoxic doses, thus sensitizing tumor cells to proapoptotic stimuli.

Animals↗
PubMed2003

Hsp90 as a therapeutic target in prostate cancer.

Prostate cancers are hormone-dependent malignancies that respond to drugs that reduce circulating testosterone levels or prevent binding of this ligand to the androgen receptor (AR). While effective, these approaches are not curative and, in almost all cases, progression to a castration-resistant state is eventually observed. The mechanisms underlying the development of hormone resistance are poorly defined but several molecular changes are commonly associated with this process. Since a common element of these resistance mechanisms is restoration of AR signaling, agents that target AR expression represent an attractive treatment option for prostate cancer patients with disease progression following castration. Prior to ligand binding, AR exists in a complex with heat shock protein 90 (Hsp90) and other co-chaperones. The AR-Hsp90 interaction maintains AR in a high-affinity ligand-binding conformation, which is necessary for efficient response to hormone. 17-Allyamino-17-demethoxygeldanamycin (17-AAG) is an inhibitor of the Hsp90 chaperone protein. Inhibition of Hsp90 function causes the proteasomal degradation of proteins that require this chaperone for maturation or stability. Hsp90 clients include several proteins of potential importance in mediating prostate cancer progression, including wild-type and mutated AR, HER2, and Akt. In murine models of prostate cancer, 17-AAG causes the degradation of these client proteins at nontoxic doses and inhibits the growth of hormone-naive and castration-resistant tumors. These data suggest that inhibitors of Hsp90 may represent a novel strategy for the treatment of patients with prostate cancer and clinical trials to test this hypothesis are currently ongoing.

Androgen Antagonists↗
PubMed2003

Microtiter cell-based assay for detection of agents that alter cellular levels of Her2 and EGFR.

Overexpression of the transmembrane tyrosine kinases Her2 and EGFR is associated with aggressive malignancies, and several therapeutic strategies targeting the two receptors are now in various stages of clinical development. Most of the known agents block the activation or inhibit the activity of the kinases; however, a more significant therapeutic outcome may result from degrading these oncoproteins. Here, we report the development of a microtiter cell-based assay that sensitively detects cellular levels of Her2 and EGFR. The assay is useful in identifying small molecules that alter cellular levels of these kinases and in quantifying their effect. The method gives results comparable to Western blot, but it is faster, less labor intensive, and amenable to high throughput.

Actins↗
PubMed2003

Development of purine-scaffold small molecule inhibitors of Hsp90.

The Hsp90 chaperones play a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype in tumor cells. Agents that interfere with the function of the chaperone may thus be beneficial in the treatment of cancers. The ansamycins (geldanamycin and herbimycin) and the unrelated natural product radicicol were found to bind to the N-terminal pocket of Hsp90 and inhibit its function. However, translation of these compounds to the clinic was impeded by stability and hepatoxicity issues. 17AAG, a derivative of geldanamycin, was found to be less hepatotoxic and is currently undergoing Phase I clinical trial. Unfortunately, 17AAG is insoluble, difficult to formulate and it is not yet clear if therapeutically effective doses can be administered without escalating non-Hsp90 associated toxicities. Additionally, for reasons not yet completely understood, a subset of tumor cells are insensitive to the action of the drug. The development of novel agents that lack the drawbacks of the natural products is thus necessary. Here we present an overview of such efforts with focus on a new class of purine-scaffold Hsp90 inhibitors developed by rational design.

Animals↗
PubMed2003

17AAG: low target binding affinity and potent cell activity--finding an explanation.

The ansamycin geldanamycin (GM) and its derivative, 17AAG, now in early clinical trials in cancer patients, have potent activity against several cancer cells at low nanomolar concentrations. The main target of these drugs is the molecular chaperone heat shock protein 90. Contrary to the high antitumor potency, the affinity of these drugs for the chaperone was determined to be approximately 1 microM. We propose that this difference can partly be explained by the physicochemical characteristics of the ansamycins. GM and 17AAG accumulate in cells, producing higher intracellular concentrations than expected. We conclude that although apparent activity for ansamycins can be seen at low nanomolar concentration, their real activity correlates with the heat shock protein 90 binding affinity and is in the low micromolar concentration range. We suggest that in the clinic, micromolar concentrations of 17AAG must accumulate in the tumor cells to achieve antitumor effects in patients comparable with ones achieved in tissue culture settings.

Antibiotics, Antineoplastic↗
PubMed2003

A novel pyridopyrimidine inhibitor of abl kinase is a picomolar inhibitor of Bcr-abl-driven K562 cells and is effective against STI571-resistant Bcr-abl mutants.

Inhibition of the constitutively active Bcr-abl tyrosine kinase(TK) by STI571 has proven to be a highly effective treatment for chronic myelogenous leukemia (CML). However, STI571 is only transiently effective in blast crisis, and drug resistance emerges by amplification of or development of mutational changes in Bcr-abl. We have screened a family of TK inhibitors of the pyrido [2,3-d]pyrimidine class, unrelated to STI571, and describe here a compound with substantial activity against STI-resistant mutant Bcr-abl proteins. This compound, PD166326, is a dual specificity TK inhibitor and inhibits src and abl in vitro with IC(50)s of 6 and 8 nM respectively. PD166326 inhibits the growth of K562 cells with IC(50) of 300 pM, leading to apoptotic G(1) arrest, whereas non-Bcr-abl cell types require >1000 times higher concentrations. We tested the effects of PD166326 on two of the clinically observed STI571-resistant Bcr-abl mutants. PD166326 potently inhibits the E255K mutant Bcr-abl protein and the growth of Bcr-ablE255K-driven cells. The T315I mutant Bcr-abl protein, which is mutated within the ATP-binding pocket, is resistant to PD166326; however, the growth of Bcr-ablT315I-driven cells is partially sensitive to this compound, likely through the inhibition of Bcr-abl effector pathways. These findings show that TK drug resistance is a structure-specific phenomenon and can be overcome by TK inhibitors of other structural classes, suggesting new approaches for future anticancer drug development. PD166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and substantial activity against STI571-resistant mutants.

Antineoplastic Agents↗
PubMed2002

BCR-ABL point mutants isolated from patients with imatinib mesylate-resistant chronic myeloid leukemia remain sensitive to inhibitors of the BCR-ABL chaperone heat shock protein 90.

Clinical resistance to imatinib mesylate is commonly observed in patients with advanced Philadelphia chromosome- positive (Ph(+)) leukemias. Acquired resistance is typically associated with reactivation of BCR-ABL due to kinase domain mutations or gene amplification, indicating that BCR-ABL remains a viable target for inhibition in these patients. Strategies for overcoming resistance can be envisioned through exploitation of other molecular features of the BCR-ABL protein, such as its dependence on the molecular chaperone heat shock protein 90 (Hsp90). To determine whether inhibition of Hsp90 could induce degradation of imatinib mesylate-resistant, mutant BCR-ABL proteins, hematopoietic cells expressing 2 mutant BCR-ABL proteins found in imatinib mesylate-resistant patients (T315I and E255K) were examined for sensitivity to geldanamycin and 17-allylaminogeldanamycin (17-AAG). Both compounds induced the degradation of wild-type and mutant BCR-ABL and inhibited cell growth, with a trend indicating more potent activity against mutant BCR-ABL proteins. These data support clinical investigations of 17-AAG in imatinib mesylate-resistant Ph(+) leukemias.

Antineoplastic Agents↗
PubMed2002

Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function.

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2002

Degradation of HER2 by ansamycins induces growth arrest and apoptosis in cells with HER2 overexpression via a HER3, phosphatidylinositol 3'-kinase-AKT-dependent pathway.

Breast cancers with high expression of HER2 are associated frequently with aggressive, poor prognosis disease and resistance to chemotherapy-induced apoptosis. Geldanamycin and its less toxic analogue, 17- (allylamino)-17-demethoxygeldanamycin (17-AAG) are ansamycin antibiotics that bind to a highly conserved pocket in the hsp 90 chaperone protein and inhibit its function. Hsp 90 is required for the refolding of proteins during environmental stress and the conformational maturation of certain signaling proteins. Among the most sensitive targets of 17-AAG are the HER kinases. Therefore, tumors that are dependent on these kinases may be especially sensitive to 17-AAG either alone or in combination with chemotherapy. In this study we demonstrate that cells that overexpress HER2 are 10-100-fold more sensitive to 17-AAG than cancer cells expressing low levels of HER2. We found that HER2 is degraded in several cell lines, but only cell lines with high levels of HER2 are sensitive to the drug. The effects of 17-AAG on growth and apoptosis are because of inhibition of signaling through HER2-HER3, phosphatidylinositol 3'- kinase. The absence of HER3 and the introduction of constitutively active p110alpha rendered cells with high HER2 expression more resistant to 17-AAG. These findings suggest that 17-AAG may be useful for the treatment of breast cancer cells with high levels of HER2. However, the overexpression of HER2 alone may not be predictive of response, because the coexpression of HER3 and the activation of phosphatidylinositol 3'-kinase may play a crucial role in the response of these cells to 17-AAG and other drugs directed against HER2. These observations have important clinical implications because they may help to identify patients that are most likely to benefit from 17-AAG and may explain resistance to Herceptin as seen in many patients.

Animals↗
PubMed2002

Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2.

Ansamycin antibiotics, such as 17-allylaminogeldanamycin (17-AAG), bind to Hsp90 and regulate its function, resulting in the proteasomal degradation of a subset of signaling proteins that require Hsp90 for conformational maturation. HER2 is a very sensitive target of these drugs. Ansamycins cause RB-dependent G1 arrest that is associated with loss of D-cyclins via a PI3 kinase, Akt dependent pathway. Downregulation of D-cyclin was due, in part, to loss of Akt expression in response to drug. Moreover, in HER2 overexpressing breast cancer cells, 17-AAG caused rapid inhibition of Akt activity prior to any change in Akt protein. Ansamycins caused rapid degradation of HER2 and a concomitant loss in HER3 associated PI3 kinase activity. This led to a loss of Akt activity, dephosphorylation of Akt substrates, and loss of D-cyclin expression. Introduction into cells of a constitutively membrane bound form of PI3 kinase prevented the effects of the drug on Akt activity and D-cyclins. Thus, in breast cancer cells with high HER2, Akt activation by HER2/HER3 heterodimers is required for D-cyclin expression. In murine xenograft models, non-toxic doses of 17-AAG markedly reduced the expression of HER2 and phosphorylation of Akt and inhibited tumor growth. Thus, pharmacological inhibition of Akt activation is achievable with ansamycins and may be useful for the treatment of HER2 driven tumors.

Animals↗
PubMed2002

Development of a purine-scaffold novel class of Hsp90 binders that inhibit the proliferation of cancer cells and induce the degradation of Her2 tyrosine kinase.

The first published synthesis and characterization of a purine-scaffold library of hsp90 inhibitors is presented. The purine-scaffold represents a platform for the creation of easily synthesizable and derivatizable soluble molecules that are amenable for oral administration. The most active compound of the series (71) exhibits binding to hsp90 comparable to the natural product derivative 17AAG that is now in Phase I clinical trial as a cancer therapeutic. Induces the degradation of Her2 tyrosine kinase and arrests the MCF-7 breast cancer cell line at low micromolar concentrations (IC50=2 microM).

Alkylation↗