Activated B-RAF Is an Hsp90 Client Protein That Is Targeted by the Anticancer Drug 17-Allylamino-17-Demethoxygeldanamycin

Dias, Silvy da Rocha; Friedlos, Frank; Light, Yvonne; Springer, Caroline J.; Workman, Paul; Marais, Richard

doi:10.1158/0008-5472.can-05-2632

Cited by 236 publications

(227 citation statements)

References 18 publications

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“…We found that the interaction of either cdc2 or cdc25c with p50 cdc37 decreased after GA treatment ( Figure 6) and that conversely, the association of cdc2 and cdc25c with Hsp70 in glioblastoma cells increased over time after GA treatment ( Figure 6). We conclude from these experiments that cdc2 and cdc25c are Hsp90 client proteins and that ansamycin treatment alters the composition of the chaperone complexes associated with either cdc2 or cdc25c, as it has been demonstrated with oncoproteins such as bcr-abl, src , Akt , or mutated B-raf (da Rocha et al, 2005;Grbovic et al, 2006). After GA treatment, the multichaperone complex tends to adopt the ADP-bound conformation, which may target cdc2 and cdc25c proteins for degradation, presumably via the proteasome.…”

Section: Discussionmentioning

confidence: 61%

Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G2/M arrest in glioblastoma cell lines

et al. 2007

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Ansamycins exert their effects by binding heat shock protein 90 (Hsp90) and targeting important signalling molecules for degradation via the proteasome pathway. We wanted to study the effect of geldanamycin (GA) and its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) on glioblastoma cell lines. We show that these cells are growth inhibited by ansamycins by being arrested in G 2 / M and, subsequently, cells undergo apoptosis. The protein levels of cell division cycle 2 (cdc2) kinase and cell division cycle 25c (cdc25c) were downregulated upon GA and 17-AAG treatment and cdc2 kinase activity was inhibited. However, other proteins involved in the G 2 /M checkpoint were not affected. The cdc2 and cdc25c mRNA levels did not show significant differences upon ansamycin treatment, but the stability of cdc2 protein was reduced. The association of cdc2 and cdc25c with p50 cdc37 , an Hsp90 cochaperone, decreased, but the interaction of cdc2 and cdc25c with the Hsp70 co-chaperone increased after ansamycin treatment. Proteasome inhibitors were able to rescue the cdc2 downregulation, but not the cdc25c reduction. However, calpain inhibitors were able to rescue the cdc25c downregulation, suggesting that cdc25c is proteolysed by calpains in the presence of ansamycins, and not by the proteasome. We conclude that ansamycins downregulate cdc2 and cdc25c by two different mechanisms.

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Section: Discussionmentioning

confidence: 61%

Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G2/M arrest in glioblastoma cell lines

et al. 2007

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“…Antibodies used are listed in Supplementary Materials. 5 Cell Cycle Analysis Cell cycle effects were determined using propidium iodide staining and flow cytometry (36).…”

Section: Methodsmentioning

confidence: 99%

“…Control, plasma, and tumor (100 AL) were analyzed for compound levels following protein precipitation with 5 Supplementary materials for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). 3 volumes of methanol containing an analogue as internal standard.…”

Section: Methodsmentioning

confidence: 99%

“…Therapeutic selectivity for cancer versus normal cells might be achieved by exploiting oncogene addiction (4). In addition, mutated oncoproteins are often hypersensitive to HSP90 inhibition (5,6), and chaperone dependence of tumors is increased by the fact that cancer cells are stressed by the effects of oncogenes and microenvironmental factors (2). Furthermore, HSP90 in cancer cells is reported to exist in a superchaperone complex that is much more sensitive to HSP90 inhibition than the uncomplexed form that predominates in normal cells (7).…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

Sharp

Prodromou

Boxall

et al. 2007

Molecular Cancer Therapeutics

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139

103

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Although the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) shows clinical promise, potential limitations encourage development of alternative chemotypes. We discovered the 3,4-diarylpyrazole resorcinol CCT018159 by high-throughput screening and used structure-based design to generate more potent pyrazole amide analogues, exemplified by VER-49009. Here, we describe the detailed biological properties of VER-49009 and the corresponding isoxazole VER-50589. X-ray crystallography showed a virtually identical HSP90 binding mode. However, the dissociation constant (K d ) of VER-50589 was 4.5 F 2.2 nmol/L compared with 78.0 F 10.4 nmol/L for VER-49009, attributable to higher enthalpy for VER-50589 binding. A competitive binding assay gave a lower IC 50 of 21 F 4 nmol/L for VER-50589 compared with 47 F 9 nmol/L for VER-49009. Cellular uptake of VER-50589 was 4-fold greater than for VER-49009. Mean cellular antiproliferative GI 50 values for VER-50589 and VER-49009 for a human cancer cell line panel were 78 F 15 and 685 F 119 nmol/L, respectively, showing a 9-fold potency gain for the isoxazole. Unlike 17-AAG, but as with CCT018159, cellular potency of these analogues was independent of NAD(P)H:quinone oxidoreductase 1/DT-diaphorase and Pglycoprotein expression. Consistent with HSP90 inhibition, VER-50589 and VER-49009 caused induction of HSP72 and HSP27 alongside depletion of client proteins, including C-RAF, B-RAF, and survivin, and the protein arginine methyltransferase PRMT5. Both caused cell cycle arrest and apoptosis. Extent and duration of pharmacodynamic changes in an orthotopic human ovarian carcinoma model confirmed the superiority of VER-50589 over VER-49009. VER-50589 accumulated in HCT116 human colon cancer xenografts at levels above the cellular GI 50 for 24 h, resulting in 30% growth inhibition. The results indicate the therapeutic potential of the resorcinylic pyrazole/isoxazole amide analogues as

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“…This shows that, in human melanocyte-derived cells, SCF is as strong an activator of the PI3K pathway as in murine melanocytes. In 501mel cells, the Erk pathway is constitutively activated by a B-Raf mutation (da Rocha Dias et al, 2005) and insensitive to SCF. To confirm these results in vivo, we examined c-Kit expression and Akt phosphorylation in situ using immunohistochemistry c-KIT mutants are oncogenic in melanocytes G Monsel et al in an acral tumor containing the D576 c-Kit mutation.…”

Section: D576pmentioning

confidence: 99%

c-Kit mutants require hypoxia-inducible factor 1α to transform melanocytes

et al. 2009

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Many studies have highlighted the critical role of c-Kit in normal melanocyte development but its role in melanoma development remains unclear. Although c-Kit expression is often lost during melanoma progression, a subset of melanoma has been found to overexpress c-Kit and mutations activating c-Kit have recently been identified in some acral and mucosal melanoma. To address the role of these c-Kit mutants in the transformation of melanocytes, we characterized the physiological responses of melanocytes expressing the most frequent c-Kit mutants found in melanoma (K642E and L576P) and a novel mutant we identified in an acral melanoma. We analysed signaling pathways activated downstream of c-Kit and showed that all three mutants led to a strong activation of the phosphatidyl-inositol-3 kinase (PI3K) pathway but only weak activation of the Ras/Raf/Mek/Erk pathway, which was not sufficient to promote uncontrolled melanocyte proliferation and transformation. However, in hypoxic conditions or coexpressed with a constitutively active form of hypoxia-inducible factor 1a (HIF-1a), c-Kit mutants activate the Ras/Raf/Mek/Erk pathway, stimulate proliferation and transform melanocytes. Proliferation of melanocytes transformed by these mutants was specifically inhibited by imatinib. These results show for the first time that melanocytes require a specific epigenetic environment to be transformed by c-Kit mutants and highlight a distinct molecular mechanism of melanocyte transformation.

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Activated B-RAF Is an Hsp90 Client Protein That Is Targeted by the Anticancer Drug 17-Allylamino-17-Demethoxygeldanamycin

Cited by 236 publications

References 18 publications

Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G2/M arrest in glioblastoma cell lines

Inhibition of Hsp90 function by ansamycins causes downregulation of cdc2 and cdc25c and G2/M arrest in glioblastoma cell lines

Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

c-Kit mutants require hypoxia-inducible factor 1α to transform melanocytes

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