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Biomedical subjects

Len Neckers

Publications and source records attributed to Len Neckers.

47 records · Page 3Linked to original sources
PubMed2002

Microtubule disruption utilizes an NFkappa B-dependent pathway to stabilize HIF-1alpha protein.

Hypoxia-inducible factor (HIF)-1alpha levels are elevated in normoxic cells undergoing physiological processes involving large scale microtubule reorganization, such as embryonic development, wound healing, and tumor cell metastasis. Although alterations in microtubules affect numerous cellular responses, no data have yet implicated microtubule dynamics in HIF-1alpha regulation. To investigate the effect of microtubule change upon HIF-1alpha regulation, we treated cells with the microtubule-depolymerizing agents (MDAs) colchicine, vinblastine or nocodazole. We demonstrate that these agents are able to induce transcriptionally active HIF-1. MDA-mediated induction of HIF-1alpha required microtubule depolymerization, since HIF-1alpha levels were unchanged in cells treated with either the microtubule-stabilizing agent paclitaxel, or an inactive form of colchicine, or in colchicine-resistant cells. HIF-1 induction was dependent upon cellular transcription, as transcription inhibitors abrogated HIF-1alpha protein up-regulation. The ability of transcriptional inhibitors to interfere with HIF-1alpha accumulation was specific to the MDA-initiated pathway, as they were ineffective in preventing hypoxia-mediated HIF-1 induction, which occurs by a distinct post-translational pathway. Moreover, we provide evidence implicating a requirement for NFkappaB transcription in the HIF-1 induction mediated by MDAs. The ability of MDAs to induce HIF-1alpha is dependent upon activation of NFkappaB, since inhibition of NFkappaB either pharmacologically or by transfection of an NFkappaB super-repressor plasmid abrogated this induction. Collectively, these data support a model in which NFkappaB is a focal point for the convergence of MDA-mediated signaling events leading to HIF-1 induction, thus revealing a novel aspect of HIF-1alpha regulation and function.

Animals↗
PubMed2002

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

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

Animals↗
PubMed2002

Drug-induced ubiquitylation and degradation of ErbB receptor tyrosine kinases: implications for cancer therapy.

Overexpression of ErbB-2/HER2 is associated with aggressive human malignancies, and therapeutic strategies targeting the oncoprotein are currently in different stages of clinical application. Tyrosine kinase inhibitors (TKIs) that block the nucleotide-binding site of the kinase are especially effective against tumors. Here we report an unexpected activity of TKIs: along with inhibition of tyrosine phosphorylation, they enhance ubiquitylation and accelerate endocytosis and subsequent intracellular destruction of ErbB-2 molecules. Especially potent is an irreversible TKI (CI-1033) that alkylates a cysteine specific to ErbB receptors. The degradative pathway stimulated by TKIs appears to be chaperone mediated, and is common to the heat shock protein 90 (Hsp90) antagonist geldanamycin and a stress-induced mechanism. In agreement with this conclusion, CI-1033 and geldanamycin additively inhibit tumor cell growth. Based upon a model for drug-induced degradation of ErbB-2, we propose a general strategy for selective destruction of oncoproteins by targeting their interaction with molecular chaperones.

Antineoplastic Agents↗
PubMed2002

Modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228.

BACKGROUND: Histone deacetylases (HDACs) modulate chromatin structure by regulating acetylation of core histone proteins. HDAC inhibitors, such as depsipeptide FR901228 (FK228), induce growth arrest and apoptosis in a variety of human cancer cells by mechanisms that cannot be attributed solely to histone acetylation. This study evaluated the mechanisms by which FK228 mediates apoptosis in non-small-cell lung cancer (NSCLC) cells. METHODS: Proliferation and apoptosis were assessed in a panel of NSCLC cell lines that vary in the expression of the growth-regulating proteins p53, pRb, and K-Ras treated with a clinically relevant dose of FK228 (25 ng/mL). Western blot and immunoprecipitation techniques were used to analyze expression of cell-cycle proteins (cyclin A, cyclin E, p53, and p21), signaling-related proteins (ErbB1, ErbB2, and Raf-1), activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2), binding of mutant p53 and Raf-1 to heat shock protein (Hsp)90, and acetylation of Hsp90. RESULTS: FK228 treatment inhibited growth and induced apoptosis in NSCLC cells expressing wild-type or mutant p53. FK228 treatment led to altered expression of cyclin A, cyclin E, and p21, and to reduced expression of mutant, but not wild-type, p53. FK228-treated cells also were depleted of ErbB1, ErbB2, and Raf-1 proteins, and exhibited lower ERK1/2 activity. FK228 treatment also inhibited the binding of mutant p53 and Raf-1 to Hsp90; this inhibition was associated with acetylation of Hsp90. CONCLUSIONS: FK228 depletes the levels of several oncoproteins that are normally stabilized by binding to Hsp90 in cancer cells. The resulting ability of FK228 to diminish signal transduction via pathways involving Raf-1 and ERK may contribute to the potency and specificity of this novel antitumor agent.

Anti-Bacterial Agents↗
PubMed2002

Hsp90 inhibitors as novel cancer chemotherapeutic agents.

Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for the stability and function of multiple mutated, chimeric and over-expressed signaling proteins that promote the growth and/or survival of cancer cells. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, Akt, ErbB2 and hypoxia-inducible factor 1 alpha (HIF-1 alpha). Hsp90 inhibitors, by interacting specifically with a single molecular target, cause the destabilization and eventual degradation of Hsp90 client proteins, and they have shown promising antitumor activity in preclinical model systems. One Hsp90 inhibitor, 17-allylaminogeldanamycin (17AAG), is currently in phase I clinical trial. Because of the chemoprotective activity of several proteins that are Hsp90 clients, the combination of an Hsp90 inhibitor with a standard chemotherapeutic agent could dramatically increase the in vivo efficacy of the therapeutic agent.

Antibiotics, Antineoplastic↗
PubMed2002

Heat shock protein 90 modulates the unfolded protein response by stabilizing IRE1alpha.

The molecular chaperone HSP90 regulates stability and function of multiple protein kinases. The HSP90-binding drug geldanamycin interferes with this activity and promotes proteasome-dependent degradation of most HSP90 client proteins. Geldanamycin also binds to GRP94, the HSP90 paralog located in the endoplasmic reticulum (ER). Because two of three ER stress sensors are transmembrane kinases, namely IRE1alpha and PERK, we investigated whether HSP90 is necessary for the stability and function of these proteins. We found that HSP90 associates with the cytoplasmic domains of both kinases. Both geldanamycin and the HSP90-specific inhibitor, 514, led to the dissociation of HSP90 from the kinases and a concomitant turnover of newly synthesized and existing pools of these proteins, demonstrating that the continued association of HSP90 with the kinases was required to maintain their stability. Further, the previously reported ability of geldanamycin to stimulate ER stress-dependent transcription apparently depends on its interaction with GRP94, not HSP90, since geldanamycin but not 514 led to up-regulation of BiP. However, this effect is eventually superseded by HSP90-dependent destabilization of unfolded protein response signaling. These data establish a role for HSP90 in the cellular transcriptional response to ER stress and demonstrate that chaperone systems on both sides of the ER membrane serve to integrate this signal transduction cascade.

Activating Transcription Factor 6↗
PubMed2002

Heat-shock protein 90 inhibitors as novel cancer chemotherapeutic agents.

Heat-shock protein 90 (Hsp90) is a molecular chaperone whose association is required for the stability and function of multiple mutated, chimeric and overexpressed signalling proteins that promote cancer cell growth and/or survival. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, Akt, HER2/Neu (ErbB2) and hypoxia inducible factor-1alpha (HIF-1alpha). Through specific interaction with a single molecular target, Hsp90 inhibitors cause the destabilisation and eventual degradation of Hsp90 client proteins, and they have shown promising antitumour activity in preclinical model systems. One Hsp90 inhibitor, 17-allylamino-geldanamycin (17-AAG), is currently in Phase I clinical trials. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple signalling pathways on which cancer cells depend for growth and survival. Further, because of the unique effect that Hsp90 inhibition has on cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent.

Journal Article↗
PubMed2002

Heat shock protein 90 is a rational molecular target in breast cancer.

Heat shock protein 90 is a molecular chaperone whose association is required for stability and function of multiple mutated, chimeric, and over-expressed signaling proteins that promote cancer cell growth and/or survival. Hsp90 client proteins important in breast cancer include the estrogen receptor, the serine-threonine kinases Raf-1 and Akt, the receptor tyrosine kinase ErbB2/Neu, and the hypoxia inducible transcription factor HIF-1alpha. Hsp90 small molecule inhibitors, by interacting specifically with a single molecular target, thus promote the destabilization and eventual degradation of multiple cancer cell survival and growth promoting proteins, and these inhibitors have shown promising anti-tumor activity in preclinical breast cancer model systems. One Hsp90 inhibitor, 17-AAG, is currently in Phase I clinical trial. Because of their unique ability to inhibit multiple survival pathways utilized by cancer cells, combination of Hsp90 inhibitors with standard chemotherapeutic agents may dramatically increase in vivo efficacy.

Journal Article↗
PubMed1999

Hsp90 as an anti-cancer target.

Heat shock protein 90 is one of the most abundant cellular proteins. Although its functions are still being characterized, it appears to serve as a chaperone for a growing list of cell signaling proteins, including many tyrosine and serine/threonine kinases, involved in cell proliferation and/or survival. The recent discovery of natural products which are able to inhibit Hsp90 function have allowed for both identification of its client proteins and for a better understanding of its role in their activity. Accumulating data have suggested that targeting Hsp90 in cancer cells may be of clinical benefit. Copyright 1999 Harcourt Publishers Ltd.

Journal Article↗
PubMedDate not supplied

Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry.

The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.

Amino Acid Sequence↗