cyclin-d1 and geldanamycin

Somatic mutations in the kinase domain of the epidermal growth factor receptor (EGFR), including L858R and exon 19 deletions, underlie responsiveness to gefitinib and erlotinib in non-small cell lung cancer (NSCLC). Acquired resistance to these tyrosine kinase inhibitors is in some cases mediated by a second mutation, T790M. Ansamycin antibiotics, such as geldanamycin, potently inhibit heat shock protein 90 (Hsp90), promoting ubiquitin-mediated degradation of oncogenic kinases that require the chaperone for proper conformational folding. Here, we show that L858R and deletion mutant EGFR proteins found in NSCLC interact with the chaperone and are sensitive to degradation following Hsp90 inhibition. In NIH/3T3 cells expressing either wild-type or mutant EGFR, diminution of expression of both L858R and EGFR delL747-S752, P753S occurred following exposure to 50 nmol/L geldanamycin over 24 hours, whereas partial diminution of wild-type EGFR required a minimum of 200 nmol/L drug. In time course experiments, mutant EGFR expression was depleted after only 4 hours of exposure to 1 micromol/L geldanamycin, whereas diminution of wild-type EGFR was less substantial and seen only following 12 hours. Similarly, EGFR proteins in NSCLC cell lines harboring EGFR mutations, including NCI-H1650, NCI-H3255, and NCI-H1975, were also more sensitive to geldanamycin-induced degradation compared with the protein in wild-type cells. Exposure of EGFR-mutant cell lines to geldanamycin induced marked depletion of phospho-Akt and cyclin D1 as well as apoptosis. These data suggest mutational activation of EGFR is associated with dependence on Hsp90 for stability and that Hsp90 inhibition may represent a novel strategy for the treatment of EGFR-mutant NSCLC.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Benzoquinones; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin D1; Cysteine Proteinase Inhibitors; ErbB Receptors; Gene Deletion; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Lung Neoplasms; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NIH 3T3 Cells; Phosphorylation; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinones

Hyperplasia of mesangial cells (MCs) is a frequent finding in glomerulonephritis. Heat shock protein 90 (HSP90) is a major cellular chaperone that assists protein folding under physiological and stress conditions.. To identify genes that are potentially involved in the pathogenesis of glomerulonephritis, we analyzed glomerular gene expression in mesangioproliferative rat anti-Thy1.1 nephritis by representational difference analysis (RDA). Expression of HSP90beta in anti-Thy1.1 nephritis was studied by Northern and Western blot analyses and immunohistochemistry. In cultured rat MCs, the requirement of HSP90 for mitogenic signaling steps and MC replication was studied by incubation with the specific HSP90 inhibitor geldanamycin.. By RDA, a cDNA fragment homologous to HSP90beta was identified. Glomerular mRNA and protein expression of HSP90beta was markedly and transiently up-regulated during the course of anti-Thy1.1 nephritis, with a maximum at day 6, coinciding with the peak of MC proliferation. By immunohistochemistry, HSP90beta expression in normal glomeruli was detected in podocytes. However, in anti-Thy1.1 nephritis, glomerular HSP90beta protein expression was strongly and transiently increased in mesangial localization. In vitro, mitogenic stimulation of rat MCs led to the induction of HSP90beta mRNA and protein. Incubation of MCs with geldanamycin dose-dependently inhibited DNA synthesis and replication. Moreover, geldanamycin interfered with mitogen-induced phosphorylation of extracellular signal-regulated kinase and transcription of c-fos and Egr-1, but not with transactivation of STAT1 transcription factor. Cell cycle analysis of serum-stimulated MCs revealed that geldanamycin inhibited kinase activity of cyclin D1/CDK4 complexes and blocked progression in the G0/G1 phase and at the S/G2 phase transition.. The up-regulation of HSP90beta in anti-Thy1.1 nephritis may reflect its functional involvement in phenotypical alterations of MCs in mesangioproliferative glomerulonephritis. Our in vitro studies indicate that HSP90 governs the capacity of MCs to respond to proliferative stimuli by regulating critical mitogenic signaling steps necessary for G1 entry and S-phase progression.

Topics: Animals; Antibodies; Benzoquinones; Blood Proteins; Cell Division; Cells, Cultured; Cicatrix; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; G1 Phase; G2 Phase; Gene Expression; Glomerular Mesangium; Glomerulonephritis; HSP90 Heat-Shock Proteins; Hyperplasia; Lactams, Macrocyclic; Male; Mitogen-Activated Protein Kinases; Protein Folding; Proto-Oncogene Proteins; Quinones; Rats; Rats, Sprague-Dawley; Resting Phase, Cell Cycle; RNA, Messenger; S Phase; STAT1 Transcription Factor; Thy-1 Antigens; Trans-Activators