Association with HSP90 Inhibits Cbl-Mediated Down-regulation of Mutant Epidermal Growth Factor Receptors

Abstract

Activating mutations in the epidermal growth factor receptor (EGFR), localized in the activation loop within the kinase domain, have been discovered in non-small cell lung cancers (NSCLC). Most of these mutants are exquisitely sensitive to EGFR tyrosine kinase inhibitors, suggesting that they generate receptor dependence in the cancers that express them. 32D cells stably expressing EGFR-L861Q and EGFR-L858R but not wild-type EGFR exhibited ligand-independent receptor phosphorylation and viability. Ligand-induced receptor downregulation (LIRD) was impaired in mutant-expressing cells. The EGFR mutants were constitutively associated with the E3 ubiquitin ligase Cbl but did not associate with the adaptor protein CIN85 on the addition of ligand. Inhibition of HSP90 activity with geldanamycin restored Cbl function as indicated by receptor ubiquitination and LIRD. These results suggest that EGFR mutants form defective endocytic complexes. In addition, HSP90 plays a role in maintaining the functional conformation of EGFR mutants and protecting activated

High-Resolution Mapping of Structural Mutations in Prostate Cancer with Single Nucleotide Polymorphism Arrays

“…Consistent with our findings in 293T cells and astrocytes, all examined EGFR ectodomain mutants showed increased tyrosine phosphorylation under serum-starved conditions and were responsive to exogenous EGF ( Figure 3C). We also noted that EGF stimulation led to a more pronounced drop of EGFR levels in Ba/F3 cells expressing wildtype EGFR than in subclones expressing EGFR ectodomain mutants (Figure 3C), reminiscent of the impaired ligand-induced receptor downregulation reported for selected EGFR kinase domain mutants [33].…”

“…We have found GLAD (31), which identifies segments with a constant copy number and averages the signal intensities across all loci in each segment, provides the most accurate results in a reasonable amount of computational time (data not shown). Several alternative software packages (dChipSNP, CNAT, CNAG, GIM) (22, 23, 32,33) also exist to convert probe-level data into overall SNP-specific signal intensities. Preliminary results seem to point to CNAG as producing the most optimal signal-to-noise ratios (data not shown).…”

“…To explore the biochemical basis for the gain of function observed with EGFR ectodomain mutants, we first examined the basal catalytic activity of A289V-EGFR in transiently transfected 293T cells using EGFR autophosphorylation as a readout for receptor activation. We also expressed selected EGFR mutants (R108K, T263P, A289V, G598V, L861Q) in murine hematopoietic cells (Ba/F3 cells) which do not express any EGF receptor family members [20] but otherwise retain functional properties of the EGF-signaling pathway [30,31] [32] [33]. Consistent with our findings in 293T cells and astrocytes, all examined EGFR ectodomain mutants showed increased tyrosine phosphorylation under serum-starved conditions and were responsive to exogenous EGF ( Figure 3C).…”

“…More recently, algorithms have been developed to reduce noise levels introduced by variations in conditions between experiments. These include Copy Number Analyzer for GeneChip (CNAG) [33 ], Genomic Imbalance Map (GIM) [34 ], and Copy Number Analysis with Regression And Tree (CARAT) [35 ]. All of these consider signal intensities at an SNP locus to be depen dent not only on the underlying copy number of the sample, but also take into account how well restriction digested fragments of varying lengths and GC contents amplify, under varying experimental conditions, during DNA preparation for hybridization to the array.…”

Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling

“…This leads to recruitment of several signaling molecules, but c-Cbl is excluded from the complex (Levkowitz et al, 1996;Graus Porta et al, 1997;Muthuswamy et al, 1999;Worthylake et al, 1999), probably because HER2 cannot trans-phosphorylate tyrosine 1113 of EGFR (Muthuswamy et al, 1999), and therefore ubiquitinylation and downregulation of mutant forms of EGFR are defective. According to a recently proposed alternative model, heat shock protein 90 uncouples mutant forms of EGFR from c-Cbl (Yang et al, 2006). Also relevant to our model is the ability of EGFR mutants to recruit another family member, namely ErbB-3 (Engelman et al, 2005), a kinasedefective receptor whose downregulation is defective .…”