Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition

The ErbB receptor family is implicated in the malignant transformation of several tumor types and is overexpressed frequently in breast, ovarian, and other tumors. The mechanism by which CI-1033 and gemcitabine, either singly or in combination, kill tumor cells was examined in two breast lines, MDA-MB-453 and BT474; both overexpress the ErbB-2 receptor. CI-1033, a potent inhibitor of the ErbB family of receptor tyrosine kinases, reduced levels of activated Akt in MDA-MB-453 cells. This effect alone, however, did not induce apoptosis in these cells. Gemcitabine treatment resulted in a moderate increase in the percentage of apoptotic cells that was accompanied by activation of p38 and MAPK (ERK1/2). CI-1033 given 24 h after gemcitabine produced a significant increase in the apoptotic fraction over treatment with either drug alone. During the combined treatment p38 remained activated, whereas Akt and activated MAPK were suppressed. Substitution of CI-1033 with the phosphatidylinositol 3-kinase inhibitor LY294002 and the MAPK/ERK kinase inhibitor PD 098059 in combination with gemcitabine produced the same results as the combination of CI-1033 and gemcitabine. p38 suppression by SB203580 prevented the enhanced cell kill by CI-1033. In contrast to MDA-MB-453, BT474 cells exhibited activated p38 under unstressed conditions as well as activated Akt and MAPK. Treatment of BT474 cells with CI-1033 inhibited both the phosphorylation of Akt and MAPK and resulted in a 47% apoptotic fraction. Gemcitabine did not cause apoptosis in the BT474 cells. These data indicate that suppression of Akt and MAPK in the presence of activated p38 results in cell death and a possible mechanism for the enhanced apoptosis produced by the combination of CI-1033 and gemcitabine in MDA-MB-453 cells. Furthermore, tumors that depend on ErbB receptor signaling for survival and exhibit activated p38 in the basal state may be susceptible to apoptosis by CI-1033 as a single agent.     

Geldanamycin induces ErbB-2 degradation by proteolytic fragmentation

Exposure of carcinoma cell lines to the antibiotic geldanamycin induces the degradation of ErbB-2, a co-receptor tyrosine kinase that is frequently overexpressed in certain tumors. Using ErbB-2 mutants expressed as chimeric receptors or green fluorescent protein fusion proteins, we report that the kinase domain of ErbB-2 is essential for geldanamycin-induced degradation. The kinase domain of the related epidermal growth factor receptor was not sensitive to this drug. The data further indicate mechanistic aspects of ErbB-2 degradation by geldanamycin. The data show that exposure to the drug induces at least one cleavage within the cytoplasmic domain of ErbB-2 producing a 135-kDa fragment and a 23-kDa fragment. The latter represents the carboxyl-terminal domain of ErbB-2, whereas the former represents the ectodomain and part of the cytoplasmic domain. Degradation of the carboxyl-terminal fragment is prevented by proteasome inhibitors, whereas degradation of the membrane-anchored 135-kDa ErbB-2 fragment is blocked by inhibitors of the endocytosis-dependent degradation pathway. Confocal microscopy studies confirm a geldanamycin-induced localization of ErbB-2 on intracellular vesicles.     

Comparison of the EGFR resistance mutation profiles generated by EGFR-targeted tyrosine kinase inhibitors and the impact of drug combinations

Recent clinical data indicates that the emergence of mutant drug-resistant kinase alleles may be particularly relevant for targeted kinase inhibitors. In order to explore how different classes of targeted therapies impact upon resistance mutations, we performed EGFR (epidermal-growth-factor receptor) resistance mutation screens with erlotinib, lapatinib and CI-1033. Distinct mutation spectra were generated with each inhibitor and were reflective of their respective mechanisms of action. Lapatinib yielded the widest variety of mutations, whereas mutational variability was lower in the erlotinib and CI-1033 screens. Lapatinib was uniquely sensitive to mutations of residues located deep within the selectivity pocket, whereas mutation of either Gly(796) or Cys(797) resulted in a dramatic loss of CI-1033 potency. The clinically observed T790M mutation was common to all inhibitors, but occurred with varying frequencies. Importantly, the presence of C797S with T790M in the same EGFR allele conferred complete resistance to erlotinib, lapatinib and CI-1033. The combination of erlotinib and CI-1033 effectively reduced the number of drug-resistant clones, suggesting a possible clinical strategy to overcome drug resistance. Interestingly, our results also indicate that co-expression of ErbB2 (v-erb-b2 erythroblastic leukaemia viral oncogene homologue 2) has an impact upon the EGFR resistance mutations obtained, suggesting that ErbB2 may play an active role in the acquisition of drug-resistant mutations.     

Ubiquitination and downregulation of ErbB2 and estrogen receptor-alpha by kinase inhibitor MP-412 in human breast cancer cells

ErbB2 has been proven to be an important target for breast cancer therapy. MP-412 is a dual ErbB2 and epidermal growth factor receptor tyrosine kinase inhibitor belonging to an irreversible-type anilinoquinazoline derivative. We demonstrate herein that along with the kinase inhibition, MP-412 has the ability to induce ubiquitination, internalization, and degradation of ErbB2 in several human breast cancer cell lines at concentrations relatively higher than those required for kinase inhibition. Another irreversible inhibitor, CI-1033, showed similar activity, while the reversible compounds were ineffective, suggesting a crucial role of covalent bonding functionality in these effects. In MCF7 cells, MP-412 depleted not only ErbB2 but also estrogen receptor (ER)-α, and to some extent, affected Raf-1, while MP-412 activated Hsp70 expression. Moreover, we observed that MP-412 increased immunocomplexing of Hsp70 with ErbB2 and ER-α, with simultaneous induction of ubiquitination of these client proteins. Furthermore, in combination with proteasome inhibitor, MP-412 resulted in the noticeable accumulation of ErbB2 and ER-α in the detergent insoluble fraction of cell lysates. These results suggest that MP-412 acts as an inhibitor of Hsp90 function, whereas MP-412 did not bind directly to ATP-binding site of Hsp90, unlike geldanamycin. We also found that new protein synthesis was involved in the activity of MP-412 on Hsp90 modulation. Since downregulation of ErbB2 and ER-α by accelerating the ubiquitin-proteolysis system will become an attractive approach for breast cancer therapy, we expect MP-412 to be a lead compound for the drug design and the development of such agents.     

Induction of apoptosis by ionizing radiation and CI-1033 in HuCCT-1 cells

CI-1033 is a quinazoline-based HER family tyrosine kinase inhibitor that is currently being evaluated as a potential anticancer agent. The present study examines the molecular mechanism by which CI-1033 induces apoptosis either as a single agent or in combination with radiation. Although CI-1033 alone did not induce apoptosis, the simultaneous exposure of cells to CI-1033 and radiation induced significant levels of apoptosis. The sequential treatment of cells with CI-1033 followed by radiation induced an even greater effect with 62.6% of cells undergoing apoptosis but this enhanced effect was not seen if cells were treated first with radiation and then CI-1033. The combination treatment induces apoptosis of HuCCT-1 via upregulation of FasL and Bid cleavage. These data suggest that modulation of the Fas-FasL pathway and activation of Bid could be useful for increasing the anti-tumor effect of CI-1033 in this type of cancer.     

Hsp90 restrains ErbB-2/HER2 signalling by limiting heterodimer formation

ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes.     

The achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated cochaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.     

The Achilles Heel of ErbB-2/HER2: Regulation by the Hsp90 Chaperone Machine and Potential for Pharmacological Intervention

Signal transduction mediated by ErbB/HER receptor tyrosine kinases is crucial for the development and maintenance of epithelial tissues, and aberrant signaling is frequently associated with malignancies of epithelial origin. This review focuses on the roles played by the Hsp90 chaperone machinery in the regulation of signaling through the ErbB/HER network, and discusses potential therapeutic strategies that disrupt chaperone functions. Hsp90 and its associated co-chaperones regulate ErbB signal transduction through multiple mechanisms. The chaperone system controls the stability of the nascent forms of both ErbB-1 (EGF-receptor) and ErbB-2/HER2, while regulation of the mature form is restricted to ErbB-2. Regulation by the Hsp90 complex extends to downstream effectors of ErbB signaling, namely Raf-1, Pdk-1 and Akt/PKB. Disrupting the function of Hsp90 results in the degradation of both the receptors and their effectors, thereby inhibiting tumor cell growth. The importance of an Hsp90-recognition motif located within the kinase domain of ErbB-2 is discussed, as well as a direct role for Hsp90 in regulating tyrosine kinase activity. In light of recent observations, we emphasize the ability of specific tyrosine kinase inhibitors to selectively target ErbB-2 to the chaperone-mediated degradation pathway. ErbB-specific drugs are already used to treat cancers, and clinical trials are underway for additional compounds that intercept ErbB signaling, including drugs that target Hsp90. Hence, the dependence of ErbB-2 upon Hsp90 reveals an Achilles heel, which opens a window of opportunity for combating cancers driven by the ErbB/HER signaling network.     

G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.     

Clinical stage EGFR inhibitors irreversibly alkylate Bmx kinase

Irreversible HER/erbB inhibitors selectively inhibit HER-family kinases by targeting a unique cysteine residue located within the ATP-binding pocket. Sequence alignment reveals that this rare cysteine is also present in ten other protein kinases including all five Tec-family members. We demonstrate that the Tec-family kinase Bmx is potently inhibited by irreversible modification at Cys496 by clinical stage EGFR inhibitors such as CI-1033. This cross-reactivity may have significant clinical implications.     

The 90-kDa heat shock protein stabilizes the polysomal ribonuclease 1 mRNA endonuclease to degradation by the 26S proteasome

The polysomal ribonuclease 1 (PMR1) mRNA endonuclease forms a selective complex with its translating substrate mRNAs where it is activated to initiate mRNA decay. Previous work showed tyrosine phosphorylation is required for PMR1 targeting to this polysome-bound complex, and it identified c-Src as the responsible kinase. c-Src phosphorylation occurs in a distinct complex, and the current study shows that 90-kDa heat shock protein (Hsp90) is also recovered with PMR1 and c-Src. Hsp90 binding to PMR1 is inhibited by geldanamycin, and geldanamycin stabilizes substrate mRNA to PMR1-mediated decay. PMR1 is inherently unstable and geldanamycin causes PMR1 to rapidly disappear in a process that is catalyzed by the 26S proteasome. We present a model where Hsp90 interacts transiently to stabilize PMR1 in a manner similar to its interaction with c-Src, thus facilitating the tyrosine phosphorylation and targeting of PMR1 to polysomes.     

A multivariate model of ErbB network composition predicts ovarian cancer cell response to canertinib

Identifying the optimal treatment strategy for cancer is an important challenge, particularly for complex diseases like epithelial ovarian cancer (EOC) that are prone to recurrence. In this study we developed a quantitative, multivariate model to predict the extent of ovarian cancer cell death following treatment with an ErbB inhibitor (canertinib, CI-1033). A partial least squares regression model related the levels of ErbB receptors and ligands at the time of treatment to sensitivity to CI-1033. In this way, the model mimics the clinical problem by incorporating only information that would be available at the time of drug treatment. The full model was able to fit the training set data and was predictive. Model analysis demonstrated the importance of including both ligand and receptor levels in this approach, consistent with reports of the role of ErbB autocrine loops in EOC. A reduced multi-protein model was able to predict CI-1033 sensitivity of six distinct EOC cell lines derived from the three subtypes of EOC, suggesting that quantitatively characterizing the ErbB network could be used to broadly predict EOC response to CI-1033. Ultimately, this systems biology approach examining multiple proteins has the potential to uncover multivariate functions to identify subsets of tumors that are most likely to respond to a targeted therapy.     

Biochemical Assay-Based Selectivity Profiling of Clinically Relevant Kinase Inhibitors on Mutant Forms of EGF Receptor

Gefitinib and erlotinib are potent EGFR tyrosine kinase inhibitors (potentially) useful for the treatment of non-small-cell lung cancer (NSCLC). Clinical responses, however, in NSCLC patients have been linked to the presence of certain activating mutations of EGFR. We used an ELISA-based biochemical assay to confirm the selective inhibitory efficacy of gefitinib and erlotinib on the activated mutant receptor. Our results are in line with the clinical observations providing evidence for the predictive power of the kinase assay. Four additional compounds were also investigated: CI-1033 and EKB-569 had dramatic inhibitory effects on all EGFR forms, whereas PD153035 and AG1478 were active on wild-type and activating mutant protein. In docking simulations with wild-type EGFR, our inhibitory data are in good agreement with the binding scores. These data confirm that anilinoquinazolines are good starting structures for the next generation of selective drugs against mutant EGFR, whereas CI-1033 and EKB-569 may represent advances for patients with both wild-type and anilinoquinazoline-resistant mutant tumors.     

Irreversible pan-ErbB tyrosine kinase inhibitor CI-1033 induces caspase-independent apoptosis in colorectal cancer DiFi cell line

The epidermal growth factor receptor (EGFR) is overexpressed in the majority of colorectal carcinomas and represents a target for therapeutic interventions with signal transduction inhibitors. We investigated the ability of CI-1033 to induce apoptosis and inhibition of proliferation in the colorectal cancer cell lines DiFi and Caco-2, which both express high levels of EGFR. While in Caco-2 cells CI-1033 treatment at a concentration 0.1 μ M for 72 hours demonstrated only antiproliferative (53.7 ± 4.3%) but no pro-apoptotic effects, treatment of DiFi cells resulted in a reduced proliferation rate (31.4 ± 3.1%) and in apoptosis (44.2 ± 8.9%). In order to define proteins involved in the regulation of apoptosis, we aimed to determine differences in the proteome profile of both cell lines before and after treatment with CI-1033. Cellular proteins were analyzed by 2-D gel electrophoresis followed by computational image analysis and mass spectrometry. Our data show that DiFi cells differ from Caco-2 cells in nine significantly upregulated proteins, and their potential role in apoptosis is described. We demonstrate that induction of apoptosis was triggered via caspase-independent pathways. Overexpression of leukocyte elastase inhibitor (LEI) and translocation of cathepsin D to the cytosol accompanied by upregulation of other defined proteins resulted in Bax-independent AIF translocation from mitochondria into the nucleus and apoptosis. Definition of these proteins can pave the way for functional studies and contribute to a better understanding of the effects of CI-1033 and the pathways of caspase-independent cell death.     

Biochemical assay-based selectivity profiling of clinically relevant kinase inhibitors on mutant forms of EGF receptor

Gefitinib and erlotinib are potent EGFR tyrosine kinase inhibitors (potentially) useful for the treatment of non-small-cell lung cancer (NSCLC). Clinical responses, however, in NSCLC patients have been linked to the presence of certain activating mutations of EGFR. We used an ELISA-based biochemical assay to confirm the selective inhibitory efficacy of gefitinib and erlotinib on the activated mutant receptor. Our results are in line with the clinical observations providing evidence for the predictive power of the kinase assay. Four additional compounds were also investigated: CI-1033 and EKB-569 had dramatic inhibitory effects on all EGFR forms, whereas PD153035 and AG1478 were active on wild-type and activating mutant protein. In docking simulations with wild-type EGFR, our inhibitory data are in good agreement with the binding scores. These data confirm that anilinoquinazolines are good starting structures for the next generation of selective drugs against mutant EGFR, whereas CI-1033 and EKB-569 may represent advances for patients with both wild-type and anilinoquinazoline-resistant mutant tumors.     

UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition

UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.     

Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).     

Constitutive tyrosine phosphorylation of ErbB-2 via Jak2 by autocrine secretion of prolactin in human breast cancer

Overexpression of the oncogene for ErbB-2 is an unfavorable prognostic marker in human breast cancer. Its oncogenic potential appears to depend on the state of tyrosine phosphorylation. However, the mechanisms by which ErbB-2 is constitutively tyrosine-phosphorylated in human breast cancer are poorly understood. We now show that human breast carcinoma samples with ErbB-2 overexpression have higher proliferative and metastatic activity in the presence of autocrine secretion of prolactin (PRL). By using a neutralizing antibody or dominant negative (DN) strategies or specific inhibitors, we also show that activation of Janus kinase Jak2 by autocrine secretion of PRL is one of the significant components of constitutive tyrosine phosphorylation of ErbB-2, its association with Grb2 and activation of mitogen-activated protein (MAP) kinase in human breast cancer cell lines that overexpress ErbB-2. Furthermore, the neutralizing anti-PRL antibody or erbB-2 antisense oligonucleotide or DN Jak2 or Jak2 inhibitor or DNRas or MAP kinase kinase inhibitor inhibits the proliferation of both untreated and PRL-treated cells. Our results indicate that autocrine secretion of PRL stimulates tyrosine phosphorylation of ErbB-2 by Jak2, provides docking sites for Grb2 and stimulates Ras-MAP kinase cascade, thereby causing unrestricted cellular proliferation. The identification of this novel cross-talk between ErbB-2 and the autocrine growth stimulatory loop for PRL may provide new targets for therapeutic and preventive intervention of human breast cancer.     

Evaluation of active recombinant catalytic domain of human ErbB-2 tyrosine kinase, and suppression of activity by a naturally derived inhibitor, ZH-4B

Human cancers frequently express high levels of ErbB-2 tyrosine kinase, which is associated with aggressive tumor behavior and poor prognosis. ErbB-2 is thus a promising target for cancer therapy. Here we express the catalytic domain of ErbB-2 as a soluble active kinase, and investigate the correlations between its activity and kinase concentration, ATP concentration, substrate concentration and divalent cation type. A simple and effective screening model is established to identify and evaluate potential inhibitors of ErbB-2 kinase. ZH-4B, a naturally derived small molecule compound that potently inhibits ErbB-2 kinase activity with an IC50 value of 2.45+/-0.56 microM, is identified. In SK-OV-3 human ovarian cancer cells and SK-BR-3 human breast carcinoma cells, ZH-4B blocks epidermal growth factor (EGF)-induced phosphorylation of ErbB-2 in a dose-dependent manner. Our data collectively indicate that ZH-4B is a potential novel anti-cancer agent that deserves further investigation.