HER2/ErbB2 receptor signaling in rat and human prolactinoma cells: strategy for targeted prolactinoma therapy

Dopamine agonist resistance or intolerance is encountered in approximately 20% of prolactinoma patients. Because human epidermal growth factor receptor 2 (HER2)/ErbB2 is overexpressed in prolactinomas and ErbB receptor ligands regulate prolactin (PRL) gene expression, we tested the role of HER2/ErbB2 in prolactinoma hormone regulation and adenoma cell proliferation to assess the rationale for targeting this receptor for prolactinoma therapy. As we showed prolactinoma HER2 overexpression, we generated constitutively active HER2-stable GH3 cell transfectants (HER2CA). PRL mRNA levels were induced approximately 250-fold and PRL secretion was enhanced 100-fold in HER2CA cells, which also exhibited increased proliferation. Lapatinib, a dual tyrosine kinase inhibitor (TKI) of both epidermal growth factor receptor (EGFR)/ErbB1 and HER2, blocked receptor signaling, and suppressed PRL expression more than gefitinib, a TKI of EGFR/ErbB1. Lapatinib also suppressed colony formation in soft agar more than gefitinib. Oral lapatinib treatment caused tumor shrinkage and serum PRL suppression both in HER2CA transfectant-inoculated Wistar-Furth rats and in estrogen-induced Fischer344 rat prolactinomas. In cultured human cells derived from resected prolactinoma tissue, lapatinib suppressed both PRL mRNA expression and secretion. These results demonstrate that prolactinoma HER2 potently induces PRL and regulates experimental prolactinoma cell proliferation. Because pituitary HER2 signaling is abrogated by TKIs, this receptor could be an effective target for prolactinoma therapy.     

Inhibition of proliferation and induction of apoptosis in breast cancer cells by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor ZD1839 ('Iressa') is independent of EGFR expression level

High expression of the epidermal growth factor receptor (EGFR) in breast carcinoma confers a growth advantage to the tumor cells. The EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 ('Iressa') has clinical activity in a wide range of tumor types, although the mechanism(s) by which it exerts its antitumor activity effects remain unclear. We analyzed the ability of ZD1839 to induce apoptosis and/or inhibition of proliferation in breast carcinoma cell lines, as well any association between this ability and the downregulation activity of MAPK and Akt, two recently proposed markers of ZD1839 activity. Proliferation, survival, and activation of Akt and MAPK were evaluated in six human breast cancer cell lines expressing various levels of EGFR and HER2 and exposed to ZD1839. EGFR and HER2 expression levels were determined using specific monoclonal antibodies and FACS analysis. The effects of ZD1839 were independent of EGFR expression levels, but were influenced by high HER2 expression. ZD1839 significantly reduced the rate of [3H]-thymidine incorporation in the four sensitive cell lines, while apoptosis was also induced in two of these cell lines. No correlation was found between the cytostatic or cytotoxic effects of ZD1839 and its ability to downregulate MAPK and Akt activity in the tumor cell lines. Our data suggest that the antitumor activity of ZD1839 is due to a cytostatic effect, and involves apoptosis induction in a subset of sensitive cells only, and that neither MAPK nor Akt is a reliable marker of ZD1839 activity.     

ErbB2 and ErbB3 do not quantitatively modulate ligand-induced ErbB4 tyrosine phosphorylation

The ErbB family of receptor tyrosine kinases consists of four members: the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER4. ErbB2 is an "orphan" for which there is no naturally occurring, soluble ligand. ErbB3 lacks tyrosine kinase activity. Thus, we hypothesized that ErbB2 enhances ligand-induced ErbB family receptor signalling through mass action. In contrast, we hypothesized that ErbB3 reduces ligand-induced ErbB family receptor signalling by forming receptor heterodimers that cannot undergo bidirectional cross-phosphorylation. We tested these hypotheses using three cell lines that express equal levels of ErbB4. One expresses ErbB4 alone, the second expresses ErbB2 and ErbB4, and the third expresses ErbB3 and ErbB4. We treated the cells with the ErbB4 ligands betacellulin (BTC) and neuregulin1beta (NRG1 beta) and assayed ErbB4 tyrosine phosphorylation. ErbB2 and ErbB3 do not affect the amount of ligand-induced ErbB4 tyrosine phosphorylation. We will discuss these findings within the context of a model for ErbB receptor signalling.     

Establishment and characterization of a novel ovarian serous adenocarcinoma cell line, TU-OS-4, that overexpresses EGFR and HER2

A new line of human ovarian serous adenocarcinoma cells, TU-OS-4, was established and characterized. The cells showed a short, spindle-shaped morphology and grew in monolayers without contact inhibition while forming an arrangement resembling a jigsaw puzzle. Chromosome numbers ranged from 55 to 73. The proliferation rate was lower than other serous adenocarcinoma cell lines tested (KF, SHIN-3, and SK-OV-3), and the doubling time was 53.3 h. Western blot analysis showed that TU-OS-4 cells overexpressed epidermal growth factor receptor, human epidermal growth factor receptor (HER) 2, and phosphorylated HER2 protein. The IC₅₀ values to cisplatin, paclitaxel, and lapatinib were 25.8 μM, 686 nM, and 183 nM, respectively. Heterotransplantation in nude mice reflected the original tumor of the cells. These results suggested that this cell line would be useful to study chemoresistant mechanisms and contribute to establishing novel treatment strategies for patients with ovarian cancer.     

Lapatinib,a Dual EGFR and HER2 Tyrosine Kinase Inhibitor,Downregulates Thymidylate Synthase by Inhibiting the Nuclear Translocation of EGFR and HER2

Background

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated.

Methodology and Principal Findings

In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib.

Conclusions and Significance

These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine.     

Structural analysis of the mechanism of inhibition and allosteric activation of the kinase domain of HER2 protein

Aberrant signaling of ErbB family members human epidermal growth factor 2 (HER2) and epidermal growth factor receptor (EGFR) is implicated in many human cancers, and HER2 expression is predictive of human disease recurrence and prognosis. Small molecule kinase inhibitors of EGFR and of both HER2 and EGFR have received approval for the treatment of cancer. We present the first high resolution crystal structure of the kinase domain of HER2 in complex with a selective inhibitor to understand protein activation, inhibition, and function at the molecular level. HER2 kinase domain crystallizes as a dimer and suggests evidence for an allosteric mechanism of activation comparable with previously reported activation mechanisms for EGFR and HER4. A unique Gly-rich region in HER2 following the α-helix C is responsible for increased conformational flexibility within the active site and could explain the low intrinsic catalytic activity previously reported for HER2. In addition, we solved the crystal structure of the kinase domain of EGFR in complex with a HER2/EGFR dual inhibitor (TAK-285). Comparison with previously reported inactive and active EGFR kinase domain structures gave insight into the mechanism of HER2 and EGFR inhibition and may help guide the design and development of new cancer drugs with improved potency and selectivity.     

Evaluation of the Antitumor Activity of Dacomitinib in Models of Human Bladder Cancer

Members of the human epidermal growth factor receptor (HER) family play a significant role in bladder cancer progression and may underlie the development of chemotherapy resistance. Dacomitinib is an irreversible tyrosine kinase inhibitor with structural specificity for the catalytic domains of epidermal growth factor receptor (EGFR), HER2 and HER4 that has exhibited vigorous efficacy against other solid tumors. We evaluated the antitumor activity of dacomitinib in human bladder cancer cell lines expressing varying levels of HER family receptors. These cell lines also were established as bladder cancer xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to assess dacomitinib activity in vivo. Significant cytotoxic and cytostatic effects were noted in cells expressing elevated levels of the dacomitinib target receptors with apoptosis and cell cycle arrest being the predominant mechanisms of antitumor activity. Cells expressing lower levels of HER receptors were much less sensitive to dacomitinib. Interestingly, dacomitinib was more active than either trastuzumab or cetuximab in vitro, and exhibited increased growth inhibition of bladder tumor xenografts compared with lapatinib. Pharmacodynamic effects of dacomitinib included decreased E-cadherin (E-cad) expression, reduction of EGFR and extracellular signal-regulated kinase (ERK) phosphorylation and reduced mitotic count. Dacomitinib also inhibited tumor growth in a chemotherapy-resistant xenograft and, when combined with chemotherapy in a sensitive xenograft, exhibited superior antitumor effects compared with individual treatments. Evaluation in xenograft-bearing mice revealed that this combination was broadly feasible and well tolerated. In conclusion, dacomitinib exhibited pronounced activity both as a single agent and when combined with chemotherapy in human bladder cancer models. Further investigation of dacomitinib in the preclinical and clinical trial settings is being pursued.     

Endothelin causes transactivation of the EGFR and HER2 in non-small cell lung cancer cells

Endothelin (ET)-1 is an important peptide in cancer progression stimulating cellular proliferation, tumor angiogenesis and metastasis. ET-1 binds with high affinity to the ET_A receptor (R) and ET_BR on cancer cells. High levels of tumor ET-1 and ET_AR are associated with poor survival of lung cancer patients. Here the effects of ET-1 on epidermal growth factor (EGF)R and HER2 transactivation were investigated using non-small cell lung cancer (NSCLC) cells. ET_AR mRNA was present in all 10 NSCLC cell lines examined. Addition of ET-1 to NCI-H838 or H1975 cells increased EGFR, HER2 and ERK tyrosine phosphorylation within 2 min. The increase in EGFR and HER2 transactivation caused by ET-1 addition to NSCLC cells was inhibited by lapatinib (EGFR and HER2 tyrosine kinase inhibitor (TKI)), gefitinib (EGFR TKI), ZD4054 or BQ-123 (ET_AR antagonist), GM6001 (matrix metalloprotease inhibitor), PP2 (Src inhibitor) or Tiron (superoxide scavenger). ET-1 addition to NSCLC cells increased cytosolic Ca²⁺ and reactive oxygen species. ET-1 increased NSCLC clonal growth, whereas BQ123, ZD4054, lapatinib or gefitinib inhibited proliferation. The results indicate that ET-1 may regulate NSCLC cellular proliferation in an EGFR- and HER2-dependent manner.     

Combined Inhibition of ErbB1/2 and Notch Receptors Effectively Targets Breast Ductal Carcinoma In Situ (DCIS) Stem/Progenitor Cell Activity Regardless of ErbB2 Status

Pathways involved in DCIS stem and progenitor signalling are poorly understood yet are critical to understand DCIS biology and to develop new therapies. Notch and ErbB1/2 receptor signalling cross talk has been demonstrated in invasive breast cancer, but their role in DCIS stem and progenitor cells has not been investigated. We have utilised 2 DCIS cell lines, MCF10DCIS.com (ErbB2-normal) and SUM225 (ErbB2-overexpressing) and 7 human primary DCIS samples were cultured in 3D matrigel and as mammospheres in the presence, absence or combination of the Notch inhibitor, DAPT, and ErbB1/2 inhibitors, lapatinib or gefitinib. Western blotting was applied to assess downstream signalling. In this study we demonstrate that DAPT reduced acini size and mammosphere formation in MCF10DCIS.com whereas there was no effect in SUM225. Lapatinb reduced acini size and mammosphere formation in SUM225, whereas mammosphere formation and Notch1 activity were increased in MCF10DCIS.com. Combined DAPT/lapatinib treatment was more effective at reducing acini size in both DCIS cell lines. Mammosphere formation in cell lines and human primary DCIS was reduced further by DAPT/lapatinib or DAPT/gefitinib regardless of ErbB2 receptor status. Our pre-clinical human models of DCIS demonstrate that Notch and ErbB1/2 both play a role in DCIS acini growth and stem cell activity. We report for the first time that cross talk between the two pathways in DCIS occurs regardless of ErbB2 receptor status and inhibition of Notch and ErbB1/2 was more efficacious than either alone. These data provide further understanding of DCIS biology and suggest treatment strategies combining Notch and ErbB1/2 inhibitors should be investigated regardless of ErbB2 receptor status.     

Constitutively active ErbB4 and ErbB2 mutants exhibit distinct biological activities.

ErbB4 is a member of the epidermal growth factor receptor(EGFR) family of tyrosine kinases, which includes EGFR/ErbB1, ErbB2/HER2/Neu, and ErbB3/HER3. These receptors play important roles both in normal development and in neoplasia. For example, deregulated signaling by ErbB1 and ErbB2 is observed in many human malignancies. In contrast, the roles that ErbB4 plays in tumorigenesis and normal biological processes have not been clearly defined. To identify the biological responses that are coupled to ErbB4, we have constructed three constitutively active ErbB4 mutants. Unlike a constitutively active ErbB2 mutant, the ErbB4 mutants are not coupled to increased cell proliferation, loss of contact inhibition, or anchorage independence in a rodent fibroblast cell line. This suggests that ErbB2 and ErbB4 may play distinct roles in tumorigenesis in vivo.     

Modeling the effects of HER/ErbB1-3 coexpression on receptor dimerization and biological response

The human epidermal growth factor receptor (HER/ErbB) system comprises the epidermal growth factor receptor (EGFR/HER1) and three other homologs, namely HERs 2-4. This receptor system plays a critical role in cell proliferation and differentiation and receptor overexpression has been associated with poor prognosis in cancers of the epithelium. Here, we examine the effect of coexpressing varying levels of HERs 1-3 on the receptor dimerization patterns using a detailed kinetic model for HER/ErbB dimerization and trafficking. Our results indicate that coexpression of EGFR with HER2 or HER3 biases signaling to the cell surface and retards signal downregulation. In addition, simultaneous coexpression of HERs 1-3 leads to an abundance of HER2-HER3 heterodimers, which are known to be potent inducers of cell growth and transformation. Our new approach to use parameter dependence analysis in experimental design reveals that measurements of HER3 phosphorylation and HER2 internalization ratio may prove to be especially useful for the estimation of critical model parameters. Further, we examine the effect of receptor dimerization patterns on biological response using a simple phenomenological model. Results indicate that coexpression of EGFR with HER2 and HER3 at low to moderate levels may enable cells to match the response of a high HER2 expresser.     

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.     

Simultaneous Inhibition of Estrogen Receptor and the HER2 Pathway in Breast Cancer: Effects of HER2 Abundance

The estrogen receptor (ER) pathway and the epidermal growth factor receptor (EGFR) pathway play pivotal roles in breast cancer progression. Targeted therapies able to intercept ER or signaling downstream to EGFR and its kin, HER2, are routinely used to treat distinct groups of breast cancer patients. However, patient responses are limited by resistance to endocrine therapy, which may be due to compensatory HER2/EGFR signaling. This raises the possibility that simultaneous interception of HER2 and ER may enhance therapeutic efficacy. To address the question, we treated breast cancer cells with both fulvestrant (ICI 182780), an ER antagonist with no agonist effects, and lapatinib, an orally available tyrosine kinase inhibitor specific to EGFR and HER2. Our results indicate that the combination of drugs is especially effective when applied to HER2-overexpressing, ER-positive cancer cells. Interestingly, fulvestrant activated the mitogen-activated protein kinase (MAPK) pathway of these cells, but complete inhibition of MAPK signaling was observed on cotreatment with lapatinib. Taken together, our observations reinforce the possibility that the effectiveness of combining anti-ER and anti-HER2/EGFR drugs may be especially effective on a relatively small subtype of HER2-overexpressing, ER-positive tumors of the breast.     

Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer

Epidermal Growth Factor Receptor (EGFR) is overexpressed on a number of human cancers, and often is indicative of a poor outcome. Treatment of EGFR/HER2 overexpressing cancers includes monoclonal antibody therapy (cetuximab/trastuzumab) either alone or in conjunction with other standard cancer therapies. While monoclonal antibody therapy has been proven to be efficacious in the treatment of EGFR/HER2 overexpressing tumors, drawbacks include the lack of long-lasting immunity and acquired resistance to monoclonal therapy. An alternative approach is to induce a polyclonal anti-EGFR/HER2 tumor antigen response by vaccine therapy. In this phase I/II open-label study, we examined anti-tumor immunity in companion dogs with spontaneous EGFR expressing tumors. Canine cancers represent an outbred population in which the initiation, progression of disease, mutations and growth factors closely resemble that of human cancers. Dogs with EGFR expressing tumors were immunized with a short peptide of the EGFR extracellular domain with sequence homology to HER2. Serial serum analyses demonstrated high titers of EGFR/HER2 binding antibodies with biological activity similar to that of cetuximab and trastuzumab. Canine antibodies bound both canine and human EGFR on tumor cell lines and tumor tissue. CD8 T cells and IgG deposition were evident in tumors from immunized dogs. The antibodies inhibited EGFR intracellular signaling and inhibited tumor growth in vitro. Additionally, we illustrate objective responses in reducing tumors at metastatic sites in host animals. The data support the approach of amplifying anti-tumor immunity that may be relevant in combination with other immune modifying therapies such as checkpoint inhibitors.     

Lysophospholipids transactivate HER2/neu (erbB-2) in human gastric cancer cells

The ligand-less receptor HER2/neu (erbB-2) has been proposed as a prognostic marker of gastric cancer that correlates with poor clinical outcome, indicating that HER2 signals play an important role in gastric cancer progression. This study demonstrated that two major natural lysophospholipids, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), induce rapid and transient phosphorylation of HER2 in two human gastric cancer cell lines, MKN28 and MKN74 cells. We also revealed that tyrosine phosphorylation of HER2 induced by both lysophospholipids was significantly attenuated by two inhibitors, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, AG1478, and a broad-spectrum matrix metalloproteinase inhibitor, GM6001. This suggests that the pathway of HER2 transactivation induced by these lysophospholipids is dependent on the proteolytically released EGFR ligands. Our results indicate that LPA and S1P act upstream of HER2 in gastric cancer cells, and thus may act as potent stimulators of gastric cancer.     

Model-Based Analysis of HER Activation in Cells Co-Expressing EGFR,HER2 and HER3

The HER/ErbB family of receptor tyrosine kinases drives critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation, and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panel of human mammary epithelial cells expressing varying levels of EGFR/HER1, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of human mammary epithelial cells lines with known HER expression levels in response to stimulations with ligands EGF and HRG. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1-HER1 and HER1-HER2 dimers, and not HER1-HER3 dimers, ii) HER1-HER2 and HER2-HER3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2-HER3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

Laser capture microdissection and protein microarray analysis of human non-small cell lung cancer: differential epidermal growth factor receptor (EGPR) phosphorylation events associated with mutated EGFR compared with wild type

Little is known about lung carcinoma epidermal growth factor (EGF) kinase pathway signaling within the context of the tissue microenvironment. We quantitatively profiled the phosphorylation and abundance of signal pathway proteins relevant to the EGF receptor within laser capture microdissected untreated, human non-small cell lung cancer (NSCLC) (n = 25) of known epidermal growth factor receptor (EGFR) tyrosine kinase domain mutation status. We measured six phosphorylation sites on EGFR to evaluate whether EGFR mutation status in vivo was associated with the coordinated phosphorylation of specific multiple phosphorylation sites on the EGFR and downstream proteins. Reverse phase protein array quantitation of NSCLC revealed simultaneous increased phosphorylation of EGFR residues Tyr-1148 (p < 0.044) and Tyr-1068 (p < 0.026) and decreased phosphorylation of EGFR Tyr-1045 (p < 0.002), HER2 Tyr-1248 (p < 0.015), IRS-1 Ser-612 (p < 0.001), and SMAD Ser-465/467 (p < 0.011) across all classes of mutated EGFR patient samples compared with wild type. To explore which subset of correlations was influenced by ligand induction versus an intrinsic phenotype of the EGFR mutants, we profiled the time course of 115 cellular signal proteins for EGF ligand-stimulated (three dosages) NSCLC mutant and wild type cultured cell lines. EGFR mutant cell lines (H1975 L858R) displayed a pattern of EGFR Tyr-1045 and HER2 Tyr-1248 phosphorylation similar to that found in tissue. Persistence of phosphorylation for AKT Ser-473 following ligand stimulation was found for the mutant. These data suggest that a higher proportion of the EGFR mutant carcinoma cells may exhibit activation of the phosphatidylinositol 3-kinase/protein kinase B (AKT)/mammalian target of rapamycin (MTOR) pathway through Tyr-1148 and Tyr-1068 and suppression of IRS-1 Ser-612, altered heterodimerization with ERBB2, reduced response to transforming growth factor beta suppression, and reduced ubiquitination/degradation of the EGFR through EGFR Tyr-1045, thus providing a survival advantage. This is the first comparison of multiple, site-specific phosphoproteins with the EGFR tyrosine kinase domain mutation status in vivo.     

Optimization of Substituted 6-Salicyl-4-Anilinoquinazoline Derivatives as Dual EGFR/HER2 Tyrosine Kinase Inhibitors

4-Anilinoquinazolines as an important class of protein kinase inhibitor are widely investigated for epidermal growth factor receptor (EGFR) tyrosine kinase or epidermal growth factor receptor 2 (HER2) inhibition. A series of novel 6-salicyl-4-anilinoquinazoline derivatives 9–27 were prepared and evaluated for their EGFR/HER2 tyrosine kinase inhibitory activity as well as their antiproliferative properties on three variant cancer cell lines (A431, MCF-7, and A549). The bioassay results showed most of the designed compounds exhibited moderate to potent in vitro inhibitory activity in the enzymatic and cellular assays, of which compound 21 revealed the most potent dual EGFR/HER2 inhibitory activity, with IC₅₀ values of 0.12 µM and 0.096 µM, respectively, comparable to the control compounds Erlotinib and Lapatinib. Furthermore, the kinase selectivity profile of 21 was accessed and demonstrated its good selectivity over the majority of the close kinase targets. Docking simulation was performed to position compound 21 into the EGFR/HER2 active site to determine the probable binding pose. These new findings along with molecular docking observations could provide an important basis for further development of compound 21 as a potent EGFR/HER2 dual kinase inhibitor.     

Activation of ErbB2 by 2-methyl-1,4-naphthoquinone (menadione) in human keratinocytes: role of EGFR and protein tyrosine phosphatases

Activation of ErbB receptor tyrosine kinases triggers multiple signaling pathways that regulate cellular proliferation and survival. We here demonstrate that ErbB2 is activated via the epidermal growth factor receptor (EGFR) upon exposure of cultured human keratinocytes to 2-methyl-1,4-naphthoquinone (menadione). Both ErbB2 and EGFR are shown to be regulated by protein tyrosine phosphatases that are inhibited by menadione, giving rise to the hypothesis that phosphatase inhibition by menadione may result in a net activation of EGFR and an enhanced ErbB2 phosphorylation. Isolated PTP-1B, a protein tyrosine phosphatase known to be associated with ErbB receptors, is demonstrated to be inhibited by menadione.