Design and Optimization of Peptide Ligands to Target Breast Cancer-Positive HER2 by Grafting and Truncation of MIG6 Peptide

The human epidermal growth factor receptor 2 (EGFR2 or HER2) has been established as a therapeutic target for HER2-positive breast cancer. Although a number of small-molecule agents have been developed to target HER2, many adverse drug reactions (ADRs) such as side effects and drug resistance are frequently observed in the chemotherapeutics. Previously, peptides derived from MIG6 protein, a natural negative regulator of EGFR and HER2, have been shown to destabilize EGFR dimerization (Zhang et al. 2007). Here, we grafted a MIG6 fragment (³³⁶KSLPSYLNGVMPPTQSFAPDPKYVSS³⁶¹) in crystal interaction site from EGFR to HER2, truncated the fragment to obtain a short segment (³⁴⁶MPPTQSFA³⁵³) with reserved binding capability (K _d = 121.6 ± 14.5 μM) to HER2 kinase domain, and optimized the segment to improve its affinity for the domain. Consequently, three peptides (MLPNQSFA, MFPNQSFA and MFPYQSFA) were successfully designed to exhibit moderate or high potency (K _d = 78.0 ± 9.8, 28.7 ± 3.9 and 65.1 ± 7.2 μM, respectively) towards HER2 kinase domain, which are expected to destabilize HER2 dimerization and then suppress the kinase activation in breast cancer.     

Structure–activity relationships of peptidomimetics that inhibit PPI of HER2‐HER3

Human epidermal growth factor receptor‐2 (HER2) is a tyrosine kinase family protein receptor that is known to undergo heterodimerization with other members of the family of epidermal growth factor receptors (EGFR) for cell signaling. Overexpression of HER2 and deregulation of signaling has implications in breast, ovarian, and lung cancers. We have designed several peptidomimetics to block the HER2‐mediated dimerization, resulting in antiproliferative activity for cancer cells. In this work, we have investigated the structure–activity relationships of peptidomimetic analogs of Compound 5. Compound 5 was conformationally constrained by N‐ and C‐terminal modification and cyclization as well as by substitution with d ‐amino acids at the N‐and C‐termini. Among the compounds studied in this work, a peptidomimetic Compound 21 with d ‐amino acid substitution and its N‐ and C‐termini capped with acetyl and amide functional groups and a reversed sequence compared to that of Compound 5 exhibited better antiproliferative activity in HER2‐overexpressed breast, ovarian, and lung cancer cell lines. Compound 21 was further evaluated for its protein–protein interaction (PPI) inhibition ability using enzyme fragment complementation assay, proximity ligation assay, and Western blot analysis. Results suggested that Compound 21 is able to block HER2:HER3 interaction and inhibit phosphorylation of the kinase domain of HER2. The mode of binding of Compound 21 to HER2 protein was modeled using a docking method. Compound 21 seems to bind to domain IV of HER2 near the PPI site of EGFR:HER2, and HER:HER3 and inhibit PPI. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 693–702, 2014.     

Heterodimerization of c-erbB2 with different epidermal growth factor receptor mutants elicits stimulatory or inhibitory responses.

Ligand-induced dimerization of growth factor receptors is crucial for stimulation of their intrinsic protein tyrosine kinase activity promoting receptor autophosphorylation by an intermolecular mechanism. Moreover, the suppressive and negative dominant action of defective epidermal growth factor receptor (EGFR) was shown to be caused by formation of inactive heterodimers with normal EGFR leading to diminished biological signaling. In this report we explore the structural requirements and functional significance of heterodimerization between EGFR and HER2. HER2 (also called c-erbB-2 or neu) is a member of the EGFR family whose natural ligand is still unknown. We show that in response to EGF, wild type EGFR and various EGFR mutants were able to undergo heterodimerization with HER2. Addition of EGF to transfected cells co-expressing HER2 with a kinase negative point mutant of EGFR (K721A) stimulated heterodimer formation, tyrosine phosphorylation of K721A and HER2, and tyrosine phosphorylation of one of their known substrates, phospholipase C gamma. However, the binding of EGF to transfected cells co-expressing HER2 together with another EGFR mutant CD533 (a deletion mutant lacking most of the cytoplasmic domain of EGFR) caused heterodimerization and inhibition of tyrosine kinase activity. It appears therefore that EGF-induced heterodimerization of EGFR and HER2 can promote either stimulatory or inhibitory influences on kinase activity. We propose that the nature of receptor interactions on the cell surface can either activate or inhibit the initiation of growth factor-controlled cellular signaling.     

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3–43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K_D = 220 pM) and HER3-expressing tumor cells with EC₅₀ values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3–43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3–43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.     

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.     

Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

MBS301, a glyco-engineered bispecific anti-human epidermal growth factor receptor 2 (HER2) antibody with a typical IgG1 monoclonal antibody structure, was developed through dual-cell expression and in vitro assembling process. MBS301 consists of two half antibodies engineered from trastuzumab and pertuzumab, respectively. Integrity and purity profiles of MB301 indicated that the heterodimerization of the two half antibodies was successful. The high and similar melting temperatures (Tm1,72.0°C and Tm2, 84.8°C) of MBS301 compared with those of its parental monoclonal antibodies trastuzumab and pertuzumab (in-house made T-mab and P-mab, respectively) revealed its structural compactness. With computer-modeling experiments and Biacore binding and competition kinetics studies, the binding stoichiometry between MBS301 and HER2-ECD was determined to be 1:1 and the two arms of MBS301 were shown to bind to domains II and IV of HER2-ECD antigen simultaneously. MBS301 displayed synergistic bioactivities as the combination of T-mab and P-mab in vitro in multiple cancer cell lines and in vivo in xenograft mouse model studies, and showed more effective activity than T-mab or P-mab used individually. Moreover, fucose-knockout dramatically increased MBS301’s binding affinity to low affinity FcγRIIIa allotype 158F (K_D = 2.35 × 10^?7M) to near the high affinity level of allotype V158 (K_D = 1.17 × 10^?7M). This resulted in far more effective ADCC activity of MBS301 than the combination of T-mab and P-mab in killing HER2-positive cancer cells. Hence, a novel fully afucosylated anti-HER2 bispecific antibody with improved antitumor activities was generated and shown to have the potential to be used for treating HER2-positive but trastuzumab-resistant solid tumors.     

A conformationally constrained peptidomimetic binds to the extracellular region of HER2 protein

Human epidermal growth factor receptor 2 (HER2) is a member of the human epidermal growth factor receptor kinases (other members include EGFR or HER1, HER3, and HER4) that are involved in signaling cascades for cell growth and differentiation. It is well established that HER2-mediated heterodimerization has important implications in cancer. Deregulation of signaling pathways and overexpression of HER2 is known to occur in cancer cells, indicating a role of HER2 in tumorigenesis. Therefore, blocking HER2-mediated signaling has potential therapeutic value. We have designed several peptidomimetics to inhibit HER2-mediated signaling for cell growth. One of the compounds (HERP5, Arg-beta Naph-Phe) exhibited antiproliferative activity with IC(50) values in the micromolar-to-nanomolar range in breast cancer cell lines. Binding of fluorescently labeled HERP5 to HER2 protein was evaluated by fluorescence assay, microscopy, and circular dichroism spectroscopy. Results indicated that HERP5 binds to the extracellular region of the HER2 protein. Structure of the peptidomimetic HERP5 was studied by NMR and molecular dynamics simulations. Based on these results a model was proposed for HER2-EGFR dimerization and possible blocking by HERP5 peptidomimetic using a protein-protein docking method.     

Design and assessment of an active anti-epidermal growth factor receptor (EGFR) single chain variable fragment (ScFv) with improved solubility

ScFv is emerging as a therapeutic alternative to the full-length monoclonal antibodies due to its small size and low production cost, but its low solubility remains a limiting factor toward wider use. Here, we increased the solubility of an Anti-epidermal growth factor receptor ScFv (Anti-EGFR ScFv) by attaching, a short 12-residue solubility enhancing peptide (SEP) tag at its C terminus. We first estimated the solubility increase by running 500-ns Brownian dynamics (BD) simulations. We then experimentally evaluated the predictions by producing recombinant Anti-EGFR ScFv with and without a SEP tag (called C9R) in E. coli. At 20?^°C, ～85% of Anti-EGFR ScFv-C9R expressed in the soluble fraction, whereas all of the Anti-EGFR ScFv remained in the insoluble fraction. The total yield of Anti-EGFR ScFv-C9R was 17.15?mg which was ～3 times higher than that of Anti-EGFR ScFv refolded from the insoluble fraction. Static and dynamic light scattering demonstrated the higher solubility of the purified Anti-EGFR ScFv-C9R, and Circular Dichroism (CD) indicated its high thermal stability, whereas the untagged protein aggregated at 37?^°C and pH 6. Finally, the binding activity of Anti-EGFR ScFv-C9R to EGFR was confirmed by surface plasmon resonance (SPR). Altogether, these results illustrate the improved biophysical and biochemical characteristics of Anti-EGFR ScFv-C9R and emphasize the potentials of SEP-tags for enhancing the solubility of aggregation-prone antibody fragments.     

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.     

Study of inhibition effect of Herceptin on interaction between Heregulin and ErbB Receptors HER3/HER2 by single-molecule force spectroscopy

Herceptin is a monoclonal antibody against HER2, which is a member of the epidermal growth factor receptor (ErbB) family and is overexpressed in many cancers. In this work, we have applied single-molecule force spectroscopy to study the effect of Herceptin on HER2 modulated ligand–receptor interaction for ErbB signaling in living cells. Heregulin β1 (HRG), the specific ligand of HER3, was used for HER2 activation as HER3 is the preferable dimerization partner of HER2 and HER3/HER2 is the most representative heterodimer found in cancer. Our results demonstrated a more stable binding of HRG to the cells co-expressing HER3 and HER2 than those expressing HER3 alone. Moreover, the binding force of Herceptin and HER2 is as strong as that of HRG and HER3/HER2. With the addition of Herceptin, the binding strength of HRG to the cells co-expressing HER3 and HER2 decreased. The presence of Herceptin changed the dynamic force spectrum of HRG-HER3/HER2 to that similar to HRG-HER3. Therefore, the enhancement in HRG-HER3 binding after recruiting HER2 was inhibited by Herceptin. The method offers a new approach to study the molecular mechanism of Herceptin anti-cancer effect.     

HER2 oncogenic function escapes EGFR tyrosine kinase inhibitors via activation of alternative HER receptors in breast cancer cells

Background

The response rate to EGFR tyrosine kinase inhibitors (TKIs) may be poor and unpredictable in cancer patients with EGFR expression itself being an inadequate response indicator. There is limited understanding of the mechanisms underlying this resistance. Furthermore, although TKIs suppress the growth of HER2-overexpressing breast tumor cells, they do not fully inhibit HER2 oncogenic function at physiological doses.

Methodology and Principal Findings

Here we have provided a molecular mechanism of how HER2 oncogenic function escapes TKIs'' inhibition via alternative HER receptor activation as a result of autocrine ligand release. Using both Förster Resonance Energy Transfer (FRET) which monitors in situ HER receptor phosphorylation as well as classical biochemical analysis, we have shown that the specific tyrosine kinase inhibitors (TKIs) of EGFR, AG1478 and Iressa (Gefitinib) decreased EGFR and HER3 phosphorylation through the inhibition of EGFR/HER3 dimerization. Consequent to this, we demonstrate that cleavage of HER4 and dimerization of HER4/HER2 occur together with reactivation of HER3 via HER2/HER3, leading to persistent HER2 phosphorylation in the now resistant, surviving cells. These drug treatment–induced processes were found to be mediated by the release of ligands including heregulin and betacellulin that activate HER3 and HER4 via HER2. Whereas an anti-betacellulin antibody in combination with Iressa increased the anti-proliferative effect in resistant cells, ligands such as heregulin and betacellulin rendered sensitive SKBR3 cells resistant to Iressa.

Conclusions and Significance

These results demonstrate the role of drug-induced autocrine events leading to the activation of alternative HER receptors in maintaining HER2 phosphorylation and in mediating resistance to EGFR tyrosine kinase inhibitors (TKIs) in breast cancer cells, and hence specify treatment opportunities to overcome resistance in patients.     

Construction and characterization of a recombinant human beta defensin 2 fusion protein targeting the epidermal growth factor receptor: in vitro study

The HER2/neu proto-oncogene encodes a 185-kDa trans-membrane glycoprotein kinase with extensive homology to the epidermal growth factor receptor and plays a key role in the transformation and growth of malignant tumors. To date, two antibody drugs targeting HER2/neu have been developed successfully. In order to reduce the cost and the time of clinical treatment, we produced a fusion protein composed of human beta defensin 2 (hBD2) and anti-HER2/neu single-chain variable fragment (scFv 4D5), which is capable of specifically targeting, significantly inhibiting, and promptly killing HER2/neu-positive cancer cells. The recombinant protein was expressed in Escherichia coli using the small ubiquitin-related modifier (SUMO) as the molecular chaperone, and the optimal expression level reached to 40.2 % of the total supernatant protein. After purifying by Ni-NTA affinity chromatography, the fusion protein was cleaved with a SUMO-specific protease to obtain hBD2–4D5, which was further purified by Ni-NTA affinity chromatography. The purity of hBD2–4D5 was higher than 95 %, and the yield was 19?±?2 mg/L in flask fermentation. The cell number count and flow cytometry results showed that hBD2–4D5 exerted cytotoxic and anti-proliferative effects on HER2/neu-positive breast cancer cell line, SKBR-3. The results of scanning electron microscope and transmission electron microscope observation indicated that hBD2–4D5 could induce intracellular ultrastructure changes and cell necrosis by disrupting the cell membrane. Immunofluorescence analysis showed that hBD2–4D5 could bind to SKBR-3 cells and further be internalized into the cytoplasm. Moreover, hBD2–4D5 could also mediate apoptosis of SKBR-3 cells by up-regulating the ratio of Bax to Bcl-2.     

Design of novel lipidated peptidomimetic conjugates for targeting EGFR heterodimerization in HER2 + cancer

The human epidermal growth factor receptor (EGFR) family is known to be involved in cell signaling pathways. The extracellular domain of EGFR consists of four domains, of which domain II and domain IV are known to be involved in the dimerization process. Overexpression of these receptors is known to play a significant role in heterodimerization of these receptors leading to the development of cancer. We have designed peptidomimetic molecules to inhibit the EGFR heterodimerization interaction that have shown antiproliferative activity and specificity for HER2-positive cancer cell lines. Among these, a peptidomimetic, compound 5, exhibited antiproliferative activity at low nanomolar concentrations in HER2-overexpressing cancer cell lines. To improve the stability of this peptidomimetic, we have designed and synthesized a novel conjugate of peptidomimetic compound 5 with a lipid, stearic acid. The antiproliferative activity of this conjugate was evaluated in HER2-positive cancer cell lines. Results suggested that the conjugate exhibited selective antiproliferative activity in HER2-overexpressing breast and lung cancer cell lines and was able to block HER2:HER3 heterodimerization. Also, the conjugate showed improved stability with a half-life of 5?h in human serum compared to the half-life of 2?h for parent compound 5. The binding affinity of the conjugate to HER2 protein was evaluated by SPR analysis, and the mode of binding of the lipid conjugate to domain IV of HER2 protein was demonstrated by docking analysis. Thus, this novel lipid conjugate can be used to target HER2-overexpressing cancers.     

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.     

Preparation and Identification of HER2 Radioactive Ligands and Imaging Study of Breast Cancer-Bearing Nude Mice

OBJECTIVE: A micro-molecule peptide TP1623 of ^99mTc-human epithelial growth factor receptor 2 (HER2) was prepared and the feasibility of using it as a HER2-positive molecular imaging agent for breast cancer was evaluated. METHODS: TP1623 was chemically synthesized and labeled with ^99mTc. The labeling ratio and stability were detected. HER2 expression levels of breast cancer cells (SKBR3 and MDA-MB-231) and cell binding activity were measured. Biodistribution of ^99mTC-TP1623 in normal mice was detected. SKBR3/MDA-MB-231-bearing nude mice models with high/low expressions of HER2 were established. Tumor tissues were stained with hematoxylin–eosin (HE) and measured by immunohistochemistry to confirm the formation of tumors and HER2 expression. SPECT imaging was conducted for HER2-overexpressing SKBR3-bearing nude mice. The T/NT ratio was calculated and compared with that of MDA-MB-231-bearing nude mice with low HER2 expression. The competitive inhibition image was used to discuss the specific binding of ^99mTc- TP1623 and the tumor. RESULTS: The labeling ratio of ^99mTc-TP1623, specific activity, and radiochemical purity (RCP) after 6 h at room temperature were (97.39 ± 0.23)%, (24.61 ± 0.06) TBq/mmol, and (93.25 ± 0.06)%, respectively. HER2 of SKBR3 and MDA-MB-231 cells showed high and low expression levels by immunohistochemistry, respectively. The in vitro receptor assays indicated that specific binding of TP1623 and HER2 was retained. Radioactivity in the brain was always at the lowest level, while the clearance rate of blood and the excretion rate of the kidneys were fast. HE staining showed that tumor cells were observed in SKBR3- and MDA-MB-231-bearing nude mice, with significant heteromorphism and increased mitotic count. The imaging of mice showed that targeted images could be made of ^99mTc-TP1623 in high HER2-expressing tumors, while no obvious development was shown in tumors in low HER2-expressing nude mice. No development was visible in tumors in competitive inhibition of imaging, which indicates the combination of ^99mTc-TP1623 and tumor was mediated by HER2. CONCLUSION: High labeling ratio and specific activity of ^99mTc-TP1623 is successfully prepared; it is a molecular imaging agent for HER2-positive tumors that has potential applicative value.     

The HER2-Binding Affibody Molecule (ZHER2∶342)2 Increases Radiosensitivity in SKBR-3 Cells

We have previously shown that the HER2-specific affibody molecule (Z_HER2∶342)₂ inhibits proliferation of SKBR-3 cells. Here, we continue to investigate its biological effects in vitro by studying receptor dimerization and clonogenic survival following irradiation. We found that (Z_HER2∶342)₂ sensitizes the HER2-overexpressing cell line SKBR-3 to ionizing radiation. The survival after exposure to (Z_HER2∶342)₂ and 8 Gy (S_8Gy 0.006) was decreased by a factor four compared to the untreated (S_8Gy 0.023). The low HER2-expressing cell line MCF-7 was more radiosensitive than SKBR-3 but did not respond to (Z_HER2∶342)₂. Treatment by (Z_HER2∶342)₂ strongly increased the levels of dimerized and phosphorylated HER2 even after 5 minutes of stimulation. The monomeric Z_HER2∶342 does not seem to be able to induce receptor phosphorylation and dimerization or sensitize cells to irradiation.     

HER2 cytoplasmic domain generates normal mitogenic and transforming signals in a chimeric receptor.

We have investigated the biological function of an unidentified human growth factor, the ligand of the putative HER2 receptor, by characterizing the signalling properties of its receptor. HER2 (or c-erbB-2), the human homolog of the rat neu proto-oncogene, encodes a transmembrane glycoprotein of the tyrosine kinase family that appears to play an important role in human breast carcinoma. Since a potential ligand for HER2 has not yet been identified, it has been difficult to analyze the biochemical properties and biological function of this cell surface protein. For this reason, we replaced the HER2 extracellular domain with the closely related ligand binding domain sequences of the epidermal growth factor (EGF) receptor, and examined the ligand-induced biological signalling potential of this chimeric HER1-2 protein. This HER1-2 receptor is targetted to the cell surface of transfected NIH 3T3 cells, forms high and low affinity binding sites, and generates normal mitogenic and cell transforming signals upon interaction with EGF or TGF alpha. The constitutive activation of wild-type HER2 in transfected NIH 3T3 cells suggests the possibility that these cells synthesize the as yet unidentified HER2 ligand and activate HER2 by an autocrine mechanism.     

Relationship between clinical data and gene expression in the HER2/ErbB2-dependent signaling pathway in patients with acute heart failure

Anticancer treatment with the human epidermal growth factor receptor (HER) 2 inhibitors can lead to significant myocardial dysfunction. The primary aim of the study was to estimate the possible association between gene expression in the ErbB signaling pathway and selected clinical event data in patients with acute heart failure. Twenty-four patients (19 males), aged 68.6?±?12.3 years, were diagnosed and treated due to acute heart failure. The globaltest method was used for the correlation between blood nuclear cells’ gene expression in the ErbB pathway (KEGG pathway id 04012) and important clinical data. Decreased expression of ErbB2/HER2 was found to be associated with the release of troponin and the need for inotropic support, whereas decreased neuregulin 1 (NRG1) expression was found to be associated with a decrease of ejection fraction below 40 % (globaltest p-value < 0.05). In summary, the ErbB signaling pathway and, especially, HER2/ErbB2 receptor expression are significantly associated with some of the recognized, clinically significant parameters of patients with acute heart failure. Evaluation of the molecular function of the HER2 receptor may be essential for the prognosis and targeted therapy of heart diseases.     

Gefitinib induces epidermal growth factor receptor dimers which alters the interaction characteristics with ¹²⁵I-EGF

The tyrosine kinase inhibitor gefitinib inhibits growth in some tumor types by targeting the epidermal growth factor receptor (EGFR). Previous studies show that the affinity of the EGF-EGFR interaction varies between hosting cell line, and that gefitinib increases the affinity for some cell lines. In this paper, we investigate possible mechanisms behind these observations. Real-time interaction analysis in LigandTracer® Grey revealed that the HER2 dimerization preventing antibody pertuzumab clearly modified the binding of ¹²⁵I-EGF to EGFR on HER2 overexpressing SKOV3 cells in the presence of gefitinib. Pertuzumab did not affect the binding on A431 cells, which express low levels of HER2. Cross-linking measurements showed that gefitinib increased the amount of EGFR dimers 3.0–3.8 times in A431 cells in the absence of EGF. In EGF stimulated SKOV3 cells the amount of EGFR dimers increased 1.8–2.2 times by gefitinib, but this effect was cancelled by pertuzumab. Gefitinib treatment did not alter the number of EGFR or HER2 expressed in tumor cell lines A431, U343, SKOV3 and SKBR3. Real-time binding traces were further analyzed in a novel tool, Interaction Map, which deciphered the different components of the measured interaction and supports EGF binding to multiple binding sites. EGFR and HER2 expression affect the levels of EGFR monomers, homodimers and heterodimers and EGF binds to the various monomeric/dimeric forms of EGFR with unique binding properties. Taken together, we conclude that dimerization explains the varying affinity of EGF – EGFR in different cells, and we propose that gefitinib induces EGFR dimmers, which alters the interaction characteristics with ¹²⁵I-EGF.     

p185HER2 signal transduction in breast cancer cells.

A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2-overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neu-overexpressing breast cancer cells (BT-474 and SK-Br-3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56-kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5- stimulated phosphorylation of p185HER2 and ptyr56 was a 5-10-fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity and a 2-fold induction of inositol 1,4,5-trisphosphate 3'-kinase activity. The increased phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and also co-eluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonistic-like signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.