A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions

Baxter Laboratory in Genetic Pharmacology, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5175, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2007; 103(50):19063-8. DOI: 10.1073/pnas.0605218103
Source: PubMed

ABSTRACT The orphan receptor tyrosine kinase ErbB2 is activated by each of the EGFR family members upon ligand binding. However, difficulties monitoring the dynamic interactions of the membrane receptors have hindered the elucidation of the mechanism of ErbB2 activation. We have engineered a system to monitor protein-protein interactions in intact mammalian cells such that different sets of protein interactions can be quantitatively compared. Application of this system to the interactions of the EGFR family showed that ErbB2 interacts stably with the EGFR and ErbB3, but fails to spontaneously homooligomerize. The widely used anti-cancer antibody Herceptin was found to effectively inhibit the interaction of the EGFR and ErbB2 but not to interfere with the interaction of ErbB2-ErbB3. Treatment of cells expressing EGFR and ErbB2 with Herceptin results in increased EGFR homooligomerization in the presence of EGF and a subsequent rapid internalization and down-regulation of the EGFR. In summary, the protein interaction system described here enabled the characterization of ErbB2 interactions within the biological context of the plasma membrane and provides insight into the mechanism of Herceptin action on cells overexpressing ErbB2.

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Available from: Tom Wehrman, Sep 29, 2015
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    • "Since trastuzumab does not prevent HER3 dimerization (Wehrman et al., 2006), the overexpression of HER3 could overcome trastuzumab-mediated inhibition of HER2 signaling. It was previously found that co-expression of HER3 and HER2 is required for the transformation of NIH-3T3 cells (Alimandi et al., 1995) and that HER2-HER3 dimerization results in increased tyrosine phosphorylation of HER3 which subsequently increases PI3K recruitment and downstream activation (Alimandi et al., 1995; Holbro et al., 2003). "
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    ABSTRACT: HER2-positive breast cancer accounts for 20-30% of all breast cancers and has the second-poorest prognosis among breast cancer subtypes. The approval of trastuzumab in 1998 has significantly improved patients' outcomes and paved the way for the beginning of advent of targeted approaches in breast cancer treatment. However, primary or acquired resistance to trastuzumab has been increasingly recognized as a major obstacle in the clinical management of this disease. In addition, in clinical practice, there are currently no conclusive biomarkers for patient response to trastuzumab. Therefore, understanding the molecular mechanism of trastuzumab and the development of resistance to this drug are of interest. Such understanding will provide the guidance critically needed for the design of better combination therapy and will allow the appropriate selection of patients who are responsive to trastuzumab-based strategies. In line with that, our review highlights the well-accepted mechanisms of action and resistance to the therapy and discusses the progress that has been made toward successfully overcoming this resistance.
    Frontiers in Oncology 06/2012; 2:62. DOI:10.3389/fonc.2012.00062
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    • "The oligomerization properties of the HER receptors confirmed that interactions between EGFR/HER2 and HER2/HER3 are detected in the presence of ligand. In addition, they found that trastuzumab is ineffective in blocking HER2/HER3 dimerization [84]. "
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    ABSTRACT: The epidermal growth factor receptor 2 (HER2) is a tyrosine kinase overexpressed in nearly 20% to 25% of invasive breast cancers. Trastuzumab is a humanized monoclonal antibody that targets HER2. The majority of patients with metastatic breast cancer initially respond to trastuzumab, however, within 1 year of treatment disease progresses. Several molecular mechanisms have been described as contributing to the development of trastuzumab resistance. They could be grouped as impaired access of trastuzumab to HER2, upregulation of HER2 downstream signaling pathways, signaling of alternative pathways, and impaired immune antitumor mechanisms. However, since many of them have overlapping effects, it would be of great clinical impact to identify the principal signaling pathways involved in drug resistance. Significant efforts are being applied to find other therapeutic modalities besides trastuzumab treatment to be used alone or in combination with current modalities.
    09/2011; 2011:352182. DOI:10.4061/2011/352182
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    • "To further investigate the role of HER2-mediated interactions, we used another HER2 blocking antibody, Herceptin [57] in our HER2+/3+ cells. Like 2C4, 4D5/Herceptin also inhibits HER2 dimerization but in a selective fashion; it disrupts the interaction between EGFR and HER2 but does not markedly interfere with the HER2-HER3 interaction [58]. This is most likely due to the differential involvement of HER2 cell surface residues in the formation of the various dimer types [59,60]. "
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    ABSTRACT: Knowledge about signaling pathways is typically compiled based on data gathered using different cell lines. This approach implicitly assumes that the cell line dependence is not important. However, different cell lines do not always respond to a particular stimulus in the same way, and lack of coherent data collected from closely related cellular systems can be detrimental to the efforts to understand the regulation of biological processes. To address this issue, we created a clone library of human mammary epithelial (HME) cells that expresses different levels of HER2 and HER3 receptors in combination with endogenous EGFR/HER1. Using our clone library, we have quantified the receptor activation patterns and systematically tested the validity of the existing hypotheses about the interaction patterns between HER1-3 receptors. Our study identified HER2 as the dominant dimerization partner for both EGFR and HER3. Contrary to earlier suggestions, we find that lateral interactions with HER2 do not lead to strong transactivation between EGFR and HER3, i.e., EGFR activation and HER3 activation are only weakly linked in HME cells. We also find that observed weak transactivation is uni-directional where stimulation of EGFR leads to HER3 activation whereas HER3 stimulation does not activate the EGFR. Repeating our experiments at lower cell confluency established that cell confluency is not a major factor in the observed interaction patterns. We have also quantified the dependence of the kinetics of Erk and Akt activation on different HER receptors. We found that HER3 signaling makes the strongest contribution to Akt activation and that, stimulation of either EGFR or HER3 leads to significant Erk activation. Our study shows that clone cell libraries can be a powerful resource in systems biology research by making it possible to differentiate between various hypotheses in a consistent cellular background. Using our constructed clone library we profiled the cell signaling patterns to establish the role of HER2 in the crosstalk between EGFR and HER3 receptors in HME cells. Our results for HME cells show that the weak linkage between EGFR and HER3 pathways can lead to distinct downstream cellular signaling patterns in response to the ligands of these two receptors.
    BMC Cell Biology 10/2009; 10(1):78. DOI:10.1186/1471-2121-10-78 · 2.34 Impact Factor
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