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Temporal Profiling of Lapatinib-suppressed Phosphorylation Signals in EGFR/HER2 Pathways*

  • Koshi Imami
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan, 997-0017;

    Present address: Centre for High-Throughput Biology, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4;
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  • Naoyuki Sugiyama
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan, 997-0017;
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  • Haruna Imamura
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan, 997-0017;

    Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan, 606-8501;
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  • Masaki Wakabayashi
    Affiliations
    Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan, 606-8501;
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  • Masaru Tomita
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan, 997-0017;
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  • Masatoshi Taniguchi
    Affiliations
    Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan, 606-8507
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  • Takayuki Ueno
    Affiliations
    Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan, 606-8507
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  • Masakazu Toi
    Affiliations
    Department of Breast Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan, 606-8507
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  • Yasushi Ishihama
    Correspondence
    To whom correspondence should be addressed: Department of Molecular and Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshidashimoadachicho, Sakyo-ku, Kyoto, Japan, 606-8501. Tel.:+81-75-753-4555; Fax: +81-75-753-4601;
    Affiliations
    Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan, 997-0017;

    Department of Molecular & Cellular BioAnalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan, 606-8501;
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  • Author Footnotes
    * This work was supported by research funds from Yamagata Prefecture and Tsuruoka City, by a grant for the Science and Technology Incubation Program in Advanced Regions from Japan Science and Technology Agency, by grants from JSPS Grant-in-Aid Scientific Research No. 215184 (KI), No. 11J04789 (HI), No. 21310129 (YI) and No. 24241062 (YI), and by a research grant from Ministry of Health, Labor and Welfare (H19–3JIGAN-IPPAN-007).
    This article contains supplemental Data, Figs. S1 to S10, and Tables S1 to S3.
Open AccessPublished:September 10, 2012DOI:https://doi.org/10.1074/mcp.M112.019919
      Lapatinib is a clinically potent kinase inhibitor for breast cancer patients because of its outstanding selectivity for epidermal growth factor receptor (EGFR) and EGFR2 (also known as HER2). However, there is only limited information about the in vivo effects of lapatinib on EGFR/HER2 and downstream signaling targets. Here, we profiled the lapatinib-induced time- and dose-dependent phosphorylation dynamics in SKBR3 breast cancer cells by means of quantitative phosphoproteomics. Among 4953 identified phosphopeptides from 1548 proteins, a small proportion (5–7%) was regulated at least twofold by 1–10 μm lapatinib. We obtained a comprehensive phosphorylation map of 21 sites on EGFR/HER2, including nine novel sites on HER2. Among them, serine/threonine phosphosites located in a small region of HER2 (amino acid residues 1049–1083) were up-regulated by the drug, whereas all other sites were down-regulated. We show that cAMP-dependent protein kinase is involved in phosphorylation of this particular region of HER2 and regulates HER2 tyrosine kinase activity. Computational analyses of quantitative phosphoproteome data indicated for the first time that protein-protein networks related to cytoskeletal organization and transcriptional/translational regulation, such as RNP complexes (i.e. hnRNP, snRNP, telomerase, ribosome), are linked to EGFR/HER2 signaling networks. To our knowledge, this is the first report to profile the temporal response of phosphorylation dynamics to a kinase inhibitor. The results provide new insights into EGFR/HER2 regulation through region-specific phosphorylation, as well as a global view of the cellular signaling networks associated with the anti-breast cancer action of lapatinib.
      Human epidermal growth factor receptor 2 (EGFR2
      The abbreviations used are:
      EGFR
      epidermal growth factor receptor
      HER
      human epidermal growth factor receptor
      HPRD
      Human Protein Reference Database
      FPR
      false-positive rate
      SPL
      shortest path length
      PPI
      protein-protein interaction
      RNP
      ribonucleoprotein.
      1The abbreviations used are:EGFR
      epidermal growth factor receptor
      HER
      human epidermal growth factor receptor
      HPRD
      Human Protein Reference Database
      FPR
      false-positive rate
      SPL
      shortest path length
      PPI
      protein-protein interaction
      RNP
      ribonucleoprotein.
      also known as HER2) is a key molecule in breast carcinoma cells. It is overexpressed and/or hyperactivated in ∼25% of human breast cancer patients (
      • Slamon D.J.
      • Clark G.M.
      • Wong S.G.
      • Levin W.J.
      • Ullrich A.
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      Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene.
      ,
      • Slamon D.J.
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      ), constitutively enhancing the downstream signaling pathways toward tumor growth and invasion by forming kinase-active dimers with EGFR family members (i.e. EGFR, HER2, HER3, and HER4) (
      • Pinkas-Kramarski R.
      • Soussan L.
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      • Alroy I.
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      • Seger R.
      • Ratzkin B.J.
      • Sela M.
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      Diversification of neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.
      ). A humanized monoclonal antibody-based drug, trastuzumab (Herceptin®) has been approved for the treatment of breast cancer patients with HER2 protein overexpression and HER2 gene amplification (
      • Harries M.
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      The development and clinical use of trastuzumab (herceptin).
      ). It has been proposed that trastuzumab inhibits the downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) pathways (
      • Nagata Y.
      • Lan K.H.
      • Zhou X.
      • Tan M.
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      • Klos K.S.
      • Li P.
      • Monia B.P.
      • Nguyen N.T.
      • Hortobagyi G.N.
      • Hung M.C.
      • Yu D.
      PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients.
      ,
      • Junttila T.T.
      • Akita R.W.
      • Parsons K.
      • Fields C.
      • Lewis Phillips G.D.
      • Friedman L.S.
      • Sampath D.
      • Sliwkowski M.X.
      Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941.
      ), and/or induces Fc receptor-mediated cytotoxity through immune response (
      • Clynes R.A.
      • Towers T.L.
      • Presta L.G.
      • Ravetch J.V.
      Inhibitory fc receptors modulate in vivo cytoxicity against tumor targets.
      ). One critical issue is that most HER2-positive patients tend to be insensitive to trastuzumab treatment or acquire drug resistance (
      • Nahta R.
      • Esteva F.J.
      HER2 therapy: Molecular mechanisms of trastuzumab resistance.
      ,
      • Lan K.H.
      • Lu C.H.
      • Yu D.
      Mechanisms of trastuzumab resistance and their clinical implications.
      ). On the other hand, small-molecular inhibitors such as gefitinib and imatinib have been launched as selective kinase inhibitors for several types of cancer (
      • Zhang J.
      • Yang P.L.
      • Gray N.S.
      Targeting cancer with small molecule kinase inhibitors.
      ). These inhibitors are generally designed to inhibit kinase activity by binding to the ATP-binding pocket of target kinase domains, thereby blocking the downstream signaling pathways that induce cell proliferation, growth, survival, and so on. However, most kinase inhibitors have multiple targets, including so-called “off targets” because of the similarity of ATP-binding pockets among more than 500 human protein kinases, and this can result in critical side effects such as cardiotoxicity (
      • Force T.
      • Kolaja K.L.
      Cardiotoxicity of kinase inhibitors: The prediction and translation of preclinical models to clinical outcomes.
      ). Although multitarget inhibitors (e.g. sunitinib and sorafenib) are effective in some cases (
      • Hopkins A.L.
      • Mason J.S.
      • Overington J.P.
      Can we rationally design promiscuous drugs?.
      ), targeting specific protein kinase(s) would be a rational approach for the development of drugs without critical side effects. Among 12 kinase inhibitors so far launched on the US market, including imatinib, gefitinib, erlotinib, and dasatinib (
      • Force T.
      • Kolaja K.L.
      Cardiotoxicity of kinase inhibitors: The prediction and translation of preclinical models to clinical outcomes.
      ), lapatinib (TykerbTM, GSK572016) exhibits the highest selectivity and has a dual inhibitory effect on EGFR/HER2 (
      • Karaman M.W.
      • Herrgard S.
      • Treiber D.K.
      • Gallant P.
      • Atteridge C.E.
      • Campbell B.T.
      • Chan K.W.
      • Ciceri P.
      • Davis M.I.
      • Edeen P.T.
      • Faraoni R.
      • Floyd M.
      • Hunt J.P.
      • Lockhart D.J.
      • Milanov Z.V.
      • Morrison M.J.
      • Pallares G.
      • Patel H.K.
      • Pritchard S.
      • Wodicka L.M.
      • Zarrinkar P.P.
      A quantitative analysis of kinase inhibitor selectivity.
      ). The dissociation constant (Kd) values of EGFR (2.4 nm) and HER2 (7 nm) are 500–1800 times higher than those of off-target molecules not belonging to the EGFR family, such as serine/threonine-protein kinase 10 and receptor-interacting serine/threonine-protein kinase 2 (
      • Karaman M.W.
      • Herrgard S.
      • Treiber D.K.
      • Gallant P.
      • Atteridge C.E.
      • Campbell B.T.
      • Chan K.W.
      • Ciceri P.
      • Davis M.I.
      • Edeen P.T.
      • Faraoni R.
      • Floyd M.
      • Hunt J.P.
      • Lockhart D.J.
      • Milanov Z.V.
      • Morrison M.J.
      • Pallares G.
      • Patel H.K.
      • Pritchard S.
      • Wodicka L.M.
      • Zarrinkar P.P.
      A quantitative analysis of kinase inhibitor selectivity.
      ). Further, because dissociation of lapatinib from EGFR/HER2 is much slower than that of other EGFR-selective inhibitors such as gefitinib and erlotinib, its inhibitory effects on EGFR/HER2 tyrosine phosphorylation are more prolonged (
      • Nahta R.
      • Esteva F.J.
      HER2 therapy: Molecular mechanisms of trastuzumab resistance.
      ). Lapatinib was reported to be effective against HER-2-overexpressing and trastuzumab-treated breast cancer cells (
      • Konecny G.E.
      • Pegram M.D.
      • Venkatesan N.
      • Finn R.
      • Yang G.
      • Rahmeh M.
      • Untch M.
      • Rusnak D.W.
      • Spehar G.
      • Mullin R.J.
      • Keith B.R.
      • Gilmer T.M.
      • Berger M.
      • Podratz K.C.
      • Slamon D.J.
      Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells.
      ). But, although lapatinib is currently one of the most potent clinical treatments for breast cancer patients (
      • Wood E.R.
      • Truesdale A.T.
      • McDonald O.B.
      • Yuan D.
      • Hassell A.
      • Dickerson S.H.
      • Ellis B.
      • Pennisi C.
      • Horne E.
      • Lackey K.
      • Alligood K.J.
      • Rusnak D.W.
      • Gilmer T.M.
      • Shewchuk L.
      A unique structure for epidermal growth factor receptor bound to GW572016 (lapatinib): Relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells.
      ), its global effect on EGFR/HER2 signaling networks in vivo is still poorly understood. Vazquez-Martin et al. performed a limited-scale phosphoproteome analysis of a lapatinib-resistant breast cancer cell line using a phospho-specific antibody array for 21 different oncogenic kinases (
      • Vazquez-Martin A.
      • Oliveras-Ferraros C.
      • Colomer R.
      • Brunet J.
      • Menendez J.A.
      Low-scale phosphoproteome analyses identify the mTOR effector p70 S6 kinase 1 as a specific biomarker of the dual-HER1/HER2 tyrosine kinase inhibitor lapatinib (tykerb) in human breast carcinoma cells.
      ). They found that the phosphorylation state of ribosomal protein S6 kinase beta-1 could be a potential marker for drug resistance. However, a proteome-wide picture of lapatinib's inhibitory effects remains to be uncovered.
      In recent years, mass spectrometry (MS)-based quantitative proteomics has been applied to large-scale drug profiling. Fabian et al. reported an in vitro competitive binding assay using recombinant kinase mixtures and defined interactions between 38 inhibitors and 317 kinases at various drug concentrations (
      • Karaman M.W.
      • Herrgard S.
      • Treiber D.K.
      • Gallant P.
      • Atteridge C.E.
      • Campbell B.T.
      • Chan K.W.
      • Ciceri P.
      • Davis M.I.
      • Edeen P.T.
      • Faraoni R.
      • Floyd M.
      • Hunt J.P.
      • Lockhart D.J.
      • Milanov Z.V.
      • Morrison M.J.
      • Pallares G.
      • Patel H.K.
      • Pritchard S.
      • Wodicka L.M.
      • Zarrinkar P.P.
      A quantitative analysis of kinase inhibitor selectivity.
      ,
      • Fabian M.A.
      • Biggs 3rd, W.H.
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      • Insko D.E.
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      • Velasco A.M.
      • Wodicka L.M.
      • Patel H.K.
      • Zarrinkar P.P.
      • Lockhart D.J.
      A small molecule-kinase interaction map for clinical kinase inhibitors.
      ). Bantscheff et al. developed a MS-based assay with a kinase-selective enrichment method (called “kinobeads,” on which nonselective kinase inhibitors are immobilized) to elucidate the mechanism of action of clinical ABL kinase inhibitors (
      • Bantscheff M.
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      • Abraham Y.
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      • Neubauer G.
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      • Rick J.
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      ). Similarly, Sharma et al. introduced a multiplex SILAC (stable isotope labeling by amino acids in cell culture)-based proteomics approach combined with kinase inhibitor-containing beads to globally characterize drug-targeted kinases (
      • Sharma K.
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      • Greff Z.
      • Kéri G.
      • Cox J.
      • Olsen J.V.
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      Proteomics strategy for quantitative protein interaction profiling in cell extracts.
      ). More recently, KAYAK (kinase activity assay for kinome profiling) has been introduced to monitor multiplex kinase activities in a cell lysate by quantifying a library of 90 synthetic peptides that mimic putative substrates of human protein kinases (
      • Yu Y.
      • Anjum R.
      • Kubota K.
      • Rush J.
      • Villen J.
      • Gygi S.P.
      A site-specific, multiplexed kinase activity assay using stable-isotope dilution and high-resolution mass spectrometry.
      ,
      • Kubota K.
      • Anjum R.
      • Yu Y.
      • Kunz R.C.
      • Andersen J.N.
      • Kraus M.
      • Keilhack H.
      • Nagashima K.
      • Krauss S.
      • Paweletz C.
      • Hendrickson R.C.
      • Feldman A.S.
      • Wu C.L.
      • Rush J.
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      ). Although these in vitro strategies are well designed for simple, rapid, and comprehensive drug profiling to determine the Kd values as well as drug target kinases and off-target molecules, the nature of the changes in in vivo phosphoproteome profiles induced by drugs remains unclear. Meanwhile, several research groups have explored quantitative phosphoproteomics approaches for in vivo profiling of kinase inhibitors (e.g. imatinib (
      • Rubbi L.
      • Titz B.
      • Brown L.
      • Galvan E.
      • Komisopoulou E.
      • Chen S.S.
      • Low T.
      • Tahmasian M.
      • Skaggs B.
      • Muschen M.
      • Pellegrini M.
      • Graeber T.G.
      Global phosphoproteomics reveals crosstalk between bcr-abl and negative feedback mechanisms controlling SRC signaling.
      ), dasatinib (
      • Li J.
      • Rix U.
      • Fang B.
      • Bai Y.
      • Edwards A.
      • Colinge J.
      • Bennett K.L.
      • Gao J.
      • Song L.
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      • Superti-Furga G.
      • Koomen J.
      • Haura E.B.
      A chemical and phosphoproteomic characterization of dasatinib action in lung cancer.
      ,
      • Pan C.
      • Olsen J.V.
      • Daub H.
      • Mann M.
      Global effects of kinase inhibitors on signaling networks revealed by quantitative phosphoproteomics.
      ), PD168393 (
      • Bose R.
      • Molina H.
      • Patterson A.S.
      • Bitok J.K.
      • Periaswamy B.
      • Bader J.S.
      • Pandey A.
      • Cole P.A.
      Phosphoproteomic analysis of Her2/neu signaling and inhibition.
      ), and PI-103 (
      • Andersen J.N.
      • Sathyanarayanan S.
      • Di Bacco A.
      • Chi A.
      • Zhang T.
      • Chen A.H.
      • Dolinski B.
      • Kraus M.
      • Roberts B.
      • Arthur W.
      • Klinghoffer R.A.
      • Gargano D.
      • Li L.
      • Feldman I.
      • Lynch B.
      • Rush J.
      • Hendrickson R.C.
      • Blume-Jensen P.
      • Paweletz C.P.
      Pathway-based identification of biomarkers for targeted therapeutics: Personalized oncology with PI3K pathway inhibitors.
      )).
      In this study, we used quantitative phosphoproteomics approaches to examine the in vivo effects of lapatinib on EGFR/HER2 as well as the downstream signaling networks. We determined the lapatinib-induced time- and dose-dependent phosphorylation dynamics of SKBR3 human breast cancer cells. In addition, computational analyses of the phosphoproteomic data were carried out to characterize the signaling networks involved in lapatinib's action.

      CONCLUSIONS

      Lapatinib, a dual tyrosine kinase inhibitor for EGFR/HER2, is of importance in the clinical treatment of breast cancer, and has the highest selectivity for EGFR/HER2 among current kinase inhibitors. To our knowledge, the present study is the first large-scale profiling of the perturbation of temporal phosphoproteome dynamics by a kinase inhibitor.
      Our quantitative phosphoproteomic profiling study revealed that lapatinib treatment induces phosphorylation in specific regions of EGFR/HER2, i.e. its regulation of EGFR/HER2 phosphorylation is region-specific. The integrative approaches used here (i.e. literature, informatics analyses of phosphorylation motif, in vitro and in vivo kinase profiling and H89 treatment) identified PKA as the putative kinase mediating HER2 serine/threonine phosphorylation, and we also demonstrated that the induced HER2 phosphorylation contributes to the regulation of HER2 tyrosine kinase activity.
      With respect to the impact of lapatinib on downstream signaling targets, as expected from the specificity of lapatinib, only a few percent of the 4953 identified phosphopeptides were regulated. The STRING PPI network analysis allowed us to identify lapatinib-regulated protein networks related to cytoskeletal organization, transcription, and translation, which have not previously been implicated in EGFR/HER2 signaling pathways. Although several proteome and phosphoproteome studies have used PPI- and pathway-based analyses for profiling the global impacts of a particular perturbation on entire protein networks, there has been no further in-depth data analysis of the obtained protein networks. We employed novel data-mining approaches with PPI network data and quantitatively showed that the uncharacterized PPI networks obtained by means of STRING form a part of the EGFR/HER2 signaling networks.
      Taken together, our results provide a novel map of the EGFR/HER2 signaling network, including new molecules, based on the integration of quantitative phosphoproteomics using a EGFR/HER2-specific inhibitor with a variety of computational approaches.

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