Advertisement

Quantitative Proteomics with siRNA Screening Identifies Novel Mechanisms of Trastuzumab Resistance in HER2 Amplified Breast Cancers*

  • Alaina P. Boyer
    Footnotes
    Affiliations
    Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110;
    Search for articles by this author
  • Timothy S. Collier
    Footnotes
    Affiliations
    Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110;
    Search for articles by this author
  • Ilan Vidavsky
    Affiliations
    Department of Chemistry, Washington University, St. Louis, MO 63130
    Search for articles by this author
  • Ron Bose
    Correspondence
    To whom correspondence should be addressed: Ron Bose, Tel.:314-747-9308, Fax: 314-747-9320, [email protected]
    Affiliations
    Division of Oncology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110;

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110
    Search for articles by this author
  • Author Footnotes
    * This research was supported by grants from Susan G. Komen for the Cure, the 'Ohana Breast Cancer Research fund, and the Foundation for Barnes-Jewish Hospital. A.P.B. and T.S.C. are supported by NIH T32 training grants (Grant No. CA113275 for A.P.B. and Grant No. 2T32HL007088–36 for T.S.C.). Mass spectrometer instrument support was provided by the National Center for Research Resources of the NIH (Grant No. 2P41RR000954 to M. L. Gross).
    This article contains supplemental material.
    § These authors contributed equally to this work.
Open AccessPublished:October 25, 2012DOI:https://doi.org/10.1074/mcp.M112.020115
      HER2 is a receptor tyrosine kinase that is overexpressed in 20% to 30% of human breast cancers and which affects patient prognosis and survival. Treatment of HER2-positive breast cancer with the monoclonal antibody trastuzumab (Herceptin) has improved patient survival, but the development of trastuzumab resistance is a major medical problem. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns, and therefore quantitative proteomics was used to examine phosphotyrosine signaling networks in trastuzumab-resistant cells. The model system used in this study was two pairs of trastuzumab-sensitive and -resistant breast cancer cell lines. Using stable isotope labeling, phosphotyrosine immunoprecipitations, and online TiO2 chromatography utilizing a dual trap configuration, ∼1700 proteins were quantified. Comparing quantified proteins between the two cell line pairs showed only a small number of common protein ratio changes, demonstrating heterogeneity in phosphotyrosine signaling networks across different trastuzumab-resistant cancers. Proteins showing significant increases in resistant versus sensitive cells were subjected to a focused siRNA screen to evaluate their functional relevance to trastuzumab resistance. The screen revealed proteins related to the Src kinase pathway, such as CDCP1/Trask, embryonal Fyn substrate, and Paxillin. We also identify several novel proteins that increased trastuzumab sensitivity in resistant cells when targeted by siRNAs, including FAM83A and MAPK1. These proteins may present targets for the development of clinical diagnostics or therapeutic strategies to guide the treatment of HER2+ breast cancer patients who develop trastuzumab resistance.
      HER2 is a member of the epidermal growth factor receptor (EGFR)/ErbB family of receptor tyrosine kinases. Under normal physiologic conditions, HER2 tyrosine kinase signaling is tightly regulated spatially and temporally by the requirement for it to heterodimerize with a ligand bound family member, such as EGFR, HER3/ErbB3, or HER4/ErbB4 (
      • Hynes N.E.
      • Lane H.A.
      ERBB receptors and cancer: the complexity of targeted inhibitors.
      ). However, in 20% to 30% of human breast cancer cases, HER2 gene amplification is present, resulting in a high level of HER2 protein overexpression and unregulated, constitutive HER2 tyrosine kinase signaling (
      • Slamon D.J.
      • Clark G.M.
      • Wong S.G.
      • Levin W.J.
      • Ullrich A.
      • McGuire W.L.
      Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
      ,
      • Slamon D.J.
      • Godolphin W.
      • Jones L.A.
      • Holt J.A.
      • Wong S.G.
      • Keith D.E.
      • Levin W.J.
      • Stuart S.G.
      • Udove J.
      • Ullrich A.
      • Press M.F.
      Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
      ). HER2 gene amplified breast cancer, also termed HER2-positive breast cancer, carries a poor prognosis, but the development of the HER2 targeted monoclonal antibody trastuzumab (Herceptin) has significantly improved patient survival (
      • Slamon D.J.
      • Clark G.M.
      • Wong S.G.
      • Levin W.J.
      • Ullrich A.
      • McGuire W.L.
      Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
      ). Despite the clinical effectiveness of trastuzumab, the development of drug resistance significantly increases the risk of patient death. This poses a major medical problem, as most metastatic HER2-positive breast cancer patients develop trastuzumab resistance over the course of their cancer treatment (
      • Burris 3rd, H.A.
      • Rugo H.S.
      • Vukelja S.J.
      • Vogel C.L.
      • Borson R.A.
      • Limentani S.
      • Tan-Chiu E.
      • Krop I.E.
      • Michaelson R.A.
      • Girish S.
      • Amler L.
      • Zheng M.
      • Chu Y.W.
      • Klencke B.
      • O'Shaughnessy J.A.
      Phase II study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy.
      ). The treatment approach for HER2+ breast cancer patients after trastuzumab resistance develops is mostly a trial-and-error process that subjects the patient to increased toxicity. Therefore, there is a substantial medical need for strategies to overcome trastuzumab resistance.
      Multiple trastuzumab-resistance mechanisms have been identified, and they alter signaling networks and protein phosphorylation patterns in either a direct or an indirect manner. These mechanisms can be grouped into three categories. The first category is the activation of a parallel signaling network by other tyrosine kinases. These kinases include the receptor tyrosine kinases, EGFR, IGF1R, Her3, Met, EphA2, and Axl, as well as the erythropoietin-receptor-mediated activation of the cytoplasmic tyrosine kinases Jak2 and Src (
      • Ritter C.A.
      • Perez-Torres M.
      • Rinehart C.
      • Guix M.
      • Dugger T.
      • Engelman J.A.
      • Arteaga C.L.
      Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network.
      ,
      • Lu Y.
      • Zi X.
      • Zhao Y.
      • Mascarenhas D.
      • Pollak M.
      Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin).
      ,
      • Nahta R.
      • Yuan L.X.
      • Zhang B.
      • Kobayashi R.
      • Esteva F.J.
      Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells.
      ,
      • Zhuang G.
      • Brantley-Sieders D.M.
      • Vaught D.
      • Yu J.
      • Xie L.
      • Wells S.
      • Jackson D.
      • Muraoka-Cook R.
      • Arteaga C.
      • Chen J.
      Elevation of receptor tyrosine kinase EphA2 mediates resistance to trastuzumab therapy.
      ,
      • Garrett J.T.
      • Olivares M.G.
      • Rinehart C.
      • Granja-Ingram N.D.
      • Sanchez V.
      • Chakrabarty A.
      • Dave B.
      • Cook R.S.
      • Pao W.
      • McKinely E.
      • Manning H.C.
      • Chang J.
      • Arteaga C.L.
      Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase.
      ,
      • Liang K.
      • Esteva F.J.
      • Albarracin C.
      • Stemke-Hale K.
      • Lu Y.
      • Bianchini G.
      • Yang C.Y.
      • Li Y.
      • Li X.
      • Chen C.T.
      • Mills G.B.
      • Hortobagyi G.N.
      • Mendelsohn J.
      • Hung M.C.
      • Fan Z.
      Recombinant human erythropoietin antagonizes trastuzumab treatment of breast cancer cells via Jak2-mediated Src activation and PTEN inactivation.
      ,
      • Zhang S.
      • Huang W.C.
      • Li P.
      • Guo H.
      • Poh S.B.
      • Brady S.W.
      • Xiong Y.
      • Tseng L.M.
      • Li S.H.
      • Ding Z.
      • Sahin A.A.
      • Esteva F.J.
      • Hortobagyi G.N.
      • Yu D.
      Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways.
      ). The second category is the activation of downstream signaling proteins. Multiple studies have demonstrated activation of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway in trastuzumab resistance, which occurs either via deletion of the PTEN lipid phosphatase or mutation of the PI3K genes (
      • Mukohara T.
      Mechanisms of resistance to anti-human epidermal growth factor receptor 2 agents in breast cancer.
      ,
      • Berns K.
      • Horlings H.M.
      • Hennessy B.T.
      • Madiredjo M.
      • Hijmans E.M.
      • Beelen K.
      • Linn S.C.
      • Gonzalez-Angulo A.M.
      • Stemke-Hale K.
      • Hauptmann M.
      • Beijersbergen R.L.
      • Mills G.B.
      • van de Vijver M.J.
      • Bernards R.
      A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.
      ). Activation of Src family kinases or overexpression of cyclin E, which increases the cyclin E–cyclin-dependent kinase 2 signaling pathway, has also been reported (
      • Scaltriti M.
      • Eichhorn P.J.
      • Cortes J.
      • Prudkin L.
      • Aura C.
      • Jimenez J.
      • Chandarlapaty S.
      • Serra V.
      • Prat A.
      • Ibrahim Y.H.
      • Guzman M.
      • Gili M.
      • Rodriguez O.
      • Rodriguez S.
      • Perez J.
      • Green S.R.
      • Mai S.
      • Rosen N.
      • Hudis C.
      • Baselga J.
      Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients.
      ). The third category includes mechanisms that maintain HER2 signaling even in the presence of trastuzumab. The production of a truncated isoform of HER2, p95HER2, which lacks the trastuzumab binding site, causes constitutive HER2 signaling (
      • Arribas J.
      • Baselga J.
      • Pedersen K.
      • Parra-Palau J.L.
      p95HER2 and breast cancer.
      ,
      • Scaltriti M.
      • Rojo F.
      • Ocana A.
      • Anido J.
      • Guzman M.
      • Cortes J.
      • Di Cosimo S.
      • Matias-Guiu X.
      • Ramon y Cajal S.
      • Arribas J.
      • Baselga J.
      Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer.
      ). Overexpression of the MUC4 sialomucin complex inhibits trastuzumab binding to HER2 and thereby maintains HER2 signaling (
      • Nagy P.
      • Friedlander E.
      • Tanner M.
      • Kapanen A.I.
      • Carraway K.L.
      • Isola J.
      • Jovin T.M.
      Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line.
      ,
      • Price-Schiavi S.A.
      • Jepson S.
      • Li P.
      • Arango M.
      • Rudland P.S.
      • Yee L.
      • Carraway K.L.
      Rat MUC4 (sialomucin complex) reduces binding of anti-ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance.
      ).
      Given that multiple trastuzumab-resistance mechanisms alter signaling networks and protein phosphorylation patterns, we reasoned that mapping phosphotyrosine signaling networks using quantitative proteomics would be a highly useful strategy for analyzing known mechanisms and identifying novel mechanisms of trastuzumab resistance. Quantitative proteomics and phosphotyrosine enrichment approaches have been extensively used to study the EGFR signal networks (
      • Olsen J.V.
      • Blagoev B.
      • Gnad F.
      • Macek B.
      • Kumar C.
      • Mortensen P.
      • Mann M.
      Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.
      ,
      • Blagoev B.
      • Ong S.E.
      • Kratchmarova I.
      • Mann M.
      Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.
      ,
      • Kratchmarova I.
      • Blagoev B.
      • Haack-Sorensen M.
      • Kassem M.
      • Mann M.
      Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation.
      ,
      • Wolf-Yadlin A.
      • Kumar N.
      • Zhang Y.
      • Hautaniemi S.
      • Zaman M.
      • Kim H.D.
      • Grantcharova V.
      • Lauffenburger D.A.
      • White F.M.
      Effects of HER2 overexpression on cell signaling networks governing proliferation and migration.
      ,
      • Kumar N.
      • Wolf-Yadlin A.
      • White F.M.
      • Lauffenburger D.A.
      Modeling HER2 effects on cell behavior from mass spectrometry phosphotyrosine data.
      ). We and others have used these approaches to map the HER2 signaling network (
      • Wolf-Yadlin A.
      • Kumar N.
      • Zhang Y.
      • Hautaniemi S.
      • Zaman M.
      • Kim H.D.
      • Grantcharova V.
      • Lauffenburger D.A.
      • White F.M.
      Effects of HER2 overexpression on cell signaling networks governing proliferation and migration.
      ,
      • 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.
      ,
      • Mukherji M.
      • Brill L.M.
      • Ficarro S.B.
      • Hampton G.M.
      • Schultz P.G.
      A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways.
      ). Multiple other tyrosine kinase signaling networks were analyzed using quantitative proteomics, including Ephrin receptor, EphB2 (
      • Zhang G.
      • Fenyo D.
      • Neubert T.A.
      Screening for EphB signaling effectors using SILAC with a linear ion trap-orbitrap mass spectrometer.
      ,
      • Zhang G.
      • Spellman D.S.
      • Skolnik E.Y.
      • Neubert T.A.
      Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC).
      ,
      • Jorgensen C.
      • Sherman A.
      • Chen G.I.
      • Pasculescu A.
      • Poliakov A.
      • Hsiung M.
      • Larsen B.
      • Wilkinson D.G.
      • Linding R.
      • Pawson T.
      Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells.
      ), platelet-derived growth factor receptor (PDGFR) (
      • Kratchmarova I.
      • Blagoev B.
      • Haack-Sorensen M.
      • Kassem M.
      • Mann M.
      Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation.
      ), insulin receptor (
      • Kruger M.
      • Kratchmarova I.
      • Blagoev B.
      • Tseng Y.H.
      • Kahn C.R.
      • Mann M.
      Dissection of the insulin signaling pathway via quantitative phosphoproteomics.
      ,
      • Schmelzle K.
      • Kane S.
      • Gridley S.
      • Lienhard G.E.
      • White F.M.
      Temporal dynamics of tyrosine phosphorylation in insulin signaling.
      ), and the receptor for hepatocyte growth factor, c-MET (
      • Hammond D.E.
      • Hyde R.
      • Kratchmarova I.
      • Beynon R.J.
      • Blagoev B.
      • Clague M.J.
      Quantitative analysis of HGF and EGF-dependent phosphotyrosine signaling networks.
      ).
      The goal of this study is to identify, quantify, and functionally screen proteins that might be involved in trastuzumab resistance. We used two pairs of HER2 gene amplified trastuzumab-sensitive (parental, SkBr3 and BT474) and -resistant (SkBr3R and BT474R) human breast cancer cell lines as models for trastuzumab resistance. These cell lines and their trastuzumab-resistant derivatives have been extensively characterized and highly cited in the breast cancer literature (
      • 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.
      ,
      • Hu X.
      • Stern H.M.
      • Ge L.
      • O'Brien C.
      • Haydu L.
      • Honchell C.D.
      • Haverty P.M.
      • Peters B.A.
      • Wu T.D.
      • Amler L.C.
      • Chant J.
      • Stokoe D.
      • Lackner M.R.
      • Cavet G.
      Genetic alterations and oncogenic pathways associated with breast cancer subtypes.
      ). Using stable isotope labeling of amino acids in cell culture (SILAC),
      The abbreviations used are:
      ER
      estrogen receptor
      IP
      immunoprecipitation
      PANTHER
      Protein Analysis through Evolutionary Relationships
      PPI
      phosphatase and protease inhibitors
      PR
      progesterone receptor
      pTyr
      phosphotyrosine
      SILAC
      stable isotope labeling by amino acids in cell culture
      siRNA
      short interfering RNA.
      1The abbreviations used are:ER
      estrogen receptor
      IP
      immunoprecipitation
      PANTHER
      Protein Analysis through Evolutionary Relationships
      PPI
      phosphatase and protease inhibitors
      PR
      progesterone receptor
      pTyr
      phosphotyrosine
      SILAC
      stable isotope labeling by amino acids in cell culture
      siRNA
      short interfering RNA.
      phosphotyrosine immunoprecipitations, and online TiO2 chromatography with dual trap configuration, we quantified the changes in phosphotyrosine containing proteins and interactors between trastuzumab-sensitive and -resistant cells. Several of the known trastuzumab-resistance mechanisms were identified, which serves as a positive control and validation of our approach, and large protein ratio changes were measured in proteins that had not been previously connected with trastuzumab resistance. We then performed a focused siRNA screen targeting the proteins with significantly increased protein ratios. This screen functionally tested the role of the identified proteins and identifies which proteins might have the largest effect on reversing trastuzumab resistance.

      DISCUSSION

      Trastuzumab resistance represents a serious medical problem, with most metastatic patients developing resistance over the course of their treatment, and this contributes to an increase in patient mortality. In this study, we used quantitative proteomics and phospho-enrichment methods to analyze the changes occurring in trastuzumab-resistant breast cancer cells. We identified both known and potentially novel trastuzumab resistance proteins. A comparison of results obtained from SkBr3 and BT474 cell pairs demonstrates that there is a high degree of diversity in trastuzumab resistance. The HER2 signaling network is known to be complex (
      • Wolf-Yadlin A.
      • Kumar N.
      • Zhang Y.
      • Hautaniemi S.
      • Zaman M.
      • Kim H.D.
      • Grantcharova V.
      • Lauffenburger D.A.
      • White F.M.
      Effects of HER2 overexpression on cell signaling networks governing proliferation and migration.
      ,
      • 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 multiple resistance mechanisms have been previously identified (
      • Mukohara T.
      Mechanisms of resistance to anti-human epidermal growth factor receptor 2 agents in breast cancer.
      ). These results demonstrate that individual breast cancers can develop trastuzumab resistance by a wide variety of means, suggesting that molecular tests to diagnose which resistance mechanisms are active in a patient could be highly clinically useful. The development of such molecular tests could potentially guide future treatment of trastuzumab-resistant HER2 gene amplified breast cancer patients.
      A focused siRNA screen was performed to determine which proteins, of those that showed a significant quantitative change, were functionally relevant to the resistant phenotype. A focused siRNA screen of the proteomic identifications is a time- and cost-effective strategy to evaluate their functional role. The siRNA strategy was designed to identify which proteins might be good drug targets for overcoming trastuzumab resistance, and it employed a library tailored to the proteins with increased ratios in the resistant cells. A converse screening strategy using siRNA to induce resistance in the sensitive cells could yield functional information on proteins with a decreased ratio. This approach is conceptually similar to the genome-wide siRNA screen performed by Berns et al. (
      • Berns K.
      • Horlings H.M.
      • Hennessy B.T.
      • Madiredjo M.
      • Hijmans E.M.
      • Beelen K.
      • Linn S.C.
      • Gonzalez-Angulo A.M.
      • Stemke-Hale K.
      • Hauptmann M.
      • Beijersbergen R.L.
      • Mills G.B.
      • van de Vijver M.J.
      • Bernards R.
      A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.
      ).
      Our quantitative phosphoproteomics-siRNA screening strategy revealed several proteins related to the Src kinase pathway, including CDCP1/Trask, embryonal Fyn-associated substrate, epiplakin, focal adhesion kinase, and Paxillin. Src has recently emerged as a promising therapeutic target for overcoming trastuzumab resistance (
      • Zhang S.
      • Huang W.C.
      • Li P.
      • Guo H.
      • Poh S.B.
      • Brady S.W.
      • Xiong Y.
      • Tseng L.M.
      • Li S.H.
      • Ding Z.
      • Sahin A.A.
      • Esteva F.J.
      • Hortobagyi G.N.
      • Yu D.
      Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways.
      ), and our finding that Src-interactors have a mitigating effect in resistant cells increases the confidence in these results. In addition, several novel proteins involved in trastuzumab resistance were identified. FAM83A is a putative prognostic marker for lung cancer, but its role in breast cancer is not known, and its ability to increase trastuzumab sensitivity in these studies warrants further investigation into its biological function. Knockdown of MAPK1 reversed trastuzumab resistance in SkBr3 cells, suggesting that combining MAP kinase pathway inhibitors with HER2 targeted drugs is a potential avenue for new therapy.
      With the diversity of mechanisms that our study and others have indicated, there is an urgent need for diagnostic markers and therapeutic targets to guide the treatment of patients with trastuzumab-resistant breast cancers. The results from these analyses warrant future investigations into the specific roles that these novel proteins play in trastuzumab resistance.

      Acknowledgments

      We thank Michael L. Gross, Henry Rohrs, Leslie Hicks, and Sophie Alvarez for mass spectrometry instrument access and support. We also thank Dennis Slamon and Gottfried Konecny for generously providing the cell lines used in this study.
      The data associated with this manuscript may be downloaded from the Proteome Commons Tranche using the following hash:
      dCdKmk7RydWT76hBKX49gCu2yFHotyzqM9BsPDjGTXYQ5pch8PgvtS5EOYENrDZDafcZhUjf2z6BlliHcPGEBg+amtQAAAAAAABKhg==
      The hash may be used to prove exactly what files were published as part of this manuscript's dataset, and the hash may also be used to check that the data have not changed since publication.

      REFERENCES

        • Hynes N.E.
        • Lane H.A.
        ERBB receptors and cancer: the complexity of targeted inhibitors.
        Nat. Rev. Cancer. 2005; 5: 341-354
        • Slamon D.J.
        • Clark G.M.
        • Wong S.G.
        • Levin W.J.
        • Ullrich A.
        • McGuire W.L.
        Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
        Science. 1987; 235: 177-182
        • Slamon D.J.
        • Godolphin W.
        • Jones L.A.
        • Holt J.A.
        • Wong S.G.
        • Keith D.E.
        • Levin W.J.
        • Stuart S.G.
        • Udove J.
        • Ullrich A.
        • Press M.F.
        Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
        Science. 1989; 244: 707-712
        • Burris 3rd, H.A.
        • Rugo H.S.
        • Vukelja S.J.
        • Vogel C.L.
        • Borson R.A.
        • Limentani S.
        • Tan-Chiu E.
        • Krop I.E.
        • Michaelson R.A.
        • Girish S.
        • Amler L.
        • Zheng M.
        • Chu Y.W.
        • Klencke B.
        • O'Shaughnessy J.A.
        Phase II study of the antibody drug conjugate trastuzumab-DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy.
        J. Clin. Oncol. 2011; 29: 398-405
        • Ritter C.A.
        • Perez-Torres M.
        • Rinehart C.
        • Guix M.
        • Dugger T.
        • Engelman J.A.
        • Arteaga C.L.
        Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network.
        Clin. Cancer Res. 2007; 13: 4909-4919
        • Lu Y.
        • Zi X.
        • Zhao Y.
        • Mascarenhas D.
        • Pollak M.
        Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin).
        J. Natl. Cancer Inst. 2001; 93: 1852-1857
        • Nahta R.
        • Yuan L.X.
        • Zhang B.
        • Kobayashi R.
        • Esteva F.J.
        Insulin-like growth factor-I receptor/human epidermal growth factor receptor 2 heterodimerization contributes to trastuzumab resistance of breast cancer cells.
        Cancer Res. 2005; 65: 11118-11128
        • Zhuang G.
        • Brantley-Sieders D.M.
        • Vaught D.
        • Yu J.
        • Xie L.
        • Wells S.
        • Jackson D.
        • Muraoka-Cook R.
        • Arteaga C.
        • Chen J.
        Elevation of receptor tyrosine kinase EphA2 mediates resistance to trastuzumab therapy.
        Cancer Res. 2010; 70: 299-308
        • Garrett J.T.
        • Olivares M.G.
        • Rinehart C.
        • Granja-Ingram N.D.
        • Sanchez V.
        • Chakrabarty A.
        • Dave B.
        • Cook R.S.
        • Pao W.
        • McKinely E.
        • Manning H.C.
        • Chang J.
        • Arteaga C.L.
        Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 5021-5026
        • Liang K.
        • Esteva F.J.
        • Albarracin C.
        • Stemke-Hale K.
        • Lu Y.
        • Bianchini G.
        • Yang C.Y.
        • Li Y.
        • Li X.
        • Chen C.T.
        • Mills G.B.
        • Hortobagyi G.N.
        • Mendelsohn J.
        • Hung M.C.
        • Fan Z.
        Recombinant human erythropoietin antagonizes trastuzumab treatment of breast cancer cells via Jak2-mediated Src activation and PTEN inactivation.
        Cancer Cell. 2010; 18: 423-435
        • Zhang S.
        • Huang W.C.
        • Li P.
        • Guo H.
        • Poh S.B.
        • Brady S.W.
        • Xiong Y.
        • Tseng L.M.
        • Li S.H.
        • Ding Z.
        • Sahin A.A.
        • Esteva F.J.
        • Hortobagyi G.N.
        • Yu D.
        Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways.
        Nat. Med. 2011; 17: 461-469
        • Mukohara T.
        Mechanisms of resistance to anti-human epidermal growth factor receptor 2 agents in breast cancer.
        Cancer Sci. 2011; 102: 1-8
        • Berns K.
        • Horlings H.M.
        • Hennessy B.T.
        • Madiredjo M.
        • Hijmans E.M.
        • Beelen K.
        • Linn S.C.
        • Gonzalez-Angulo A.M.
        • Stemke-Hale K.
        • Hauptmann M.
        • Beijersbergen R.L.
        • Mills G.B.
        • van de Vijver M.J.
        • Bernards R.
        A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer.
        Cancer Cell. 2007; 12: 395-402
        • Scaltriti M.
        • Eichhorn P.J.
        • Cortes J.
        • Prudkin L.
        • Aura C.
        • Jimenez J.
        • Chandarlapaty S.
        • Serra V.
        • Prat A.
        • Ibrahim Y.H.
        • Guzman M.
        • Gili M.
        • Rodriguez O.
        • Rodriguez S.
        • Perez J.
        • Green S.R.
        • Mai S.
        • Rosen N.
        • Hudis C.
        • Baselga J.
        Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 3761-3766
        • Arribas J.
        • Baselga J.
        • Pedersen K.
        • Parra-Palau J.L.
        p95HER2 and breast cancer.
        Cancer Res. 2011; 71: 1515-1519
        • Scaltriti M.
        • Rojo F.
        • Ocana A.
        • Anido J.
        • Guzman M.
        • Cortes J.
        • Di Cosimo S.
        • Matias-Guiu X.
        • Ramon y Cajal S.
        • Arribas J.
        • Baselga J.
        Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer.
        J. Natl. Cancer Inst. 2007; 99: 628-638
        • Nagy P.
        • Friedlander E.
        • Tanner M.
        • Kapanen A.I.
        • Carraway K.L.
        • Isola J.
        • Jovin T.M.
        Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line.
        Cancer Res. 2005; 65: 473-482
        • Price-Schiavi S.A.
        • Jepson S.
        • Li P.
        • Arango M.
        • Rudland P.S.
        • Yee L.
        • Carraway K.L.
        Rat MUC4 (sialomucin complex) reduces binding of anti-ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance.
        Int. J. Cancer. 2002; 99: 783-791
        • Olsen J.V.
        • Blagoev B.
        • Gnad F.
        • Macek B.
        • Kumar C.
        • Mortensen P.
        • Mann M.
        Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.
        Cell. 2006; 127: 635-648
        • Blagoev B.
        • Ong S.E.
        • Kratchmarova I.
        • Mann M.
        Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.
        Nat. Biotechnol. 2004; 22: 1139-1145
        • Kratchmarova I.
        • Blagoev B.
        • Haack-Sorensen M.
        • Kassem M.
        • Mann M.
        Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation.
        Science. 2005; 308: 1472-1477
        • Wolf-Yadlin A.
        • Kumar N.
        • Zhang Y.
        • Hautaniemi S.
        • Zaman M.
        • Kim H.D.
        • Grantcharova V.
        • Lauffenburger D.A.
        • White F.M.
        Effects of HER2 overexpression on cell signaling networks governing proliferation and migration.
        Mol. Syst. Biol. 2006; 2: 54
        • Kumar N.
        • Wolf-Yadlin A.
        • White F.M.
        • Lauffenburger D.A.
        Modeling HER2 effects on cell behavior from mass spectrometry phosphotyrosine data.
        PLoS Comput. Biol. 2007; 3: e4
        • 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.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9773-9778
        • Mukherji M.
        • Brill L.M.
        • Ficarro S.B.
        • Hampton G.M.
        • Schultz P.G.
        A phosphoproteomic analysis of the ErbB2 receptor tyrosine kinase signaling pathways.
        Biochemistry. 2006; 45: 15529-15540
        • Zhang G.
        • Fenyo D.
        • Neubert T.A.
        Screening for EphB signaling effectors using SILAC with a linear ion trap-orbitrap mass spectrometer.
        J. Proteome Res. 2008; 7: 4715-4726
        • Zhang G.
        • Spellman D.S.
        • Skolnik E.Y.
        • Neubert T.A.
        Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC).
        J. Proteome Res. 2006; 5: 581-588
        • Jorgensen C.
        • Sherman A.
        • Chen G.I.
        • Pasculescu A.
        • Poliakov A.
        • Hsiung M.
        • Larsen B.
        • Wilkinson D.G.
        • Linding R.
        • Pawson T.
        Cell-specific information processing in segregating populations of Eph receptor ephrin-expressing cells.
        Science. 2009; 326: 1502-1509
        • Kruger M.
        • Kratchmarova I.
        • Blagoev B.
        • Tseng Y.H.
        • Kahn C.R.
        • Mann M.
        Dissection of the insulin signaling pathway via quantitative phosphoproteomics.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2451-2456
        • Schmelzle K.
        • Kane S.
        • Gridley S.
        • Lienhard G.E.
        • White F.M.
        Temporal dynamics of tyrosine phosphorylation in insulin signaling.
        Diabetes. 2006; 55: 2171-2179
        • Hammond D.E.
        • Hyde R.
        • Kratchmarova I.
        • Beynon R.J.
        • Blagoev B.
        • Clague M.J.
        Quantitative analysis of HGF and EGF-dependent phosphotyrosine signaling networks.
        J Proteome Res. 2010; 9: 2734-2742
        • 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.
        Cancer Res. 2006; 66: 1630-1639
        • Hu X.
        • Stern H.M.
        • Ge L.
        • O'Brien C.
        • Haydu L.
        • Honchell C.D.
        • Haverty P.M.
        • Peters B.A.
        • Wu T.D.
        • Amler L.C.
        • Chant J.
        • Stokoe D.
        • Lackner M.R.
        • Cavet G.
        Genetic alterations and oncogenic pathways associated with breast cancer subtypes.
        Mol. Cancer Res. 2009; 7: 511-522
        • Pimienta G.
        • Chaerkady R.
        • Pandey A.
        SILAC for global phosphoproteomic analysis.
        Methods Mol. Biol. 2009; 527: 107-116
        • Pinske M.W.H.
        • Uitto P.M.
        • Hilhorst M.J.
        • Ooms B.
        • Heck A.J.R.
        Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-nanoLC-ESI-MS/MS and titanium dioxide precolumns.
        Anal. Chem. 2006; 76: 3935-3943
        • Larsen M.R.
        • Thingholm T.E.
        • Jensen O.N.
        • Roepstorff P.
        • Jorgenson T.J.D.
        Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns.
        Mol. Cell. Proteomics. 2005; 4: 873-886
        • Thingholm T.E.
        • Jorgensen T.J.D.
        • Jensen O.N.
        • Larsen M.R.
        Highly selective enrichment of phosphorylated peptides using titanium dioxide.
        Nat. Protoc. 2006; 1: 1929-1935
        • Syka J.E.P.
        • Marto J.A.
        • Bai D.L.
        • Horning S.
        • Senko M.W.
        • Schwartz J.C.
        • Ueberheide B.
        • Gacia B.
        • Busby S.
        • Muratore T.
        • Shabanowitz J.
        • Hunt D.F.
        Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post-translational modifications.
        J. Proteome Res. 2004; 3: 621-626
        • Cox J.
        • Mann M.
        MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide quantification.
        Nat. Biotechnol. 2008; 26: 1367-1372
        • Cox J.
        • Neuhauser N.
        • Michalski A.
        • Scheltema R.A.
        • Olsen J.V.
        • Mann M.
        Andromeda: a peptide search engine integrated into the MaxQuant environment.
        J. Proteome Res. 2011; 10: 1794-1805
        • Thomas P.D.
        • Campbell M.J.
        • Kejariwal A.
        • Mi H.
        • Karlak B.
        • Daverman R.
        • Diemer K.
        • Muruganujan A.
        • Narechania A.
        PANTHER: a library of protein families and subfamilies indexed by function.
        Genome Res. 2003; 13: 2129-2141
        • Thomas P.D.
        • Kejariwal A.
        • Guo N.
        • Mi H.
        • Campbell M.J.
        • Muruganujan A.
        • Lazareva-Ulitsky B.
        Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools.
        Nucleic Acids Res. 2006; 34: W645-W650
        • Page B.
        • Page M.
        • Noel C.
        A new fluorometric assay for cytotoxicity measurements in-vitro.
        Int. J. Oncol. 1993; 3: 473-476
        • Ong S.E.
        • Blagoev B.
        • Kratchmarova I.
        • Kristensen D.B.
        • Steen H.
        • Pandey A.
        • Mann M.
        Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.
        Mol. Cell. Proteomics. 2002; 1: 376-386
        • Pinkse M.W.
        • Mohammed S.
        • Gouw J.W.
        • van Breukelen B.
        • Vos H.R.
        • Heck A.J.
        Highly robust, automated, and sensitive online TiO2-based phosphoproteomics applied to study endogenous phosphorylation in Drosophila melanogaster.
        J. Proteome Res. 2008; 7: 687-697
        • Kao J.
        • Salari K.
        • Bocanegra M.
        • Choi Y.L.
        • Girard L.
        • Gandhi J.
        • Kwei K.A.
        • Hernandez-Boussard T.
        • Wang P.
        • Gazdar A.F.
        • Minna J.D.
        • Pollack J.R.
        Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery.
        PLoS One. 2009; 4: e6146
        • Liu H.
        • Ong S.E.
        • Badu-Nkansah K.
        • Schindler J.
        • White F.M.
        • Hynes R.O.
        CUB-domain-containing protein 1 (CDCP1) activates Src to promote melanoma metastasis.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 1379-1384
        • Razorenova O.V.
        • Finger E.C.
        • Colavitti R.
        • Chernikova S.B.
        • Boiko A.D.
        • Chan C.K.
        • Krieg A.
        • Bedogni B.
        • LaGory E.
        • Weissman I.L.
        • Broome-Powell M.
        • Giaccia A.J.
        VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC{delta}-driven migration.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 1931-1936
        • Spassov D.S.
        • Wong C.H.
        • Sergina N.
        • Ahuja D.
        • Fried M.
        • Sheppard D.
        • Moasser M.M.
        Phosphorylation of Trask by Src kinases inhibits integrin clustering and functions in exclusion with focal adhesion signaling.
        Mol. Cell. Biol. 2011; 31: 766-782
        • Guan J.L.
        Role of focal adhesion kinase in integrin signaling.
        Int. J. Biochem. Cell Biol. 1997; 29: 1085-1096
        • Mitra S.K.
        • Schlaepfer D.D.
        Integrin-regulated FAK-Src signaling in normal and cancer cells.
        Curr. Opin. Cell Biol. 2006; 18: 516-523
        • Thelemann A.
        • Petti F.
        • Griffin G.
        • Iwata K.
        • Hunt T.
        • Settinari T.
        • Fenyo D.
        • Gibson N.
        • Haley J.D.
        Phosphotyrosine signaling networks in epidermal growth factor receptor overexpressing squamous carcinoma cells.
        Mol. Cell. Proteomics. 2005; 4: 356-376
        • Ucar D.A.
        • Dang L.H.
        • Hochwald S.N.
        Focal adhesion kinase signaling and function in pancreatic cancer.
        Front. Biosci. (Elite Ed.). 2011; 3: 750-756
        • Zhao X.
        • Guan J.L.
        Focal adhesion kinase and its signaling pathways in cell migration and angiogenesis.
        Adv. Drug Deliv. Rev. 2011; 63: 610-615
        • Surawska H.
        • Ma P.C.
        • Salgia R.
        The role of ephrins and Eph receptors in cancer.
        Cytokine Growth Factor Rev. 2004; 15: 419-433
        • Korpal M.
        • Kang Y.
        Targeting the transforming growth factor-beta signalling pathway in metastatic cancer.
        Eur. J. Cancer. 2004; 46: 1232-1240
        • Schniewind B.
        • Groth S.
        • Sebens Muerkoster S.
        • Sipos B.
        • Schafer H.
        • Kalthoff H.
        • Fandrich F.
        • Ungefroren H.
        Dissecting the role of TGF-beta type I receptor/ALK5 in pancreatic ductal adenocarcinoma: Smad activation is crucial for both the tumor suppressive and prometastatic function.
        Oncogene. 2007; 26: 4850-4862
        • Siegel P.M.
        • Shu W.
        • Cardiff R.D.
        • Muller W.J.
        • Massague J.
        Transforming growth factor beta signaling impairs Neu-induced mammary tumorigenesis while promoting pulmonary metastasis.
        Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 8430-8435
        • Zheng W.
        Genetic polymorphisms in the transforming growth factor-beta signaling pathways and breast cancer risk and survival.
        Methods Mol. Biol. 2009; 472: 265-277
        • Xia X.
        • Cheng A.
        • Lessor T.
        • Zhang Y.
        • Hamburger A.W.
        Ebp1, an ErbB-3 binding protein, interacts with Rb and affects Rb transcriptional regulation.
        J. Cell. Physiol. 2001; 187: 209-217
        • Zhang Y.
        • Woodford N.
        • Xia X.
        • Hamburger A.W.
        Repression of E2F1-mediated transcription by the ErbB3 binding protein Ebp1 involves histone deacetylases.
        Nucleic Acids Res. 2003; 31: 2168-2177
        • Li Y.
        • Dong X.
        • Yin Y.
        • Su Y.
        • Xu Q.
        • Zhang Y.
        • Pang X.
        • Chen W.
        BJ-TSA-9, a novel human tumor-specific gene, has potential as a biomarker of lung cancer.
        Neoplasia. 2005; 7: 1073-1080
        • Liu L.
        • Liao G.Q.
        • He P.
        • Zhu H.
        • Liu P.H.
        • Qu Y.M.
        • Song X.M.
        • Xu Q.W.
        • Gao Q.
        • Zhang Y.
        • Chen W.F.
        • Yin Y.H.
        Detection of circulating cancer cells in lung cancer patients with a panel of marker genes.
        Biochem. Biophys. Res. Commun. 2008; 372: 756-760
        • Du Q.
        • Zhang Y.
        • Tian X.X.
        • Li Y.
        • Fang W.G.
        MAGE-D1 inhibits proliferation, migration and invasion of human breast cancer cells.
        Oncol. Rep. 2009; 22: 659-665
        • Kobayashi D.
        • Kumagai J.
        • Morikawa T.
        • Wilson-Morifuji M.
        • Wilson A.
        • Irie A.
        • Araki N.
        An integrated approach of differential mass spectrometry and gene ontology analysis identified novel proteins regulating neuronal differentiation and survival.
        Mol. Cell. Proteomics. 2009; 8: 2350-2367
        • Chapman M.A.
        • Lawrence M.S.
        • Keats J.J.
        • Cibulskis K.
        • Sougnez C.
        • Schinzel A.C.
        • Harview C.L.
        • Brunet J.P.
        • Ahmann G.J.
        • Adli M.
        • Anderson K.C.
        • Ardlie K.G.
        • Auclair D.
        • Baker A.
        • Bergsagel P.L.
        • Bernstein B.E.
        • Drier Y.
        • Fonseca R.
        • Gabriel S.B.
        • Hofmeister C.C.
        • Jagannath S.
        • Jakubowiak A.J.
        • Krishnan A.
        • Levy J.
        • Liefeld T.
        • Lonial S.
        • Mahan S.
        • Mfuko B.
        • Monti S.
        • Perkins L.M.
        • Onofrio R.
        • Pugh T.J.
        • Rajkumar S.V.
        • Ramos A.H.
        • Siegel D.S.
        • Sivachenko A.
        • Stewart A.K.
        • Trudel S.
        • Vij R.
        • Voet D.
        • Winckler W.
        • Zimmerman T.
        • Carpten J.
        • Trent J.
        • Hahn W.C.
        • Garraway L.A.
        • Meyerson M.
        • Lander E.S.
        • Getz G.
        • Golub T.R.
        Initial genome sequencing and analysis of multiple myeloma.
        Nature. 2011; 471: 467-472
        • Sundvall M.
        • Iljin K.
        • Kilpinen S.
        • Sara H.
        • Kallioniemi O.P.
        • Elenius K.
        Role of ErbB4 in breast cancer.
        J. Mammary Gland Biol. Neoplasia. 2008; 13: 259-268
        • Muraoka-Cook R.S.
        • Feng S.M.
        • Strunk K.E.
        • Earp 3rd, H.S.
        ErbB4/HER4: role in mammary gland development, differentiation and growth inhibition.
        J. Mammary Gland Biol. Neoplasia. 2008; 13: 235-246
        • Suo Z.
        • Risberg B.
        • Kalsson M.G.
        • Willman K.
        • Tierens A.
        • Skovlund E.
        • Nesland J.M.
        EGFR family expression in breast carcinomas. c-erbB-2 and c-erbB-4 receptors have different effects on survival.
        J. Pathol. 2002; 196: 17-25
        • Barnes N.L.
        • Khavari S.
        • Boland G.P.
        • Cramer A.
        • Knox W.F.
        • Bundred N.J.
        Absence of HER4 expression predicts recurrence of ductal carcinoma in situ of the breast.
        Clin. Cancer Res. 2005; 11: 2163-2168