Abstract
Despite the initial effectiveness of the tyrosine kinase inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, implying that tumors acquire mechanisms of drug resistance. To discover these mechanisms, we generated six lapatinib-resistant HER2-overexpressing human breast cancer cell lines. In cells that grew in the presence of lapatinib, HER2 autophosphorylation was undetectable, whereas active phosphoinositide-3 kinase (PI3K)-Akt and mitogen-activated protein kinase (MAPK) were maintained. To identify networks maintaining these signaling pathways, we profiled the tyrosine phosphoproteome of sensitive and resistant cells using an immunoaffinity-enriched mass spectrometry method. We found increased phosphorylation of Src family kinases (SFKs) and putative Src substrates in several resistant cell lines. Treatment of these resistant cells with Src kinase inhibitors partially blocked PI3K-Akt signaling and restored lapatinib sensitivity. Further, SFK mRNA expression was upregulated in primary HER2+ tumors treated with lapatinib. Finally, the combination of lapatinib and the Src inhibitor AZD0530 was more effective than lapatinib alone at inhibiting pAkt and growth of established HER2-positive BT-474 xenografts in athymic mice. These data suggest that increased Src kinase activity is a mechanism of lapatinib resistance and support the combination of HER2 antagonists with Src inhibitors early in the treatment of HER2+ breast cancers in order to prevent or overcome resistance to HER2 inhibitors.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Amin DN, Sergina N, Ahuja D, McMahon M, Blair A, Wang D et al. (2010). Resiliency and vulnerability in the HER2-HER3 tumorigenic driver. Sci Transl Med 2: 16ra17.
Belsches-Jablonski AP, Biscardi JS, Peavy DR, Tice DA, Romney DA, Parsons SJ . (2001). Src family kinases and HER2 interactions in human breast cancer cell growth and survival. Oncogene 20: 1465–1475.
Berns K, Horlings HM, Hennessy BT, Madiredjo M, Hijmans EM, Beelen K et al. (2007). A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 12: 395–402.
Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ . (1999). c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J Biol Chem 274: 8335–8343.
Bose R, Molina H, Patterson AS, Bitok JK, Periaswamy B, Bader JS et al. (2006). Phosphoproteomic analysis of Her2/neu signaling and inhibition. Proc Natl Acad Sci USA 103: 9773–9778.
Burris HA, Hurwitz HI, Dees EC, Dowlati A, Blackwell KL, O'Neil B et al. (2005). Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas. J Clin Oncol 23: 5305–5313.
Dave B, Migliaccio I, Gutierrez MC, Wu MF, Chamness GC, Wong H et al. (2011). Loss of phosphatase and tensin homolog or phosphoinositol-3 kinase activation and response to trastuzumab or lapatinib in human epidermal growth factor receptor 2-overexpressing locally advanced breast cancers. J Clin Oncol 29: 166–173.
Eichhorn PJA, Gili M, Scaltriti M, Serra V, Guzman M, Nijkamp W et al. (2008). Phosphatidylinositol 3-kinase hyperactivation results in lapatinib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235. Cancer Res 68: 9221–9230.
Erlichman C, Menefee ME, Northfelt DW, Qin R, Reid JM, Lingle WL et al. (2009). Abstract B56: a phase I trial of the combination of dasatinib and lapatinib—Erlichman et al. 8 (1001): B56—Molecular Cancer Therapeutics. Mol Cancer Ther 8: B56.
Finn R . (2008). Targeting Src in breast cancer. Ann Oncol 19: 1379.
Finn RS, Dering J, Ginther C, Wilson CA, Glaspy P, Tchekmedyian N et al. (2007). Dasatinib, an orally active small molecule inhibitor of both the src and abl kinases, selectively inhibits growth of basal-type/’triple-negative’ breast cancer cell lines growing in vitro. Breast Cancer Res Treat 105: 319–326.
Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski T et al. (2006). Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 355: 2733–2743.
Green T, Fennell M, Whittaker R, Curwen J, Jacobs V, Allen J et al. (2009). Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530. Mol Oncol 3: 248–261.
Hennequin LF, Allen J, Breed J, Curwen J, Fennell M, Green TP et al. (2006). N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor. J Med Chem 49: 6465–6488.
Hochgrafe F, Zhang L, O'Toole SA, Browne BC, Pinese M, Porta Cubas A et al. (2010). Tyrosine phosphorylation profiling reveals the signaling network characteristics of basal breast cancer cells. Cancer Res 70: 9391–9401.
Huang F, Reeves K, Han X, Fairchild C, Platero S, Wong TW et al. (2007). Identification of candidate molecular markers predicting sensitivity in solid tumors to dasatinib: rationale for patient selection. Cancer Res 67: 2226–2238.
Hubbard SR, Till JH . (2000). Protein tyrosine kinase structure and function. Annu Rev Biochem 69: 373–398.
Ishizawar RC, Miyake T, Parsons SJ . (2007). c-Src modulates ErbB2 and ErbB3 heterocomplex formation and function. Oncogene 26: 3503–3510.
Jones RJ, Young O, Renshaw L, Jacobs V, Fennell M, Marshall A et al. (2009). Src inhibitors in early breast cancer: a methodology, feasibility and variability study. Breast Cancer Res Treat 114: 211–221.
Junttila TT, Akita RW, Parsons K, Fields C, Lewis Phillips GD, Friedman LS et al. (2009). Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941. Cancer Cell 15: 429–440.
Kim H, Chan R, Dankort DL, Zuo D, Najoukas M, Park M et al. (2005). The c-Src tyrosine kinase associates with the catalytic domain of ErbB-2: implications for ErbB-2 mediated signaling and transformation. Oncogene 24: 7599–7607.
Konecny GE, Pegram MD, Venkatesan N, Finn R, Yang G, Rahmeh M et al. (2006). Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res 66: 1630–1639.
Lachmann A, Ma'ayan A . (2009). KEA: kinase enrichment analysis. Bioinformatics 25: 684–686.
Lee-Hoeflich ST, Crocker L, Yao E, Pham T, Munroe X, Hoeflich KP et al. (2008). A central role for HER3 in HER2-amplified breast cancer: implications for targeted therapy. Cancer Res 68: 5878–5887.
Lenferink AE, Busse D, Flanagan WM, Yakes FM, Arteaga CL . (2001). ErbB2/neu kinase modulates cellular p27(Kip1) and cyclin D1 through multiple signaling pathways. Cancer Res 61: 6583–6591.
Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC, Behnia K et al. (2004). Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J Med Chem 47: 6658–6661.
Luo W, Slebos RJ, Hill S, Li M, Brábek J, Amanchy R et al. (2008). Global impact of oncogenic Src on a phosphotyrosine proteome. J Proteome Res 7: 3447–3460.
Luttrell DK, Lee A, Lansing TJ, Crosby RM, Jung KD, Willard D et al. (1994). Involvement of pp60c-src with two major signaling pathways in human breast cancer. Proc Natl Acad Sci USA 91: 83.
Maira S-M, Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C et al. (2008). Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 7: 1851–1863.
Marcotte R, Zhou L, Kim H, Roskelly CD, Muller WJ . (2009). c-Src associates with ErbB2 through an interaction between catalytic domains and confers enhanced transforming potential. Mol Cell Biol 29: 5858–5871.
Moasser MM . (2007). Targeting the function of the HER2 oncogene in human cancer therapeutics. Oncogene 26: 6577–6592.
Nagata Y, Lan K-H, Zhou X, Tan M, Esteva FJ, Sahin AA et al. (2004). PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6: 117–127.
Old WM, Meyer-Arendt K, Aveline-Wolf L, Pierce KG, Mendoza A, Sevinsky JR et al. (2005). Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 4: 1487–1502.
Ritter CA, Perez-Torres M, Rinehart C, Guix M, Dugger T, Engelman JA et al. (2007). 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 13: 4909–4919.
Roskoski R . (2005). Src kinase regulation by phosphorylation and dephosphorylation. Biochem Biophys Res Commun 331: 1–14.
Rush J, Moritz A, Lee KA, Guo A, Goss VL, Spek EJ et al. (2005). Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotechnol 23: 94–101.
Rusnak DW, Lackey K, Affleck K, Wood ER, Alligood KJ, Rhodes N et al. (2001). The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 1: 85–94.
Sergina NV, Rausch M, Wang D, Blair J, Hann B, Shokat KM et al. (2007). Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3. Nature 445: 437–441.
Serra V, Markman B, Scaltriti M, Eichhorn PJA, Valero V, Guzman M et al. (2008). NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Cancer Res 68: 8022–8030.
Sheffield LG . (1998). C-Src activation by ErbB2 leads to attachment-independent growth of human breast epithelial cells. Biochem Biophys Res Commun 250: 27–31.
Tabb DL, Fernando CG, Chambers MC . (2007). MyriMatch: highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis. J Proteome Res 6: 654–661.
Tan M, Li P, Klos KS, Lu J, Lan KH, Nagata Y et al. (2005). ErbB2 promotes Src synthesis and stability: novel mechanisms of Src activation that confer breast cancer metastasis. Cancer Res 65: 1858–1867.
Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB et al. (2006). Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Mol Cancer Ther 5: 2512–2521.
Tice DA, Biscardi JS, Nickles AL, Parsons SJ . (1999). Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc Natl Acad Sci USA 96: 1415–1420.
Vadlamudi RK, Sahin AA, Adam L, Wang RA, Kumar R . (2003). Heregulin and HER2 signaling selectively activates c-Src phosphorylation at tyrosine 215. FEBS Lett 543: 76–80.
Wang SE, Narasanna A, Perez-Torres M, Xiang B, Wu FY, Yang S et al. (2006). HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors. Cancer Cell 10: 25–38.
Xu W, Yuan X, Beebe K, Xiang Z, Neckers L . (2007). Loss of Hsp90 association up-regulates Src-dependent ErbB2 activity. Mol Cell Biol 27: 220–228.
Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL . (2002). Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 62: 4132–4141.
Yarden Y, Sliwkowski MX . (2001). Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2: 127–137.
Yeatman TJ . (2004). A renaissance for SRC. Nat Rev Cancer 4: 470–480.
Zhang B, Chambers MC, Tabb DL . (2007). Proteomic parsimony through bipartite graph analysis improves accuracy and transparency. J Proteome Res 6: 3549–3557.
Zhang B, VerBerkmoes NC, Langston MA, Uberbacher E, Hettich RL, Samatova NF . (2006). Detecting differential and correlated protein expression in label-free shotgun proteomics. J Proteome Res 5: 2909–2918.
Zhang HT, O'Rourke DM, Zhao H, Murali R, Mikami Y, Davis JG et al. (1998). Absence of autophosphorylation site Y882 in the p185neu oncogene product correlates with a reduction of transforming potential. Oncogene 16: 2835–2842.
Acknowledgements
This work was supported by NIH R01 CA80195 (CLA), Dinah Armstrong Kukes Fund/Phi Mu foundation (BNR), T32 CA119910 (BNR), Department of Defense BC087465 Post-Doctoral Fellowship (BNR), ASCO Young Investigator Award (BNR), Kleberg Center for Molecular Markers at MD Anderson Cancer Center, ASCO Career Development Award (AMG), NCI 1K23CA121994-01 (AMG), Susan G Komen Foundation FAS0703849 (AMG, GBM), ACS Clinical Research Professorship CRP-07-234 (CLA), Lee Jeans Translational Breast Cancer Research Program (CLA), Breast Cancer SPORE P50 CA98131 and Vanderbilt-Ingram Cancer Center Support Grant P30 CA68485.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies the paper on the Oncogene website
Supplementary information
Rights and permissions
About this article
Cite this article
Rexer, B., Ham, AJ., Rinehart, C. et al. Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition. Oncogene 30, 4163–4174 (2011). https://doi.org/10.1038/onc.2011.130
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/onc.2011.130
Keywords
This article is cited by
-
Trastuzumab-resistant breast cancer cells-derived tumor xenograft models exhibit distinct sensitivity to lapatinib treatment in vivo
Biological Procedures Online (2023)
-
The emerging role of mass spectrometry-based proteomics in drug discovery
Nature Reviews Drug Discovery (2022)
-
Src family kinases, adaptor proteins and the actin cytoskeleton in epithelial-to-mesenchymal transition
Cell Communication and Signaling (2021)
-
Adipocyte-conditioned medium induces resistance of breast cancer cells to lapatinib
BMC Pharmacology and Toxicology (2020)
-
Monitoring protein communities and their responses to therapeutics
Nature Reviews Drug Discovery (2020)