Oncogene (2011) 30, 4163–4174 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition BN Rexer1,A-JLHam2,3,CRinehart1, S Hill2, N de Matos Granja-Ingram4, AM Gonza´lez-Angulo5,6, GB Mills5,7, B Dave8, JC Chang9, DC Liebler2,3 and CL Arteaga1,10,11 1Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA; 2Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA; 3Ayers Institute of Precancer Detection and Diagnosis, Vanderbilt University School of Medicine, Nashville, TN, USA; 4Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN, USA; 5Department of Systems Biology, MD Anderson Cancer Center, Houston, TX, USA; 6Department of Breast Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA; 7Department of Gynecologic Medical Oncology, MD Anderson Cancer Center, Houston, TX, USA; 8Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, USA; 9Department of Medicine, The Methodist Hospital, Houston, TX, USA; 10Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, USA and 11Department of Breast Cancer Research Program, Vanderbilt-Ingram Cancer Center, Nashville, TN, USA Despite the initial effectiveness of the tyrosine kinase Introduction inhibitor lapatinib against HER2 gene-amplified breast cancers, most patients eventually relapse after treatment, HER2 (ErbB2) is a member of the ErbB family of implying that tumors acquire mechanisms of drug receptor tyrosine kinases that includes the epidermal resistance. To discover these mechanisms, we generated growth factor receptor (EGFR, HER1), HER3 and six lapatinib-resistant HER2-overexpressing human breast HER4. Dimerization of HER2 with ligand-activated cancer cell lines. In cells that grew in the presence of EGFR or HER3 activates signaling for growth, lapatinib, HER2 autophosphorylation was undetectable, differentiation and survival through multiple down- whereas active phosphoinositide-3 kinase (PI3K)-Akt and stream effectors including the phosphoinositide-3 kinase mitogen-activated protein kinase (MAPK) were maintained. (PI3K)-Akt pathway (Yarden and Sliwkowski, 2001). To identify networks maintaining these signaling pathways, Amplification of the HER2 oncogene occurs in approxi- we profiled the tyrosine phosphoproteome of sensitive and mately 25% of human breast cancers and not only resistant cells using an immunoaffinity-enriched mass confers a poor prognosis but also renders tumors spectrometry method. We found increased phosphoryla- susceptible to HER2-targeted therapies (Moasser, tion of Src family kinases (SFKs) and putative Src 2007). Lapatinib, a small-molecule, adenosine triphos- substrates in several resistant cell lines. Treatment of phate (ATP)-competitive tyrosine kinase inhibitor of these resistant cells with Src kinase inhibitors partially HER2 (Rusnak et al., 2001), is an effective therapy for blocked PI3K-Akt signaling and restored lapatinib patients with HER2-overexpressing metastatic breast sensitivity. Further, SFK mRNA expression was upregu- cancer (Geyer et al., 2006). However, most patients lated in primary HER2 þ tumors treated with lapatinib. treated with lapatinib eventually relapse after treatment, Finally, the combination of lapatinib and the Src inhibitor suggesting that tumors acquire or intrinsically possess AZD0530 was more effective than lapatinib alone at mechanisms for escape from HER2 inhibition. inhibiting pAkt and growth of established HER2-positive In HER2-overexpressing cells, the major mechanism BT-474 xenografts in athymic mice. These data suggest of PI3K activation is heterodimerization with kinase- that increased Src kinase activity is a mechanism of deficient HER3, which when phosphorylated couples to lapatinib resistance and support the combination of the p85 regulatory subunit of PI3K (Yakes et al., 2002; HER2 antagonists with Src inhibitors early in the Lee-Hoeflich et al., 2008). Treatment of HER2-over- treatment of HER2 þ breast cancers in order to prevent expressing cells with lapatinib blocks HER3 phosphor- or overcome resistance to HER2 inhibitors. ylation and uncouples p85 from HER3, thus inhibiting Oncogene (2011) 30, 4163–4174; doi:10.1038/onc.2011.130; PI3K-Akt (Ritter et al., 2007; Junttila et al., 2009). published online 18 April 2011 Sustained inhibition of HER2/HER3 output to PI3K- Akt has been proposed to be essential for the antitumor Keywords: breast cancer; HER2; lapatinib; Src kinases; effect of HER2 inhibitors. Recently, inhibition of HER2 tyrosine phosphorylation phosphorylation by the EGFR tyrosine kinase inhibitor gefitinib in HER2-overexpressing human breast cancer cells was shown to be followed by feedback upregulation of activated HER3 and Akt, thus limiting the inhibitory Correspondence: Dr CL Arteaga, Division of Oncology, VUMC, 2220 effect of gefitinib (Sergina et al., 2007). Therapeutic Pierce Avenue, 777 PRB, Nashville, TN 37232-6307, USA. E-mail: [email protected] doses of lapatinib are also followed by feedback Received 15 October 2010; revised 16 March 2011; accepted 18 March upregulation of phosphorylated HER3 in HER2-depen- 2011; published online 18 April 2011 dent breast cancer cells that is only abrogated by pulsed Src kinases mediate lapatinib resistance BN Rexer et al 4164 supra-pharmacological doses (Amin et al., 2010). concentration is readily achieved in the serum of Furthermore, aberrant activation of the PI3K pathway patients treated with lapatinib (Burris et al., 2005). We has been associated with resistance to the HER2 next investigated activation of HER2 and the down- inhibitors trastuzumab and lapatinib (Yakes et al., stream PI3K-Akt and mitogen-activated protein kinase 2002; Nagata et al., 2004; Berns et al., 2007; Eichhorn (MAPK) pathways in sensitive and resistant cells by et al., 2008; Serra et al., 2008). immunoblot. In lapatinib-resistant cells, HER2 Y1248 Src family kinases (SFKs) are intracellular tyrosine phosphorylation remained suppressed to levels compar- kinases implicated in signal transduction downstream of able with lapatinib-treated parental cells. However, multiple signaling networks including the ErbB recep- despite pHER2 inhibition in resistant cells, PI3K-Akt tors. Src association with HER2 has been shown in activity, indicated by S473 pAkt, and Erk activity, indi- human breast cancer cell lines and primary tumors cated by T202/Y204 pErk, were maintained (Figure 1b). (Sheffield, 1998; Belsches-Jablonski et al., 2001). The The reactivation of these downstream pathways despite interaction is specific for the HER2 kinase domain (Kim continued HER2 inactivation by lapatinib suggested et al., 2005; Marcotte et al., 2009) and results in enhanced the engagement of alternative compensatory signaling Src kinase activity and protein stability (Luttrell et al., networks to mediate drug resistance. 1994; Vadlamudi et al., 2003; Tan et al., 2005). Interestingly, Lapatinib-resistant cells showed levels of HER2 inhibition of a Src-mediated inhibitory phosphoryla- amplification by fluorescence in situ hybridization tion of phosphatase and tensin homolog (PTEN) has comparable with parental lines (Supplementary Figure been suggested as part of the antitumor mechanism 1). Reactivation of PI3K-Akt signaling appears causal of trastuzumab (Nagata et al., 2004). Because of its to lapatinib resistance as all resistant derivatives were involvement in multiple signaling cascades, Src has sensitive to the PI3K inhibitor BEZ235 (Maira et al., become an attractive therapeutic target with several Src 2008) but not to the MEK1/2 inhibitor CI-1040 inhibitors in clinical development (Finn, 2008). (Supplementary Figure 2). To identify pathways that We generated lapatinib-resistant derivatives of could maintain PI3K-Akt signaling, we used reverse- HER2-overexpressing human breast cancer cell lines. phase protein microarray analysis, an approach analo- All these lines exhibit HER2 amplification and sensitiv- gous to high-throughput dot blotting (Tibes et al., 2006). ity to lapatinib with submicromolar IC50s (Konecny We found upregulation of pS6, p70S6K, pmTor and et al., 2006). Lapatinib-resistant cells exhibited recovery pGSK3a/b, transducers of PI3K-Akt signaling, in the of PI3K-Akt signaling despite continued inhibition of resistant cells despite continued inhibition of pHER2 the HER2 tyrosine kinase. Using a mass spectrometry (Figure 1c; Supplementary Figure 3). (MS)-based phosphoproteomic approach in BT-474 cells, we found upregulation of SFK activity in the resistant cells. This upregulation was observed in three Global phosphotyrosine profiling identifies upregulation of of six lapatinib-resistant cell lines. Treatment of these SFKs in lapatinib-resistant cells cells with Src inhibitors arrested cell proliferation, To identify upregulated signaling pathways in resistant partially blocked PI3K-Akt signaling and reversed cells, we used shotgun MS coupled with immunoaffinity lapatinib resistance in these cells. Treatment of HER2- enrichment of phosphotyrosine (pTyr)-containing pep- positive xenografts with the combination of lapatinib tides. Mass spectra of phosphopeptides were generated and a small-molecule inhibitor of Src was more effective from pTyr pulldowns of tryptic digests of parental± than either drug alone. Together, these data support Src lapatinib and resistant BT-474 cells.