title: Phosphoproteomic mass spectrometry profiling links Src family kinases to escape from HER2 tyrosine kinase inhibition.
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.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA.
authors.forename: B N
authors.initials: BN
authors.lastname: Rexer
authors.forename: A-J L
authors.initials: AJ
authors.lastname: Ham
authors.forename: C
authors.initials: C
authors.lastname: Rinehart
authors.forename: S
authors.initials: S
authors.lastname: Hill
authors.forename: N de Matos
authors.initials: Nde M
authors.lastname: Granja-Ingram
authors.forename: A M
authors.initials: AM
authors.lastname: González-Angulo
authors.forename: G B
authors.initials: GB
authors.lastname: Mills
authors.forename: B
authors.initials: B
authors.lastname: Dave
authors.forename: J C
authors.initials: JC
authors.lastname: Chang
authors.forename: D C
authors.initials: DC
authors.lastname: Liebler
authors.forename: C L
authors.initials: CL
authors.lastname: Arteaga
mesh_terms: D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D040901:Proteomics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; D019061:src-Family Kinases; C508053:ERBB2 protein, human