Intracellular Src Family Kinases Mediate PI3K-Akt Pathway Activation and Resistance to Lapatinib in HER2-Overexpressing Human Breast Cancer Cells.

We investigated mechanisms of resistance to the HER2 tyrosine kinase inhibitor (TKI) lapatinib (Lap) in breast cancer cells that overexpress HER2. Inhibition of phosphoinositide-3 kinase (PI3K)-Akt signaling downstream of HER2 has been proposed as required for the antitumor activity of HER2 inhibitors. Resistance to HER2 inhibitors has been associated with aberrant activation of the PI3K pathway. HER2-overexpressing breast cancer cells (BT-474, SKBR-3, HCC1954, MDA-MB-361, and SUM190) were made resistant to Lap by exposure to increasing concentrations of the drug. Lapatinib-resistant (LR) cells exhibited the same level of HER2 gene amplification as parental, sensitive cells as measured by fluorescent in situ hybridization. HER2 activity measured by Y1248 P-HER2 immunoblots was undetectable in LR cells. However, they consistently showed recovery of PI3K-Akt activity as demonstrated by phosphorylation of Akt at Ser473. We hypothesized that resistance to HER2 inhibitors results from reactivation of the PI3K-Akt axis through upregulation of alternate compensatory signaling pathways. To identify these putative escape pathways, we profiled the tyrosine (tyr) phosphoproteome using an immunoaffinity method of purification of tyr-phosphorylated peptides and identification by tandem liquid chromatography-mass spectrometry (LC-MS). Cell lysates from LR and sensitive cells were digested with trypsin and precipitated with a P-tyr antibody followed by tandem LC-MS. Peptide sequences were assigned from mass spectra using Myrimatch software and protein identities were determined by analysis with IDPicker. In this analysis, 820 spectra corresponding to 173 tyr-phosphorylated peptides were identified. Of these, 29 peptides were isolated more frequently or uniquely in LR cells based on spectral counts. To identify kinases that might be responsible for the unique phosphopeptides in the resistant cells, we used Kinase Enrichment Analysis with the 21 protein subtrates from these 29 peptides. This analysis suggested that Src or a Src family kinase (SFK) activity was enriched in LR cells. By western blot, we observed increased Y416 P-SFK in BT-474 and HCC1954 LR cells compared to drug-sensitive cells. The MS phosphotyrosine profiling identified Yes in BT-474 LR cells. This was confirmed by western analysis with Yes specific antibodies and qRT-PCR with SFK-specific primers. In HCC1954 cells, western analysis with Src and Lyn antibodies and qRT-PCR suggested that Lyn and/or Src are the active kinases. Treatment of LR cells with the small molecule Src inhibitors AZD0530 or dasatinib in combination with Lap resulted in inhibition of Y416 P-Src, partial inhibition of S473 P-Akt, and inhibition of cell growth. Treatment of HCC1954 sensitive cells with AZD0530 and Lap for 14 days partially abrogated the emergence of lapatinib-resistant colonies. These data suggest that combination of Src inhibitors with Lap will prevent or overcome therapeutic resistance to the HER2 TKI. Studies to determine 1) the mechanism whereby PI3K-Akt is activated by SFKs in the resistant cells, and 2) the synergistic effect of combination treatment with Src inhibitors and lapatinib against HER2-overexpressing xenografts are ongoing at this time. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 707.