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.

Figure 1. Lapatinib-resistant cells recover PI3K-Akt and MAPK signaling despite continued HER2 inhibition

(A) Parental and lapatinib-resistant cells were treated with 1–2 µM lapatinib as follows: 1 µM: SK-BR-3, MDA-MB-361, UACC-893; 2 µM: BT-474, HCC1954, SUM190PT. Cell proliferation was measured by WST-1 assay after 2, 3, 6, and 9 days of treatment. Shown is the mean WST-1 absorbance of 4 replicate wells. (B) Whole cell lysates were prepared from parental cells (P) ± 1 or 2 µM lapatinib or lapatinib-resistant cells (R) maintained in lapatinib as in (A). Lysates were immunoblotted with the indicated antibodies. (C) Lysates were similarly prepared from parental cells after 1 or 24 h lapatinib treatment and from resistant cells and analyzed by RPPA with antibodies to PI3K signaling pathway proteins. The intensity for each protein in each of 6 cell lines is displayed (filled shapes); columns indicate the grand mean of all proteins in all cell lines (bars, S.E.M.). Individual PI3K protein values are in Supplementary Figure 3.

Figure 2. Persistent Y877 HER2 phosphorylation and increased SFK activity in lapatinib-resistant cells

(A) Representative mass spectrum of the LIEDNEYTAR YES1 tryptic peptide (+2 charge state) containing amino acids 420–429 with a phosphorylation at Y426. This peptide also exists in SRC and FYN. Asterisks denote fragment ions that contain the mass shift as a result of phosphorylation. (B) Lysates of parental cells (P) after 2 h lapatinib treatment or lapatinib-resistant cells (R) were immunoblotted with antibodies as indicated. (C) Lysates from parental cells treated or not with lapatinib, and from resistant cells in the presence of lapatinib were immunoprecipitated with a pTyr antibody. Immune complexes were analyzed by immunoblot with site-specific phosphoantibodies or total HER2 antibody as indicated.

Figure 3. SFK expression is increased in cells and tumors after lapatinib and activity is blocked by SFK inhibitors

(A) Lysates from parental and resistant cells were immunoblotted with antibodies to Src, Yes, Fyn, and Lyn as indicated. (B) Lysates from BT-474 or UACC-893 cells transfected with siRNA oligonucleotides specific for Src, Yes, Fyn, or Lyn were immunoblotted for Y416 pSFK 72 h after transfection. (C) Schema of the neoadjuvant trial of lapatinib followed by trastuzumab + chemotherapy. Biopsies were obtained pre-treatment and after 6 weeks of lapatinib treatment. (D) Heatmap showing relative expression levels of 7 probesets for Src family kinases (blue = low expression; red = high expression). (E) Lysates from parental (Par) cells ± lapatinib (2 h) and resistant cells (Res) maintained in lapatinib ± the Src inhibitors AZD0530 or dasatinib or (2 h) were analyzed by immunoblot with the indicated antibodies.

Figure 4. Src inhibitors restore sensitivity to lapatinib

Parental and lapatinib-resistant cells were seeded in growth-factor reduced Matrigel; parental cells were treated or not with lapatinib while lapatinib-resistant cells were treated with lapatinib with or without AZD0530. Media and inhibitors were replenished every 3 days. Acini were photographed at 400× magnification 10–14 days after plating. Quantification of mean acini area is shown in Supplementary Figure 6.

Figure 5. Lapatinib and the Src inhibitor AZD0530 synergize against HER2-overexpressing xenografts

(A) BT-474 xenografts were established in female athymic nude mice. When tumors reached a volume ≥250 mm³, mice were randomly allocated to four treatment groups: vehicle alone, lapatinib (100 mg/kg daily by orogastric gavage), AZD0530 (50 mg/kg daily by orogastric gavage), or the combination. Tumors were measured twice weekly; mean tumor volume (n=10) in mm³ ± SEM for each treatment group is displayed. (B) Mice were weighed twice weekly during treatment and average weight ± SEM of mice in each treatment group is displayed. (C) Tumor fragments were fixed and analyzed by IHC with antibodies to Y416 pSrc and S473 pAkt. Representative sections are shown. (D) IHC was scored as described in the Methods. For pSFKs, columns indicate mean membrane H-score; for pAkt, columns indicate mean cytoplasmic H-score and percent of cells with nuclear staining (bars: mean ± S.E.M.; asterisks: *, p<0.05; **, p<0.005; ***, p<0.000005, Student’s t-test).