1] WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and College of Medicine or College of Pharmacy, Seoul National University, Seoul, Republic of Korea [2] Wide River Institute of Immunology, Seoul National University, Seoul, Republic of Korea.
Abstract
Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO2) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/α-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.
Figures
Figure 1Differentially expressed phosphoproteins in SNU216-LR and their functional annotations. (a) Volcano plot showing the P-value (−log10) versus signal-to-noise ratio (S/N) of phosphoproteins determined by power law global error model (PLGEM) statistical analysis for label-free quantification. (b) Molecular functions of identified phosphoproteins by the Ingenuity Pathway Analysis (IPA) tool. (c) Subcellular localization of identified phosphoproteins by the IPA tool.Figure 2Biological network analysis of phosphoproteins. Associations among phosphoproteins are shown by solid or dashed lines, which represent direct or indirect interactions, respectively. Upregulated proteins are shown in red, and downregulated proteins are shown in green.Figure 3Merged signaling pathways in SNU216-LR. (a) Reconstructed EGFR/HER2 and MET signaling pathways with two downstream signaling pathways (PI3K/AKT and MAPK/ERK). Red, blue and yellow closed circles on each protein represent increased, decreased and newly identified phosphorylation sites in SNU216-LR, respectively. Specific phosphorylated sites (marked with an asterisk) of HER2, EGFR, MET, SRC, C-RAF and MAPK were also identified from immunoblot analysis with antibodies against p-EGFR (pY1068), p-HER2 (pY1221/1222), p-MET (pY1234/1235), p-AKT (pS473), p-MAPK (pY202/pY204), p-SRC (pY416) and p-RAF (pS338). Protein names for symbols are as follows: AKT, α-serine/threonine-protein kinase; C-RAF, RAF proto-oncogene serine/threonine-protein kinase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HER2, human epidermal growth factor receptor 2; MAPK, mitogen-activated protein kinase 1; GRB2, growth factor receptor-bound protein 2; MET, hepatocyte growth factor/mesenchymal–epithelial transition factor; PI3K, phosphatidylinositide 3-kinase; RAC1, Ras-related C3 botulinum toxin substrate 1; RAS, Ras-related protein R-Ras; SOS, son of sevenless homolog; SRC, proto-oncogene tyrosine protein kinase Src. (b) Immunoblot analysis of SNU216 and three different resistant clones (SNU216-LR 1, 2 and 3) with phospho-tyrosine or serine-specific antibodies. α-Tubulin was used as a loading control. Data are representative of three independent experiments.Figure 4Antiproliferative effects of selected tyrosine kinase inhibitors on SNU216-LR. SNU216-LR cells were treated via single or combined treatment of lapatinib (Lap; 1 μM), NVP-BEZ235 (BEZ; 100 nM), selumetinib (Selu; 1 μM), saracatinib (Sara; 1 μM) and crizotinib (Crizo; 1 μM). (a) Viable cells were measured after 72 h. The percentage of viable cells is shown relative to untreated controls. Data represent the mean±s.d. (*P<0.05 for single vs dimethyl sulfoxide (DMSO); **P<0.05 for combination vs single) from three independent experiments. (b) Cells were fixed with 70% (v/v) ethanol, stained with propidium iodide and analyzed using flow cytometry. The proportion of cells in the G1, S and G2/M phases were quantified using the ModFit LT program (Verity Software House). Columns represent the means of three independent experiments. (c) SNU216-LR cells were treated with the indicated drug for 48 h. Whole-cell extracts were analyzed by western blot with antibodies recognizing phosphorylated and total EGFR, HER2, MET, AKT, MAPK, SRC, cyclin D and p27. Data are representative of three independent experiments. AKT, α-serine/threonine-protein kinase; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; MAPK, mitogen-activated protein kinase 1; MET, hepatocyte growth factor/mesenchymal–epithelial transition factor; SRC, proto-oncogene tyrosine protein kinase Src.
그림 출처: PubMed Central 공개 논문 (PMC3849569)
그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
Experimental & molecular medicine · eISSN 2092-6413
