Abstract
Comprehensive characterization of signaling in pancreatic ductal adenocarcinoma (PDAC) promises to enhance our understanding of the molecular aberrations driving this devastating disease, and may identify novel therapeutic targets as well as biomarkers that enable stratification of patients for optimal therapy. Here, we use immunoaffinity-coupled high-resolution mass spectrometry to characterize global tyrosine phosphorylation patterns across two large panels of human PDAC cell lines: the ATCC series (19 cell lines) and TKCC series (17 cell lines). This resulted in the identification and quantification of over 1800 class 1 tyrosine phosphorylation sites and the consistent segregation of both PDAC cell line series into three subtypes with distinct tyrosine phosphorylation profiles. Subtype-selective signaling networks were characterized by identification of subtype-enriched phosphosites together with pathway and network analyses. This revealed that the three subtypes characteristic of the ATCC series were associated with perturbations in signaling networks associated with cell-cell adhesion and epithelial-mesenchyme transition, mRNA metabolism, and receptor tyrosine kinase (RTK) signaling, respectively. Specifically, the third subtype exhibited enhanced tyrosine phosphorylation of multiple RTKs including the EGFR, ERBB3 and MET. Interestingly, a similar RTK-enriched subtype was identified in the TKCC series, and 'classifier' sites for each series identified using Random Forest models were able to predict the subtypes of the alternate series with high accuracy, highlighting the conservation of the three subtypes across the two series. Finally, RTK-enriched cell lines from both series exhibited enhanced sensitivity to the small molecule EGFR inhibitor erlotinib, indicating that their phosphosignature may provide a predictive biomarker for response to this targeted therapy. These studies highlight how resolution of subtype-selective signaling networks can provide a novel taxonomy for particular cancers, and provide insights into PDAC biology that can be exploited for improved patient management.
Highlights
From the ‡Cancer Division and Kinghorn Cancer Centre, Garvan Institute of Medical Research, 384 Victoria St, Sydney, NSW 2010, Australia; §St Vincent’s Hospital Clinical School, Faculty of Medicine, University of New South Wales, NSW 2052, Australia; ¶Cancer Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Level 1, Building 77, Monash University, VIC 3800, Australia; ʈCompetence Center Functional Genomics, University of Greifswald, F.-L-Jahnstr. 15, 17489 Greifswald, Germany; **Division of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience, The University of Queensland, St
Determination of the pancreatic ductal adenocarcinoma (PDAC) Tyrosine Phosphoproteome— Tyrosine phosphorylation profiling was undertaken across 19 PDAC cell lines sourced from the American Type Culture Collection (ATCC), as well as normal pancreatic duct epithelial (HPDE) cells and HPDE cells expressing oncogenic KRAS (25)
Our study identifies ϳ1800 tyrosine phosphorylation sites across two large, independent PDAC cell line cohorts, provides new insights into dysregulated pathways and processes in PDAC, greatly extends our knowledge regarding the suite of receptor tyrosine kinase (RTK) activated in this disease, and enables a novel subclassification based on tyrosine phosphorylation profiles
Summary
Cell Culture—Two cohorts of pancreatic cancer cell lines were used. Cohort 1 cell lines (termed the ATCC Cohort) were purchased from and authenticated by the American Type Culture Collection (ATCC) (AsPC-1, BxPC-3, CFPAC-1, Capan-1, Capan-2, HPAC, HPAF-II, Hs700T, Hs766T, Panc 02.03, Panc 03.27, Panc 04.03, Panc05.04, Panc 08.13, Panc 10.05, Panc-1, PL45, MiaPaca-2, SU.86.86, SW1990) and were used and cultured according to ATCC protocols. Phosphoproteomic Profiling— Generation of Protein Lysates—Cells were plated, allowed to reach ϳ70% confluence, and placed into base culture medium, without the addition of serum or additional growth factors, for 6 h prior to lysate collection This was undertaken in order to minimize differences in signaling because of culture conditions and enable comparison of the inherent signaling network properties of the different cell lines. LC-MS/MS, Identification and Quantification—For MS analyses, dissolved peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Thermo Scientific, Waltham, MA, USA), and mass spectra were acquired on Q Exactive Plus for the TKCC Cohort samples or Orbitrap Velos for the ATCC Cohort samples. Subtype-Specific Site Identification, Pathway Enrichment and Protein-Protein Interaction Network Analyses—To identify subtype-specific sites, a multiple-sample ANOVA test was performed with multiple test correction This identifies pTyr sites that have statistically significant (p Ͻ 0.05) specificity to one of the subtypes. Mutation screening of OncoCarta Panels v1.0, v2.0 and v3.0 (Sequenom, San Diego, CA, USA) was conducted by the manufacturer to detect and quantify mutation frequencies in the cell line panel
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