Resolution of Novel Pancreatic Ductal Adenocarcinoma Subtypes by Global Phosphotyrosine Profiling
2016; Elsevier BV; Volume: 15; Issue: 8 Linguagem: Inglês
10.1074/mcp.m116.058313
ISSN1535-9484
AutoresEmily S. Humphrey, Shih‐Ping Su, Adnan Nagrial, Falko Hochgräfe, Marina Pajic, Gillian M. Lehrbach, Robert G. Parton, Alpha S. Yap, Lisa G. Horvath, David K. Chang, Andrew V. Biankin, Jianmin Wu, Roger J. Daly,
Tópico(s)Cancer Genomics and Diagnostics
ResumoComprehensive 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. 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. Pancreatic ductal adenocarcinoma (PDAC) 1The abbreviations used are:PDACpancreatic ductal adenocarcinomaAJsadheren junctionsATCCAmerican Type Culture CollectionEMTepithelial to mesenchyme transitionHPDEhuman pancreatic duct epithelialIAPimmunoaffinity purificationLC-MS/MSLiquid chromatography-tandem mass spectrometryMDAmean decrease accuracyMSmass spectrometryPCAprincipal component analysisPINAProtein Interaction Network AnalysispTyrphosphotyrosineQMquasimesenchymalRFRandom ForestRTRoom temperatureRTKreceptor tyrosine kinaseTBSTTris Buffered Saline with TweenTCEPtris (2-carboxyethyl) phosphineTFAtrifluoroacetic acidTJstight junctionsTKCCThe Kinghorn Cancer Centre. remains one of the most deadly solid cancers, characterized by extremely poor survival rates and limited therapeutic options (1.Vogelzang N.J. Benowitz S.I. Adams S. Aghajanian C. Chang S.M. Dreyer Z.E. Janne P.A. Ko A.H. Masters G.A. Odenike O. Patel J.D. Roth B.J. Samlowski W.E. Seidman A.D. Tap W.D. Temel J.S. Von Roenn J.H. Kris M.G. Clinical cancer advances 2011: Annual Report on Progress Against Cancer from the American Society of Clinical Oncology.J. Clin. Oncol. 2012; 30: 88-109Crossref PubMed Scopus (85) Google Scholar). Postoperative treatment of patients is largely limited to chemotherapeutics, such as gemcitabine, nab-paclitaxel or Folfirinox, although the addition of the EGFR-directed kinase inhibitor erlotinib to gemcitabine results in a modest improvement in patient survival (2.Paulson A.S. Tran Cao H.S. Tempero M.A. Lowy A.M. Therapeutic advances in pancreatic cancer.Gastroenterology. 2013; 144: 1316-1326Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). In addition, patient stratification for therapy remains in its infancy. These factors highlight an urgent need to better understand the molecular mechanisms that contribute to PDAC development, progression and heterogeneity. pancreatic ductal adenocarcinoma adheren junctions American Type Culture Collection epithelial to mesenchyme transition human pancreatic duct epithelial immunoaffinity purification Liquid chromatography-tandem mass spectrometry mean decrease accuracy mass spectrometry principal component analysis Protein Interaction Network Analysis phosphotyrosine quasimesenchymal Random Forest Room temperature receptor tyrosine kinase Tris Buffered Saline with Tween tris (2-carboxyethyl) phosphine trifluoroacetic acid tight junctions The Kinghorn Cancer Centre. Over the last two decades, a deeper understanding of the genetic and molecular basis of cancer has led to the development of targeted therapeutics and personalized treatment strategies that combine such approaches with companion biomarkers. This paradigm has yet to be successfully applied to PDAC, which likely explains its poor overall response to adjuvant therapy. Although almost all PDACs harbor activating mutations in KRAS, and inactivating mutations in TP53, SMAD4 and CDKN2A occur at a frequency of > 30% (3.Biankin A.V. Waddell N. Kassahn K.S. Gingras M.C. Muthuswamy L.B. Johns A.L. Miller D.K. Wilson P.J. Patch A.M. Wu J. Chang D.K. Cowley M.J. Gardiner B.B. Song S. Harliwong I. Idrisoglu S. Nourse C. Nourbakhsh E. Manning S. Wani S. Gongora M. Pajic M. Scarlett C.J. Gill A.J. Pinho A.V. Rooman I. Anderson M. Holmes O. Leonard C. Taylor D. Wood S. Xu Q. Nones K. Fink J.L. Christ A. Bruxner T. Cloonan N. Kolle G. Newell F. Pinese M. Mead R.S. Humphris J.L. Kaplan W. Jones M.D. Colvin E.K. Nagrial A.M. Humphrey E.S. Chou A. Chin V.T. Chantrill L.A. Mawson A. Samra J.S. Kench J.G. Lovell J.A. Daly R.J. Merrett N.D. Toon C. Epari K. Nguyen N.Q. Barbour A. Zeps N. Kakkar N. Zhao F. Wu Y.Q. Wang M. Muzny D.M. Fisher W.E. Brunicardi F.C. Hodges S.E. Reid J.G. Drummond J. Chang K. Han Y. Lewis L.R. Dinh H. Buhay C.J. Beck T. Timms L. Sam M. Begley K. Brown A. Pai D. Panchal A. Buchner N. De Borja R. Denroche R.E. Yung C.K. Serra S. Onetto N. Mukhopadhyay D. Tsao M.S. Shaw P.A. Petersen G.M. Gallinger S. Hruban R.H. Maitra A. Iacobuzio-Donahue C.A. Schulick R.D. Wolfgang C.L. Morgan R.A. Lawlor R.T. Capelli P. Corbo V. Scardoni M. Tortora G. Tempero M.A. Mann K.M. Jenkins N.A. Perez-Mancera P.A. Adams D.J. Largaespada D.A. Wessels L.F. Rust A.G. Stein L.D. Tuveson D.A. Copeland N.G. Musgrove E.A. Scarpa A. Eshleman J.R. Hudson T.J. Sutherland R.L. Wheeler D.A. Pearson J.V. McPherson J.D. Gibbs R.A. Grimmond S.M. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 4.Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. Johns A.L. Miller D. Nones K. Quek K. Quinn M.C. Robertson A.J. Fadlullah M.Z. Bruxner T.J. Christ A.N. Harliwong I. Idrisoglu S. Manning S. Nourse C. Nourbakhsh E. Wani S. Wilson P.J. Markham E. Cloonan N. Anderson M.J. Fink J.L. Holmes O. Kazakoff S.H. Leonard C. Newell F. Poudel B. Song S. Taylor D. Waddell N. Wood S. Xu Q. Wu J. Pinese M. Cowley M.J. Lee H.C. Jones M.D. Nagrial A.M. Humphris J. Chantrill L.A. Chin V. Steinmann A.M. Mawson A. Humphrey E.S. Colvin E.K. Chou A. Scarlett C.J. Pinho A.V. Giry-Laterriere M. Rooman I. Samra J.S. Kench J.G. Pettitt J.A. Merrett N.D. Toon C. Epari K. Nguyen N.Q. Barbour A. Zeps N. Jamieson N.B. Graham J.S. Niclou S.P. Bjerkvig R. Grutzmann R. Aust D. Hruban R.H. Maitra A. Iacobuzio-Donahue C.A. Wolfgang C.L. Morgan R.A. Lawlor R.T. Corbo V. Bassi C. Falconi M. Zamboni G. Tortora G. Tempero M.A. Gill A.J. Eshleman J.R. Pilarsky C. Scarpa A. Musgrove E.A. Pearson J.V. Biankin A.V. Grimmond S.M. Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1687) Google Scholar), mutations in these genes are not associated with clinically 'actionable' phenotypes. Evidence for the existence of different molecular phenotypes of PDAC has been found through exploration of the genomic landscape of this disease. Exome sequencing identified an unsuspected role for axon guidance pathway genes in ∼20% of PDAC patients (3.Biankin A.V. Waddell N. Kassahn K.S. Gingras M.C. Muthuswamy L.B. Johns A.L. Miller D.K. Wilson P.J. Patch A.M. Wu J. Chang D.K. Cowley M.J. Gardiner B.B. Song S. Harliwong I. Idrisoglu S. Nourse C. Nourbakhsh E. Manning S. Wani S. Gongora M. Pajic M. Scarlett C.J. Gill A.J. Pinho A.V. Rooman I. Anderson M. Holmes O. Leonard C. Taylor D. Wood S. Xu Q. Nones K. Fink J.L. Christ A. Bruxner T. Cloonan N. Kolle G. Newell F. Pinese M. Mead R.S. Humphris J.L. Kaplan W. Jones M.D. Colvin E.K. Nagrial A.M. Humphrey E.S. Chou A. Chin V.T. Chantrill L.A. Mawson A. Samra J.S. Kench J.G. Lovell J.A. Daly R.J. Merrett N.D. Toon C. Epari K. Nguyen N.Q. Barbour A. Zeps N. Kakkar N. Zhao F. Wu Y.Q. Wang M. Muzny D.M. Fisher W.E. Brunicardi F.C. Hodges S.E. Reid J.G. Drummond J. Chang K. Han Y. Lewis L.R. Dinh H. Buhay C.J. Beck T. Timms L. Sam M. Begley K. Brown A. Pai D. Panchal A. Buchner N. De Borja R. Denroche R.E. Yung C.K. Serra S. Onetto N. Mukhopadhyay D. Tsao M.S. Shaw P.A. Petersen G.M. Gallinger S. Hruban R.H. Maitra A. Iacobuzio-Donahue C.A. Schulick R.D. Wolfgang C.L. Morgan R.A. Lawlor R.T. Capelli P. Corbo V. Scardoni M. Tortora G. Tempero M.A. Mann K.M. Jenkins N.A. Perez-Mancera P.A. Adams D.J. Largaespada D.A. Wessels L.F. Rust A.G. Stein L.D. Tuveson D.A. Copeland N.G. Musgrove E.A. Scarpa A. Eshleman J.R. Hudson T.J. Sutherland R.L. Wheeler D.A. Pearson J.V. McPherson J.D. Gibbs R.A. Grimmond S.M. Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar), whereas whole genome sequencing of PDAC specimens provided the basis for classification into four subtypes based upon patterns of genomic structural variation (4.Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. Johns A.L. Miller D. Nones K. Quek K. Quinn M.C. Robertson A.J. Fadlullah M.Z. Bruxner T.J. Christ A.N. Harliwong I. Idrisoglu S. Manning S. Nourse C. Nourbakhsh E. Wani S. Wilson P.J. Markham E. Cloonan N. Anderson M.J. Fink J.L. Holmes O. Kazakoff S.H. Leonard C. Newell F. Poudel B. Song S. Taylor D. Waddell N. Wood S. Xu Q. Wu J. Pinese M. Cowley M.J. Lee H.C. Jones M.D. Nagrial A.M. Humphris J. Chantrill L.A. Chin V. Steinmann A.M. Mawson A. Humphrey E.S. Colvin E.K. Chou A. Scarlett C.J. Pinho A.V. Giry-Laterriere M. Rooman I. Samra J.S. Kench J.G. Pettitt J.A. Merrett N.D. Toon C. Epari K. Nguyen N.Q. Barbour A. Zeps N. Jamieson N.B. Graham J.S. Niclou S.P. Bjerkvig R. Grutzmann R. Aust D. Hruban R.H. Maitra A. Iacobuzio-Donahue C.A. Wolfgang C.L. Morgan R.A. Lawlor R.T. Corbo V. Bassi C. Falconi M. Zamboni G. Tortora G. Tempero M.A. Gill A.J. Eshleman J.R. Pilarsky C. Scarpa A. Musgrove E.A. Pearson J.V. Biankin A.V. Grimmond S.M. Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1687) Google Scholar). In the latter study, a subtype of patients characterized by unstable genomes and/or a BRCA mutational signature was demonstrated to have increased sensitivity to platinum-based therapy. In addition, transcript profiling subclassifies PDAC into subtypes exhibiting contrasting histopathogical characteristics, mutation patterns and patient outcome (5.Bailey P. Chang D.K. Nones K. Johns A.L. Patch A.M. Gingras M.C. Miller D.K. Christ A.N. Bruxner T.J. Quinn M.C. Nourse C. Murtaugh L.C. Harliwong I. Idrisoglu S. 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This new molecular taxonomy for PDAC provides important insights into disease mechanisms and highlights potential biomarkers to help guide patient stratification for therapy. 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. Cohort 2 cell lines (termed the TKCC Cohort) were isolated from primary patient-derived pancreatic ductal adenocarcinoma xenografts in house (TKCC 2.1, TKCC 04, TKCC 05, TKCC 06, TKCC 07, TKCC 09, TKCC 10, TKCC 12, TKCC 14, TKCC 15, TKCC 16, TKCC 17, TKCC 18, TKCC 19, TKCC 22, TKCC 26, TKCC 27) (4.Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. Johns A.L. Miller D. Nones K. Quek K. Quinn M.C. Robertson A.J. Fadlullah M.Z. Bruxner T.J. Christ A.N. Harliwong I. Idrisoglu S. Manning S. Nourse C. Nourbakhsh E. Wani S. Wilson P.J. Markham E. Cloonan N. Anderson M.J. Fink J.L. Holmes O. Kazakoff S.H. Leonard C. Newell F. Poudel B. Song S. Taylor D. Waddell N. Wood S. Xu Q. Wu J. Pinese M. Cowley M.J. Lee H.C. Jones M.D. Nagrial A.M. Humphris J. Chantrill L.A. Chin V. Steinmann A.M. Mawson A. Humphrey E.S. Colvin E.K. Chou A. Scarlett C.J. Pinho A.V. Giry-Laterriere M. Rooman I. Samra J.S. Kench J.G. Pettitt J.A. Merrett N.D. Toon C. Epari K. Nguyen N.Q. Barbour A. Zeps N. Jamieson N.B. Graham J.S. Niclou S.P. Bjerkvig R. Grutzmann R. Aust D. Hruban R.H. Maitra A. Iacobuzio-Donahue C.A. Wolfgang C.L. Morgan R.A. Lawlor R.T. Corbo V. Bassi C. Falconi M. Zamboni G. Tortora G. Tempero M.A. Gill A.J. Eshleman J.R. Pilarsky C. Scarpa A. Musgrove E.A. Pearson J.V. Biankin A.V. Grimmond S.M. Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1687) Google Scholar). Animal experimentation for cell line generation was approved by the Garvan Institute/St Vincent's Hospital Animal Ethics Committee (approval number: ARA 12/21). Culture conditions used are summarized in supplemental Table S1. Cells were plated, allowed to reach ∼70% 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. For each cell line, two independent biological replicates of ∼2 × 108 cells were generated. ATCC cells were homogenized in 8 m Urea lysis buffer containing 20 mm HEPES (pH 8), 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm sodium orthovanadate and the reducing agent 1 mm tris (2-carboxyethyl) phosphine (TCEP). The procedure for TKCC cells was similar except the lysis buffer was based on 6 m Guanidine hydrochloride containing 50 mm Tris-HCL (pH 8). Samples were sonicated on ice, cleared by centrifugation, then alkylated for 45 min with 4 mm iodoacetamide (in the dark) at room temperature (RT). These lysates were subsequently used to conduct pTyr profiling, as well as Western blotting. 20 mg (TKCC panel) or 30 mg (ATCC panel) of cell line lysate was diluted to 1 mm guanidine hydrochloride or urea respectively with 50 mm ammonium bicarbonate, and digested at 1:200 (w/w) with Lysyl Endopeptidase (WAKO Chemicals, Cape Charles, VA, USA) for 4 h (RT) and then at 1:100 (w/w) overnight (RT) with TPCK treated trypsin (Worthington, Lakewood, NJ, USA, ratio of 1:100). Two "spike-in" heavy peptides with alanine (al) C13N15 modifications were added prior to tC-18 clean-up (Waters, Milford, MA, USA). These peptides were EF1A1 (EHA(al)LLApYTLGVK) (500 fM) and MAPK14 (HTDDEMTGpYVA(al)TR) (50 pm) (Mimotopes, Nottinghill, Vic, Aus). Purified peptides were lyophilized overnight and dissolved in immunoaffinity purification (IAP) buffer (50 mm MOPS (pH 7.4) with 10 mm sodium phosphate dibasic, 130 mm sodium chloride and either 0.5% (v/v) Nonidet P-40 (ATCC panel) or 1% (v/v) n-octyl-b-d-glucopyranoside (TKCC panel)). For the ATCC panel, Sepharose 4B beads pre-conjugated to 100 μg of PY100 antibody (Cell Signaling Technology, Danvers, MA, USA) were then added. Peptides were incubated with antibody overnight at 4 °C on a rotating apparatus. For the TKCC panel, the flowthrough from the PY100 IP was collected and immunoprecipitated again with 150 μg of Purified Mouse Anti-Phosphotyrosine (pY20) antibody (BD Transduction Labs, San Jose, CA, USA). Peptides exhibiting nonspecific binding were removed by three washes with IAP buffer and three washes with dH2O. Bound peptides were eluted with 0.15% (v/v) trifluoroacetic acid (TFA) in 40% (v/v) acetonitrile. 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. Samples were desalted by loading onto a C18 pre-column (100 μm, 2 cm column; particle size 5 μm; pore size 100 Å; Acclaim PepMap RSLC, Thermo Scientific), and separated on a 12 cm 75 μm ID analytical column pulled to an internal diameter of 5 μm by a P-2000 laser puller (Sutter Instruments Co, Novato, CA, USA) packed with C18 Magic reverse phase material, using a Dionex Ultimate 3000 LC system. For the Q Exactive, peptides were eluted at 250 nL/min using a gradient of acetonitrile in 0.1% (v/v) aqueous formic acid as follows: 2–10% in 1 min, 10–26% in 27 min, 26–34% in 2 min, and 34–80% in 5 min. The eluent was directed into a nano-electrospray ion source (Nanospray Flex, Thermo Scientific) with a spray voltage of 1.7 kV. Survey scans in the mass range of 375–1600 m/z were acquired with a resolution of 70,000 at m/z 200 and an AGC target of 3e6 ions (max IIT 120 ms). The top 12 most intense ions (ion selection threshold > 1000 ions, charge state 2–5, preferred peptide match, exclude isotopes) were sequentially isolated (isolation window 1.8 m/z) at a target of 1e5 ions (max IIT 100 ms), and fragmented in the HCD cell (normalized collision energy 27). MS/MS spectra were acquired in the Orbitrap mass analyzer at a resolution of 17,500 at m/z 200 and ions were excluded from selection for a further 15 s. For the Orbitrap Velos, peptides were electrosprayed directly into the MS using a spray voltage of 1.8 kV, survey scans in the mass range of 350–1750 m/z were acquired with a resolution of 60,000 at m/z 400 and an AGC target of 1e6 ions. The top 10 most intense ions (ion selection threshold > 500 ions, charge state ≥2) were sequentially isolated (isolation window 2.5 m/z) at a target of 1e5 ions and fragmented in
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