title: Identifying novel targets of oncogenic EGF receptor signaling in lung cancer through global phosphoproteomics.
abstract: Mutations in the epidermal growth factor receptor (EGFR) kinase domain occur in 10-30% of lung adenocarcinoma and are associated with tyrosine kinase inhibitor (TKI) sensitivity. We sought to identify the immediate direct and indirect phosphorylation targets of mutant EGFRs in lung adenocarcinoma. We undertook SILAC strategy, phosphopeptide enrichment, and quantitative MS to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring EGFR(L858R) and EGFR(L858R/T790M) , the TKI-sensitive, and TKI-resistant mutations, respectively. Top canonical pathways that were inhibited upon erlotinib treatment in sensitive cells, but not in the resistant cells include EGFR, insulin receptor, hepatocyte growth factor, mitogen-activated protein kinase, mechanistic target of rapamycin, ribosomal protein S6 kinase beta 1, and Janus kinase/signal transducer and activator of transcription signaling. We identified phosphosites in proteins of the autophagy network, such as ULK1 (S623) that is constitutively phosphorylated in these lung adenocarcinoma cells; phosphorylation is inhibited upon erlotinib treatment in sensitive cells, but not in resistant cells. Finally, kinase-substrate prediction analysis from our data indicated that substrates of basophilic kinases from, AGC and Calcium and calmodulin-dependent kinase groups, as well as STE group kinases were significantly enriched and those of proline-directed kinases from, CMGC and Casein kinase groups were significantly depleted among substrates that exhibited increased phosphorylation upon EGF stimulation and reduced phosphorylation upon TKI inhibition. This is the first study to date to examine global phosphorylation changes upon erlotinib treatment of lung adenocarcinoma cells and results from this study provide new insights into signaling downstream of mutant EGFRs in lung adenocarcinoma. All MS data have been deposited in the ProteomeXchange with identifier PXD001101 (http://proteomecentral.proteomexchange.org/dataset/PXD001101).
authors.affiliation: Thoracic and Gastrointestinal Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Xu
authors.initials: X
authors.lastname: Zhang
authors.forename: Natalya
authors.initials: N
authors.lastname: Belkina
authors.forename: Harrys Kishore Charles
authors.initials: HK
authors.lastname: Jacob
authors.forename: Tapan
authors.initials: T
authors.lastname: Maity
authors.forename: Romi
authors.initials: R
authors.lastname: Biswas
authors.forename: Abhilash
authors.initials: A
authors.lastname: Venugopalan
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Shaw
authors.forename: Min-Sik
authors.initials: MS
authors.lastname: Kim
authors.forename: Raghothama
authors.initials: R
authors.lastname: Chaerkady
authors.forename: Akhilesh
authors.initials: A
authors.lastname: Pandey
authors.forename: Udayan
authors.initials: U
authors.lastname: Guha
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D001343:Autophagy / Q000187:drug effects; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000069347:Erlotinib Hydrochloride; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017354:Point Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D040901:Proteomics; D011799:Quinazolines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D010748:Phosphopeptides; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D000069347:Erlotinib Hydrochloride; D066246:ErbB Receptors
keywords: Autophagy; EGFR; Erlotinib; Mass spectrometry; NSCLC; Phosphoproteomics; SILAC