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).

Phosphotyrosine-based-phosphoproteomics scaled-down to biopsy level for analysis of individual tumor biology and treatment selection

“…The power of this approach was shown in 2007 by the identification of previously unknown ALK, ROS and PDGFRα activated NSCLC subtypes [7]. This research strategy has further matured and applied in the past years [8,[15][16][17][18][19].…”

Dysregulation of splicing proteins in head and neck squamous cell carcinoma

“…2,3 Studies have shown that aberrant activation of kinase signaling pathways contribute to abnormality in cellular processes which results in tumorigenesis. 4,5 Oncogenic activation of PI3K, Notch and hedgehog signaling pathways have been reported in HNSCC and they are being tested in clinical studies as potential targets. [6][7][8] Mass spectrometry-based proteome/phosphoproteome profiling has become a reliable method for studying the signaling pathways/events that are dysregulated in any given system.…”

Analysis of contribution of protein phosphorylation in the development of the diseases

“…Однако совместное использование 2-DG и ингибитора IGF1R значительно увеличивает смертность раковых клеток [50]. Во многих работах были показаны достоверные различия в профилях ПТМ для чувствительных и резистентных к противоопухолевым препаратам клеток, некоторые из этих ПТМ предлагается использовать в качестве маркеров для типирования опухолей и выбора подходящего лечения [51][52][53]. Две независимые группы исследователей продемонстрировали зависимость эффективности терапии глиобластомы ингибитором тирозинкиназ эрлотинибом от уровня фосфорилирования PKB/Akt (рис.…”