Utilising the EGFR interactome to identify mechanisms of drug resistance in non-small cell lung cancer - Proof of concept towards a systems pharmacology approach.
Drug treatment of epidermal growth factor receptor (EGFR) positive non-small cell lung cancer has improved substantially by targeting activating mutations within the receptor tyrosine kinase domain. However, the development of drug resistance still limits this approach. As root causes, large heterogeneity between tumour entities but also within tumour cells have been suggested. Therefore, approaches to identify these multitude and complex mechanisms are urgently required. Affinity purification coupled with high resolution mass spectrometry was applied to isolate and characterise the EGFR interactome from HCC4006 non-small cell lung cancer cells and their variant HCC4006rERLO0.5adapted to grow in the presence of therapeutically relevant concentrations of erlotinib. Bioinformatics analyses were carried out to identify proteins and their related molecular functions that interact differentially with EGFR in the untreated state or when incubated with erlotinib prior to EGFR activation. Across all experimental conditions 375 proteins were detected to participate in the EGFR interactome, 90% of which constituted a complex protein interaction network that was bioinformatically reconstructed from literature data. Treatment of HCC4006rERLO0.5cells carrying a resistance phenotype to erlotinib was associated with an increase of protein levels of members of the clathrin-associated adaptor protein family AP2 (AP2A1, AP2A2, AP2B1), structural proteins of cytoskeleton rearrangement as well as signalling molecules such as Shc. Validation experiments confirmed activation of the Ras-Raf-Mek-Erk (MAPK)-pathway, of which Shc is an initiating adaptor molecule, in HCC4006rERLO0.5cells. Taken together, differential proteins in the EGFR interactome of HCC4006rERLO0.5cells were identified that could be related to multiple resistance mechanisms including alterations in growth factor receptor expression, cellular remodelling processes suggesting epithelial-to-mesenchymal transition as well as alterations in downstream signalling. Knowledge of these mechanisms is a pivotal step to build an integrative model of drug resistance in a systems pharmacology manner and to be able to investigate the interplay of these mechanisms and ultimately recommend combinatorial treatment strategies to overcome drug resistance.
Insitute of Pharmacy, Clinical Pharmacy, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Hisham
H
Saafan
Insitute of Pharmacy, Clinical Pharmacy, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Sarah
S
Foerster
Institute of Pharmacy, Department of Clinical Pharmacy and Biochemistry, Freie Universitaet Berlin, Germany.
Zinnia P
ZP
Parra-Guillen
Interfaculty Institute of Genetics and Functional Genomics, University Medicine, Ernst-Moritz-Arndt-University of Greifswald, Germany.
Elke
E
Hammer
Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, UK.
Martin
M
Michaelis
Institut für Medizinische Virologie, Klinikum der Goethe-Universität, Frankfurt/Main, Germany.
Jindrich
J
Cinatl
Interfaculty Institute of Genetics and Functional Genomics, University Medicine, Ernst-Moritz-Arndt-University of Greifswald, Germany.
Uwe
U
Völker
Algorithmic Bioinformatics, University of Bonn, Germany.
Holger
H
Fröhlich
Institute of Pharmacy, Department of Clinical Pharmacy and Biochemistry, Freie Universitaet Berlin, Germany. Electronic address: charlotte.kloft@fu-berlin.de.
Charlotte
C
Kloft
Insitute of Pharmacy, Clinical Pharmacy, Ernst-Moritz-Arndt-University, Greifswald, Germany. Electronic address: ritter@uni-greifswald.de.
Christoph A
CA
Ritter
D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology / Q000627:therapeutic use; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000502:physiology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D010601:Pharmacology, Clinical / Q000379:methods; D049490:Systems Biology / Q000379:methods*
D000970:Antineoplastic Agents; C512478:EGFR protein, human; D066246:ErbB Receptors
Drug resistance; EGFR; Interactome; Lung cancer; Systems pharmacology; Tyrosine kinase inhibitors