Anti-AP2A1 monoclonal antibody, clone BD2-N22

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 HCC4006(r)ERLO(0.5) adapted 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 HCC4006(r)ERLO(0.5) cells 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 HCC4006(r)ERLO(0.5) cells. Taken together, differential proteins in the EGFR interactome of HCC4006(r)ERLO(0.5) cells 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. (C) 2016 Elsevier B.V. All rights reserved.

IL-33 has emerged as a central mediator of immune, inflammatory, and fibrotic responses. Many studies have focused on mature IL-33, but elevated expression of the precursor, full-length IL-33 (FLIL33), has also been implicated in a spectrum of diseases, including tissue fibrosis. We previously reported and now confirmed that overexpression of FLIL33 induced phosphorylation of the key profibrotic signaling mediator of TGF-beta, Smad3, in primary human lung fibroblasts from healthy donors and idiopathic pulmonary fibrosis patients. Presently, we demonstrate that FLIL33-induced Smad3 phosphorylation was not abrogated by anti-TGF-beta antibody but was abrogated by ALK5/TGFBR1-specific and Smad3-specific inhibition, indicating that FLIL33 effect was in- dependent of TGF-beta but dependent on its receptor, TGFBR. Western blotting analyses revealed that FLIL33 overexpression increased levels, but did not affect subcellular distribution, of the AP2A1 and AP2B1 subunits of the adaptor protein complex 2 (AP2), a known TGFBR binding partner. siRNA-mediated inhibition of these subunits blocked FLIL33-induced Smad3 phosphorylation, whereas AP2 subunit overexpression induced Smad3 phosphorylation even in the absence of FLIL33. RNA-Seq transcriptomic analyses revealed that fibroblast stimulation with TGF-beta induced major changes in expression levels of numerous genes, whereas overexpression of FLIL33 induced modest expression changes in a small number of genes. Furthermore, qRT-PCR tests demon- strated that despite inducing Smad3 phosphorylation, FLIL33 did not induce collagen gene transcription and even mildly attenuated TGF-beta-induced levels of collagen I and III mRNAs. We conclude that FLIL33 induces Smad3 phosphorylation through a TGF-beta-independent but TGF-beta receptor- and AP2- dependent mechanism and has limited downstream transcriptomic consequences.