Abstract B19: Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition
Authors: de Boer Harmen R. , Joosten Esméé , Fusetti Fabrizia , Fehrmann Rudolf S.N. , de Vries Elisabeth G.E. , van Vugt Marcel A.T.M. 
Published: May 2016

Abstract
Introduction
The epidermal growth factor receptor (EGFR) is often hyperactivated through overexpression or somatic mutation in multiple cancer types, driving tumor growth and survival. Targeted therapies against EGFR show clinical benefit, but resistance to these agents invariably develops. Thus, there is need for markers of effective treatment, termed ‘effect sensors’, to monitor the effective treatment with EGFR therapeutics during therapy. Therefore, we investigated the cellular rewiring at the proteomic level in response to EGFR inhibition, with the aim of identifying proteins that can be used as effect sensors. These effect sensors potentially constitute powerful targets for the development of tracers to monitor EGFR-directed therapies.
Materials and Methods
Human breast cancer cell lines were tested for cell viability in presence of the EGFR inhibitor erlotinib and sensitive cell lines (SKBR3, SUM149, and BT474) were identified. These cell lines were labeled with stable (SILAC) isotopes and subsequently control-treated or treated with erlotinib for 48 hours. Membranes were then isolated and analyzed with quantitative mass-spectrometry (MS). Results were validated in erlotinib-sensitive (SKBR3, SUM149, and BT474) and resistant breast cancer cell lines (MB-231 and BT549), as well as erlotinib-sensitive human non-small cell lung cancer (NSCLC) cell lines (H292 and HCC827).
Results
In total, 1,485 overlapping proteins were quantitatively determined in all cell lines, containing 256 plasma membrane proteins as determined using Gene Ontology. Of those, 8 proteins showed at least 1.5 fold up-regulation and were selected for validation. We found MUC1 to be significantly upregulated in response to erlotinib treatment. Upregulation of MUC1 was detected both at protein and mRNA levels in all erlotinib-sensitive breast cancer cell lines, but not in erlotinib-resistant cell lines. In addition, MUC1 levels were elevated upon erlotinib treatment in NSCLC cell lines H292 (EGFR-WT) and HCC827 (EGFR-exon-19 deletion). MUC1 upregulation was observed as early as 48 hours after treatment, and persisted until 14 days with continuous erlotinib treatment. Upregulation of MUC1 was also observed in response to other clinical EGFR inhibitors, including lapatinib and afatinib, as well as in response to the monoclonal EGFR-directed antibody cetuximab. Erlotinib-induced MUC1 expression was dependent on PI3K/AKT/mTOR signaling, since MUC1 expression was completely blocked when EGFR inhibition was combined with either BEZ235, MK2206, or everolimus, which inhibit PI3K, AKT, and mTOR respectively. Importantly, MUC1 upregulation could be measured by flow cytometry, indicating that elevated MUC1 levels are expressed at plasma membranes and underscoring that MUC1 is a potential target for tracer development for medical imaging.
Conclusion
We identified MUC1 to be specifically upregulated in response to EGFR-directed treatments in breast and lung cancer cell lines. These results warrant in vivo assessment of MUC1 as an effect sensor, to ultimately test whether this strategy can be employed to stratify patients for treatment with EGFR therapeutics.
Supported by ERC grant OnQview-2011-293445 to EGE de Vries.
Citation Format: Harmen R. de Boer, Esméé Joosten, Fabrizia Fusetti, Rudolf S.N. Fehrmann, Elisabeth G.E. de Vries, Marcel A.T.M. van Vugt. Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B19.