Dataset Information

A systematic comparison of label-free, SILAC and TMT techniques to study early adaption towards inhibition of EGFR signaling in the colorectal cancer cell line Difi

ABSTRACT: The epithelial growth factor receptor (EGFR) signalling network plays an essential role inproliferation and survival of colorectal cancer cells. For this reason, EGFR is targeted using therapeutic antibodies blocking EGFR signalling (e.g. cetuximab). However, anti-EGFR therapy is rarely curative as subpopulations of tumor cells survive the initial treatment and develop therapy-resistance. To analyse cellular signalling and assess the quantitative changes of the proteome and the phosphoproteome several proteomics approaches have been established. Here, we compared the widely used quantification strategies label-free (LF), SILAC and TMT and systematically evaluated their technical characteristics including coverage, technical variability, complementarity and the ability to assess statistical significant differences. We studied the dynamics of the EGFR signalling network upon treatment withcetuximab after 0 h, 3 h and 24 h, representing the first cellular adaption towards anti-EGFR therapy, with the aim to identify the most powerful approach for monitoring cellular signalling in this setup. We demonstrate that LF had a superior coverage regarding quantification of both, the proteome and phosphoproteome, while SILAC exhibited the highest precision resulting in superior assessment of differentially abundant proteins and class I phosphosites (p-sites). TMT was outperformed by the other two approaches, illustrating its critical dependency on the applied labelling-design. On the protein level, we observed only little regulation upon cetuximab treatment, whereas a great fraction of p-sites was significantly regulated after 3 h and 24 h. The dynamics of significantly regulated p-sites illustrated an initial downregulation of the EGFR and MAPK signalling pathways, which was partially rescued as soon as after 24h. We identified upregulation and signalling via ERBB3 as a possible mechanism bypassing the blockage of EGFR. Moreover, phosphorylation motifs associated to calcium signalling were continuously upregulated, thereby representing compensatory signalling presumably resulting in the observed reactivation of MAPK1/3 (Erk1/2).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Colon

DISEASE(S): Colon Cancer

SUBMITTER: Markus Stepath

LAB HEAD: Barbara Sitek

PROVIDER: PXD014565 | Pride | 2019-12-11

REPOSITORIES: Pride

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Systematic Comparison of Label-Free, SILAC, and TMT Techniques to Study Early Adaption toward Inhibition of EGFR Signaling in the Colorectal Cancer Cell Line DiFi.

Stepath Markus M Zülch Birgit B Maghnouj Abdelouahid A Schork Karin K Turewicz Michael M Eisenacher Martin M Hahn Stephan S Sitek Barbara B Bracht Thilo T

Journal of proteome research 20200114 2

We evaluated the quantification strategies label-free (LF), stable isotope labeling by amino acids in cell culture (SILAC), and tandem mass tags (TMT) and their performance in quantification of proteins and phosphosites (p-sites) to identify the most powerful approach for monitoring cellular signaling. We analyzed the epidermal growth factor receptor (EGFR) signaling network, which plays an essential role in colorectal cancer, and studied its dynamics within 24 h upon treatment with the EGFR-blo ...[more]

PMID: 31814417

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Project description:Lung cancer continues to be the leading cause of cancer mortality worldwide. The treatment of lung cancer patients harboring mutant EGFR with orally administered EGFR TKIs has been a paradigm shift. Osimertinib and rociletinib are two 3rd generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance to it. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here, we have characterized the proteome and phosphoproteome of a series of isogenic EGFR mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs. To our knowledge, this is the most comprehensive proteomic dataset resource to date to investigate 3rd generation EGFR TKI resistance in lung adenocarcinoma. We have interrogated this unbiased global quantitative proteomic and phosphoproteomic dataset to uncover alterations in signaling pathways, and to reveal a proteomic signature of EMT and kinases / phosphatases with altered protein expression and phosphorylation in the TKI resistant cells. We validated the significant role of SHP2 in the activation of RAS/MAPK and PI3K/AKT signaling pathways. Furthermore, we performed anticorrelation analyses of this phosphoproteomic dataset with the published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures (LINCS) program to predict drugs with the potential to overcome EGFR TKI resistance. We identified that dactolisib, a PI3K/mTOR inhibitor, in combination with osimertinib, may overcome osimertinib resistance both in vitro and in vivo. Introduction Lung cancer continues to be the leading cause of cancer mortality in the world (1). Many lung adenocarcinoma patients with activating epidermal growth factor receptor (EGFR) mutations initially respond dramaticlly to the first- or second-generation EGFR tyrosine kinase inhibitors (TKIs). However, they eventually develop resistance. The most common mechanism of acquired resistance is the EGFR T790M gatekeeper site residue mutation (2). Osimertinib, a third generation irreversible EGFR TKI has been approved by the FDA to treat patients harboring the EGFR T790M mutation who have developed resistance to first- and second- generation EGFR TKIs (3). Recently, osimertinib was also approved for the front-line treatment of patients harboring EGFR mutations (4). Rociletinib is another irreversible inhibitor targeting the EGFR T790M mutation, which has minimal activity against wild-type EGFR. Both drugs have therapeutic benefits and have demonstrated activity in tumors with T790M-mediated resistance to other EGFR tyrosine kinase inhibitors (5, 6). Further development of rociletinib was ceased in 2016 due to less than expected efficacy, poor brain penetration leading to tumor progression in brain tissues and off-target effects on IGFR activation leading to hyperglycemia (7, 8). Although 3rd-generation TKIs provide clinical benefit to most patients with EGFR mutations, some patients, demonstrating primary resistance, still do not respond to these inhibitors. Complete responses are rare, and all patients eventually develop resistance, suggesting primary and acquired resistance mechanisms decrease the efficacy of the drugs (9, 10). Genomic approaches have been used primarily to interrogate osimertinib resistance mechanisms (9, 11-15). Several mechanisms of osimertinib resistance have been identified (16), including novel second site EGFR mutations, activated bypass pathways such as MET amplification, HER2 amplification, RAS mutations, BRAF mutations, PIK3CA mutations, and novel fusion events (17). However, the resistance mechanism is complex and still not fully understood. Previously, we have used SILAC-based quantitative phosphoproteomics to identify the global dynamic modification which occur upon treatment of TKI-sensitive and -resistant lung adenocarcinoma cells with the 1st and 2nd generation EGFR TKIs, erlotinib and afatinib. Utilizing this strategy, we identified the targets of mutant EGFR signaling pathways responsible for TKI resistance, and possible off-target effects of the drugs (18, 19). In this study, we employed SILAC-based quantitative mass spectrometry to characterize alterations in the proteome and phosphoproteome which occur upon acquired resistance and sought to identify novel mechanisms of resistance to the third generation EGFR TKIs, osimertinib and rociletinib. To our knowledge, this is the most comprehensive 3rd generation EGFR TKI resistant proteome and phosphoproteome analysis resource available to date.

			Action	DRS
	MaxQuant_1.6.1.0.zip	Other
	MaxQuant_Output_LF.zip	Other
	MaxQuant_Output_SILAC.zip	Other
	MaxQuant_Output_TMT.zip	Other
	QEXI22082.raw	Raw