Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysi
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RESEARCH ARTICLE
Open Access
Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines Karolin Ebert1, Gwen Zwingenberger1, Elena Barbaria1, Simone Keller1, Corinna Heck1, Rouven Arnold1, Vanessa Hollerieth1, Julian Mattes2, Robert Geffers3, Elba Raimúndez4,5, Jan Hasenauer4,5,6 and Birgit Luber1*
Abstract Background: Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies. Methods: A time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCIN87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3. Results: The Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments. (Continued on next page)
* Correspondence: [email protected] 1 Fakultät für Medizin, Technische Universität München, Klinikum rechts der Isar, Institut für Allgemeine Pathologie und Pathologische Anatomie, 81675 München, Germany Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative
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