title: The pan-HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR-mutated and -wildtype non-small cell lung cancer cells.
abstract: BACKGROUND
  The receptor tyrosine kinase (RTK) EGFR is overexpressed and mutated in NSCLC. These mutations can be targeted by RTK inhibitors (TKIs) such as erlotinib. Chromatin-modifying agents may offer a novel therapeutic approach by sensitizing tumor cells to TKIs.
  
  
  METHODS
  The NSCLC cell lines HCC827 (EGFR mutant, adenocarcinoma), A549 (EGFR wt, adenocarcinoma) and NCI-H460 (EGFR wt, large cell carcinoma) were analyzed by SNP6.0 array. Changes in proliferation after panobinostat (LBH-589, PS) and erlotinib treatment were quantified by WST-1 assay and apoptosis by Annexin V/7-AAD flow cytometry. Abundance of target proteins and histone marks (acH3, H3K4me1/2/3) was determined by immunoblotting.
  
  
  RESULTS
  As expected, the EGFR wt cell lines A549 and NCI-H460 were quite insensitive to the growth-inhibitory effect of erlotinib (IC50 70-100 μM), compared to HCC827 (IC50<0.02 μM). All three cell lines were sensitive to PS treatment (IC50: HCC827 10 nM, A549 20 nM and NCI-H460 35 nM). The combination of both drugs further reduced proliferation in HCC827 and in A549, but not in NCI-H460. PS alone induced differentiation and expression of p21WAF1/CIP1 and p53 and decreased CHK1 in all three cell lines, with almost no further effect when combined with erlotinib. In contrast, combination treatment additively decreased pEGFR, pERK and pAKT in A549. Both drugs synergistically induced acH3 in the adenocarcinoma lines. Surprisingly, we also observed induction of H3K4 methylation marks after erlotinib treatment in HCC827 and in A549 that was further enhanced by combination with PS.
  
  
  CONCLUSIONS
  PS sensitized lung adenocarcinoma cells to the antiproliferative effects of erlotinib. In these cell lines, the drug combination also had a robust, not previously described effect on histone H3 acetylation and H3K4 methylation.
authors.affiliation: University of Freiburg Medical Center, Freiburg, Germany. gabriele.greve@uniklinik-freiburg.de.|University of Freiburg, Faculty of Biology, Freiburg, Germany. gabriele.greve@uniklinik-freiburg.de.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Greve
authors.affiliation: University of Freiburg Medical Center, Freiburg, Germany. insa.schiffmann@uniklinik-freiburg.de.|University of Freiburg, Faculty of Medicine, Freiburg, Germany. insa.schiffmann@uniklinik-freiburg.de.
authors.forename: Insa
authors.initials: I
authors.lastname: Schiffmann
authors.affiliation: University of Freiburg Medical Center, Freiburg, Germany. dietmar.pfeifer@uniklinik-freiburg.de.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Pfeifer
authors.affiliation: University of Freiburg Medical Center, Freiburg, Germany. milena.pantic@uniklinik-freiburg.de.
authors.forename: Milena
authors.initials: M
authors.lastname: Pantic
authors.affiliation: Department for in vivo Tumorbiology, Oncotest GmbH, Freiburg, Germany. julia.schueler@oncotest.de.
authors.forename: Julia
authors.initials: J
authors.lastname: Schüler
authors.affiliation: University of Freiburg Medical Center, Freiburg, Germany. michael.luebbert@uniklinik-freiburg.de.|DKTK, German Consortium for Translational Cancer Research, Freiburg, Germany. michael.luebbert@uniklinik-freiburg.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Lübbert
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004357:Drug Synergism; D066246:ErbB Receptors / Q000235:genetics; D000069347:Erlotinib Hydrochloride / Q000494:pharmacology; D018773:Genes, erbB-1; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000235:genetics*; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D000077767:Panobinostat
chemical_list: D000970:Antineoplastic Agents; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D007211:Indoles; D000077767:Panobinostat; D000069347:Erlotinib Hydrochloride; C512478:EGFR protein, human; D066246:ErbB Receptors