Unknown,Transcriptomics,Genomics,Proteomics

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The HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR mutated and wildtype NSCLC cells

ABSTRACT: 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 offer a novel therapy approach by sensitizing tumor cells to TKIs. 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 were quantified by WST-1 assay, apoptosis by Annexin V/7-AAD flow cytometry and histone marks (acH3, H3K4me1,-2,-3) by immunoblotting. Expectedly, the EGFR wt cell lines A549 and NCI-H460 were insensitive to the growth-inhibiting effect of single-agent erlotinib (IC50 70-100µM), compared to HCC827 (IC50 <0.02μM). Treatment with panobinostat diminished growth to <50% in both EGFR wt and <30% in HCC827 cells. The combination of both drugs significantly reduced proliferation by ≥70% in A549, >95% in HCC827, but not further in NCI-H460. Panobinostat alone induced differentiation and expression of p21WAF1/CIP1 and p53 in all three cell lines, with almost no further increase when combined with erlotinib. In contrast, combination treatment additively decreased pERK, pAKT and pEGFR in A549, and synergistically induced acH3 in both adenocarcinoma lines. Surprisingly, we also saw an induction of H3K4 methylation marks in all three cell lines. In conclusion, panobinostat synergistically sensitized lung adenocarcinoma cells to the antiproliferative effects of erlotinib. Since single-agent erlotinib has only modest clinical effects in adenocarcinoma EGFR wt patients, combination therapy with an HDACi might offer a promising therapy approach to extend this activity. Copy-number analysis of three NSCLC cell lines HCC827, A549 and NCI-H460 (in unicates) was performed according to protocol by Affymetrix Genome-Wide Human SNP-Array 6.0.

ORGANISM(S): Homo sapiens

SUBMITTER: Gabriele Greve

PROVIDER: E-GEOD-63784 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Introduction: The clinical benefit of EGFR tyrosine kinase inhibitor (TKI) treatment in non-small cell lung cancer (NSCLC) patients with activating EGFR mutations is temporary, as virtually all patients develop acquired EGFR TKI resistance that occurs via diverse mechanisms. Here, we identified increased FGFR1 expression as such a resistance mechanism and using pathways analysis and drug combination testing we identified a novel combination treatment to control growth of these resistant tumors. Methods: Novel erlotinib-resistant NSCLC cell lines were generated and analyzed by mass spectrometry-based proteomics to identify altered pathways associated with erlotinib resistance. The altered pathways were further analyzed in gefitinib and osibenib resistant cell lines. Small molecule inhibitor combinations were used to block the altered pathways and investigate growth reduction in vitro and in two xenograft mouse models. FGFR1 mRNA levels were examined in pre- and (post?)- EGFR TKI treatment clinical tumor samples. Results: Proteomic analysis revealed increased expression of FGFR1 and AXL as well as increased Akt and ERK1/2 activation in a panel of novel erlotinib-resistant HCC827 cell lines. Combined treatment with erlotinib or osimertinib and a panel of small molecule inhibitors targeting AXL/MET, FGFRs, Akt, PI3K/mTOR, MEK or ERK1/2 showed that the most prominent re-sensitization to EGFR TKI occurred with the pan-FGFR inhibitor, PD173074. Interestingly, simultaneous blockade of components of the Akt pathway using specific Akt or dual PI3K-mTOR inhibitors combined with inhibitors targeting the FGFR family exhibited the most efficient growth inhibition of FGFR1 overexpressing EGFR TKI-resistant cell lines. Phosphorylation of proteins downstream of Akt, including PRAS40, FOXO and S6 ribosomal protein, were completely abrogated by PD173074 combined with the Akt inhibitor GSK2141795 . Combination treatment with PD173074 and an Akt inhibitor exhibited synergistic growth inhibition in vivo in two FGFR1 overexpressing NSCLC EGFR TKI-resistant animal models. Conclusion: The significant growth inhibition in vitro and in vivo observed with PD173074 combined with Akt compared to either drug alone imply that inhibition of several key targets may be beneficial in controlling erlotinib-resistant NSCLC. The complete abrogation of PRAS40, FOXO and S6 phosphorylations by PD173074 combined with an Akt inhibitor indicates that the Akt pathway is no longer active.

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The HDAC inhibitor panobinostat acts as a sensitizer for erlotinib activity in EGFR mutated and wildtype NSCLC cells

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