Background: Epithelial-mesenchymal-transition (EMT) is an epigenetic-based mechanism contributing to the acquired treatment resistance against receptor tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR)-mutations. Delineating the exact epigenetic and gene-expression alterations in EMT-associated EGFR TKI-resistance (EMT-E-TKI-R) is vital for improved diagnosis and treatment of NSCLC patients. Methods: We characterized genome-wide changes in mRNA-expression, DNA-methylation and the histone-modification H3K36me3 in EGFR-mutated NSCLC HCC827 cells in result of acquired EMT-E-TKI-R. CRISPR/Cas9 was used to functional examine key findings from the omics analyses. Results: Acquired EMT-E-TKI-R was analyzed with three omics approaches. RNA-sequencing identified 2,233 and 1,972 up- and down-regulated genes, respectively, and among these were established EMT-markers. DNA-methylation EPIC array analyses identified 14,163 and 7,999 hyper- and hypo-methylated, respectively, differential methylated positions of which several were present in EMT-markers. Finally, H3K36me3 chromatin immunoprecipitation (ChIP)-sequencing detected 2,873 and 3,836 genes with enrichment and depletion, respectively, and among these were established EMT-markers. Correlation analyses showed that EMT-E-TKI-R mRNA-expression changes correlated better with H3K36me3 changes than with DNA-methylation changes. Moreover, the omics data supported the involvement of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1 signaling axis for acquired EMT-E-TKI-R. CRISPR/Cas9-mediated analyses corroborated the importance of ZEB1 in acquired EMT-E-TKI-R, MIR200C and MIR141 to be in an EMT-E-TKI-R-associated auto-regulatory loop with ZEB1, and FGFR1 to mediate cell survival in EMT-E-TKI-R. Conclusions: The current study describes the synchronous genome-wide changes in mRNA-expression, DNA-methylation, and H3K36me3 in NSCLC EMT-E-TKI-R. The omics approaches revealed potential novel diagnostic markers and treatment targets. Besides, the study consolidates the functional impact of the MIR141/MIR200C-ZEB1/ZEB2-FGFR1-signaling axis in NSCLC EMT-E-TKI-R.
Bibliografisk noteFunding Information:
The project was supported by Kræftfonden, Dagmar Marshalls Mindelegat, Fabrikant Einar Willumsens Mindelegat, Marie og Børge Kroghs Fond, P. A. Messerschmidt og Hustrus Fond, Thora og Viggo Grove's Mindelegat, Familien Erichsens Familiefond, Axel Muusfeldts Fond, Direktør Emil C. Hertz og Hustru Inger Hertz' Fond, Købmand Sven Hansen og Hustru Ina Hansens Fond, Peetz legat, Frimodt-Heineke Fonden, Else og Mogens Wedell-Wedellsborgs Fond, and Harboefonden.
© Translational Lung Cancer Research. All rights reserved.