Abstract LB-399: Chronic inhibition of mutant EGFR in NSCLC leads to EGFR TKI resistance by TGF-β1 mediated epithelial to mesenchymal transition
Authors: Shimamura Takeshi , Carretero Julian , Xu Chunxiao , Capelletti Marzia , Rikova Klarisa , Gu Ting-Lei , Kobayashi Susumu , Rogers Andrew , Janne Pasi A. , Wong Kwok-Kin , Shapiro Geoffrey I. 
Published: August 2014

Abstract
In NSCLC, activating EGFR mutations underlie responsiveness of NSCLCs to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite initial responses, acquired resistance invariably develops, mediated by the emergence of the secondary T790M mutation and by focal amplification of MET, in approximately 50% and 30% of patients, respectively. The resistance mechanisms for the remaining 20% of cases remain elusive. EGFR TKI-sensitive HCC827 cells were exposed to graded concentrations of erlotinib for 6 months. Approximately 70% of the isolated clones were resistant to erlotinib and harbored MET amplification, and were sensitive to dual EGFR/MET inhibition. The remaining 30% of clones were resistant to EGFR/MET dual-inhibition and demonstrated less than 1.4-fold increase in MET copy number and no secondary T790M mutation. Interestingly, 38 plex Luminex growth factor assays revealed that the 30% of clones for which the mechanism of resistance to erlotinib is unknown secrete high levels of TGF-1. In NSCLC, TGF-β1 has been identified as a potent inducer of epithelial to mesenchymal transition (EMT). Subsequent gene expression profiles of HCC827 erlotinib-resistant clones without MET amplification and secondary EGFR T790M mutation exhibit significant enrichment of genes unique to EMT and clustered separately from gene expression profiles of clones with MET amplification. In agreement with genomic analyses, Western blots confirmed the EMT phenotype in the subset of clones. Additionally, phosphoprotein arrays of the clones demonstrated markedly decreased expression of EGFR, ERBB3, and MET receptors, while maintaining high level of PI3-K MAPK, and SRC activities. Importantly, clones from HCC827 cells with stable EGFR knockdown (HCC827shEGFR) was established and all clones displayed EMT markers, suggesting induction of the EMT phenotype is not due to off-target effects of erlotinib, but rather is primarily due to the sustained loss of EGFR signaling in HCC827 cells. Multiplex Luminex growth factor assays identified TGF-β1 as the most abundant growth factor secreted from HCC827shEGFR clones. Overexpression of mutant EGFR in HCC827shEGFR clones re-sensitized the clones to EGFR TKIs and partially reversed EMT phenotype. To test the roles of TGF-β1in the emergence of EGFR TKI resistance, HCC827 cells were cultured in the media supplemented with TGF-1 for 30 days. MTS assays revealed that HCC827 cells became insensitive to EGFR TKIs after chronic exposure to TGF-β1 for 30 days. However, subsequent removal of TGF-β1 for 30 days re-sensitizes the cells to EGFR TKIs. Consequently, it is hypothesized that sustained suppression of EGFR in HCC827 cells promotes TGF-β1 secretion and subsequent induction of EMT processes, which modulate signaling and apoptosis pathways contributing to the development of resistance to erlotinib.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-399. doi:10.1158/1538-7445.AM2011-LB-399