Cells resistant to EGFR inhibitors have stem-cell like properties
medwireNews: The mechanism by which resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) is acquired in vitro seems to be influenced by the method of EGFR-TKI exposure used during culture, report researchers.
"The microenvironment is a determining factor for the mechanisms underlying acquisition of resistance to EGFR-TKI," say Shinichi Toyooka (Okayama University Hospital, Japan) and colleagues.
In a study of cell lines resistant to EGFR-TK1 in EGFR mutant lung cancers that were exposed to gefitinib, use of the stepwise escalation method established the majority of previously reported gefitinib-resistant cells (GR-step series). In addition, they established these resistant cells with the high-concentration exposure method, but the use of this approach also revealed novel features of the cells.
As reported in Cancer Research, nine GR sublines were established: HCC827-GR-step, PC-9-GR-step, HCC4006-GR-step, HCC4011-GR-step, HCC827-GR-high1, HCC827-GR-high2, PC-9-GR-high, HCC4006-GR-high, and HCC4011-GR-high.
In sublines established with the stepwise escalation method, real-time quantitative polymerase chain reaction (qPCR) showed that HCC827-GR-step and HCC4011-GR-step exhibited MET amplification and PC-9-GR step showed the presence of EGFR T790M mutation. Among sublines identified using the high-concentration method, only HCC4011-GR-high showed MET amplification.
Each of the sublines HCC827-GR-high1, HCC827-GR-high2, HCC4006-GR-high, and HCC4006-GR-step displayed epithelial to mesenchymal transition features, while PC-9 and HCC4011 cells and the derived resistant sublines did not.
After acquiring resistance to EGFR-TKIs, all sublines except HCC827-GR-high2 retained EGFR mutations. Furthermore, examination of the mRNA expression profile for HCC827-GR-high2 showed the upregulation of many genes and particularly aldehyde dehydrogenase isoform 1 (ALDH1A1) and ABC transporters, which were observed in the cells with stem-like properties.
Furthermore, the upregulation of ALDH1A1 in HCC827-GR-high was confirmed by Western blotting and immunofluorescent immunocytochemistry.
"Up-regulation of ALDH1A1 and ABC transporters suggests that HCC827-GR-highs may acquire stem cell-like properties," say the researchers who went on to quantify the side-population (SP) using dual wavelength flow cytometry.
The researchers found a significant increase in the number of SP cells in HCC827-GR-highs, with the majority of the SP fraction eliminated in the presence of verapamil, demonstrating the specificity of the SP.
Furthermore, a sphere formation assay showed that the HCC827-GR-highs acquired a higher ability to form spheres in suspension culture, compared with HCC827 and HCC827-GR-step.
The researchers also report that the resistant sublines with stem cell-like properties were resistant to chemotherapy agents but equally sensitive to histone deacetylase and proteasome inhibitors, compared with parental cells.
medwireNews (www.medwirenews.com) is an independent clinical news service provided by Springer Healthcare Limited. © Springer Healthcare Ltd; 2013
By Sally Robertson, medwireNews Reporter