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07-05-2013 | Article

Cells resistant to EGFR inhibitors have stem-cell like properties


Free abstract

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 ( is an independent clinical news service provided by Springer Healthcare Limited. © Springer Healthcare Ltd; 2013

By Sally Robertson, medwireNews Reporter