Abstract
BackgroundEpidermal growth factor receptor (EGFR) signaling plays an important role in non-small cell lung cancer (NSCLC) and therapeutics targeted against EGFR have been effective in treating a subset of patients bearing somatic EFGR mutations. However, the cancer eventually progresses during treatment with EGFR inhibitors, even in the patients who respond to these drugs initially. Recent studies have identified that the acquisition of resistance in approximately 50% of cases is due to generation of a secondary mutation (T790M) in the EGFR kinase domain. In about 20% of the cases, resistance is associated with the amplification of MET kinase. In the remaining 30-40% of the cases, the mechanism underpinning the therapeutic resistance is unknown.MethodsAn erlotinib resistant subline (H1650-ER1) was generated upon continuous exposure of NSCLC cell line NCI-H1650 to erlotinib. Cancer stem cell like traits including expression of stem cell markers, enhanced ability to self-renew and differentiate, and increased tumorigenicity in vitro were assessed in erlotinib resistant H1650-ER1 cells.ResultsThe erlotinib resistant subline contained a population of cells with properties similar to cancer stem cells. These cells were found to be less sensitive towards erlotinib treatment as measured by cell proliferation and generation of tumor spheres in the presence of erlotinib.ConclusionsOur findings suggest that in cases of NSCLC accompanied by mutant EGFR, treatment targeting inhibition of EGFR kinase activity in differentiated cancer cells may generate a population of cancer cells with stem cell properties.
Highlights
Epidermal growth factor receptor (EGFR) signaling plays an important role in non-small cell lung cancer (NSCLC) and therapeutics targeted against EGFR have been effective in treating a subset of patients bearing somatic EFGR mutations
Sequencing of the EGFR gene revealed the persistence of the deletion mutation ΔE746-A750 within the EGFR kinase domain in both H1650 and the resistant H1650-ER1 subline; no additional mutation was observed in the EGFR open reading frame in H1650-ER1 cells
Taken together our observations suggest that H1650-ER1 cells have undergone a partial epithelial to mesenchymal transition (EMT)
Summary
Cells Human lung cancer cell line NCI-H1650 ( forth referred to as H1650) was obtained from ATCC (Manassas, VA). Human head and neck squamous cell carcinoma cell line SCC-1 and erlotinib and gefitinib resistant sublines (SCC-1-Erl-R and SCC-1-Gef-R) were maintained in DMEM supplemented with 10% FBS, and 1 μg/mL hydrocortisone. Growth medium (RPMI1640 supplemented with 10% FBS and 2 mM glutamine) was changed every 3 days. In order to investigate the ability of SP cells to differentiate, sorted SP and non SP cells were cultured in RPMI 1640 for 10 days. Flow analysis H1650 and H1650-ER1 cells (2 × 105) were fixed in 1% paraformaldehyde for 10 min at 37°C and incubated overnight with Alexa647-CD24 (BD Biosciences), FITCCD44 (BD Biosciences), APC-CD133 (Miltenyi Biotec), PE-anti-SSEA-3 (BD Pharmingen), SSEA-4 (Santa Cruz), Tra-1-60 (Santa Cruz), and Tra-1-80 antibodies (Santa Cruz) (1:500 in PBS with 2% FBS and 0.1% NaN3) at 4°C. Control samples were incubated with only secondary antibody or APC-mouse IgG and PE-rat IgM antibodies
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