Effect of siRNAs targeting the EGFR T790M mutation in a non-small cell lung cancer cell line resistant to EGFR tyrosine kinase inhibitors and combination with various agents

Abstract

a b s t r a c tThe epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, some mutations confer resistance to current available agents, especially the frequently occurring T790M mutation. In the current study, we have examined, in a NSCLC cell line H1975 containing both L858R and T790M mutations, the effect of T790M-specific-siRNAs versus other EGFR-specific-siRNAs. T790M-specific-siRNAs were able to inhibit T790M and EGFR mRNA, to reduce EGFR protein expression, as well as to reduce the cell growth and induce cell caspase activity in H1975 cells. However, this effect showed less potency compared to the other EGFR-specific-siRNAs. EGFR-specific-siRNAs strongly inhibited cell growth and induced apoptosis in H358, H1650, H292, HCC827 and also in H1975 cells, which showed weak response to tyrosine kinase inhibitors (TKIs) or cetuximab. The addition of T790M-specific-siRNAs could rescue the sensitivity of T790M mutant H1975 cells to TKIs. The combination of T790M-specific-siRNAs and cetuximab also additively enhanced cell growth inhibition and induction of apoptosis in H1975 cells. Among the anti-EGFR agents tested, the strongest biological effect was observed when afatinib was combined with T790M-specific-siRNAs. Afatinib also offered extra effect when combined with cetuximab in H1975 cells. In conclusion, knock-down of T790M transcript by siRNAs further decreases the cell growth of T790M mutant lung cancer cells that are treated with TKIs or cetuximab. The combination of a potent, irreversible kinase inhibitor such as afatinib, with T790M-specific-siRNAs should be further investigated as a new strategy in the treatment of lung cancer containing the resistant T790M mutation.

Combined inhibition of rho-associated protein kinase and EGFR suppresses the invasive phenotype in EGFR-dependent lung cancer cells

“…EGFR T790M2600 -specific mediated-knockdown effected a ∼25% reduction in cell growth 72 h post-transfection in concordance with our previous findings [17], while EGFR WT2600 siRNA reduced cell growth by ∼15% at the same time point (Fig. 2c).…”

“…24 h later, cells were transfected with siRNA against EGFR WT2600 , EGFR T790M2600 and scrambled EGFR T790M2600 [17]; EGFR, HER2, Genome Non-Targeting siRNA (Thermo Scientific); and ROCK (Qiagen) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.…”

Serum CEA Level Change and Its Significance Before and after Gefitinib Therapy on Patients with Advanced Non-small Cell Lung Cancer

“…not only regulate cell proliferation, but also has critical impact on the development of tumors, formation of vessels as well as metastasis and spreading of tumor cells, whereas over-expression of EGFR is insensitive to chemotherapy and radiotherapy, which can easily bring about distant metastasis and poor prognosis Karachaliou et al, 2013;Lopez-Rios et al, 2013). Results of other studies have indicated that EGFR is over expressed in 40~80% of NSCLC patients with poor prognosis but it can be activated by selective inhibition of targeted therapy on the activity of tyrosine kinase (TK) to inhabit proliferation, invasion and metastasis of tumor cells and strengthen chemotherapeutic effect Chen et al, 2013).…”

Expression and Prognostic Significance of lncRNA MALAT1 in Pancreatic Cancer Tissues

“…Amplification of the appropriate product was confirmed by melting curve analysis and gel electrophoresis. Relative mRNA expression of MALAT1 was calculated with the comparative threshold cycle (CT) (2 −ΔΔCT ) method ( Livak and Schmittgen, 2001;Chen et al, 2013a;2013b).…”