Combined inhibition of MEK and PI3K pathways overcomes acquired resistance to EGFR-TKIs in non-small cell lung cancer

Abstract

Compensatory activation of the signal transduction pathways is one of the major obstacles for the targeted therapy of non-small cell lung cancer (NSCLC). Herein, we present the therapeutic strategy of combined targeted therapy against the MEK and phosphoinositide-3 kinase (PI3K) pathways for acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in NSCLC. We investigated the efficacy of combined trametinib plus taselisib therapy using experimentally established EGFR-TKI-resistant NSCLC cell lines. The results showed that the feedback loop between MEK/ERK and PI3K/AKT pathways had developed in several resistant cell lines, which caused the resistance to single-agent treatment with either inhibitor alone. Meanwhile, the combined therapy successfully regulated the compensatory activation of the key intracellular signals and synergistically inhibited the cell growth of those cells in vitro and in vivo. The resistance mechanisms for which the dual kinase inhibitor therapy proved effective included (MET) mesenchymal-epithelial transition factor amplification, induction of epithelial-to-mesenchymal transition (EMT) and EGFR T790M mutation. In further analysis, the combination therapy induced the phosphorylation of p38 MAPK signaling, leading to the activation of apoptosis cascade. Additionally, long-term treatment with the combination therapy induced the conversion from EMT to mesenchymal-to-epithelial transition in the resistant cell line harboring EMT features, restoring the sensitivity to EGFR-TKI. In conclusion, our results indicate that the combined therapy using MEK and PI3K inhibitors is a potent therapeutic strategy for NSCLC with the acquired resistance to EGFR-TKIs.

Keywords: MEK inhibitor; PI3K inhibitor; acquired resistance; compensatory activation; non-small cell lung cancer.

Figure 1

Growth‐inhibitory effect of combined trametinib plus taselisib therapy on non‐small cell lung cancer (NSCLC) cell lines with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). (A) Survival curves of each resistant cell line treated with trametinib, taselisib, or a combination of the two drugs. When the drug combination was used, equal concentrations of the two drugs were used. (B) Synergistic effect of the combined kinase inhibitor therapy. CI, combination index; Fa, fraction affected

Figure 2

Effect of combined trametinib plus taselisib therapy on the intracellular transduction pathways. Cells were treated with trametinib (100 nmol/L), taselisib (100 nmol/L), or a combination of the two drugs. After 48 h, the cell extracts were analyzed by western blot analysis

Figure 3

Therapeutic effect of combined trametinib plus taselisib therapy on the tumor growth in vivo. (A,B) The mean volumes of the subcutaneous xenograft tumors were calculated for 5 tumors in each group. The combined inhibitor therapy significantly inhibited the tumor growth in the mouse xenograft models of PC9‐GRS (A) and HCC827‐GRS (B). Time‐dependent changes of the tumor volumes are shown on the left side and the appearances of the tumors at the time of sacrifice are shown on the right. (C) At the time point of sacrifice, protein was extracted from the HCC827‐GRS tumor specimens and subjected to western blot analysis

Figure 4

Mechanism underlying the growth inhibition induced by combined trametinib plus taselisib therapy. (A) Cells were treated with trametinib (100 nmol/L), taselisib (100 nmol/L), or a combination of the two drugs for 48 h. The effect of the combined inhibitor therapy on cellular apoptosis was analyzed by western blot analysis. (B) Nuclear staining of HCC827‐GRS, HCC4006‐GRS and PC9‐GRS cells with Hoechst. The percentage of apoptotic cells was calculated by counting the number of cells with condensed chromatin. (C) The results of the phospho‐MAPK protein array analysis using HCC827‐GRS and HCC4006‐GRS cells treated or not treated with the drug combination of trametinib plus taselisib. The phosphorylation statuses of JNK, four p38 isoforms, and het shock protein (HSP)27 were evaluated by densitometric analysis. (D) The combined inhibitor therapy induced the phosphorylation of p38, and p38 inhibitor, SB203580, suppressed the expression of c‐PARP (poly[ADP‐ribose] polymerase) in HCC827‐GRS and HCC4006‐GRS cells. Cells were treated with the combination of trametinib plus taselisib (100 nmol/L each) ± SB203580 (1 μmol/L) for 48 h. (E) The combined inhibitor therapy induced significant G1 cycle arrest in the HCC827‐ARS and HCC4006‐ARS cells. (F) Representative enriched pathways in each group are shown. FDR, false discovery rate; NES, normalized enrichment score

Figure 5

Effects of combined trametinib plus taselisib therapy in HCC827‐ARS cells harboring epithelial‐to‐mesenchymal transition (EMT) features. (A) HCC827‐ARS cells were treated with trametinib plus taselisib (100 nmol/L) for 7 d. Thereafter, the cells were cultured in a drug‐free condition for 7 d. The morphologies of the cells at the indicated times (* Day 0, ** Day 7, *** Day 14) are shown. Scale bars, 100 μm. (B) Alterations of EMT‐related markers. Combined trametinib plus taselisib treatment, but not treatment with either inhibitor used alone, induced conversion from EMT to mesenchymal‐epithelial transition (MET) in the HCC827‐ARS cells. (C) Relative miR‐200c expression level measured by quantitative reverse‐transcription PCR (qRT‐PCR) assay in the HCC827‐ARS cells. Treatment with the drug combination restored the expression of miR‐200c. (D) Relative aldehyde dehydrogenase 1 family member A1 (ALDH1A1) expression level measured by qRT‐PCR assay in the HCC827‐ARS cells. Exposure of the cells to the drug combination did not affect the expression levels of ALDH1A1. (E) The sensitivity of cells (HCC827, HCC827‐ARS [no treatment], HCC827‐ARS (after the 7‐d treatment]) to afatinib were examined. For HCC827‐ARS cells after the 7‐d combined treatment, afatinib sensitivity was investigated in the presence of trametinib (100 nmol/L) and taselisib (100 nmol/L) to maintain MET features. The 50% inhibitory concentration (IC₅₀) values of afatinib were <1 nmol/L for the HCC827 cells, 3.82 μmol/L for the HCC827‐ARS cells (no treatment), and 0.72 μmol/L for the HCC827‐ARS cells (treated with trametinib and taselisib)