MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30-40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI

Analysis of the <i>EGFR</i> gene in the RA2 ERL-resistant cell line.

<p>A) Analysis of <i>EGFR</i> exon 19 nucleotides sequence. The pherogram of the parental cell line with peaks corresponding to the <i>EGFR</i> mutated sequence (ΔE746-A750) and the pherogram of the RA2 resistant cells with peaks corresponding to the mutated and wild type (WT) EGFR nucleotides sequence are shown. B) qPCR analysis. Relative <i>EGFR</i> gene copy number (GCN) in genomic DNA, normalized to the <i>Rnase P</i> gene, is expressed relative to the levels in parental cell lines (P) set as 1 (mean ± SD of triplicate determinations). Results are representative of those obtained from 2 independent analysis.</p

MET analysis in ERL-resistant cell lines.

<p>A) qPCR analysis of gene copy numbers of <i>MET;</i> B) western blots of total cell lysates with the antibodies indicated of parental (P) and ERL-resistant cell lines; C) qPCR analysis of MET mRNA expression in parental (P) and ERL-resistant cell lines. <i>MET</i> gene and mRNA in A) and C) are normalized to RNaseP gene and rp-L31 mRNA respectively and both are expressed relative to the levels in parental cell lines set as 1 (mean ± SD of triplicate determinations). qPCR data are representative of those obtained from 2 independent analysis; D) Confocal microscopy analysis of MET receptor (green) expression in xenograft nodes of mice subcutaneously injected with the parental HCC827 cells and the ERL-resistant RA1, RB1 and RA2 cell lines. Images show representative xy-plane maximum projection of the specimens. Scale bars correspond to 15 μm.</p

Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.

<p>Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.</p

HER2/HER3 and AXL expression and phosphorylation analysis.

<p>A) Representative western blots of total cell lysates of HCC827 and HCC4006 parental cell lines (P) and their derived ERL-resistant cell lines. Arrows indicate the expected molecular weight size. Total cell lysates loaded were 40 μg for AXL and pAXL analyses and 25 μg for the others. B) qPCR analysis of AXL mRNA normalized to rp-L31 mRNA and expressed relative to the levels in parental cell lines set as 1 (mean ± SD of triplicate determinations). Western blots and qPCR data are representative of those obtained respectively from 3 and 2 independent analysis. C) Dose-effect curves were calculated using CompuSyn software and plotting the entered Fa values against the entered dose values. For combination treatments, the combined drugs dose was entered. Each data point represents the mean of 3 replicates.</p

Cell inhibition growth analysis of ERL-resistant NSCLC cell lines.

<p>A) Representative dose-effect curve plots of HCC827 and HCC4006 parental cell lines to the indicated TKIs. Cell viability was determined by MTT assays. The results are expressed as the percentage of cell viability in drug-treated cultures relative to DMSO-treated control samples; B) Dose effect curve plots of derived ERL-resistant HCC827 and HCC4006 cell lines. The results are expressed as described above. Data (mean ± s.e.m) in A and B are representative of more than three independent experiments.</p

Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.

<p>Dm₅₀ of single agent and drugs combination for parental and ERL-resistant NSCLC cell lines.</p

Erlotinib impairs EGFR and ERK1/2 phosphorylation in ERL-resistant cell lines.

<p>A) Representative western blots with EGFR and ERK1/2 antibodies and B) pEGFR (Y1068) and pERK1/2 (T202/Y204) antibodies in the indicated parental and ERL-resistant cell lines treated with EGF (100 μg/ml), ERL (Erlotinib, 100 nM) or vehicle (DMSO) at different time points (8’, 30’, 1hr, 3hrs). Densitometric analyses of band signals were normalized with GAPDH, the number indicates the signals quantification at 30’ upon ERL-treatment. For RA2 cell line double amount of total cell lysate was loaded to analyze EGFR expression.</p

IC₅₀ values of TKIs against parental and ERL-resistant NSCLC cell lines.

<p>IC₅₀ values of TKIs against parental and ERL-resistant NSCLC cell lines.</p