An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance

Abstract

Purpose: Epithelial-mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non-small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study.

Experimental design: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified.

Results: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies.

Conclusion: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype.

Fig 1. The EMT gene expression signature separates NSCLC cell lines into distinct epithelial-like and mesenchymal-like groups independent of microarray platform

(A) Affymetrix probes corresponding to the EMT signature genes were clustered by two-way hierarchical clustering using Pearson correlation distance between genes (rows), Euclidean distance between cell lines (columns), and the Ward’s linkage rule. NSCLC cell lines separated into distinct epithelial (green bar) and mesenchymal (red bar) groups at the first major branching of the dendrogram. Mutation status for EGFR and KRAS are indicated by the color bars above the heatmap (dark blue=mutated, light blue=wild-type, white=unknown). EGFR mutations were seen only in the epithelial group. KRAS mutations were more common in the mesenchymal group and expressed higher levels of FN1 and FN1-associated genes. (B) Cell line classifications were concordant across platforms, with the exception of H1395 which switched from epithelial to mesenchymal group when arrayed on the Illumina WG v2 platform. First principal component analysis shows good separation of the epithelial and mesenchymal groups on both Affymetrix and Illumina platforms. (C) Characteristic differences in morphology are seen between lines characterized as epithelial or mesenchymal by the EMT signature. (D) In an independent set of 39 NSCLC cell lines profiled on a third platform (Illumina WGv3), the EMT signature separated cell lines into distinct epithelial (green) and mesenchymal (red) groups by hierarchical clustering and principal component analysis.

Fig 2. Integrated analysis of protein expression and the EMT signature

(A) RPPA analysis reveals that cell lines dichotomize into epitheial and mesenchymal groups based on their overall proteomic signature. (B) E-cadherin protein levels quantified by RPPA were strongly correlated with the EMT signature first principal component in the training and testing cell line sets. (C) Hierarchical clustering of proteins strongly associated with an epithelial or mesenchymal signature showed higher expression of EGFR pathway proteins and Rab25 in epithelial lines. (D) Axl expression was significantly higher in a subset of mesenchymal cell lines at the mRNA and protein levels.

Fig 3. Mesenchymal lines are significantly more resistant to EGFR inhibition and PI3K pathway inhibition but sensitive to Axl inhibition by SGI-7079

(A) Relative IC₅₀ levels of targeted agents are shown with p-values corresponding to Wilcoxon rank sum test. (B) Fold difference between mean IC50s in epithelial (E) versus mesenchymal (M) cell lines. (C) SGI-7079 inhibits Gas6-induced Axl phosphorylation as shown by Western analysis. Densitometry histogram of the Western blot is graphed as the percentage of no drug treatment control of p-Axl relative to total Axl. The EC₅₀ for SGI-7079 is < 100 nM. (D-E) Mesenchymal cell lines (red bars) are relatively more sensitive to SGI7079 whereas epithelial cell lines (black bars) are more sensitive to erlotinib. Gray bar (C) denotes 1uM concentration.

Fig 4. Axl blockade by SGI-7079 inhibits the growth of mesenchymal NSCLC xenograft tumors

(A) Mean tumor volume for A549 xenografts implanted in mice treated with vehicle or SGI-7079. (B) Mean tumor volume for A549 xenografts in mice treated with vehicle and the combination of SGI-7079 plus erlotinib.

Fig 5. Improved 8-week disease control in BATTLE patients with epithelial signatures treated with erlotinib

(A) BATTLE patients (all treatment arms) were classified as mesenchymal or epithelial-like based on the EMT signature. (B) Among patients with wild type EGFR and KRAS treated with erlotinib, 8-week disease control appeared superior in patients with more epithelial tumors (p=0.052). (C) There was no significant difference in 8 week disease control between epithelial and mesenchymal tumors in other treatment arms.