PTEN loss contributes to erlotinib resistance in EGFR-mutant lung cancer by activation of Akt and EGFR

Abstract

Clinical resistance to epidermal growth factor receptor (EGFR) inhibition in lung cancer has been linked to the emergence of the EGFR T790M resistance mutation or amplification of MET. Additional mechanisms contributing to EGFR inhibitor resistance remain elusive. By applying combined analyses of gene expression, copy number, and biochemical analyses of EGFR inhibitor responsiveness, we identified homozygous loss of PTEN to segregate EGFR-dependent and EGFR-independent cells. We show that in EGFR-dependent cells, PTEN loss partially uncouples mutant EGFR from downstream signaling and activates EGFR, thereby contributing to erlotinib resistance. The clinical relevance of our findings is supported by the observation of PTEN loss in 1 out of 24 primary EGFR-mutant non-small cell lung cancer (NSCLC) tumors. These results suggest a novel resistance mechanism in EGFR-mutant NSCLC involving PTEN loss.

Figure 1

An EGFR independence signature in H1650 cells. A, hierarchical clustering of 53 NSCLC cells according to gene expression. Erlotinib sensitivity (IC₅₀ < 1 μmol/L, red; IC₅₀ > 1 μmol/L, gray) and EGFR mutations (EGFR-mutant, black; T790M, red; EGFR wild-type, gray) as well as MET amplification (black). B, left, cellular viability as a function of erlotinib dose for all three cell lines studied. Right, mutation status and IC₅₀ values. C, cells were treated with different doses of erlotinib. Activation of EGFR and downstream signaling pathways was determined by analyzing the amount of phosphorylated versions of the respective proteins in comparison with their total levels using phosphorylation-specific antibodies.

Figure 2

Genomic characterization of PTEN loss in H1650 cells. A, list of genes affected by differential lesions between H1650 cells and EGFR-mutant and erlotinib-sensitive cell lines. B, left, screenshot showing chromosomal aberrations at chromosome 10 (Integrative Genomics Viewer; http://www.broad.mit.edu/igv/) of all EGFR-mutant cells. Middle, 3′-region mapping of PTEN using quantitative PCR reveals a homozygous deletion deleting parts of exon 8 and the entire exon 9. Right, the sequence bridging the breakpoint. C, left, PTEN protein status determined using immunoblotting in different NSCLC cell lines. Right, NH₂-terminal and COOH-terminal PTEN detection by immunoblotting. LNCAP cells, known to express a truncated version of PTEN, served as controls. D, analysis of EGFR mutations (red) and homozygous deletions of PTEN (black) and PTEN mutations (blue) in 140 lung cancer biopsy specimens.

Figure 3

Erlotinib resistance in EGFR-mutated NSCLC with PTEN loss. A, left, in H1650^PTEN cells, PTEN levels were determined by immunoblotting. Right, levels of phospho-EGFR and phospho-AKT were assessed by immunoblotting in H1650, H1650^MOCK, and H1650^PTEN cells treated with erlotinib. B, left, in PC9^PTENkd cells, PTEN levels were determined by immunoblotting. Right, levels of phospho-EGFR and phospho-AKT were assessed in PC9, PC9^CONTkd, and PC9^PTENkd cells treated with erlotinib. C, left, percentage of apoptotic cells (in %, analyzed by measuring the fraction of cells positive for Annexin V and/or propidium iodide by flow cytometry) after treatment with either erlotinib (1 μmol/L) or control. Right, cumulative histograms of apoptosis induction. D, levels of Bim (EL, extra long; L, long; S, short), phospho-ERK, phospho-pAKT, and actin were measured after serum starvation (serum starvation “+”), EGF stimulation (EGF “+”), or treatment with erlotinib (1 μmol/L erlotinib “+”) for 24 h.

Figure 4

PTEN loss activates EGFR. A, phospho-EGFR was detected by immunoblotting after short exposure (SE) and long exposure (LE) in PC9, PC9^CONTkd, and PC9^PTENkd cells. Actin levels served as a loading control. B, left, levels of phospho-EGFR of PC9^PTENkd and PC9 cells treated with erlotinib were determined (+/− EGF) under serum starvation. Right, apoptosis (%) after erlotinib treatment (0.5 μmol/L) in the given cells. C, left, phospho-EGFR and phospho-AKT in H3255 and H3255^MyrAKT cells were assessed by immunoblotting. Right, the fraction of apoptotic cells (in %) in the given cells. D, a simplified model explaining our observations: in EGFR-mutant cells, EGFR is the sole input for production of PIP3. Inhibiting EGFR dramatically reduces the input into PIP3 production. Therefore, the lack of negative regulation of PIP3 production by loss of PTEN is limited.