Journal of Thoracic Oncology:
Tumor Dependence on the EGFR Signaling Pathway Expressed by the p-EGFR:p-AKT Ratio Predicts Erlotinib Sensitivity in Human Non-small Cell Lung Cancer (NSCLC) Cells Expressing Wild-Type EGFR Gene
Li, Tianhong MD, PhD; Ling, Yi-He PhD; Perez-Soler, Roman MD
Department of Oncology, Montefiore Medical Center and the Albert Einstein Cancer Center, Bronx, NY.
Disclosure: Tianhog Li: none; Yi-He Ling: none; Roman Perez-Soler:Grants/Research support from OSIP and NIH; Stockholder: OSIP, Genentech, Amgen, Novartis, Lilly, Transave, AstraZeneca; Consultant: OSIP, AstraZeneca, Genentech, Amgen, Novartis, Alza, Lilly, BMS, Transave.
Address for correspondence: Tianhong Li, MD, PhD, Department of Oncology, Montefiore Medical Center, Albert Einstein College of Medicine, 1825 Eastchester Road, 2 South, Room 55, Bronx, NY 10461. E-mail: email@example.com
Supported by National Cancer Institute grants CA91784 and CA96515 and a grant from OSI Pharmaceuticals, Inc.
Introduction: This study was undertaken to identify molecular determinants of tumor dependency on the epidermal growth factor receptor (EGFR) signaling pathway for predicting clinical benefit from erlotinib monotherapy in non-small cell lung cancer (NSCLC) patients with tumors expressing wild-type EGFR gene.
Methods: The effect of erlotinib on the total and phosphorylated protein expression of EGFR and key downstream signaling molecules was determined by immunoblots in a panel of NSCLC cells expressing wild-type EGFR gene. The parameters that correlate with cell sensitivity and resistance to erlotinib was analyzed.
Results: Individual assessment of total or phosphorylated protein expression of EGFR or a downstream signaling molecule does not correlate with sensitivity to erlotinib in these NSCLC tumors. Resistance of NSCLC cells to erlotinib is associated with failed inhibition of at least one phosphorylated downstream signaling molecule. The dependency of NSCLC cells on the activated EGFR axis was measured by the ratio of p-EGFR to a phosphorylated downstream protein. A high ratio should indicate that activation of a downstream signaling molecule primarily results from the activation of upstream EGFR; and a low ratio should indicate that activation of a downstream signaling molecule primarily results from the activation of a upstream receptors other than EGFR. The p-EGFR:p-AKT ratio was 10-fold higher in erlotinib-sensitive cells than erlotinib-resistant cells (p = 0.03). It was the best predictor of erlotinib sensitivity among all parameters analyzed in this panel of NSCLC cell lines.
Conclusions: The p-EGFR:p-AKT ratio deserves further investigation as a predictive parameter for clinical response to erlotinib in NSCLC tumors expressing wild-type EGFR gene.
Erlotinib, an oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was approved by the US FDA in 2004 for unselected, advanced non-small cell lung cancer (NSCLC) patients who have failed first-line combination chemotherapy.1,2 About 10% of NSCLC patients whose tumors express high levels of EGFR protein as a result of the presence of tyrosine kinase-activating somatic mutations in the EGFR gene and/or high EGFR gene copy number exhibit a higher degree of dependence on the EGFR pathway and a higher tumor response to EGFR TKIs.3,4 However, acquired resistance to EGFR TKIs emerges in these tumors by at least two mechanisms: (1) the presence of a second somatic mutation T790M, which prevents drug binding to the TK domain of EGFR; and (2) activation of alternative signaling pathway(s) as an escape or survival response.5,6 The molecular mechanisms of sensitivity and resistance of NSCLC tumors expressing wild-type EGFR gene to EGFR TKIs are less clear, although a survival benefit is also observed in approximately 35% of NSCLC patients who have a robust and long-lasting stable disease (i.e., cytostatic effect) from erlotinib treatment.2,7 These patients, who achieve stable disease from erlotinib or gefitinib treatment have similar time to disease progression and overall survival compared with those NSCLC patients who achieve an objective response.8
To select this subgroup of NSCLC patients, this study was undertaken to identify molecular determinants of tumor dependency on the EGFR pathway and hence of erlotinib sensitivity in a panel of human NSCLC cell lines expressing the wild-type EGFR gene. The activity of EGFR signal transduction pathway was assessed by the expression of total and phosphorylated protein of EGFR and downstream signaling proteins by immunoblots. The parameters that could correlate with cell sensitivity and resistance to erlotinib were explored.
MATERIALS AND METHODS
Drug, Antibodies, and Cell Lines.
Erlotinib (Tarceva, formerly OSI 774) was obtained from OSI pharmaceuticals and processed as described before.9 Most of the antibodies used for immunoblots were purchased from Cell Signaling (Beverly, MA). Antibodies against extracellular signal-responsive kinase (ERK)1/2 and p-ERK1/2 were obtained from Promega (Seattle, WA). All human tumor cell lines used in this study, except HN5 human head and neck carcinoma cell line (a generous gift from OSI Pharmaceuticals) were purchased from the American Type Culture Collection (Manassas, VA) and maintained routinely in RPMI 1640 medium supplemented with 10% fetal bovine serum.
Growth inhibition assay and immunoblot analysis were performed as previously described.9
RESULTS AND DISCUSSION
Neither Total EGFR nor p-EGFR Expression Predicts Erlotinib Sensitivity
We confirmed our previous findings that NSCLC cell lines expressing wild-type EGFR gene displayed a variable, dose-dependent sensitivity to erlotinib9 in a panel of nine human NSCLC cell lines with different clinical and histologic features (H23, H322, H358, H460, H522, A549, H596, H661, and H1299) (IC50: 0.8 to >20 μM, data not shown). There was no absolute intrinsic resistance to erlotinib observed, although at 2 μM, the median plasma concentration in patients taking a standard dose of erlotinib 150 mg daily,10 only two of nine NSCLC cell lines showed a >50% growth inhibition (data not shown). We further characterized the effect of EGF and erlotinib on the EGFR expression in four erlotinib-sensitive tumor cell lines (H322, H358, HN5 and A431; IC50 <2 μM) and four erlotinib-resistant NSCLC cell lines (H460, A549, H596 and H1299; IC50 ≥2 μM) by immunoblots (Figure 1). The cytotoxicity of HN5 and A431 cells to erlotinib was used as positive controls since they express high level of EGFR protein and are known to be sensitive to EGFR TKIs.11–13
The expression of p-EGFR at tyrosine (Y1068) was measured as it is the binding site for the PI3K and MAPK interacting protein Gab1, which mediates the activation of downstream PI3K/AKT and MAP/ERK signal pathways. The baseline expression of p-EGFR was barely detectable in all tested cell lines deprived of serum (Figure 1A, lane 1), suggesting that these NSCLC cells have low constitutive EGFR activation in the absence of ligands. Addition of EGF to the cell culture media dramatically induced the phosphorylation of EGFR in both erlotinib-sensitive and erlotinib-resistant tumor cell lines (Figure 1A, lane 2), suggesting that the EGFR pathway can be activated in response to EGF in all tested NSCLC cell lines although this modulation does not alter the sensitivity to erlotinib. With increasing concentrations of erlotinib, the expression of p-EGFR (upper panels) was inhibited to a much greater extent as compared with that of t-EGFR (lower panels) in each cell line (Figure 1A, lanes 3–6). Two μM of erlotinib inhibited p-EGFR expression by about 90% in all NSCLC cell lines, except for H1299, regardless of their sensitivity to erlotinib (Figure 1B). Of note, 20 μM of erlotinib only inhibited p-EGFR expression by only approximately 80% in HN5 cells that were sensitive to erlotinib (Figure 1B, legend ♢). Thus, neither the expression of p-EGFR nor t-EGFR correlates with the sensitivity of these human NSCLC cells to erlotinib. We did not find any correlation between the level of total and phosphorylated ErbB-2 or ErbB-3 protein expression and erlotinib sensitivity in the cell lines studied (data not shown), although only a few cell lines were analyzed.
Lack of Inhibition of Phosphorylated Downstream Signaling Proteins of EGFR Predicts Resistance of NSCLCL Cells to Erlotinib
Three major signaling pathways mediate the downstream effects of EGFR activation: the cell proliferation pathway (Ras/ERK) and survival pathways [PI3K/AKT and Jak/STAT]. Figure 2 illustrates that the concentrations of erlotinib required to inhibit the activity of downstream p-AKT, p-ERK1/2, and p-STAT3 proteins were higher than those required to inhibit p-EGFR protein in both erlotinib-sensitive and erlotinib-resistant cells (p < 0.05), although they varied considerably. Compared with erlotinib-sensitive cell lines, erlotinib-resistant NSCLC cell lines had at least one phosphorylated downstream signaling protein that was refractory to erlotinib inhibition: A549 (p-AKT, p-ERK1/2, p-STAT3), H1299 (p-AKT, p-ERK1/2), H596 (p-AKT), and H460 (p-STAT3). These data suggest that alternative activation of growth and survival mechanisms via an EGFR-independent pathway(s) might also be a common mechanism of erlotinib resistance in these NSCLC cells harboring wild-type EGFR gene. Among the three major activated downstream EGFR signaling proteins, lack of inhibition of p-AKT expression was most commonly associated with erlotinib resistance in these NSCLC cell lines. This is consistent with the key role of p-AKT in mediating the effect of erlotinib alone and in combination with at least some cytotoxic agents.9,14–16
Ratios Quantifying the Dependency of NSCLC Tumors on the EGFR Signaling Pathway Predict Erlotinib Sensitivity
We explored the ratios of activated upstream to downstream signaling proteins to quantify the dependency of NSCLC tumors on the EGFR signaling pathway. The assumption was that in cases of high EGFR dependence, the likelihood that the activation of a downstream signaling protein is mainly from the activation of upstream EGFR should be higher than in cases of low EGFR dependence, where downstream activation occurs not as a consequence of upstream EGFR activation. Figure 3A illustrates the expression of phosphorylated and total EGFR and AKT by immunoblots in erlotinib-sensitive tumor cell lines (n = 4) and erlotinib-resistant NSCLC cell lines (n = 7) treated with EGF. The mean of relative amount of each protein expression was normalized to the expression in H322 (as 1) and illustrated under each band. The ratio of p-EGFR expression to p-AKT alone (i.e., p-EGFR:p-AKT) was 10-fold higher in erlotinib-sensitive cells than in erlotinib-resistant cells (6.0 ± 2.8 and 0.6 ± 0.5, respectively, p = 0.03, Mann-Whitney U test), followed by the ratio of their levels relative to total EGFR and total AKT, respectively [i.e., (p-EGFR:EGFR):(p-AKT:AKT)] (6-fold higher: 2.4 ± 1.1 and 0.4 ± 0.2, respectively, p = 0.02) and the ratio of p-EGFR to t-EGFR (3-fold higher) (1.4 ± 0.1 and 0.4 ± 0.1, respectively, p = 0.02) (Figure 3B). Thus, the ratio of p-EGFR:p-AKT best correlates with the sensitivity of these NSCLC cells to erlotinib.
In summary, we here report that only the activated (phosphorylated) form of EGFR and downstream signaling proteins are the targets of erlotinib inhibition. The expression levels of total EGFR and downstream AKT or ERK or STAT3 proteins are not significantly altered by erlotinib treatment and do not correlate with the sensitivity of NSCLC cells to erlotinib. These data do not support determining the expression of total amount of a protein to predict the clinical efficacy of EGFR TKI.17,18 Effective inhibition of all activated EGFR downstream signaling molecules (such as p-AKT, p-ERK, and p-STAT3) by erlotinib was associated with sensitivity to erlotinib, whereas lack of inhibition of at least one downstream signaling protein by erlotinib was associated with resistance to erlotinib, reflecting the cell’s ability to use or trigger an EGFR-independent pathway to maintain cell growth and survival. The p-EGFR:p-AKT ratio, which vertically couples the activity of upstream and downstream EGFR signaling molecules, is a parameter indicative of tumor dependency on the EGFR pathway. Although there are still technical hurdles to overcome, these results support our current effort in developing assays for clinical application and validation of the p-EGFR:p-AKT ratio as a predictive biomarker in retrospective and prospective clinical studies in NSCLC.
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