Epithelial membrane protein-1 is a biomarker of gefitinib resistance - PubMed (original) (raw)

Epithelial membrane protein-1 is a biomarker of gefitinib resistance

Anjali Jain et al. Proc Natl Acad Sci U S A. 2005.

Abstract

We describe a molecular resistance biomarker to gefitinib, epithelial membrane protein-1 (EMP-1). Gefitinib is a small-molecule inhibitor that competes for the ATP-binding site on EGF receptor (EGFR) and has been approved for patients with advanced lung cancers. Treatment with gefitinib has resulted in clinical benefit in patients, and, recently, heterozygous somatic mutations within the EGFR catalytic domain have been identified as a clinical correlate to objective response to gefitinib. However, clinical resistance to gefitinib limits the utility of this therapeutic to a fraction of patients, and objective clinical responses are rare. We aimed to assess the molecular phenotype and mechanism of in vivo gefitinib resistance in xenograft models and in patient samples. We generated in vivo gefitinib-resistance models in an adenocarcinoma xenograft model by serially passaging tumors in nude mice in presence of gefitinib until resistance was acquired. EMP-1 was identified as a surface biomarker whose expression correlated with acquisition of gefitinib resistance. EMP-1 expression was further correlated with lack of complete or partial response to gefitinib in lung cancer patient samples as well as clinical progression to secondary gefitinib resistance. EMP-1 expression and acquisition of gefitinib clinical resistance was independent of gefitinib-sensitizing EGFR somatic mutations. This report suggests the role of the adhesion molecule, EMP-1, as a biomarker of gefitinib clinical resistance, and further suggests a probable cross-talk between this molecule and the EGFR signaling pathway.

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Figures

Fig. 1.

Gefitinib treatment of prostate cancer xenograft tumors. The response of androgen-independent CWR22R GP (A) and GR (B) xenograft tumor to gefitinib (open diamonds) administered at a dosage of 100 mg/kg QD, oral gavage 5 days/week, or vehicle-treated control (filled diamonds). Arrow indicates initiation of therapy. Results are presented as mean tumor volume (n = 10) ± SE, and P values are indicated.

Fig. 2.

EMP-1 is up-regulated in the GR model. (A) Representation of EMP-1 mRNA expression as determined in the gene chip microarray analysis. The white bars represent the gefitinib-sensitive (GP or F0) tumors, light gray bars are the gefitinib-sensitive tumors treated with vehicle alone for 12 h, dark gray bars are the gefitinib-sensitive tumors treated with gefitinib for 12 h, and the black bars represent two different generations of the GR tumors. The y axis represents the absolute EMP-1 mRNA expression. (B) Representative real-time quantitative RT-PCR analysis of EMP-1 mRNA expression in tumor generations F0–F9. The absolute EMP-1 mRNA expression was normalized to β-actin mRNA expression, and the data are presented as relative receptor expression to that observed in the F0 generation. (C) Representative real-time quantitative RT-PCR analysis of EMP-1 mRNA expression in ex vivo cells derived from the gefitinib-sensitive and -resistant tumors. The absolute EMP-1 mRNA expression was normalized to β-actin mRNA expression, and the data are presented as relative receptor expression to that observed in sensitive ex vivo cells.

Fig. 3.

EMP-1 expression in tumor samples from NSCLC patients receiving gefitinib monotherapy. (A) Probability of clinical response to gefitinib decreases as EMP-1 expression increases. The x axis represents the reference normalized cycle threshold for EMP-1 expression, and the y axis represents the probability of response. The red dashed curves indicate the 95% confidence interval. (B) EMP-1 expression in individual samples. The red dashed line represents an arbitrary threshold of 10%. The red circles represent the samples from patients who responded to gefitinib therapy, whereas the blue diamonds represent the nonresponders. The yellow circle highlights the gefitinib responder who eventually acquired gefitinib resistance, and the sample was further characterized. (C) Normal human organ distribution of EMP-1 mRNA expression. A representative graph showing the EMP-1 mRNA expression in normal body tissues, compared with that in the GP and GR tumors.

Fig. 4.

EMP-1 expression in NSCLC clinical samples. Real-time quantitative RT-PCR analysis of EMP-1 mRNA expression in NSCLC patient sample who acquired gefitinib resistance (gray bar) is shown. The expression in the NSCLC sample is compared to that in three GP tumors (white bars) and three GR tumors (black bars). One of the GR tumors was assigned as a calibrator sample (expression value = 1), and the relative EMP-1 mRNA expression was normalized to GAPDH mRNA expression.

Fig. 5.

A model for in vivo resistance to gefitinib therapy. (A) In untreated cells, EGF stimulation (shown by a blue triangle) causes EGFR dimerization followed by phosphorylation events that leads to productive downstream signaling events. The core kinase domain extends from amino acid residues 688 to 950, and the ATP-binding site, R817, is shown in red. Positions of certain key tyrosine residues (Y845, Y1068, Y1086, Y1148, and Y1173) are shown. (B) When tumors are treated with a standard dose of gefitinib, gefitinib preferentially competes for its high-affinity binding site on the kinase domain, i.e., the ATP-binding site (gefitinib is shown as “G” in the yellow circle). We hypothesize that the major dephosphorylation events may occur only at a few key tyrosine residues such as Y845 within the kinase domain, Y1148, which is an EGF-stimulated phosphorylation site, and Y1173, which is the major autophosphorylation site. The other tyrosine residues may continue to signal under these conditions, albeit less robustly, and therefore, a reduction in tumor growth, and not an inhibition in tumor growth, is observed upon gefitinib treatment. (C) Cells that acquire resistance to gefitinib or the GR cells that are enriched by selection express high levels of integrin family members (shown in green) and EMP-1 (shown in red). Treating these cells with a standard dose of gefitinib can efficiently block probably only the high-affinity ATP-binding site and the related autophosphorylation sites on EGFR. The activation of integrins and adhesion molecules such as EMP-1 may enhance increased interactions with the extracellular matrix and constitutive signaling of the EGFR signaling pathway despite the presence of gefitinib. Actin (shown as red and blue beaded filaments) can increase EGFR signaling by binding to EGFR between residues 984 and 996, and EMP-1 may also enhance this process.

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Epithelial membrane protein-1 is a biomarker of gefitinib resistance - PubMed (original) (raw)