Intratumoral epiregulin is a marker of advanced disease in non-small cell lung cancer patients and confers invasive properties on EGFR-mutant cells

Abstract

Non-small cell lung cancer (NSCLC) cells with activating epidermal growth factor receptor (EGFR) somatic mutations have unique biological properties, including high expression of the ErbB ligand epiregulin; however, the biological role of epiregulin in these cells has not been elucidated. To examine its role, we used an immunohistochemical approach to detect epiregulin expression in NSCLC biopsy samples and pharmacologic and genetic approaches to inhibit epiregulin in cultured NSCLC cells. In NSCLC biopsy samples, epiregulin was detected in 237 of 366 (64.7%) tumors, which correlated with nodal metastasis and a shorter duration of survival. In EGFR-mutant NSCLC cell lines, treatment with a small-molecule EGFR tyrosine kinase inhibitor diminished mRNA levels of the gene encoding epiregulin (EREG). The ability of EGFR-mutant NSCLC cells to invade through Matrigel in vitro was inhibited by treatment with an anti-epiregulin neutralizing antibody or by transfection with an EREG short hairpin RNA. Collectively, these findings show that epiregulin expression correlated with advanced disease, was EGFR dependent, and conferred invasive properties on NSCLC cells. Additional studies are warranted in NSCLC patients to evaluate whether epiregulin expression predicts the metastatic potential of primary tumors and whether anti-epiregulin treatment strategies are efficacious in the prevention of metastasis.

Fig. 1

Intratumoral epiregulin expression correlates with poor prognosis in NSCLC. A, epiregulin detection by immunohistochemistry. a to c, anti-epiregulin–specific staining. Immunohistochemical analysis of a NSCLC biopsy sample in the presence (a) and absence (b) of primary antibody and in the presence of primary antibody preincubated with blocking peptide (c). d to g, intratumoral variability in epiregulin staining. Two adenocarcinomas (d and e) and two squamous cell carcinomas (f and g) in which epiregulin was detectable (d and f) or undetectable (e and g). B, intratumoral epiregulin expression correlated with a shorter duration of survival. Plots show Kaplan-Meier survival curves for tumors that were positive (+) or negative (−) for epiregulin staining (scores ≥100 were considered positive). E/N, deaths/total number of patients.

Fig. 2

EREG expression is EGFR dependent. A, EREG mRNA was detected by semiquantitative PCR analysis of RNA prepared from a panel of NSCLC cell lines in which EGFR is mutant (HCC827, H3255, and HCC2279) or wild-type (H1299, H460, A549, H322, and H596). Actin was used as the control for RNA integrity. B, kinase-specific regulation of EREG expression. HCC827 cells were treated with inhibitors of EGFR (gefitinib), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (CI-1040), p38 (SB202190), mammalian target of rapamycin (CCI-779), c-jun NH₂-terminal kinase (SP600125), or AKT (A838450.3). Reverse transcription-PCR analysis of EREG and actin (top). EREG bands were quantified by densitometric analysis, normalized for loading differences based on actin, and expressed relative to that of untreated cells, which was set at 1.0. To evaluate dose-dependent target inhibition by the inhibitors, p38 kinase assays (KA) and Western blot analyses were done (bottom) of phosphorylated Y1068-EGFR (p-EGFR), T202/204-extracellular signal–regulated kinase (p-ERK), S235/236-S6 (p-S6), S21/9-glycogen synthase kinase 3 (p-GSK), S63-c-Jun (p-cJun), and corresponding total proteins.

Fig. 3

Pharmacologic inhibition of epiregulin in NSCLC cell lines. A, EGFR phosphorylation is epiregulin dependent. Western blotting of HCC827 cells treated for 24 h with an anti-epiregulin neutralizing antibody (+) or IgG control (−). Bands were quantified by densitometric scanning and expressed relative to those in control cells, which was set at 1.0. B, NSCLC cell invasion is epiregulin dependent. NSCLC cell lines were seeded into Transwell invasion chambers and treated for 20 h with the indicated antibodies. Invasive cells were photographed (left) and quantified by manual counting (right). Columns, mean of replicate (triplicate) wells; bars, SD.

Fig. 4

Genetic inhibition of epiregulin in NSCLC cells. A, efficient EREG depletion by shRNA constructs. Quantitative PCR analysis of EREG mRNA in NSCLC cell lines transiently transfected with retroviral vectors expressing EREG shRNA constructs (–4) or scrambled control. B, EREG depletion inhibits NSCLC cell proliferation. WST-1 assays of transfectants done at the indicated time points. C, EREG depletion induces apoptosis. Quantification of TUNEL-positive HCC827 cells by flow cytometric analysis. D, EREG depletion inhibits NSCLC cell invasion. Transfectants were seeded into Transwell invasion chambers. Invasive cells were photographed (top) and quantified by manual counting (bottom). Columns, mean of replicate (triplicate) wells; bars, SD.