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. 2013 Aug 22;32(34):4034-42.
doi: 10.1038/onc.2012.402. Epub 2012 Sep 10.

Oncogenic KRAS-induced epiregulin overexpression contributes to aggressive phenotype and is a promising therapeutic target in non-small-cell lung cancer

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Oncogenic KRAS-induced epiregulin overexpression contributes to aggressive phenotype and is a promising therapeutic target in non-small-cell lung cancer

N Sunaga et al. Oncogene. .

Abstract

KRAS mutations are one of the most common driver mutations in non-small-cell lung cancer (NSCLC) and finding druggable target molecules to inhibit oncogenic KRAS signaling is a significant challenge in NSCLC therapy. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in both KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the current study, we found that EREG is overexpressed in NSCLCs harboring KRAS, BRAF or EGFR mutations compared with NSCLCs with wild-type KRAS/BRAF/EGFR. Small interfering RNAs (siRNAs) targeting mutant KRAS, but not an siRNA targeting wild-type KRAS, significantly reduced EREG expression in KRAS-mutant and EREG-overexpressing NSCLC cell lines. In these cell lines, EREG expression was downregulated by MEK and ERK inhibitors. Importantly, EREG expression significantly correlated with KRAS expression or KRAS copy number in KRAS-mutant NSCLC cell lines. Further expression analysis using 89 NSCLC specimens showed that EREG was predominantly expressed in NSCLCs with pleural involvement, lymphatic permeation or vascular invasion and in KRAS-mutant adenocarcinomas. In addition, multivariate analysis revealed that EREG expression is an independent prognostic marker and EREG overexpression in combination with KRAS mutations was associated with an unfavorable prognosis for lung adenocarcinoma patients. In KRAS-mutant and EREG overexpressing NSCLC cells, siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis. Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

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Figures

Figure 1
Figure 1
(a) Expression of EREG mRNA in human bronchial epithelial cell lines (noncancerous cells; N = 5), NSCLC cell lines with wild-type EGFR/BRAF/KRAS (EGFR/BRAF/KRAS WT; N = 10), NSCLC cell lines harboring EGFR mutations (EGFR Mut; N = 9), BRAF mutations (BRAF Mut; N = 4) or KRAS mutations (KRAS Mut; N = 12). Significant differences were observed among all groups (P < 0.0001, Kruskal–Wallis test). The points represent the mean EREG levels from four independent experiments. The lines represent the median levels in each group. (b) The mutant KRAS transcripts and the wild-type KRAS transcripts were specifically reduced by mutant KRAS siRNAs and a wild-type KRAS siRNA, respectively. BstNI digestion produces a 156 bp DNA fragment (WT) in cells that have wild-type KRAS alleles (for example, H1299 cells), whereas a 186 bp DNA fragment (Mut) remained uncut in cells that have a mutant KRAS allele but no wild-type alleles. (c) siRNA-mediated EREG silencing in KRAS-mutant NSCLC cell lines, H1792, HCC4017, H441 and H358. For KRAS mutant-specific knockdown, an siRNA against KRAS G12C mutant was used for H358, HCC4017 and H1792, and an siRNA against KRAS G12V mutant was used for H441. NT, non-treatment; siControl, Tax siRNA-transfected cells; siKRAS-Mut, siRNA against mutant KRAS transfected cells; siKRAS-WT, siRNA against wild-type KRAS transfected cells. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison with NT by the Kruskal–Wallis test with Dunn’s Multiple Comparison.
Figure 2
Figure 2
The effects of U0126 (MEK inhibitor) and FR180204 (ERK inhibitor) on EREG expression in KRAS-mutant NSCLC cell lines. Twenty-four hours after 5 × 105 cells were plated in each well of six-well plates, cultured medium was replaced with 2 ml of the growth medium with U0126 (10 μM) or FR180204 (10 μM). After culture for an additional 6 h, the cells were harvested for subsequent quantitative RT–PCR analysis. The columns represent the means ± s.d. of 8 determinants from two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 for comparison with mock treatment (DMSO alone) by the Kruskal–Wallis test with Dunn’s Multiple Comparison.
Figure 3
Figure 3
Significant correlations were observed (a) between EREG expression and KRAS expression (Pearson r = 0.7043, P = 0.0106), (b) between KRAS expression and KRAS copy number (Pearson r = 0.7256, P = 0.0076) and (c) between EREG expression and KRAS copy number (Spearman r = 0.6970, P = 0.0142) in KRAS-mutant NSCLC cell lines.
Figure 4
Figure 4
Representative figures of the immunohistochemical staining of EREG protein are shown in (a, b) an EREG-overexpressing tumor (EREG mRNA level = 142.1 a.u.; EREG protein score = 4) and (c, d) an EREG-undetectable tumor (EREG mRNA level = 0.0 a.u.; EREG protein score = 0). In Figures 4(a, b), cytoplasmic and nuclear staining of epiregulin was observed in the tumor, consistent = with a previous study. (e) Comparisons of EREG mRNA expression levels between lung adenocarcinomas versus squamous cell carcinomas (P = 0.0265), between tumors with or without pleural involvement (P = 0.0013), between tumors with or without lymphatic permeation (P = 0.0224), and between tumors with or without vascular invasion (P = 0.0034). The differences between groups were statistically analyzed by the Mann–Whitney test. (f) The comparison of EREG expression between tumors of lung adenocarcinomas with wild-type EGFR/KRAS (EGFR/KRAS WT), EGFR mutations (EGFR Mut) or KRAS mutations (KRAS Mut). P < 0.001 for differences among the three groups by the Kruskal–Wallis test, and P < 0.01 for differences between EGFR Mut and KRAS Mut or between EGFR Mut and EGFR/KRAS WT by the Kruskal–Wallis test with Dunn’s Multiple Comparison. (g) The comparison of EREG expression among the adenocarcinoma groups classified according to KRAS mutation status and smoking status. P < 0.05 for differences among the four groups by the Kruskal–Wallis test, and P < 0.05 for differences between KRAS WT/nonsmoker and KRAS Mut/Smoker by the Kruskal-Wallis test with Dunn’s Multiple Comparison. EREG expression levels in NSCLC tumors were normalized to the mean ( = 1 a.u.) of values obtained from nine different noncancerous lung tissues. The points represent the mean EREG levels obtained from four independent experiments. The lines represent the median EREG levels in each group.
Figure 5
Figure 5
Kaplan–Meier analysis of overall survival (month) in lung adenocarcinoma patients who were classified (a) according to the EREG expression levels or (b) according to EREG expression levels and KRAS mutational status. KRAS-WT, KRAS wild-type; KRAS-Mut, KRAS mutant; EREG-Low, ≤2.127 a.u.; EREG-High, >2.127 a.u. (the median of EREG levels in all tumor specimens is 2.127 a.u.). There is a significant difference in overall survival between EREG-High/KRAS Mut and EREG-Low/KRAS-WT groups (P = 0.0031, log-rank test with Bonferroni’s correction for multiple comparisons).
Figure 6
Figure 6
(a) siRNA-mediated EREG silencing in H358 cells as evaluated by quantitative RT–PCR. NT, non-treatment; siControl, treatment with Tax siRNA. siEREG-1 and siEREG-2: two siRNAs targeting different sites of EREG mRNA were used. *P < 0.01; **P < 0.001 by the Kruskal–Wallis test with Dunn’s multiple comparison. siRNA-mediated EREG silencing inhibits cell proliferation and colony formation as evaluated by (b) MTT assay (*P < 0.05; **P < 0.001, Kruskal–Wallis test with Dunn’s multiple comparison), (c) colony-formation assay in liquid culture and (d) soft-agar colony-formation assay in H358 cells (*P < 0.0001, ANOVA with Bonferroni’s multiple comparison). The columns represent the mean ± s.d. from three independent experiments, and NT was set at 100%. (e) Representative figures for annexin-V-positive apoptotic H358 cells (green fluorescence) with/without nuclear staining with Hoechst 33342 (blue fluorescence) and (f) the percentage of both annexin-V and Hoechst 33342-positive H358 cells after treatments with EREG siRNAs or the control siRNA. The columns represent the means ± s.d. of 12 determinants from two independent experiments. *P < 0.001 by the Kruskal–Wallis test with Dunn’s Multiple Comparison. (g) siRNA-mediated EREG silencing induces DNA fragmentation in H358 cells. *P < 0.0001 by ANOVA with Bonferroni’s multiple comparison. The enrichment factor was used as a parameter of apoptosis. The columns represent the mean ± s.d. from four independent experiments, and NT control was set at 1. All statistical analyses in Figure 6 were performed for comparison between NT control and each treatment.

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