Overexpression of wildtype EGFR is tumorigenic and denotes a therapeutic target in non-small cell lung cancer

Abstract

Current guidelines for lung cancer treatment with EGFR tyrosine kinase inhibitors (TKI) include only patients with mutated EGFR, although some patients with wildtype EGFR (wt-EGFR) have exhibited positive responses to this therapy as well. Biomarkers predicting the benefit from EGFR-TKIs treatment remain to be determined for patients with wild-type EGFR.Here, we report that wt-EGFR overexpression transformed cells in vitro and induced tumorigenesis in vivo in transgenic mouse models. Wt-EGFR driven lung cancer was hypersensitive to TKI treatment in mouse model. Lung cancer patients with high-expression of wt-EGFR showed longer Overall Survival in comparison to low-expression patients after TKI treatment. Our data therefore suggest that treatment with EGFR inhibitors should be extended to include not only patients with mutated EGFR but also a subset of patients with overexpression of wt-EGFR.

Keywords: EGFR; lung cancer; transgenic mouse model; tyrosine kinase inhibitor.

Figure 1. Lung cancer patients overexpressing wt-EGFR respond to TKI treatment

A. Representative CT images for responders before and after Gefitinib treatment. Tumor is highlighted with read arrows. PreRx for before treatment; PostRx for after treatment. B. H&E examination revealed poorly differentiated lung adenocarcinoma pathological type in responders. C. IHC staining of representative responders and non-responders for EGFR expression level (scale bars, 200μm). D. Statistics of EGFR expression level comparison between responders and non-responders. Statistics was done on randomly picked 6 responders and 6 non-responders.

Figure 2. Overexpression of wt-EGFR is transforming

A. Overexpression of wt-EGFR is transforming in Beas-2B cell line in presence of EGF by soft-agar assay. Left panel for typical pictures of soft-agar colony; Right panel for statistics of soft-agar colony. B. Overexpression of wt-EGFR activates downstream signaling. High- and low- wt-EGFR expressing Beas-2B cell line were subjected to western analysis to assess activation of EGFR, Akt, and Erk. Erlotinib is applied in high-expressing cells to check the ability to inhibit above mentioned signals. All the gels were run under the same experimental conditions. C. Overexpression of wt-EGFR maintains high protein level in cells. wt-EGFR overexpressing Beas-2B cells were stimulated with EGF for indicated time (minutes) and western blot analysis was done with antibody for total EGFR, p-Y1173 EGFR and actin (upper panel). Expression level of EGFR in Beas-2b overexpressing wt-EGFR were compared to widely used lung cancer cell lines (lower panel). D. Overexpression of wt-EGFR is transforming in NIH-3T3 cell line in presence of EGF. Left panel for typical pictures of soft-agar colony; Right panel for statistics of soft-agar colony. (scale bar, 400μm) E. Overexpression of wt-EGFR activates downstream signaling. High- and low- wt-EGFR expressing 3T3 cell line were subjected to western analysis to assess activation of EGFR, Akt, and Erk. Erlotinib is applied in high-expressing cells to check the ability to inhibit above mentioned signals. All the gels were run under the same experimental conditions.

Figure 3. overexpression of wt-EGFR in lung epithelium is tumorigenic and necessary for tumor maintenance in vivo

A. Wt-EGFR expression is tightly controlled in the transgenic mice. Upper panel for mRNA expression in the lung. Lower panel for protein expression in lung tissue before and 5 days after doxycycline withdrawal. All the gels were run under the same experimental conditions. Days for on doxycycline treatment are indicated by number. Withdrawal of doxycycline for 5 days is indicated by “-5”. B. Tumor nodules formed in lungs from CC10rtTA/TetO-wtEGFR bitransgenic mice. Left panel, mice not fed with doxycycline. Right panel, mice fed with doxycycline for around 8 months. Large tumor nodules highlighted with arrow-head. C. Tumors formed in CC10rtTA/TetO-wtEGFR bitransgenic mice are moderately to poorly differentiated lung adenocarcinoma. D. Tumors formed in CC10rtTA/TetO-wtEGFR bitransgenic mice depend on continuous expression of wt-EGFR for maintenance. upper panel: MRI images of mice after 8 months doxycycline induction (PreRx); lower panel: MRI images of same mice after doxycycline withdrawal (PostRx). Tumor region highlighted with green arrow-head. E. Histology of the lung of mice withdrawn doxycycline for 2 weeks. Histology (H&E staining) showed nearly normal lung, with occasional area of fibrosis. (scale bar for high magnification 50μm; scale bar for low magnification 200μm). F. TetO-wt-EGFR tumors express high amount of EGFR. Tumors from different mice (T1 & T2) were compared against HCC827 (EGFR exon19 deletion) and H3255 (EGFR L858R). Result showed mice tumors express high amount of EGFR.

Figure 4. Lung cancers driven by wt-EGFR overexpression are sensitive to erlotinib treatment

A. Tumor regression in CC10rtTA/TetO-wtEGFR mice in response to erlotinib treatment. Left panel: MRI images showing tumor regression of 2 representative mice (M#1 and M#2), Pre for before erlotinib treatment and Post for after erlotinib treatment; middle panel: quantification of tumor regression on 4 erlotinib treated mice; right panel. Histology of the lung in CC10rtTA/TetO-wtEGFR mice treated for 2 weeks. Lungs are nearly normal. Occasional areas of fibrosis are shown. B. wt-EGFR phosphorylation is sensitive to erlotinib treatment for 48 hours. IHC analysis of the lung tumors of mice treated with vehicle and erlotinib is shown. C. wt-EGFR phosphorylation is sensitive to erlotinib treatment for 48 hours. Western analysis of the lungs of mice treated with vehicle and erlotinib is shown. All the gels were run under the same experimental conditions. D. Erlotinib treatment resulted in arrest of proliferation and promotion of apoptosis in tumors. IHC staining shown for Ki-67 and TUNEL in the tumors from mice treated with vehicle and erlotinib. (scale bar 50 μm) E. Statistics of TUNEL staining. F. Statistics of Ki-67 staining.

Figure 5. Kaplan-Meier analysis of overall survival according to EGFR IHC score in NSCLC patients

Patients were all treated with gefitinib. The H scores of 100 was chosen as the cutoff point for separating tumors with high or low EGFR expression. The P value for the difference between the two curves was determined by the log-rank test (P = 0.031).