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. 2014 Oct 15;74(20):5891-902.
doi: 10.1158/0008-5472.CAN-14-0184. Epub 2014 Aug 27.

β-catenin contributes to lung tumor development induced by EGFR mutations

Sohei Nakayama  1 , Natasha Sng  1 , Julian Carretero  2 , Robert Welner  1 , Yuichiro Hayashi  3 , Mihoko Yamamoto  1 , Alistair J Tan  1 , Norihiro Yamaguchi  1 , Hiroyuki Yasuda  4 , Danan Li  5 , Kenzo Soejima  6 , Ross A Soo  7 , Daniel B Costa  1 , Kwok-Kin Wong  5 , Susumu S Kobayashi  8
Affiliations

β-catenin contributes to lung tumor development induced by EGFR mutations

Sohei Nakayama et al. Cancer Res. .

Abstract

The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here, we show that β-catenin is essential for development of EGFR-mutated lung cancers. β-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated β-catenin, leading to an increase in β-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth, and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest:

D.L. is currently an employee of Pfizer. D.B.C has previously received consulting fees from AstraZeneca, Roche and Pfizer.

Figures

Figure 1
Figure 1. β-catenin is highly expressed in cells with EGFR activating mutations
(A) Immunoblots of extracts from cancer cell lines harboring wild type or mutant EGFR. (B) Confocal images of A549, NCI-H1650, NCI-H1975 cells stained with anti β-catenin antibody and visualized by Alexa Fluor 488. Scale Bar = 10 µm. (C) Immunohistochemical analysis of lungs isolated from a CCSP-rtTA mouse (I, II, III, IV) and lung tumors from an EGFR-L858R (LR)-T790M (TM) (EGFR-LR-TM)/CCSP-rtTA double transgenic mouse (V, VI, VII, VIII). Both animals were sacrificed after 20 weeks of doxycycline administration. Scale bar = 50 µm.
Figure 2
Figure 2. β-catenin is stabilized and activated by EGFR mutants
(A) Immunoblots of fractionated extracts from 293T cells co-transfected with constructs containing FLAG-tagged β-catenin and either HA-tagged wild type EGFR (WT), EGFR-L858R (LR), or EGFR-L858R-T790M (LR-TM). (B) Immunoblots of whole extracts from BEAS-2B cells stably expressing HA-tagged wild type EGFR (WT), EGFR-L858R (LR), or EGFR-L858R-T790M (LR-TM). BEAS-2B cells infected with MigR1 empty vector were used as a control. (C) Confocal images of stable BEAS-2B cells described in (B). Cells were incubated with anti- β-catenin antibody and visualized by Alexa Fluor 488. Scale bar = 10 µm. (D) Transactivation of β-catenin measured by luciferase activity. Data represents mean ± standard deviation from eight independent experiments. * indicates p ≤ 0.05. ** indicates p ≤ 0.01. (E) Immunohistochemical analysis of β-galactosidase (I, III, V) and X-gal staining (II, IV, VI). Lungs isolated from CCSP-rtTA/TOPGAL (I, II), EGFRTL/CCSP-rtTA (III, IV), and EGFRTL/CCSP-rtTA/TOPGAL (V, VI) are shown. Scale bar = 50 µm.
Figure 3
Figure 3. β-catenin is activated in human NSCLC tumors harboring EGFR mutants
(A) Frequency distribution of nuclear β-catenin based on immunohistochemistry on human tissue microarray. P-value was calculated using Fisher's exact test. (B) Frequency of nuclear β-catenin in 4 pairs of pre- and post-TKIs NSCLC tumors. Each symbol represents an individual patient. The detailed information can be found in Supplementary Table. P-value was calculated using Student’s t-test.
Figure 4
Figure 4. EGFR mutants tyrosine-phosphorylate β-catenin and increase expression and activity
(A) Immunoblots of 293T cell extracts transiently transfected with constructs containing HA-tagged EGFR (wild type [WT], L858R [LR], or L858R-T790M [LR-TM]) and FLAG-tagged β-catenin. (B) Immunoprecipitation (IP) and immunoblot (IB) analyses showing binding of β-catenin and mutant EGFR. (C) IP and IB analyses showing tyrosine-phosphorylation of β-catenin by mutant EGFR. 293T cells were transiently transfected with constructs containing HA-tagged EGFR-L858R-T790M and FLAG-tagged β-catenin and treated with either 0.1% DMSO, 1 µM erlotinib, or 1 µM afatinib. (D) Immunoblots of nuclear extracts from 293T transiently transfected with constructs containing HA-tagged EGFR-L858R-T790M and FLAG-tagged β-catenin. Cells were treated with either 0.1% DMSO, 1 µM afatinib, or 1 µM SU6656. (E) Transactivation of β-catenin measured by luciferase activity. Cells were treated with either 0.1% DMSO, 1 µM afatinib, or 1 µM SU6656. Data represents mean ± standard deviation from three independent experiments. * indicates p ≤ 0.05. N.S.= not significant. (F) Transactivation of β-catenin measured by luciferase activity. A plasmid encoding for WT β-catenin and its tyrosine-to-phenylalanine (Y-to-F) mutants Y86F, Y654F, Y86F-Y654F, and Y333F were transiently transfected with EGFR-L858R-T790M together with either pTOPFLASH or pFOPFLASH plasmids. Data represents mean ± standard deviation from four independent experiments. * indicates p ≤ 0.05. N.S. indicates not significant.
Figure 5
Figure 5. Pharmacological inhibition of β-catenin suppresses tumor growth
(A) Immunoblots of β-catenin knockdown NCI-H1975. (B) Colony formation assay of β-catenin knockdown NCI-H1975 as described in (A). Colony numbers were counted after 14 days. Data represents mean ± standard deviation from three independent experiments. * indicates p ≤ 0.05. (C) Relative AXIN2 mRNA levels in NCI-H1975 and NCI-H460 cells treated with either 0.1% DMSO or 0.3125 µM ICG-001 were determined by real-time PCR. Data represents mean ± standard deviation from three independent experiments. * indicates p ≤ 0.05. (D) Cell proliferation assay of NCI-H1975 and NCI-H460 cells treated as in (C). Data represents mean ± standard deviation from three independent experiments. * indicates p ≤ 0.05 vs cells treated with 0.1% DMSO. (E) Representative MRI photographs of mice. Mice were treated with doxycycline for 11–15 weeks and treated with either vehicle or ICG-001. Arrows indicate lung tumors. Note that tumor size remained unchanged in mice treated with ICG-001, whereas tumors grew in control mice. “H” indicates location of the heart. (F) Changes in tumor volume were compared to baseline in mice treated with either vehicle (n=8) or ICG-001(n=12). P-value was calculated using Wilcoxon signed-rank test.
Figure 6
Figure 6. Deletion of Ctnnb1 impairs tumor formation in vivo
(A) Scheme of EGFR-TL, Cre, and Ctnnb1construct for conditional transgenic mice. (B–E) Generation of lung specific conditional Ctnnb1knockout / EGFR-L858R-T790M transgenic mice. I: CCSP-rtTA/Ctnnb1 F/+ ; II: EGFRTL/CCSP-rtTA/teto-Cre/Ctnnb1 F/F; III: EGFRTL/CCSP-rtTA/teto-Cre/Ctnnb1 F/+; IV: CCSP-rtTA/teto-Cre/Ctnnb1 F/F. (B) MRI images of transgenic mice treated with doxycycline for 10 weeks. Arrows indicate lung tumors. “H” indicates location of the heart. (C) Lung weight isolated from mice treated with doxycycline for 8–28 weeks. Data represents mean ± standard deviation. * indicates p ≤ 0.01. (D) H&E staining of lungs isolated from mice. Scale bar = 100 µm. (E) Survival curves after administration of doxycycline. P-value was determined by the log-rank test.
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