YAP1 is essential for tumor growth and is a potential therapeutic target for EGFR-dependent lung adenocarcinomas - PubMed (original) (raw)

YAP1 is essential for tumor growth and is a potential therapeutic target for EGFR-dependent lung adenocarcinomas

Ting-Fang Lee et al. Oncotarget. 2017.

Abstract

Epidermal growth factor receptor (EGFR) mutations are found in lung adenocarcinomas leading to tumor cells proliferation and survival. EGFR tyrosine kinase inhibitors (TKIs) that block EGFR activity are effective therapeutics for EGFR-mutant lung adenocarcinoma patients, but TKI-resistance inevitably occurs. The YES-associated protein (YAP1) transcription coactivator has been implicated as an oncogene and is amplified in human cancers and provides tumor cells strong proliferation and survival cues. This study investigated the roles of YAP1 in lung adenocarcinoma by exploring its regulation and functions mediated by EGFR signaling. In this study, we detected a correlation between YAP1 level and EGFR mutation status in lung adenocarcinoma tissues. Using lung adenocarcinoma cell lines, enhanced YAP1 expression and activity mediated by EGFR signaling was detected through enhanced protein stability. A SRC family protein, YES, was involved in EGFR-regulated YAP1 expression and this pathway was crucial for proliferation in EGFR-dependent cells. Small molecules that reduced YAP1 levels by mechanisms bypassing EGFR signaling were effective in reducing viability in EGFR-dependent cells including those with EGFR T790M, the major cause of TKI-resistance. These observations unveiled the significance of YAP1 in EGFR mutant lung adenocarcinomas and identified YAP1 as a promising therapeutic target for EGFR-dependent lung adenocarcinoma patients, including those with EGFR T790M-caused TKI resistance.

Keywords: EGFR mutation; TKI-resistance; YAP1; lung adenocarcinoma.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflict of interest.

Figures

Figure 1

Figure 1. YAP1 expression correlated to EGFR mutation status

A. YAP1 expression and EGFR mutation status were analyzed in 164 cases of NSCLC tissue. Positive YAP1 staining was observed in 58% (95/164) cases while EGFR active-mutation detected in 55% (90/164). YAP1 expression was correlated with EGFR mutation status (p = 0.03). Comparison of EGFR mutation status vs YAP1 positivity was analyzed by Fisher's exact test. B. Representative images showed YAP1 (brown staining) expression in lung adenocarcinoma tissue. C. After 4h serum starvation, ten lung adenocarcinoma cell lines: five EGFR wild-type and five EGFR active-mutant, were collected. EGFR mutant cell lines showed endogenous EGFR phosphorylation and upregulated YAP1 expression. D. The EGFR active-mutant lung adenocarcinoma cell lines H1975 and HCC827 had stronger YAP1 signal and nuclear localization compared to EGFR wild type A549 detected by immunocytostaining.

Figure 2

Figure 2. EGFR signaling promoted YAP1 expression and activity

A. Knocking down EGFR using shRNAs reduced EGFR phosphorylation and total YAP1 expression. B. Gefitinib, an EGFR TKI, blocked EGFR phosphorylation and reduced YAP1 protein levels. C. 8XGTIIC luciferase activity assays demonstrated reduced YAP1 activity in the presence of gefitinib in HCC827 cells. The error bars represent the S.E. of 3 independent experiments. *P < 0.05. D. EGF (1 h) triggered an increase of YAP1 in A549 cells while gefitinib blocked both EGFR phosphorylation and YAP1 level. E. Increased luciferase activity triggered by EGF (2 h) indicated increased YAP1 activity. The data are presented as mean±S.E. of three independent experiments. *P < 0.05. F. Upregulated mRNA expressions of CTGF and ANKRD1, the TEAD target genes, were stimulated by EGF (2 h). The error bars represent the S.E. of four independent experiments. *P < 0.05.

Figure 3

Figure 3. EGFR promoted YAP1 stabilization

A. MG132 (10 μM, 3 h) induced YAP1 accumulation in A549 cells in the absence of EGF (lanes 1 and 2); while no further increase of YAP1 level induced by MG132 (3 h) in the presence of EGF (1 h) (lanes 3 and 4). CHX (20 μg/ml) treatment caused decreased YAP1 levels in A549 cells B. while YAP1 levels maintained in H1975 and HCC827 cells C. with CHX treatment. D. By immunoprecipitating YAP1, its binding with β-TRCP was detected in A549 cells. EGF treatment reduced the binding between YAP1 and β-TRCP. E. In the presence of gefitinib (0.1 μM, 24 h), enhanced YAP1/β-TRCP binding was detected in HCC827 cells.

Figure 4

Figure 4. Roles of YAP1 in cell proliferation, survival and tumor growth

A. Cell proliferation assay for H1975, HCC827 or PC9 cells knocking down scramble control or YAP1. Cells knocking down YAP1 showed significantly reduced proliferation. The error bars represent the S.E. of four independent experiments. *P < 0.05. B. Knocking down EGFR in the EGFR-dependent H1975 cells reduced cell viability and forced YAP1 expression rescued loss-of-EGFR-caused viability loss. The error bar represents the S.E. (n = 4). *P < 0.05 control compared with shEGFR, and #P < 0.05 shEGFR compared with YAP1+shEGFR. Subcutaneous injection of H1975 cells knocking down scramble control or YAP1 into nude mice. Reduced tumor size C. and reduced tumor weight D. was detected in YAP1 knockdown groups. The data are presented as the mean±S.E. (n = 6 per group).*P < 0.05.

Figure 5

Figure 5. Role of YES in EGFR-mediated YAP1 expression and function

A. Knockdowns of YES in EGFR mutant cell lines, H1975 and HCC827 caused reduced YAP1 expression. B. Knockdowns of YES did not reduce YAP1 level in A549 (left panel). EGF (1 h) triggered YAP1 expression was diminished in YES knockdown groups (right panel). C. Immunoprecipitated EGFR can detect YES expression in H1975 cells but not in A549. D. Knockdowns of YES caused decreased cell proliferation in H1975 cells. The error bars represent the S.E. of three independent experiments. *P < 0.05. Subcutaneous tumor xenograft model showed knockdowns of YES in H1975 cells reduced tumor size E. and tumor weight F. The data are presented as the mean±S.E. (n = 6 per group).*P < 0.05.

Figure 6

Figure 6. EGFR-dependent cells were sensitive to YAP1 inhibitors

A. HCC827 and B. PC9 cells (exon 19 deletion) were sensitive to EGFR TKIs, afatinib and gefitinib, and were also sensitive to dasatinib. C. H1975 (L858R and T790M) and D. human primary culture CLH21 (L858R and T790M) cells were insensitive to gefitinib due to T790M but were sensitive to afatinib and dasatinib. The error bar represents the S.E. (n = 3). *P < 0.05 vehicle control compared with gefitinib; #P<0.05 compared with afatinib and *P < 0.05 compared with dasatinib treatment. E. and F. Gefitinib and afatinib reduced YAP1 expression in an EGFR-dependent manner in HCC827 cells, while gefitinib had little effects on H1975 cells EGFR phosphorylation and YAP1 expression. Dasatinib reduced YAP1 level in an EGFR-independent manner in both HCC827 and H1975 cells. EGFR-dependent cells were also sensitive to YAP1 inhibitors, verteporfin or fluvastatin with reduced cell viability in G. HCC827 or H. H1975 cells. The error bar represents the S.E. (n = 3). *P < 0.05 vehicle control compared with verteporfin; #P < 0.05 compared with fluvastatin. Reduced YAP1 protein expressions in the presence of verteporfin (1 μM) or fluvastatin (1 μM) were detected in I. HCC827 and J. H1975 cells 48 h after treatment.

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