Enhanced YAP expression leads to EGFR TKI resistance in lung adenocarcinomas - PubMed (original) (raw)

Enhanced YAP expression leads to EGFR TKI resistance in lung adenocarcinomas

Ting-Fang Lee et al. Sci Rep. 2018.

Abstract

Epidermal growth factor receptor (EGFR) mutation is prevalently expressed in lung adenocarcinoma cases and acts as one of the major driving oncogenes. EGFR tyrosine kinase inhibitors (TKIs) have been used in patients with EGFR-mutant as an effective targeted therapy in lung adenocarcinoma, but drug resistance and tumor recurrence inevitably occurs. Recently, Yes-associate protein (YAP) has been reported to promote multiple cancer cell properties, such as promoting cell proliferation, epithelial-mesenchymal transition and drug resistance. This study investigated the roles of YAP in TKI-resistant lung adenocarcinoma. In TKI-sensitive cells, enhanced YAP expression leads to TKI resistant. Also, upregulated YAP expression and activation were detected in long-term TKI-induced resistant cells. With reduced YAP expression using shRNA or YAP inhibitors, TKI-resistant cells become TKI-sensitive. reduced xenograft tumor size in nude mice and Moreover, combined EGFR TKI and a YAP inhibitor, statin, prolonged survival among lung cancer patients analyzed by Taiwan National Health Insurance Research database. These observations revealed the importance of YAP in promoting TKI-resistance and combined YAP inhibition can be a potential therapy delaying the occurrence of TKI-resistance in lung adenocarcinoma.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Figure 1

Enhanced YAP expression and activity promotes drug resistance. HCC827 cells overexpressed with wild-type (YAP WT) or constitutively active YAP (YAP 5SA) were cultured in the presence of (A) gefitinib or (B) afatinib. (C) H1975 cells with YAP expression were treated with afatinib. The error bars represent the S.E. of 3 independent experiments. *P < 0.05, empty compared with YAP 5SA, and #P < 0.05, empty compared with YAP WT. Upregulated YAP protein levels and activity were detected in both YAP overexpressed groups in (D) HCC827 and (E) H1975 cells. The original blot of these samples was shown in Supplementary Figure S5A. The error bars represent the S.E. of 3 independent experiments. *P < 0.05, empty compared with YAP 5SA or YAP WT.

Figure 2

Figure 2

Enhanced YAP expression and activity in TKI-resistance cells. Compared to the parental HCC827 cells, the two resistant lines, HCC827AR and HCC827GR cells showed enhanced (A) YAP protein levels, (B) promoter activity and (C) ANKRD1 and CTGF mRNA levels. Upregulated YAP expression and activity were also found in H1975AR cells compared to H1975 cells (D, E and F). The error bars represent the S.E. of 3 independent experiments. *P < 0.05, parental compared with resistant lines. (G) Primary cells collected from plural effusion. Cells collected from TKI-resistant patient showed upregulated YAP level. The original blots were shown in Supplementary Figure S5B.

Figure 3

Figure 3

TKI that reduced YAP levels caused cytotoxicity in the resistant lines. Cell viability assays performed in three resistant lines: (A) HCC827GR (B) HCC827AR or (C) HCC827GR cells in the presence of gefitinib afatinib or dasatinib. Immunoblotting demonstrated YAP protein levels (B, C,F,G,J and K) or activity (D,H and L) in the presence of different TKIs in resistant lines. The original blots were shown in Supplementary Figure S6.

Figure 4

Figure 4

Reduce YAP expression by shRNAs re-sensitized the resistant lines. Gefitinib or afatinib-induced cytotoxicity was performed in 4 TKI-resistant cell lines: (A) HCC827GR, (B) HCC827AR (C) H1975 or (D) H1975AR in the presence of scramble or YAP shRNAs. The error bars represent the S.E. of 3 independent experiments. *P < 0.05, shSC compared with YAP knockdowns. Reduced YAP expression was detected in the YAP knockdown grouped (E–H). The original blots were shown in Supplementary Figure S7A.

Figure 5

Figure 5

Pharmaceutical suppression of YAP re-sensitized the resistant lines. Gefitinib or afatinib-induced cytotoxicity was performed in 4 TKI-resistant cell lines: (A) HCC827GR, (B) HCC827AR (C) H1975 or (D) H1975AR in the presence of vehicle control (0.01% DMSO), verteporfin (VP; 1 μM) or fluvastatin (Flu; 1 μM). The error bars represent the S.E. of 3 independent experiments. *P < 0.05, vehicle control compared with VP, and #P < 0.05, vehicle compared with Flu. TKI-resistant cells cultured in the presence or absence of gefitinib or afatinib combined with verteporfin (E–H) or fluvastatin (I-L) for 48 h. Immunoblotting demonstrated YAP levels. The original blots were shown in Supplementary Figure S7B and C.

Figure 6

Figure 6

Effects of combined therapy of TKI and statin, as a YAP inhibitor. Subcutaneous xenograft model demonstrated the effect of combined therapy of afatinib and fluvastatin in the inhibition of TKI-resistant tumor. (A) Tumor size and (B) tumor weight measured in either vehicle or drug treated groups. (C) Analysis of the Taiwan National Health Insurance data sources revealed prolonged survival in TKI prescribed patients with regular statin use compared to no statin used patients.

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