Overexpression of yes-associated protein contributes to progression and poor prognosis of non-small-cell lung cancer

Abstract

Yes-associated protein (YAP), the nuclear effector of the Hippo pathway, is a key regulator of organ size and a candidate human oncogene. This study aimed to assess the clinical significance and biological functions of YAP in non-small-cell lung cancer (NSCLC). We investigated the expression of YAP in 92 cases of NSCLC tissue by immunohistochemistry and found that YAP was expressed in 66.3% (61/92) cases and predominantly presented in the nucleus. The expression of YAP in NSCLC was significantly correlated with p-TNM stage (P = 0.0037) and lymph node metastasis (P = 0.0093). Importantly, YAP expression was associated with short overall survival. Further study in NSCLC cell lines in which YAP was either overexpressed or depleted confirmed that YAP markedly promoted cell proliferation and invasion. These results indicate that YAP plays an important role in NSCLC and might be a useful therapeutic target of NSCLC.

Figure 1

Expression of yes‐associated protein (YAP) in normal lung and non‐small‐cell lung cancer (NSCLC). (a) High YAP expression in NSCLC with both cytoplasm and nucleus present. (b) High YAP expression mainly in the nucleus of NSCLC. (c) Low YAP expression in NSCLC, staining in both cytoplasm and nucleus of tumor cells. (d) Low YAP nuclear expression in NSCLC. (e) Negative YAP expression in NSCLC. (f) YAP expression in normal lung epithelium with strong nuclear YAP present in the type II pneumocytes. Original magnification, ×400; scale bar, 50 μm.

Figure 2

Survival curves of patients with positive and negative yes‐associated protein (YAP) expression.

Figure 3

Relative yes‐associated protein (YAP) mRNA expression in cell lines. Seven non‐small‐cell lung cancer (NSCLC) cell lines, one small‐cell lung cancer cell line (H446), and one colon cancer cell line (SW480) were measured by real‐time PCR. The fold change of YAP expression was calculated by the 2^−ΔΔCt Method. Columns, mean; bars, SD.

Figure 4

Yes‐associated protein (YAP) expression in lung cancer cells transfected with YAP or YAP specific siRNA. (a) Real‐time PCR; and (b) western blot analysis showed the effects of YAP overexpression and YAP silencing. Left, A549 cells transfected with pcDNA3.1‐hYAP and empty vector. Right, H157 cells transfected with specific YAP siRNA and negative control siRNA.

Figure 5

Effects of yes‐associated protein (YAP) on the proliferation of non‐small‐cell lung cancer (NSCLC) cell lines. (a) MTT assay indicated that overexpression of YAP in A549 cells promoted proliferation and that knockdown of YAP in H157 cells suppressed proliferation. Points, mean results of three independent experiments; bars, SD. (b) Colony formation assay indicated that YAP overexpression significantly promoted cell proliferation in A549 cells (YAP overexpression vs control: 299 ± 21 vs 68 ± 8, P < 0.001) and that YAP down‐regulation markedly suppressed proliferation in H157 cells (control vs YAP siRNA: 194 ± 17 vs 102 ± 12, P < 0.001). (c) Columns, mean for three experiments; bars, SD; *P < 0.05.

Figure 6

Yes‐associated protein (YAP) promotes cell invasion in vitro. (a) Transwell assay indicated that overexpression of YAP in A549 cells significantly promoted cell invasion. Knockdown of YAP in H157 cells greatly suppressed tumor cell invasion. Original magnification, ×200. (b) Graphs show the number of cells invaded through the Transwell after 16 h of incubation. The number of invaded cells was counted in 10 randomly selected high power fields (×400) under microscope. Columns, mean for three experiments; bars, SD; *P < 0.05.

Figure 7

Effects of yes‐associated protein (YAP) on epithelial–mesenchymal transition (EMT)‐related molecules. (a) Real‐time PCR and (b) western blotting showed the expression changes after overexpressing YAP in A549 cells and silencing YAP in H157 cells. The expression of E‐cadherin (E‐cad), N‐cadherin (N‐cad), Vimentin (VIM), matrix metalloproteinase 2 (MMP2), MMP9, transforming growth factor‐β1 (TGFB1), and transforming growth factor‐β2 (TGFB2) were explored. Columns, mean; bars, SD.