Role of YAP in lung cancer resistance to cisplatin

Abstract

Yes-associated protein (YAP) serves a critical role in the initiation and progression of a variety of types of cancer via modulating the expression of genes involved in cell proliferation and the downregulation of apoptosis. Recent studies have suggested that YAP is responsible for the development of drug resistance and cancer metastasis and recurrence. However, the association between YAP and chemoresistance in lung cancer, particularly in lung cancer stem cells (LCSCs) remains largely unknown. In the current study, lung cancer cell spheres were established using the A549 cell line, which demonstrated stem cell properties. It was revealed that YAP was overexpressed in lung cancer spheres compared with normal A549 adherent cells and was associated with enhanced cisplatin (CDDP) resistance. Knockdown of YAP effectively sensitized the adherent A549 and tumor spheres to CDDP treatment and resulted in enhanced cell death. These results suggest that YAP serves a critical role in LCSCs drug resistance and YAP targeting could become a promising adjuvant to current the chemotherapy for lung cancer.

Keywords: Yes-associated protein; chemoresistance; cisplatin; lung cancer stem cell.

Figure 1.

Morphologies of A549 adherent cells and A549 spheres. Adherent A549 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and floating A549 spheres were cultured in serum-free DMEM/F-12 medium. DMEM, Dulbecco's modified Eagle's medium.

Figure 2.

YAP expression in A549 cells and A549 spheres. (A) YAP expression at the mRNA level was determined by reverse transcription-polymerase chain reaction. (B) YAP expression at the protein level was determined by western blotting. The band intensity of (C) YAP mRNA and (D) YAP protein were normalized against β-actin. Data are presented as the mean ± standard deviation of three replicates. **P<0.01 and ***P<0.001. YAP, Yes-associated protein; si, short interfering; NC, negative control.

Figure 3.

YAP silencing significantly enhances the efficacy of CDDP. (A) Dose response curve to determine the IC₅₀ of CDDP in A549 cells and A549 tumor spheres with YAP knockdown or control knockdown. (B) The inhibitory effect of 2.5 µg/ml CDDP on A549 cells and A549 tumor spheres with YAP knockdown or control knockdown. Data are presented as the mean ± standard deviation of three replicates. ***P<0.001. CDDP, cisplatin; YAP, Yes-associated protein; si, short interfering; NC, negative control.

Figure 4.

YAP silencing significantly induces apoptosis. (A) A549 adherent cells and A549 spheres under different treatments were collected, stained by Annexin V/propidium iodide and analyzed by flow cytometry. (B) The percentage of A549 cells and A549 sphere cells undergoing apoptosis upon different treatments. Data are presented as the mean ± standard deviation of three replicates. **P<0.01 and ***P<0.001. YAP, Yes-associated protein; CDDP, cisplatin; si, short interfering; NC, negative control.

Figure 5.

Dependency of ABCB1 expression on YAP. Reverse transcription polymerase chain reaction results demonstrating the relative ABCB1 expression in A549 adherent cells and A549 spheres with YAP knockdown or control knockdown. Data are presented as the mean ± standard deviation of three replicates. **P<0.01 and ***P<0.001. YAP, Yes-associated protein; si, short interfering; NC, negative control; ABCB1, ATP-binding cassette subfamily B member 1.