Yes-associated protein (YAP) increases chemosensitivity of hepatocellular carcinoma cells by modulation of p53

Abstract

The yes-associated protein (YAP) transcription co-activator has been reported either as an oncogene candidate or a tumor suppressor. Liver tissue chips revealed that about 51.4% human hepatocellular carcinoma (HCC) samples express YAP and 32.9% HCC samples express phosphorylated YAP. In this study, we found that chemotherapy increased YAP protein expression and nuclear translocation in HepG2 cells, as well as p53 protein expression and nuclear translocation. However, little is known about YAP functions during chemotherapy. Our results show that overexpression of YAP increases chemosensitivity of HepG2 cells during chemotherapy. Dominant negative transfection of Flag-S94A (TEAD binding domain mutant) or Flag-W1W2 (WW domain mutant) to HepG2 cells decreases p53 expression/ nuclear translocation and chemosensitivity when compared with control HepG2 cells. Furthermore, rescue transfection of Flag-5SA-S94A or Flag-5SA-W1W2, respectively to HepG2 cells regains p53 expression/nuclear translocation and chemosensitivity. These results indicate that YAP promotes chemosensitivity by modulating p53 during chemotherapy and both TEAD and WW binding domains are required for YAP-mediated p53 function. ChIP assay results also indicated that YAP binds directly to the p53 promoter to improve its expression. In addition, p53 could positively feedback YAP expression through binding to the YAP promoter. Taken together, our current data indicate that YAP functions as a tumor suppressor that enhances apoptosis by modulating p53 during chemotherapy.

Keywords: YAP; hepatocellular carcinoma; p53.

Figure 1. Evaluation of YAP expression in hepatocellular carcinoma and normal liver samples. (A) A–L show YAP expression in HCC and normal liver samples. A’–L’ reveal p-YAP expression in HCC and normal liver samples. A, A’, B and B’ represent Grade I HCC; C, C’, D and D’ represent Grade II HCC; E, E’, F and F’ represent Grade III HCC; G, G’, H and H’ represent Grade II cholangiohepatoma; I, I’, J and J’ represent Grade IV HCC; K and K’ represent sarcomas hepatocellular carcinoma; L,L; represent normal liver samples. (B) Statistics of positive expression of YAP and p-YAP in liver cancer samples. (C) Statistics of YAP and p-YAP expression levels in liver cancer samples.

Figure 2. YAP is expressed in both HCC and normal liver cell lines. (A) YAP proteins are widely expressed in both cytoplasm and nucleus. (B) Test of YAP expression by western blots.

Figure 3. YAP is activated when treated with doxorubicin. HepG2 cells were treated with 0.7 μg/ml doxorubicin from 0 to 24 h; then cells were harvested to test mRNA (A) and protein expression (B) levels of YAP at different time point. The pixel density from the western blot results of Yap (C) and p-YAP (D) were analyzed and plotted as Means + SD (n = 3). (E) The ratio of protein expression level of YAP/ p-YAP were plotted as Means+SD (n = 3). (F) The same as for (B) except that analysis was done by immunofluorescence.

Figure 4. YAP2 improves the apoptosis of HepG2 cells after treatment with DDP and doxorubicin. HepG2 cells were transiently transfected with WT-YAP or YAP-mutants, after 48 h, cells were harvested and seeded into 96-well plate. Once cells attached to the plates, they were treated with different concentration of (A) doxorubicin and (B) DDP for 72 h and then subjected to WST-1 analysis. (C) HepG2 cells were transiently transfected with sh-YAP or scramble control; after 48 h, cells were harvested and seeded into 96-well plate. Once cells attached to the plates, they were treated with different concentration of doxorubicin.

Figure 5. p53 is activated when treated with Doxorubicin. (A) HepG2 cells were treated with 0.7 μg/ml doxorubicin from 0 to 24 h; then cells were harvested for testing p53 by immunofluorescence (A) or testing p53 and p-p53 by western blots (B).

Figure 6. YAP improves apoptosis by binding to the p53 promoter and enhances p53 expression. (A) HepG2 cells were transiently transfected with Flag-YAP or Flag-YAP mutants and subjected to western blots. After 48 h, the overexpression of Flag-YAP was confirmed by using flag antibody. (B) Western blot results of p53 in HepG2-Flag-YAP and HepG2-Flag-YAP mutants after treatment with 0.7 μg/ml doxorubicin for 24 h. (C) Real-time result of p53 in HepG2 cell transfected with empty vector control or WT-YAP plasmid and incubated with doxorubicin for 8 h. (D) ChIP was used to assay the binding of YAP to p53 promoter in HepG2 cells treated with doxorubicin for 8 h. (E and F) DNA-damage induced apoptosis and cell-cycle regulation proteins were examined. (G) Immunofluorescence of p53 (green) HepG2-Flag-YAP and HepG2-Flag-YAP mutants after treatment with doxorubicin for 24 h.

Figure 7. p53 could positively feedback YAP expression through binding to YAP promoter. (A) Real-time RT-PCR and (B) western blot results of YAP in HepG2 cells transiently transfected with empty vector control or WT-p53 after treatment with doxorubicin for 24 h. (C) Western blot results of p53, YAP and their phosphorylation forms in HepG2 cells after 2 hours’ pretreatment with p53 inhibitor pifichrin-α and then 24 h treatment with doxorubicin in the presence of pifichrin-α. (D) HepG2 cells were treated with doxorubicin for 24 h and harvested for ChIP assay with normal rabbit IgG, p53 antibody or RNA polymerase II. PCR were done by using the primers of the YAP promoter.