Targeting Yes1 Associated Transcriptional Regulator Inhibits Hepatocellular Carcinoma Progression and Improves Sensitivity to Sorafenib: An in vitro and in vivo Study

Abstract

Purpose: The aim of this study was to investigate the role of Yes1 associated transcriptional regulator (YAP1) in the pathology of hepatocellular carcinoma (HCC) and its potential as a therapeutic target.

Methods: YAP1 expression in HCC and adjacent tissues was determined via immunohistochemistry; in HCC and human normal liver cell lines, expression was examined via Western blotting. The effects of YAP1 knockdown and overexpression were detected following transfection of HCC cells with siRNA-YAP1 recombinants or pcDNA3.1-YAP1 plasmids. A tumor xenograft model was constructed by implanting YAP1-knockdown lentivirus-infected Hep-3B cells into nude mice, and the animals were treated with sorafenib.

Results: In patients with HCC, YAP1 was upregulated in tumor tissue compared with adjacent tissue, and its high expression in the tumor was associated with increased Edmonson grade. In vitro, YAP1 expression was increased in Hep-3B, SK-HEP-1 and Huh7 cells, while it was similar in SMMC-7721 cells and LO2 cells. Meanwhile, YAP1 increased cell proliferation and invasion, promoted the progression of epithelial-mesenchymal transition, and inhibited cell apoptosis in HCC cells; furthermore, YAP1 knockdown combined with the administration of sorafenib decreased cell viability and increased cell apoptosis compared with YAP1 knockdown or treatment with sorafenib alone. In vivo, YAP1 knockdown inhibited tumor growth and metastasis, whereas it promoted apoptosis; meanwhile, YAP1 knockdown synergized with sorafenib to suppress tumor progression in HCC mice.

Conclusion: YAP1 is upregulated in both HCC tumor tissues and cell lines. Moreover, it promotes cell proliferation and invasion and promoted the progression of epithelial-mesenchymal transition in vitro. Furthermore, targeting YAP1 inhibits HCC progression and improves sensitivity to sorafenib in vitro and in vivo.

Keywords: Yes1 associated transcriptional regulator; apoptosis; hepatocellular carcinoma; invasion; proliferation; sorafenib.

Figure 1

YAP1 was upregulated in HCC tumor tissues compared with adjacent tissues. Representative images of low YAP1 expression in adjacent tissue and high YAP1 expression in tumor tissue (A). Comparison of YAP1 expression between adjacent tissue and tumor tissue (B).

Figure 2

YAP1 was upregulated, whereas pYAP1 was downregulated, in HCC cell lines compared with normal liver cells. Comparison of YAP1 and pYAP1 expression between human HCC cell lines (Hep-3B, SMMC-7721, SK-HEP-1, Huh-7) and a human normal liver cell line (LO2) (A and B). P value was displayed as **P < 0.01, ***P < 0.001, and NS (P > 0.05).

Figure 3

Establishment of YAP1 knockdown and overexpression in HCC cells. Comparison of YAP1 expression between cells transfected with siRNA-YAP1 recombinants and those transfected with siRNA-NC (HEP-3B cells) (A and B). Comparison of YAP1 expression between cells transfected with the pcDNA3.1-YAP1 plasmid and those transfected with the pcDNA3.1-NC plasmid (SMMC-7721 cells) (C and D). P value was displayed as **P < 0.01, ***P < 0.001, and NS (P > 0.05).

Figure 4

YAP1 promoted proliferation in HCC cells. Comparison of OD450 nm absorbance by CCK-8 (A) and Ki67 protein expression (B and C) between Hep-3B cells transfected with siRNA-YAP1 recombinants and those transfected with siRNA-NC at 24 h, 48 h, and 72h after transfection. Comparison of OD450 nm absorbance by CCK-8 (D) and Ki67 protein expression (E and F) between SMMC-7721 cells transfected with the pcDNA3.1-YAP1 plasmid and those transfected with the pcDNA3.1-NC plasmid at 24 h, 48 h, and 72 h after transfection. P value was displayed as *P < 0.05 and NS (P > 0.05).

Figure 5

YAP1 decreased apoptosis in HCC cells. Comparison of the cell apoptosis rate by the AV/PI assay between Hep-3B cells transfected with siRNA-YAP1 recombinants and those transfected with siRNA-NC at 48 h after transfection (Aand B). Comparison of the cell apoptosis rate by the AV/PI assay between SMMC-7721 cells transfected with the pcDNA3.1-YAP1 plasmid and those transfected with the pcDNA3.1-NC plasmid at 48 h after transfection (Cand D). P value was displayed as ***P < 0.001, and NS (P > 0.05).

Figure 6

YAP1 promoted cell invasion in HCC cells. Comparison of invasive cell number by Transwell assay between siRNA-YAP1 recombinants transfected cells and siRNA-NC transfected cells in Hep-3B cells at 48 h after transfection (Aand B). Comparison of invasive cell number by Transwell assay between pcDNA3.1-YAP1 plasmid transfected cells and pcDNA3.1-NC plasmid transfected cells in SMMC-7721 cells at 48h after transfection (Cand D). P value was displayed as **P < 0.01 and *** P < 0.001.

Figure 7

YAP1 increased Tead expression and promoted EMT markers in HCC cells. Comparison of Tead, E-cadherin, and N-cadherin expression between Hep-3B cells transfected with siRNA-YAP1 and those transfected with siRNA-NC (A and B), and between SMMC-7721 cells transfected with the pcDNA3.1-YAP1 plasmid and those transfected with the pcDNA3.1-NC plasmid (C and D). P value was displayed as *P < 0.05 and **P < 0.01.

Figure 8

YAP1 overexpression promoted proliferation and invasion, but inhibited apoptosis of Hep-3B cells. Comparison of YAP1 expression (A and B), OD450 nm absorbance (C), Ki67 expression (D and E), apoptosis rate (F and G) and number of invasive cells (H and I) between cells transfected with the pcDNA3.1-YAP1 plasmid and those transfected with the pcDNA3.1-NC plasmid. P value was displayed as *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 9

YAP1 increased Tead expression and promoted EMT in Hep-3B cells. Comparison of Tead, E-cadherin, and N-cadherin expression (A and B) between cells transfected with the pcDNA3.1-YAP1 plasmid and those transfected with the pcDNA3.1-NC plasmid. P value was displayed as **P < 0.01.

Figure 10

Combination of YAP1 knockdown and administration of sorafenib inhibited cell viability, but promoted apoptosis of HCC cells. Comparison of relative cell viability (A) and cell apoptosis rate (B and C) among control cells, sorafenib cells, YAP1-KD-LV cells, and sorafenib and YAP1-KD-LV cells. P value was displayed as *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 11

Combination of YAP1 knockdown and administration of sorafenib inhibited tumor growth in HCC. Comparison of tumor size (A) and tumor weight (B) among the Control, Sorafenib, YAP1-KD-LV, and Sorafenib & YAP1-KD-LV groups. Comparison of tumor apoptosis, YAP1 expression, MMP3 expression, and MMP9 expression among the Control, Sorafenib, YAP1-KD-LV, and Sorafenib & YAP1-KD-LV groups (C). P value was displayed as *P < 0.05, ***P < 0.001 and NS (P > 0.05).