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YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin

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Abstract

Background

Sorafenib is the standard first-line treatment for advanced hepatocellular carcinoma (HCC), but its use is hampered by secondary drug resistance. Yes-associated protein (YAP) is a downstream effector of the Hippo signaling pathway, which is crucial for liver tumorigenesis. As yet, however, the mechanism underlying sorafenib resistance and the role of YAP therein is not fully understood and needs to be explored further.

Methods

Western blotting, flow cytometry and CCK-8 assays were used to assess the role of YAP in HCC sorafenib resistance. Next, qRT-PCR and Western blotting were performed to identify survivin as a YAP downstream effector, and rescue experiments were performed to confirm that YAP induces sorafenib resistance via survivin. Additionally, Western blotting, flow cytometry and in vivo xenograft models were used to evaluate the effect of verteporfin in combination with sorafenib on HCC.

Results

We found that sorafenib enhances YAP nuclear accumulation and activation, thereby promoting sorafenib resistance through inhibiting apoptosis in HCC cells. In addition, we found that survivin acts as a downstream mediator of YAP to resist sorafenib-induced apoptosis. Pharmacological inhibition of YAP by verteporfin increased the sensitivity of HCC cells to sorafenib and reversed sorafenib resistance. Moreover, verteporfin in combination with sorafenib significantly suppressed in vivo HCC tumor growth.

Conclusions

Our data indicate that YAP promotes sorafenib resistance through upregulation of survivin expression in HCC cells. Targeting YAP may be a therapeutic strategy to improve the antitumor effects of sorafenib in HCC.

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Data availability

The data and materials supporting the conclusion of this manuscript are included in the article.

Abbreviations

HCC:

Hepatocellular carcinoma

IHC:

Immunohistochemistry

QRT-PCR:

quantitative real-time polymerase chain reaction

siRNA:

Small interfering RNA

YAP:

Yes-associated protein

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Funding

This project was supported by the National Natural Science Foundation of China (No. 81874188) and the Science and Technology Project of Henan province of China (No. 202102310119).

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Authors and Affiliations

  1. Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China

    Ting Sun, Wenhao Mao & Hui Peng

  2. Department of Pharmacy, Kaifeng Hospital of Traditional Chinese Medicine, 475000, Kaifeng, China

    Qi Wang

  3. Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, 610041, China

    Lin Jiao

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  1. Ting Sun
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  2. Wenhao Mao
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  3. Hui Peng
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  4. Qi Wang
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Contributions

TS designed the study and prepared the manuscript; WM and HP performed most of the experiments; TS and LJ analyzed the data and competed the figures; QW revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ting Sun.

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All experiments were performed in accordance with the standards of the ethics committee of the First Affiliated Hospital of Zhengzhou University.

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Supplementary Information

Supplemental Fig. 1

The protein levels of YAP were analyzed by western blotting assay in liver cancer cell lines and in a normal human hepatic cell line LO2 (PNG 39 kb) (PNG 24 kb)

High Resolution Image (TIF 248 kb)

Supplemental Fig. 2

Huh-7R and HepG2R were resistant to sorafenib induced apoptosis. (a, b) Huh-7R and HepG2R cells were exposed to the indicated doses of sorafenib for 24 h. Cells were collected for western blotting. (c, d) Proteins from the cytoplasm and nucleus were separated, and the localization of YAP were detected by western blotting. (e, f) The mRNA expressions of CTGF and CYR61 were analyzed by qRT-PCR. Results were presented as mean ± sem (n = 3) for each treatment (PNG 114 kb)

High Resolution Image (TIF 1460 kb)

Supplemental Fig. 3

Knockdown YAP expression using shRNAs in Huh-7 and HepG2 cells. Huh-7 and HepG2 cells were transfected with shYAP. (a, b) The protein levels of YAP were analyzed with western blotting. (c, d) The mRNA levels of YAP were analyzed with RT-PCR (PNG 83 kb)

High Resolution Image (TIF 2678 kb)

Supplemental Fig. 4

Sorafenib had no significant effect on YAP mRNA expression. (a-b) Huh-7 and HepG2 cells transfected with shYAP were treated with sorafenib for 24 h. The mRNA levels of YAP were analyzed with RT-PCR (PNG 39 kb)

High Resolution Image (TIF 2478 kb)

Supplemental Fig. 5

YAP attenuated the cytotoxicity of sorafenib. (a, b) Huh-7 and HepG2 cells transfected with shRNA were treated with different concentration of sorafenib for 72 h. Cell viability was measured by CCK-8. (c) LO2 cells transfected with flag-YAP or vector were treated with different concentration of sorafenib for 72 h. Cell viability was measured by CCK-8 (PNG 35 kb)

High Resolution Image (TIF 1040 kb)

Supplemental Fig. 6

YAP inhibitor verteporfin reversed sorafenib resistance. (a, b) Huh-7R and HepG2R cells were treated with sorafenib in combination with or without verteporfin for 24 hours. Cells were analyzed using western blotting. (c, d) Huh-7R and HepG2R cells were treated with different combinations of sorafenib and verteporfin for 72 h. Cell viability was measured by CCK-8. (e, f) Huh-7R and HepG2R cells were treated with different combinations of sorafenib and verteporfin. Cells viability was analyzed by colony formation assay. Columns were the average of three independent experiments. (g) Huh-7R and HepG2R cells were treated with different combinations of sorafenib and verteporfin for 48 h. Cells were analyzed by flow cytometry. Columns were the average of three independent experiments. Results were presented as mean ± sem (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (PNG 629 kb)

High Resolution Image (TIF 7173 kb)

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Sun, T., Mao, W., Peng, H. et al. YAP promotes sorafenib resistance in hepatocellular carcinoma by upregulating survivin. Cell Oncol. 44, 689–699 (2021). https://doi.org/10.1007/s13402-021-00595-z

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