Dataset Information

Nek2 augments sorafenib resistance by regulating the ubiquitination and localization of β-catenin in hepatocellular carcinoma.

ABSTRACT:

Background

Sorafenib is the first-line treatment for advanced-stage hepatocellular carcinoma (HCC). Several studies have shown that the up-regulation of β-catenin plays a role in sorafenib resistance in HCC; however, the mechanism associated with this phenomenon remains elusive.

Methods

Western blotting, flow cytometry, and an evaluation of IC₅₀ values were used to confirm the role of β-catenin in HCC sorafenib resistance. Immunoprecipitation and western blotting were then performed to identify regulatory interactions between β-catenin and Nek2. Further, western blotting, flow cytometry, and an in vivo xenograft model were used to evaluate the function of Nek2 in HCC sorafenib resistance, whereas rescue experiments were performed to confirm that Nek2 induces sorafenib resistance via β-catenin. Finally, western blotting and immunohistochemistry were used to evaluate the expression level of Nek2 in paired HCC and non-tumor tissues.

Results

We showed that β-catenin could suppress sorafenib-induced apoptosis and cell growth inhibition in HCC cell lines. By screening β-catenin-interacting proteins, we found that Nek2 could bind β-catenin in sorafenib-treated HCC cell lines. Our results also showed that Nek2 stabilizes β-catenin and promotes its translocation to the nucleus, consequently activating the transcription of downstream target genes. We further confirmed that Nek2 could induce sorafenib resistance in HCC cell lines, and that β-catenin was the key element involved in this process. Further, a xenograft tumor model showed that Nek2 knockdown could improve the anti-tumor effect of sorafenib, whereas an analysis of tumor proteins showed that Nek2 regulates β-catenin protein levels and its nuclear translocation in vivo. In addition, Nek2 was found to be up-regulated in HCC tissue, and especially in advanced-stage disease.

Conclusions

Our study proves that Nek2 induces HCC sorafenib resistance via β-catenin and suggests a novel therapeutic strategy to improve the anti-tumor effects of sorafenib in HCC.

SUBMITTER: Deng L

PROVIDER: S-EPMC6639974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeSorafenib is a multi-kinase inhibitor that is used as a standard treatment for advanced hepatocellular carcinoma (HCC). However, the mechanism of sorafenib resistance in HCC is still unclear. It has been shown that CISD2 expression is related to the progression and poor prognosis of HCC. Here, we show a new role for CISD2 in sorafenib resistance in HCC.MethodsBioinformatic analysis was used to detect the expression of negative regulatory genes of ferroptosis in sorafenib-resistant samples. The concentration gradient method was used to establish sorafenib-resistant HCC cells. Western blot was used to detect the protein expression of CISD2, LC3, ERK, PI3K, AKT, mTOR, and Beclin1 in HCC samples. Quantitative real-time PCR (qPCR) was used to detect gene expression. CISD2 shRNA and Beclin1 shRNA were transfected to knock down the expression of the corresponding genes. Cell viability was detected by a CCK-8 assay. ROS were detected by DCFH-DA staining, and MDA and GSH were detected with a Lipid Peroxidation MDA Assay Kit and Micro Reduced Glutathione (GSH) Assay Kit, respectively. Flow cytometry was used to detect apoptosis and the levels of ROS and iron ions.ResultsCISD2 was highly expressed in HCC cells compared with normal cells and was associated with poor prognosis in patients. Knockdown of CISD2 promoted a decrease in the viability of drug-resistant HCC cells. CISD2 knockdown promoted sorafenib-induced ferroptosis in resistant HCC cells. The levels of ROS, MDA, and iron ions increased, but the change in GSH was not obvious. Knockdown of CISD2 promoted uncontrolled autophagy in resistant HCC cells. Inhibition of autophagy attenuated CISD2 knockdown-induced ferroptosis. The autophagy promoted by CISD2 knockdown was related to Beclin1. When CISD2 and Beclin1 were inhibited, the effect on ferroptosis was correspondingly weakened.ConclusionInhibition of CISD2 promoted sorafenib-induced ferroptosis in resistant cells, and this process promoted excessive iron ion accumulation through autophagy, leading to ferroptosis. The combination of CISD2 inhibition and sorafenib treatment is an effective therapeutic strategy for resistant HCC.