Hypoxia-Inducible Ubiquitin Specific Peptidase 13 Contributes to Tumor Growth and Metastasis via Enhancing the Toll-Like Receptor 4/Myeloid Differentiation Primary Response Gene 88/Nuclear Factor-κB Pathway in Hepatocellular Carcinoma.

Shan Gao,Tianxiang Chen,Yang Liu,Junjun Zhao,Kangsheng Tu,Xin Liu,Dongsheng Huang,Zhi Zeng,Qiliang Lu,Lijie Li,Qiuran Xu

doi:10.3389/fcell.2020.587389

Abstract

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. The activation of the toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-κB (TLR4/MyD88/NF-κB) pathway contributes to the development and progression of HCC. The ubiquitin–proteasome system regulates TLR4 expression. However, whether ubiquitin specific peptidase 13 (USP13) stabilizes TLR4 and facilitates HCC progression remains unclear. Here, quantitative real-time PCR (qRT-PCR) and immunohistochemistry analysis revealed that USP13 expression in HCC tissues was higher than in non-tumor liver tissues. Moreover, the elevated expression of USP13 was detected in HCC cells (SK-HEP-1, HepG2, Huh7, and Hep3B) compared to LO2 cells. Interestingly, the positive staining of USP13 was closely correlated with tumor size ≥ 5 cm and advanced tumor stage and conferred to significantly lower survival of HCC patients. Next, USP13 knockdown prominently reduced the proliferation, epithelial–mesenchymal transition (EMT), migration, and invasion of Hep3B and Huh7 cells, while USP13 overexpression enhanced these biological behaviors of HepG2 and LO2 cells. The silencing of USP13 significantly restrained the growth and lung metastasis of HCC cells in vivo. Mechanistically, the USP13 depletion markedly inhibited the TLR4/MyD88/NF-κB pathway in HCC cells. USP13 interacted with TLR4 and inhibited the ubiquitin-mediated degradation of TLR4. Significantly, TLR4 re-expression remarkably reversed the effects of USP13 knockdown on HCC cells. USP13 expression was markedly upregulated in HCC cells under hypoxia conditions. Notably, USP13 knockdown repressed hypoxia-induced activation of the TLR4/MyD88/NF-κB pathway in HCC cells. In conclusion, our study uncovered that hypoxia-induced USP13 facilitated HCC progression via enhancing TLR4 deubiquitination and subsequently activating the TLR4/MyD88/NF-κB pathway.

Highlights

Hepatocellular carcinoma is a dominating histological subtype of primary liver cancer and the second most prevalent cause of cancer-related deaths in China (Chen et al, 2016; Forner et al, 2018)
We found that UPS13 mRNA expression in hepatocellular carcinoma (HCC) tissues was higher than that in non-tumor liver tissues (P = 0.0001, Figure 1A)
Since hypoxia has been recognized as an inducer of the activation of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) pathway in HCC (Won et al, 2015; Zhang et al, 2016), we aimed to investigate whether ubiquitin specific peptidase 13 (USP13) mediated the hypoxia-induced activation of the TLR4/MyD88/NF-κB pathway in HCC

Summary

Introduction

Hepatocellular carcinoma is a dominating histological subtype (about 90%) of primary liver cancer and the second most prevalent cause of cancer-related deaths in China (Chen et al, 2016; Forner et al, 2018). The TLR4/myeloid differentiation factor 88 (MyD88) pathway has been recognized as oncogenic signaling in human cancers and is correlated with patients’ poor survival (Chen et al, 2008; Lupi et al, 2020). The inactivation of the TLR4/MyD88 pathway by geniposide reduced signal transducer and activator of transcription 3 (STAT3)/Sp1-dependent vascular endothelial growth factor (VEGF) production in HCC cells (Zhang et al, 2020). Hypoxia-inducible factor-1α (HIF-1α)mediated TLR4/MyD88 pathway participates in hypoxia-induced HCC cell proliferation, migration, and invasion (Zhang et al, 2016). Immunohistochemistry (IHC) analysis shows that TLR4 is positively expressed in 56.7% of HCC cases (Eiro et al, 2014). These data suggest that TLR4 is regulated by post-translational modifications, such as ubiquitylation

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