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

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.

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

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). HCC commonly occurs under the conditions of hepatitis B/C (HBV/HCV)-mediated chronic inflammation and metabolic syndrome (Han et al., 2020;McGlynn et al., 2020). Currently, only one third of cases are eligible for curative treatments, and the therapeutic strategies for advanced HCC are limited, which leads to the poor prognosis of patients (Gunasekaran et al., 2020). Therefore, there is an urgent need to thoroughly understand the molecular mechanisms involved in the occurrence and progression of HCC.
In this study, we explored the clinical significance of USP13 in HCC and investigated the biological role of USP13 in tumor growth and metastasis in vitro and in vivo. We also identified the underlying mechanism whereby USP13 promoted the progression of HCC and revealed the regulatory effect of hypoxia on UPS13 expression. Our data indicated that hypoxiainduced USP13 facilitated the proliferation, migration, and invasion of HCC cells via enhancing the TLR4/MyD88/NFκ B pathway.

Clinical Samples
Hepatocellular carcinoma tissues and corresponding tumoradjacent tissues were harvested from 80 patients who received hepatectomy at The First Affiliated Hospital of Xi'an Jiaotong University after signing written informed consent. The enrolled participants were pathologically diagnosed with HCC and did not receive preoperative treatment. The tissue samples were obtained and maintained at −80 • C for subsequent experiments. The follow-up time was defined from the date of surgical resection to the date of patient death or the last followup. The clinicopathologic characters of HCC patients are presented in Table 1.

Transwell Migration and Invasion Assays
Twenty-four hours after transfection, HCC cells were seeded at a density of 1 × 10 4 cells per well in the upper chamber of transwell insert (8 µm, Corning, Corning, NY, United States). Each upper chamber was covered with or without Matrigel (BD Biosciences, Franklin Lakes, NJ, United States). Moreover, each of the lower chambers contained 500 µl of medium with 10% FBS. The migrated/invaded cells were stained with 0.1% crystal violet and photographed.

Immunohistochemistry
Immunohistochemistry analysis was performed as previously described (Dou et al., 2019b). The primary USP13 antibody (ab99421) and Ki-67 antibody (ab15580) were obtained from Abcam (Cambridge, MA, United States). Not detected and low staining of USP13 were recognized as negative expression, and medium and high staining of USP13 were defined as positive expression. The percentage of positive staining cells was calculated for quantifying Ki-67 staining.

Co-immunoprecipitation Assay
The specific antibody against TLR4 (sc-293072, Santa Cruz Biotechnology) was used for the co-immunoprecipitation (co-IP) assay, which was performed according to the previously described protocols (Tu et al., 2014).

In vivo Experiments
Four-week BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Company (Shanghai, China). The in vivo tumor growth and lung metastasis experiments were performed using Hep3B cells with or without USP13 knockdown according to the protocols as previously described (Dou et al., 2019a;Guo et al., 2019). The tumor volume (V) was calculated as follows: V = (L × D2)/2 where L and D represent the tumor length and width, respectively (in mm). Three weeks after subcutaneous injection, the mice were euthanized, and tumor tissues were harvested for IHC staining of USP13 and Ki-67. Eight weeks after tail vein injection, the mice were sacrificed under euthanization, and lung tissues were collected for hematoxylin and eosin (H&E) staining. The animal study was approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

Microarray
Hep3B cells were cultured under normoxic and hypoxic conditions, respectively, for 48 h. Then, total RNA was isolated from Hep3B cells using TRIzol reagent (Invitrogen). RNA quantity and quality were measured by NanoDrop ND-1000 (Thermo Scientific). The Arraystar Human LncRNA Arrays V5 (Arraystar, Rockville, MD, United States) was used to identify differently expressed lncRNA and mRNA based on the manufacturer's standard protocols (Aksomics, Shanghai, China). Arraystar Human LncRNA Microarray V5.0 is designed for the global profiling of human LncRNAs and protein-coding transcripts. This third-generation LncRNA microarray can detect about 39,317 LncRNAs and 21,174 coding transcripts. The microarray data (GSE155505) was uploaded into the Gene Expression Omnibus (GEO).

Statistical Analysis
The date of three independent experiments is shown as mean ± standard deviation (SD). Comparison among two groups or >2 groups was executed with one-way analysis of variance (ANOVA) or Student's t-test. The survival of HCC patients was analyzed using the Kaplan-Meier's method and log-rank test. Chi-square test was employed to explore the correlations between clinical variables and USP13 expression. All statistical analyses were carried out with GraphPad Prism 8.0 (GraphPad Inc., San Diego, CA, United States). P < 0.05 was defined to be statistically significant.

USP13 Is Highly Expressed in HCC
To determine the expression difference of USP13 between HCC and tumor-adjacent tissues, qRT-PCR and IHC analysis were performed to detect USP13 mRNA and protein levels, respectively. We found that UPS13 mRNA expression in HCC tissues was higher than that in non-tumor liver tissues (P = 0.0001, Figure 1A). TCGA data analysis using GEPIA web tool (Tang et al., 2017) consistently revealed a significantly higher level of USP13 mRNA in HCC (P < 0.0001, Supplementary Figure S1A). Furthermore, IHC analysis indicated that USP13 positive expression was confirmed in 52 of 80 (65.0%) HCC cases, while only 34 of 80 (42.5%) non-tumor samples showed USP13 positive expression (P = 0.0043, Figure 1B). Besides, the level of USP13 in HCC cell lines (SK-HEP-1, HepG2, Huh7, and Hep3B) was prominently higher than that in LO2 cells (P < 0.05, Figure 1C). These data indicated an oncogenic role of USP13 in HCC.

The Positive Expression of USP13 Correlates With Poor Prognosis of HCC
Next, the clinical significance of USP13 in HCC was determined in this study. As shown in Table 1, the positive expression of USP13 was significantly associated with tumor size ≥ 5 cm (P = 0.003) and advanced tumor-node-metastasis (TNM) stage (III + IV, P = 0.024), as analyzed by chi-square test. Moreover, Frontiers in Cell and Developmental Biology | www.frontiersin.org HCC patients with USP13 positive expression had a significantly lower overall survival compared to cases with USP13 negative expression (P = 0.009, Figure 1D). More importantly, TCGA data analysis using the GEPIA web tool (Tang et al., 2017) also indicated that the high USP13 mRNA level indicated an apparent shorter overall survival and disease-free survival of HCC patients (P = 0.0026 and 0.058, respectively, Supplementary Figure S1B). Thus, our results suggested that USP13 might be a promising prognostic biomarker for HCC.

USP13 Knockdown Suppresses HCC Cell Proliferation and Invasion in vitro and in vivo
Hep3B and Huh7 cells, which expressed a relatively higher level of USP13, were transfected with two independent shRNAs to specifically downregulate USP13 (P < 0.05, Figure 2A). Both CCK-8 and EdU assays consistently indicated that USP3 knockdown markedly reduced the proliferation of HCC cells (P < 0.05, Figures 2B,C). Furthermore, the silencing of USP13 remarkably repressed HCC cell migration and invasion as determined by transwell assay (P < 0.05, Figure 3A). Western blotting data indicated that USP13 knockdown increased E-cadherin expression and reduced N-cadherin and vimentin levels in HCC cells (P < 0.05, Figure 3B). Conversely, USP13 overexpression significantly enhanced the proliferation, epithelial-mesenchymal transition (EMT), migration, and invasion of HepG2 and LO2 cells (P < 0.05, Supplementary Figures S2, S3). Then, the effects of USP13 knockdown on HCC cells were further confirmed in vivo. The growth curves of subcutaneous tumors formed by Hep3B cells suggested that USP13 silencing inhibited tumor growth in mice (P < 0.05, Figure 4A). Furthermore, tumor tissues from the USP13 knockdown group had a prominently lower USP13 and Ki-67 staining density compared to samples from the control group (P < 0.05, Figure 4B). H&E staining of lung tissues collected from the mouse HCC metastasis model indicated that the depletion of USP13 markedly reduced the number of lung metastases in vivo (P < 0.05, Figure 4C). Collectively, our data identified USP13 as an oncogene in HCC.

USP13 Regulates TLR4/MyD88/NF-κB Pathway via Enhancing TLR4 Stabilization
Since TLR4 stabilization is regulated by ubiquitination-mediated degradation (Chuang and Ulevitch, 2004;Lu et al., 2020), TCGA data analysis using the GEPIA web tool (Tang et al., 2017)  indicated that the expression of TLR4 mRNA in HCC tissues was significantly lower than that in normal liver tissues (P < 0.0001, Supplementary Figure S4A). We aimed to know whether deubiquitinase USP13 participated in regulating TLR4 stabilization in HCC. Interestingly, we found that USP13 knockdown significantly reduced the level of TLR4 protein in Hep3B and Huh7 cells (P < 0.05, Figure 5A). The co-IP assay revealed that USP13 directly interacted with TLR4 in Hep3B cells ( Figure 5B). Notably, USP13 knockdown increased the ubiquitination of TLR4 in Hep3B cells ( Figure 5C). Hep3B cells were treated with CHX to block new protein synthesis. Then, we found that TLR4 protein was degraded faster in the USP13 group than in the control group ( Figure 5D). However, proteasome inhibitor MG132 treatment could reduce the degradation of TLR4 induced by USP13 knockdown (Figure 5D). These results indicated that USP13 increased TLR4 abundance via enhancing the deubiquitination of TLR4 in HCC cells. Next, we determined the effects of USP13 knockdown on the TLR4/MyD88/NF-κB pathway in HCC cells. As expected, USP13 knockdown markedly reduced the levels of MyD88 and P-NF-κB p65 in HCC cells (P < 0.05, Figure 5A). The levels of TLR4 and MyD88 protein in HCC cells were markedly higher than those in LO2 cells (P < 0.05, Supplementary Figure S4B). Moreover, the expressions of TLR4, MyD88, and P-NF-κB p65 in subcutaneous tumor tissues from the USP13 knockdown group were prominently lower than those in the control group (P < 0.05, Supplementary Figure S4C).

Hypoxia Activates the TLR4/MyD88/NF-κB Pathway via Inducing USP13 in HCC Cells
Since hypoxia has been recognized as an inducer of the activation of the TLR4/MyD88/NF-κB pathway in HCC (Won et al., 2015;Zhang et al., 2016), therefore, we aimed to investigate whether USP13 mediated the hypoxia-induced activation of the TLR4/MyD88/NF-κB pathway in HCC. Our microarray data indicated that hypoxia increased the expression of 10 HIF target-gene mRNAs and resulted in significantly increased expression of USP13 mRNA in Hep3B cells (Figure 7A), while the expression of TLR4 mRNA was not prominently impacted under hypoxia ( Figure 7A). Furthermore, either hypoxia or CoCl 2 treatment markedly upregulated the levels of HIF-1α and USP13 protein in Hep3B and Huh7 cells (Figures 7B,C). Interestingly, the USP13 knockdown repressed the hypoxiainduced activation of the TLR4/MyD88/NF-κB pathway in HCC cells ( Figure 7D). Altogether, our results suggested that USP13 played an essential role in the hypoxia-mediated TLR4/MyD88/NF-κB pathway in HCC.

DISCUSSION
Ubiquitination-mediated protein degradation participates in several physiological and pathological processes, including cancer (Popovic et al., 2014;Xu et al., 2017b). Deubiquitinase USP13 is aberrantly expressed in several human cancers and correlates with poor prognosis (Zhang et al., 2013;Han et al., 2016;Fang et al., 2017;Man et al., 2019). For example, USP13 is overexpressed in ovarian cancer, and its overexpression significantly predicts poor clinical outcomes (Han et al., 2016). USP13 expression is upregulated in glioblastoma (GBM) tissues and inversely correlated with patients' overall survival (Fang et al., 2017). However, the underexpression of USP13 is observed in breast cancer and bladder cancer (Zhang et al., 2013;Man et al., 2019). Our data and TCGA data consistently revealed the upregulated expression of USP13 in HCC tissues compared to non-tumor liver tissues in the current study. Notably, the positive expression of USP13 was associated with unfavorable clinical features, such as tumor size ≥ 5 cm and advanced TNM stage (III + IV), and indicated poor prognosis of HCC patients. Thus, USP13 overexpression in HCC tissues might be a potential indicator of poor clinical outcomes of patients. The expression of USP13 in HCC cells was significantly higher than that in a normal hepatic cell line. Tumor heterogeneity leads to differential expression of USP13 in different HCC cell lines.
A previous study reports that USP13 knockdown suppresses ovarian cancer cell proliferation in vitro and tumor formation in vivo (Han et al., 2016). Moreover, USP13 depletion represses the proliferation of glioma stem cells (GSCs) and restrains tumor growth in mice (Fang et al., 2017). In contrast, the loss of USP13 facilitates the proliferation, glycolysis, and anchorageindependent growth of breast cancer cells (Zhang et al., 2013). The bladder cell proliferation, migration, and invasion potentials are enhanced by USP13 knockdown (Man et al., 2019). The upregulated expression of USP13 in tumor tissues indicated that it might function as an oncogenic in HCC. As expected, we found that USP13 knockdown markedly inhibited the proliferation, EMT, migration, and invasion of HCC cells and significantly repressed tumor growth and lung metastasis of HCC in vivo. Besides, the ectopic expression of USP13 facilitated the proliferation, EMT, migration, and invasion of HepG2 and LO2 cells in vitro.
As a deubiquitinase, USP13 regulates protein abundance via enhancing deubiquitylation and stabilization of the substrate. Several proteins, such as MCL1 , PTEN (Zhang et al., 2013), MITF (Zhao et al., 2011), Myc (Fang et al., 2017), USP10 (Liu et al., 2011), and RAP80 , have been demonstrated to be regulated by USP13 for deubiquitylation and stabilization. However, it is still unknown whether USP13 deubiquitinates and thus stabilizes TLR4 in HCC. Here, we demonstrated that USP13 enhanced the stabilization of TLR4 via direct binding and deubiquitylation of TLR4. Accordingly, TLR4 was recognized as a novel substrate of USP13 in HCC. The structural features of TLR4 include an ectodomain consisting of multiple leucine-rich repeats and a cysteine-rich domain, a transmembrane region, and a highly conserved cytoplasmic Tollinterleukin 1 receptor (IL-1R) domain (TIR domain) (Chuang and Ulevitch, 2001). The putative ligand-binding occurs within the ectodomain, whereas the TIR domain provides a key site for interactions with intracellular proteins such as members of the MyD88 adaptor protein family (Aderem and Ulevitch, 2000;Takeda et al., 2003). An E3 ubiquitin-protein ligase Triad3A binds to the cytoplasmic tail (TIR domain) of TLR4 and targets TLR4 for ubiquitination and proteolytic degradation (Chuang and Ulevitch, 2004). In this study, a co-IP assay demonstrated the interaction between USP13 and TLR4 in HCC cells. Since USP13 is an intracellular protein, we suggest that USP13 binds to the TIR domain of TLR4 in HCC cells. TLR4 and its adaptor MyD88 have been reported as oncogenic signaling in several human cancers, including HCC (Apetoh et al., 2007;Rathore et al., 2019;Zhang et al., 2020). Recent studies have shown that the TLR4/MyD88 pathway regulates NF-κB signaling and VEGF, IL-23, and IL-17A expression in HCC (Kang et al., 2018;Ding et al., 2019;Zhang et al., 2020). The inhibition of the TLR4/MyD88 pathway represses the occurrence and progression of HCC (Ding et al., 2019;Zhang et al., 2020). NF-κB-mediated EMT process contributes to HCC progression, and the suppression of the NF-κB pathway represses EMT and invasion of tumor cells (Song et al., 2014;Zhu et al., 2016). Importantly, USP13 knockdown markedly resulted in the inactivation of the TLR4/MyD88/NF-κB pathway in HCC cells. TLR4 re-expression reversed the inhibitory effects of USP13 knockdown on the proliferation, migration, and invasion of HCC cells. Thus, USP13 contributed to HCC progression possibly by targeting the TLR4/MyD88/NF-κ B pathway.
Hypoxia, which resulted from rapid tumor growth and vascular abnormality, has been identified as a critical driver of HCC progression (Jing et al., 2019). Our previous studies have demonstrated that hypoxia contributes to the growth and metastasis of HCC via regulating protein-coding genes and non-coding RNAs, such TUFT1 (Dou et al., 2019b), VASP (Liu et al., 2018), miR-1296 (Xu et al., 2017a), and RUNX1-IT1 (Sun et al., 2020). Hypoxia has been recognized as an inducer of the activation of the TLR4/MyD88/NF-κB pathway in hepatic ischemia/reperfusion injury and liver fibrosis Du et al., 2019). Moreover, HIF-1α knockdown inactivates the TLR4/MyD88 pathway and abrogates hypoxiainduced proliferation, migration, and invasion of HCC cells . Our data showed that hypoxia increased the expression of USP13 mRNA, while it did not impact the level of TLR4 mRNA in Hep3B cells, which further support the post-transcriptional regulation of TLR4 by USP13. Besides, either hypoxia or CoCl 2 treatment upregulated the level of USP13 protein in HCC cells. Significantly, USP13 knockdown inhibited hypoxia-induced activation of the TLR4/MyD88/NF-κB pathway in HCC cells. These results provide new insight into the underlying mechanism involved in hypoxia-induced TLR4/MyD88/NF-κB pathway activation in HCC.
In summary, our findings elucidated that the upregulated expression of USP13 in HCC tissues conferred to poor clinical outcome. We provided evidence to support that USP13, induced by hypoxia, promoted HCC progression by maintaining the TLR4/MyD88/NF-κB pathway. These data might provide novel insights into the pathogenesis of HCC.

DATA AVAILABILITY STATEMENT
All datasets presented in this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Research Ethics Committee of The First Affiliated Hospital of Xi'an Jiaotong University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Xi'an Jiaotong University.

AUTHOR CONTRIBUTIONS
QX, DH, and KT conceived and designed the experiments. SG, TC, LL, XL, YL, and JZ performed the experiments. SG, TC, and LL analyzed the data. QL and ZZ contributed reagents, materials, and analysis tools. SG and KT wrote the manuscript. All authors read and approved the final manuscript.