Comprehensive analysis of ubiquitin‐specific protease 1 reveals its importance in hepatocellular carcinoma

Abstract Objectives In this study, we comprehensively analysed the role of ubiquitin‐specific protease 1(USP1) in hepatocellular carcinoma (HCC) using data from a set of public databases. Materials and Methods We analysed the mRNA expression of USP1 in HCC and subgroups of HCC using Oncomine and UALCAN. Survival analysis of USP1 in HCC was conducted with the Kaplan‐Meier Plotter database. The mutations of USP1 in HCC were analysed using cBioPortal and the Catalogue of Somatic Mutations in Cancer database. Differential genes correlated with USP1 and WD repeat domain 48 (WDR48) were obtained using LinkedOmics. USP1 was knocked down with small interfering RNA (siRNA) or pharmacologically inhibited by ML‐323 in MHCC97H or SK‐Hep‐1 cell lines for function analysis. Results High USP1 expression predicted unfavourable overall survival in HCC patients. USP1 showed positive correlations with the abundances of macrophages and neutrophils. We identified 98 differential genes that were positively correlated with both USP1 and WDR48. These genes were mainly involved in the cell cycle, aldosterone synthesis and secretion and oestrogen signalling pathways. Interactions between USP1 and WDR 48 were confirmed using co‐immunoprecipitation. USP1 knockdown or ML‐323 treatment reduced the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E1. Conclusions Overall, USP1 is a promising target for HCC treatment with good prognostic value. USP1 and WDR48 function together in regulating cancer cell proliferation via the cell cycle.

Ubiquitination, a type of dynamic protein posttranslational modification, is critically involved in various physiological processes. 5 The dysregulation of ubiquitination leads to several disorders. In recent years, accumulating evidence has revealed the critical role of ubiquitination in tumorigenesis. 6 In cancer, the effects of ubiquitination are diverse, leading to the suppression or progression of tumorigenic pathways. Components of ubiquitination systems, including the proteasome, ubiquitin, E1/E2/E3 ligases and deubiquitinases, function differently according to their substrates. 7 Of these, deubiquitinases mediate substrate ubiquitination by removing ubiquitin moieties, thus preventing the degradation of substrate proteins. 8 In the human genome, more than 100 deubiquitinases are divided into ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases, ovarian tumour proteases, Machado-Joseph disease protein domain proteases and JAB1/MPN/MOV34 metalloenzymes. 9,10 If their substrates function as tumour suppressors, deubiquitinases prevent their degradation and function as tumour suppressors. However, if their substrates are promoters of tumour progression, deubiquitinases preserve their characteristics and promote tumour progression. 8,11 Therefore, targeting deubiquitinases has been introduced as a novel therapeutic approach for HCC; however, more data are needed to show the efficacy of this strategy. 7,12 USPs are cysteine-dependent proteases and constitute the largest subfamily of deubiquitinases, thus they have gained much interest. 11 Several high-quality reviews have summarized the critical roles of USPs in cancer. 10,11 USP1, a well-known deubiquitinase, is essential in cellular homoeostasis and the response to DNA damage. 13,14 As previously reported, USP1 is involved in diverse cellular functions. 15 USP1 and its cofactor USP1-associated factor 1, also called WD repeat domain 48 (WDR48), function as regulators in the processes of the DNA damage response, especially in the translation synthesis process and the Fanconi anaemia pathway. 13,16,17 In general, USP1 and WDR48 form a complex and function together, and WDR48 significantly enhances USP1 activity by stabilizing its expression and mediating its access to substrates. 16,18 Moreover, USP1 stabilizes inhibitors of DNA binding proteins, which are overexpressed in tumours. 19,20 USP1 is also involved in the cell cycle. The expression of USP1 is cell cycle dependent, and it reduces the degradation of phosphorylated checkpoint kinase 1 and maintains its activity. 21 In addition, USP1 is linked to treatment response in cancers. Sourisseau et al reported that USP1 was vital in cis-diamminedichloroplatinum (II) resistance in non-small-cell lung cancer, mainly due to the shortening of the USP1 mRNA 5'UTR. 14 Sonego et al demonstrated that USP1 in ovarian cancer cells was linked to the platinum response. 22 They found that USP1 mediated resistance to platinum by stabilizing Snail and then promoting tumour dissemination. 22 Overall, USP1 is a promising therapeutic target in cancers. However, the current knowledge about its role in HCC is limited. Thus, determining whether USP1 is pivotal in HCC is of great interest.
In this study, several informatics tools were used to evaluate the expression profile and the prognostic significance of USP1 in HCC.
We explored the correlation between USP1 expression and immune infiltration. Moreover, we also investigated the underlying mechanisms of USP1 in HCC by analysing the coexpressed genes of USP1 and its cofactor WDR48. The findings of this study may improve our understanding of USP1 in HCC.

| Expression analysis and survival analysis
We searched the Oncomine database (http://www.oncom ine.org) with the gene symbol 'USP1'. The primary filters were set as follows: Analysis type: Differential Analysis; Cancer vs Normal: Liver cancer vs Normal analysis, Hepatocellular Carcinoma vs Normal analysis. Datasets were screened with thresholds of P-value (1E-4), fold change (2) and gene rank (top 10%). Box plots of the expression data (log 2 median-centred intensity) obtained from datasets were generated using GraphPad software. Then, subgroup analysis of the mRNA expression of USP1 was conducted using the UALCAN database (http://ualcan.path.uab.edu). 23

| Protein-protein interaction (PPI) network analysis
We employed the LinkedOmics database (http://www.linke domics. org/login.php) to find differentially expressed genes correlated with USP1 and WDR 48. 32 RNA-seq data in the TCGA-LIHC dataset were selected for analysis (Subset: histological type-hepatocellular carcinoma, n = 371). The correlation coefficients of the differentially expressed genes and USP1 or WDR48 were analysed using Spearman tests. The PPI network was constructed using both the STRING database (http://strin g-db.org) (interaction score > 0.4) and Cytoscape software (version 3.7.1). 33,34

| Hub gene analysis
To identify the hub genes in the network, we first analysed the clusters of the network with several criteria (degree cut-off: 2; kcore: 2; node score cut-off: 0.2; and max depth: 100). Then, we calculated the node scores using the cytoHubba plug-in (version 0.1) and ranked the nodes based on degree. Finally, we enriched the hub genes by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in the Database for Annotation, Visualization, and Integrated Discovery (DAVID 6.8, v6.8, https://david.ncifc rf.gov/home.jsp), and the results were visualized with the bioinformatics online tool (http:// www.bioin forma tics.com.cn). Validation of the correlation between USP1 and WDR48 was conducted using the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cance r-pku.cn). 35

| Cell culture, transfection and reagents
The human HCC MHCC97H cell lines were purchased from

| High expression of USP1 in HCC
After data mining in the Oncomine database, we found that the mRNA expression of USP1 was elevated in various types of cancers (cancer vs normal), such as liver cancer, sarcoma and bladder cancer ( Figure 1A). Then, we further focused on its expression in HCC, which is the most prevalent primary liver cancer. Data from 4 datasets (Roessler liver 2, Roessler liver, Chen liver and Wurmbach liver) were selected ( Figure 1B). [36][37][38] We performed a meta-analysis of USP1 expression in the 4 studies with the following thresholds: p-value (1E-4), fold change (2) and gene rank (top 10%) ( Figure 1C). All of the results showed that USP1 was significantly upregulated in HCC tissues compared with normal tissues (P < .05). In the Roessler liver 2, Roessler liver, Chen liver here. USP1 was not detected in normal liver and showed weak to medium staining in HCC liver ( Figure 1D). To increase the reliability of the results, we further validated the significant overexpression of USP1 in LIHC data from TCGA using the UALCAN database. As shown in Figure 2A, the mRNA expression of USP1 was elevated in the LIHC samples (n = 371) compared with the normal samples (n = 50). Subgroup analysis showed that USP1 was also upregulated in different subgroups of HCC, including the subgroups of sex, age, race and weight ( Figure 2B-E). Regarding cancer stage and tumour grade, we found that USP1 was overexpressed in stages 1-3 and grades 1-4 ( Figure 2F,G). In addition, USP1 was overexpressed in HCC patients without regional lymph node metastasis but not in patients with metastasis ( Figure 2H).
USP1 showed a positive association with TP53 mutation status and was significantly overexpressed in HCC patients with TP53 mutations ( Figure 2I). We also evaluated the promoter methylation level of USP1 in LIHC; however, no significance was found between LIHC and normal samples ( Figure S1). Taken together, these results indicated that the high expression of USP1 was closely associated with tumour progression.

| The prognostic significance of USP1 in HCC patients
Thus, we postulated whether USP1 could function as a prognostic hall- consumed ( Figure 3E-G), but not those patients who were female, White race and alcohol consumed ( Figure 3I-K). For patients who were hepatitis virus infected or non-hepatitis virus infected, high USP1 expression predicted their poor survival ( Figure 3H,L). In conclusion, high USP1 expression was associated with poor prognosis of HCC patients.
This mutation frequency was relatively low, only 3 in 1000 samples.
Therefore, we failed to find a relationship between USP1 mutation and the prognosis of HCC patients ( Figure S2). Furthermore, the mutation types of USP1 were further evaluated in another database, COSMIC.
For clarity, two pie charts of the mutation types are shown in Figure 4.

| The association of USP1 expression and immune infiltration in HCC
We investigated the association of USP1 expression and immune infiltration using the TIMER database. The correlation coefficients between  Table 1). Taken together, these results suggest that USP1 is critically involved in immune infiltration during the progression of HCC.

| High WDR48 expression correlated with USP1 and predicted unfavourable prognosis in HCC patients
To reveal the role of WDR48, the cofactor of USP1, in HCC, we evaluated its expression and prognostic significance. Using the UALCAN database, we found that WDR48 was also significantly  Figure 6B). Intriguingly, we found that high WDR48 expression was also associated with poor OS and RFS in HCC patients ( Figure 6C,D).

| Differential genes correlated with both USP1 and WDR48 in HCC
The LinkedOmics database was used to identify differentially expressed genes that were correlated with USP1 and WDR48 in HCC.

TA B L E 1 Correlations between USP1 and immune cells' gene markers in HCC
Based on the Spearman test, the differentially expressed genes correlated with USP1 and WDR48 were identified ( Figure 7A,D). The top 50 positively (r > 0) and top 50 negatively (r < 0) correlated genes are shown in heat maps ( Figure 7B,C,E,F). Based on the Spearman test, we selected the positively correlated genes with coefficient > 0.4.
Finally, 1175 genes positively correlated with USP1 and 199 genes positively correlated with WDR48 were selected. Among these, 98 genes showed positive correlations with both USP1 and WDR48, and these genes were selected for further analysis ( Figure 8A).
The 98 differentially expressed genes were input into STRING and Cytoscape to construct a PPI network ( Figure 8B) and were used for GO and KEGG enrichment analysis using DAVID. The following biological processes were significantly affected: transcription, regulation of transcription, covalent chromatin modification, etc ( Figure 8C). The cellular component terms were mainly enriched in the nucleoplasm, nucleus, centrosome, etc ( Figure 8D). The molecular function terms were mainly enriched in DNA binding, chromatin binding, protein binding, etc ( Figure 8E). The KEGG results showed that the coexpressed genes were mainly involved in the cell cycle, aldosterone synthesis and secretion and oestrogen signalling pathways ( Figure 8F).

| Identification of hub genes and their prognostic value in HCC
First, the most important clusters in the PPI network were identified using MCODE ( Figure 8B, shown in yellow). The top ten hub genes of the network were identified using cytoHubba (ranked by degree) (Figure 9A). GO analysis results showed that biological processes, such as chromatin remodelling, covalent chromatin modification and chromatin binding, were significantly affected and enriched ( Figure 9B). Then, the prognostic value of the hub genes was evaluated in Kaplan-Meier Plotter. Among these ten genes, the high expression of seven genes was significantly related to poor OS (BPTF, SETD2, SMARCC1, UBXN7, SMC3, PBRM1 and SF3B1) ( Figure 9C), while the other three genes showed no significance (ATRX, SIN3A and USP34) ( Figure S3).

| Validation of the interaction between USP1 and WDR48 in HCC cell lines
First, we detected the expression of USP1 and WDR48 using Western blotting. We found that protein levels of USP1 and WDR48 were highly expressed in HCC cell lines, both in MHCC97H and in SK-Hep-1 ( Figure 10A). Then, we knocked down USP1 in these cell lines using siRNA targeting USP1. After siRNA-USP1 transfection, USP1 was significantly down-regulated compared with the NC-siRNA transfected cells, both at protein and mRNA levels ( Figure 10B,C,E,F).
In addition, we confirmed the interaction between USP1 and F I G U R E 6 WDR48 is overexpressed in HCC and predicts poor prognosis. A, WDR48 mRNA express is overexpressed in HCC (UALCAN). B, WDR48 is positively correlated with USP1 in HCC (the Gene Expression Profiling Interactive Analysis database, GEPIA). C-D, High WDR48 expression predicts poor OS and RFS in HCC. HR: hazard ratio WDR48 in HCC cell lines using co-immunoprecipitation, and the results showed that USP1 interacted with WDR48 in MHCC97H and SK-Hep-1 cells ( Figure 10H).

| SiRNA-USP1 transfection or ML-323 treatment decreased the proliferation of HCC cells
As mentioned above, the Figure 8F

| D ISCUSS I ON
Currently, HCC remains a worldwide health problem with poor prognosis and high death rates. Late diagnosis, metastasis and quick progression are the main causes of cancer-induced death in patients with HCC. 46 If the patients are diagnosed at an early stage and given effective therapies, their survival may significantly improve. 47 Thus, on the one hand, identifying hallmarks for the early diagnosis and tumour progression of HCC is urgently needed. 48 On the other hand, it is crucial to find novel therapeutic targets and develop new therapeutic strategies. 49 In recent years, with the development of sequencing and omics, we can further understand the underlying mechanisms of HCC. 50 As key regulators of ubiquitination, deubiquitinating family enzymes play important roles in tumour diseases. 51,52 In recent years, our understanding of deubiquitinases has made great progress, espe- Third, what is the underlying mechanism of USP1 in HCC? For these reasons, we systemically analysed the role of USP1 in HCC using a set of informatics tools. We believe our findings in this study could at least partially explain the abovementioned questions.
In this study, we confirmed the higher expression of USP1 in HCC than in normal tissues. High expression of USP1 showed clinical significance and was associated with unfavourable survival in HCC patients. These results suggest that USP1 is a potential therapeutic target in HCC. However, the promoter methylation level of USP1 did not significantly change between HCC and normal tissues, which meant that the alteration of USP1 expression was not due to this type of posttranslational modification. In present, the mechanisms underlying USP1 overexpression in human cancer is still not fully understood. In general, USP1 is phosphorylated by CDK1 at S313, and then binding with WDR48 for activation. 60 As previously reported, USP1 could be degraded by APC/C Cdh1 during G1 phase. Moreover, calpain inhibits Cdh1, and thus inhibits USP1 degradation. 44  in addition to the expressional alteration of USP1, we should also pay attention to the alteration of its activity. Nevertheless, we can conclude that USP1 is a promising therapeutic target in the treatment of HCC.

| CON CLUS IONS
In this study, we found that USP1 was highly expressed in HCC and

ACK N OWLED G EM ENTS
We thank all the contributors of the public databases used in this study for providing the data for analysis.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N
LJ Li conceived and designed this study. YL Zhao and C Xue searched the databases and analysed the data. YL Zhao drafted the manuscript. C Xue prepared the figures and tables. ZY Xie and XX Ouyang reviewed and revised the manuscript. All authors read and approved the final manuscript for publication.

E TH I C A L A PPROVA L
The study was conducted based on data in the public databases, there is no ethical statement to be declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
All of the data involved in this study are available in the public databases which are listed in the Materials and methods section.