Dysregulation and oncogenic activities of ubiquitin specific peptidase 2a in the pathogenesis of hepatocellular carcinoma

Abstract

Ubiquitin specific peptidase 2a (USP2a) plays critical roles in protein degradation and other cellular activities. Currently, our understanding on USP2a dysregulation in subjects with hepatocellular carcinoma (HCC) and its roles in HCC pathogenesis is limited. In this study, we found that USP2a mRNA and protein levels were significantly upregulated in HCC tumors from both human and mice. USP2a overexpression in HepG2 and Huh 7 cells significantly increased cell proliferation while inhibition of USP2a activity by chemical inhibitor or stable knockout of USP2 by CRISPR markedly reduced cell proliferation. In addition, USP2a overexpression significantly augmented the resistance while knockout of USP2a markedly increased the susceptibility of HepG2 cells to bile acid-induced apoptosis and necrosis. Consistent with the oncogenic activities detected in vitro, overexpression of USP2a promoted de novo HCC development in mice with significantly increased tumor occurrence rates, tumor sizes and liver/body ratios. Further investigations with unbiased co-immunoprecipitation (Co-IP)-coupled proteomic analysis and Western blot identified novel USP2a target proteins involved in cell proliferation, apoptosis, and tumorigenesis. Analysis of those USP2a target proteins revealed that USP2a's oncogenic activities are mediated through multiple pathways, including modulating protein folding and assembling through regulating protein chaperones/co-chaperones HSPA1A, DNAJA1 and TCP1, promoting DNA replication and transcription through regulating RUVBL1, PCNA and TARDBP, and altering mitochondrial apoptotic pathway through regulating VDAC2. Indeed, those newly identified USP2a target proteins were markedly dysregulated in HCC tumors. In summary, USP2a was upregulated in HCC subjects and acted as an oncogene in the pathogenesis of HCC through multiple downstream pathways. The findings provided molecular and pathogenesis bases for developing interventions to treat HCC by targeting USP2a or its downstream pathways.

Keywords: HCC; USP2a; apoptosis; cell proliferation; ubiquitination and deubiquitination.

Figure 1

USP2a expression was dysregulated in HCC subjects. (A) The expression levels of USP2a mRNA in human normal liver (n=12), HCC-NT (n=8) and HCC-T (n=21) were detected by real-time PCR. (B) USP2a protein levels in human normal liver (n=14), HCC-NT (n=8) and HCC-T (n=17) were detected and quantified by Western blot. The results from three representative tissue samples from each of the three groups (normal, HCC-NT and HCC-T) were presented in the upper panel. (C) The expression levels of USP2a mRNA in individual paired HCC-T and HCC-NT tissues (n=8). (D) The expression levels of Usp2a mRNA in mouse HCC-NT (n=26) and HCC-T tissues (n=26) collected from 12 female (F1 to F12) and 14 male (M1 to M14) mice were detected by real-time PCR as groups and (E) individual pairs. (F) Usp2a protein levels in mouse paired HCC-NT and HCC-T tissues (n=9) were detected and quantified by Western blot as groups and (G) individual pairs. The experiments with real-time were performed twice with two repeats for each sample while Western blots were performed once or twice depending on the quality of the results. *P < 0.05 with one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Figure 2

USP2a promoted cell proliferation. (A) HepG2 and (B) Huh 7 cells were transfected with USP2a or pcDNA vector as a control, followed by monitoring cell growth for a period of 48 h by the MTS assays. (C) HepG2 cells were transfected with USP2a or pcDNA vector in the presence or absence of USP2 inhibitor ML364 (2 µM) for 48 h, followed by detection of cell proliferation by MTS assays. (D) Relative proliferation of parental HepG2 wt and two independent USP2 knockout cell clones, USP2-KO (3A) and (E) USP2-KO (3E), over a period of 96 h. (F) USP2-KO (3A) and (G) USP2-KO (3E) cells were transfected with USP2a or pcDNA vector, followed by detection of the cell proliferation at 48 h post-transfection. All the experiments were performed at least twice with 4-6 repeats for each treatment or condition. *P < 0.05, **P < 0.01, ***P < 0.001 in students’ t-test for pair-wise comparison or one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.

Figure 3

USP2a exhibited anti-apoptotic and anti-necrotic activities. A. HepG2 wt and USP2-KO cells were treated with increasing concentrations of CDCA or vehicle DMSO for 4 h, followed by detection of apoptotic activities with the RealTime-Glo Annexin V Apoptosis assays. B. HepG2 wt and USP2-KO cells were treated with CDCA (500 µM) and apoptotic activities were detected at various time points over a period of 48 h. C. HepG2 wt and USP2-KO cells were treated with increasing concentrations of CDCA or vehicle DMSO for 8 h, followed by detection of necrotic activities with the RealTime-Glo Necrosis assays. D. HepG2 wt and USP2-KO cells were treated with CDCA (500 µM) and necrotic activities were detected at various time points over a period of 48 h. E. USP2-KO cells were transfected with USP2a or pcDNA vector, followed by treatment of the transfected cells with increasing concentrations of CDCA for 4 h. Apoptotic activities were then quantified. F. USP2-KO cells were transfected with USP2a or pcDNA vector, followed by treatment of the transfected cells with increasing concentrations of CDCA for 8 h. Necrotic activities were then quantified. All the experiments were performed at least twice with 4-6 repeats for each treatment or condition. *P < 0.05, **P < 0.01, ***P < 0.001 with students’ t-test for pair-wise comparison.

Figure 4

USP2a promoted HCC development in vivo in mice. (A) C57BL/6 mice at the age of 2-3 weeks were peritoneally injected with a single dose of DEN (50 mg/kg). At the age of 3-4 months, the mice were hydrodynamically injected with USP2a (n=7) or pcDNA vector (n=11) plasmid DNA at a dose of 0.5 µg/g via tail vein. The mice were fed with regular chow diet during the entire study. The mice were euthanized at the age of 13-14 months. The liver tissues of mice treated with USP2a or pcDNA were presented. (B) The percentages of mice developed visible tumors in mice treated with USP2a or pcDNA. (C) Total visible tumor numbers and (D) volumes of individual mice treated with USP2a or pcDNA. (E) Body weight, (F) liver weight and (G) liver/body weight ratios of mice treated with USP2a or pcDNA. This long-term mouse study was performed once. *P < 0.05 with students’ t-test for pair-wise comparison.

Figure 5

Identification of USP2a target proteins by Co-IP coupled MS/MS proteomic analysis and Co-IP coupled Western blot confirmation. (A) USP2-KO cells were transfected with USP2a or pcDNA vector, followed by Co-IP to pull down USP2a-associated protein complexes, which were subjected to MS/MS proteomic analysis to identify USP2a target proteins. Four replicates for each treatment were subjected to proteomic analysis. The pathway/cellular process enrichment analysis of the identified proteins was carried out with KEGG and ShinyGo. (B) The heat map analysis and (C) relative abundances of USP2a and its seven target proteins in the immunoprecipitated protein complexes detected by MS/MS and (D) confirmed by Western blot. The Co-IP assays were performed four times while the MS/MS proteomic detection was performed once with four repeats in each group. *P < 0.05, **P < 0.01, ***P < 0.001 with students’ t-test for pair-wise comparison.

Figure 6

Dysregulated expression of USP2a target proteins in HCC subjects. (A) The expression levels of USP2a target proteins in human normal liver (n=12), HCC-NT (n=12) and HCC-T (n=9 or 10) tissues were detected and quantified by Western blot, including VDAC2, (B) HSPA1A, (C) DNAJA1, (D) RUVBL1, (E) TARDBP, (F) TCP1 and (G) PCNA. The results from four representative tissue samples from each of the three groups (normal, HCC-NT and HCC-T) were presented in the left panel. The Western blots were performed once or twice dependent on the quality of the results. *P < 0.05, **P < 0.01, ***P < 0.001 in one-way ANOVA, followed by Tukey post-hoc test for multiple comparisons.