
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00231-6
10.1016/j.tranon.2024.102104
102104
Original Research
The role of deubiquitinase USP2 in driving bladder cancer progression by stabilizing EZH2 to epigenetically silence SOX1 expression
Xu Fanghua a
Xu Xiangda b
Deng Huanhuan b
Yu Zhaojun b
Huang Jianbiao b
Deng Leihong dengleihongvip@163.com
c#⁎
Chao Haichao chaohaichaovip@sina.com
b#⁎
a Department of Pathology, Ping Xiang People's Hospital, Pingxiang Economic and Technological Development District, Ping Xiang, Jiangxi 337000, China
b Department of Urology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi 330006, China
c Department of Ultrasonic medicine, The First Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, Jiangxi 330006, China
⁎ Corresponding authors. dengleihongvip@163.comchaohaichaovip@sina.com
# Leihong Deng and Haichao Chao contributed equally to this work and should be considered as co-corresponding authors.

27 8 2024
11 2024
27 8 2024
49 10210424 2 2023
26 7 2024
15 8 2024
© 2024 Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• Aberrantly highly expressed USP2 in bladder cancer tissues and cells negatively correlates with the survival of bladder cancer patients.

• USP2 silencing weakens the capacities of cell growth, migration and invasion in vitro.

• USP2 interacts with and stabilizes EZH2 by inhibiting its ubiquitination.

• USP2-mediated EZH2 stabilization promotes epigenetic suppression on SOX1.

• USP2-EZH2-SOX1 axis enhances bladder cancer tumor growth in vivo.

Background

The Ubiquitin-proteasome system (UPS) is known to participate in multiple cellular events. The deubiquitinating enzyme USP2 (ubiquitin-specific protease 2) is involved in the vasculature remodeling process associated with bladder cancer (BLCA). However, the role of USP2 in BLCA progression has not been clearly defined and whether its regulatory mechanism involving EZH2 (Enhancer of Zeste Homolog 2) remains elusive yet.

Methods

Differential expression patterns of USP2 and EZH2 were examined in 46 pairs of BLCA and adjacent normal tissues. USP2 knockdown plasmids were transfected into 5637 and J82 cells to detect its impact on cell proliferation, migration and invasion using CCK-8, EdU, wound healing and transwell assays. The USP2-EZH2-SOX1 cascade was confirmed through Co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays. An in vivo verification was conducted using a xenograft model of nude mice.

Results

USP2 was significantly upregulated in BLCA tissues and cells, which was associated with poor clinical prognosis in BLCA patients. USP2 depletion resulted in decreased cell proliferation, migration and invasion in BLCA cells. USP2 stabilized the EZH2 protein by directly binding to it, thereby reducing its ubiquitination. Ectopic introduction of EZH2 restored cell growth and invasion of BLCA cells, which had been inhibited by USP2 silencing. USP2-mediated stabilization of EZH2 promoted the enrichment of histone H3K27me3 and repression of SOX1. Involvement of the USP2-EZH2-SOX1 axis in tumor formation was ultimately verified in vivo.

Conclusion

Our findings reveal that a USP2-EZH2-SOX1 axis orchestrates the interplay between dysregulated USP2 and EZH2-mediated gene epigenetic silencing in BLCA progression.

Graphical abstract

Image, graphical abstract

Keywords

Bladder cancer
USP2
EZH2
SOX1
Stabilization
Abbreviations

EdU 5-Ethynyl-2′-deoxyuridine

BLCA Bladder cancer

ChIP Chromatin Immunoprecipitation

Co-IP Co-immunoprecipitation

CHX Cycloheximide

EZH2 Enhancer of Zeste Homolog 2

H3K27me3 histone H3 trimethylation at lysine 27

IHC Immunohistochemistry

SOX SRY-related high mobility group box

USP Ubiquitin specific protease

USP2 Ubiquitin specific protease 2
==== Body
pmcIntroduction

Bladder cancer (BLCA) is one of the most common malignancies affecting the urinary system, primarily occurring on the bladder mucosa. It is associated with various factors, such as environmental and genetic factors, as well as smoking [1]. In recent years, there has been a gradual increase in the incidence and mortality rates of BLCA [2]. Currently, surgery and chemotherapy are the main clinical treatment options for patients with BLCA [3]. However, the 5-year overall survival rate of BLCA patients is only 50–60 % [4]. Meanwhile, chemotherapy often leads to significant side effects, further exacerbating the discomfort experienced by patients during treatment [5]. Therefore, exploring the signaling molecular mechanism of the occurrence and development of BLCA is of great importance for enhancing the survival and prognosis of patients.

Ubiquitin-specific protease 2 (USP2), a well-known deubiquitinase, can remove ubiquitin to stabilize a range of protein substrates [6]. Previous studies have confirmed that USP2 functions as an oncogene, influencing tumor cell proliferation, migration and invasion in various cancers. For example, inhibition of USP2 has been shown to accelerate cyclin D1 degradation and induce cell cycle arrest in colorectal cancer and mantle cell lymphoma [7]. Moreover, USP2 has been found to promote cell migration and invasion in triple negative breast cancer cells [8]. However, limited research has investigated the potential involvement of USP2 in the progression of BLCA. A recent report revealed that in BLCA, tight junction protein 1 can recruit USP2 to facilitate the deubiquitination of TWIST1, inhibiting its degradation. This, in turn, promotes the transcriptional activation of CCL2 and affects the process of vasculature remodeling [9]. These findings suggest that USP2 may also act as an oncogene in BLCA, emphasizing the necessity for further exploring its role in BLCA.

Enhancer of Zeste Homolog 2 (EZH2), a catalytic subunit of the polycomb repressive complex 2, plays a role in gene silencing by regulating the trimethylation of histone H3 at lysine 27 (H3K27me3) modification [10]. Furthermore, overexpressed EZH2 has been observed in various types of malignant tumors, including breast cancer, colon cancer and ovarian cancer [[11], [12], [13]]. It is known that several USPs mediate the deubiquitination of EZH2. For example, USP21 has been shown to promote cell proliferation and metastasis in BLCA by suppressing EZH2 ubiquitination [14]. USP7 can enhance the stability of EZH2 by affecting its deubiquitination modification and regulate the progression of prostate cancer [15]. USP1 has been shown to mediate the stabilization of EZH2, and the downregulation of USP1/EZH2 has been found to suppress glioma cell proliferation [16]. Notably, EZH2 has also been implicated in the development of BLCA [17]. For instance, lncRNA GAS5 can promote BLCA cell apoptosis by suppressing EZH2 [18]. LncRNA SPRY4-IT1 was found to induce BLCA cell proliferation and metastasis by upregulating EZH2 [19]. In our previous work, we demonstrated a positive correlation between the expression levels of USP2 and EZH2 in BLCA tissues. Therefore, we predicted that EZH2 might be involved in the USP2-mediated BLCA progression.

In the present study, we mainly investigated and demonstrated the oncogenic role of USP2 in BLCA tumorigenesis. We observed aberrant overexpression of USP2 in both BLCA tissues and cell lines. Functional experiments further revealed that depletion of USP2 suppressed BLCA cell proliferation, migratory and invasive abilities. Additionally, mechanistic investigations unveiled an USP2-EZH2-SOX1 cascade involved in BLCA tumorigenesis, potentially offering novel therapeutic targets for BLCA treatment.

Materials and methods

Patient specimens

Forty-six BLCA patients (24 males and 22 females, aged 45–70 years) who underwent surgical treatment at The Second Affiliated Hospital of Nanchang University between March 2020 and May 2021 were selected. Paired cancer tissues and matched normal adjacent tissues were resected intraoperatively. The normal adjacent tissues referred to the tissues that were 2 cm away from the lesion. Samples were snap frozen and stored at - 80 °C for further analysis. The present study was approved by the Ethics Committee of The Second Affiliated Hospital of Nanchang University (2023R015-KS02). All patients signed informed consent. Table 1, Table 2 present the correlation between the expression levels of USP2 and EZH2 and clinicopathological parameters in BLCA. Clinical data of BLCA patients were acquired from the TCGA database (https://portal.gdc.cancer.gov/).Table 1 The association between USP2 expression and clinicopathological parameters in bladder cancer patients.

Table 1Characteristics	Number	USP2	P value	
High (n = 27)	Low (n = 19)	
Age				0.216	
≤50	21	12	9		
≥50	25	15	10		
Gender				0.116	
Male	24	14	10		
Female	22	13	9		
Grade				0.011a	
Well-moderate	22	10	12		
Poor	24	17	7		
Lymphaic metastasis				0.010 a	
Yes	23	16	7		
No	23	11	12		
Distant metastasis				0.018 a	
Yes	23	18	5		
No	23	9	14		
TNM stage				0.004 a	
I-II	22	10	12		
III-IV	24	17	7		
TNM, Tumor Node Metastasis. aP < 0.05 was considered to indicate a statistical significant difference.

Table 2 The association between EZH2 expression and clinicopathological parameters in bladder cancer patients.

Table 2Characteristics	Number	EZH2	P value	
High (n = 30)	Low (n = 16)	
Age				0.326	
≤50	21	15	6		
≥50	25	15	10		
Gender				0.216	
Male	24	12	8		
Female	22	18	8		
Grade				0.019a	
Well-moderate	22	13	9		
Poor	24	17	7		
Lymphaic metastasis				0.010 a	
Yes	24	20	4		
No	22	10	12		
Distant metastasis				0.026 a	
Yes	23	19	4		
No	23	11	12		
TNM stage				0.014 a	
I-II	19	13	6		
III-IV	27	17	10		
TNM, Tumor Node Metastasis. aP < 0.05 was considered to indicate a statistical significant difference.

Cell culture

Normal human urinary tract epithelial cell line SV-HUC-1 and BLCA cell lines (T24, 5637, UM-UC-3, J82, SW780) were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultivated in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1 % penicillin-streptomycin mixture (Gibco). The cultures were maintained in a 37 °C incubator with 5 % CO2. Cells were passaged when reaching 80 % confluency, and cells in the logarithmic growth phase were selected for experiments. For cell treatment, the transfected cells were treated with 20 μM MG132 (Sigma, St. Louis, MO, USA) for 6 h or exposed to 50 ng/ml Cycloheximide (CHX, Sigma) for 3, 6, or 9 h.

Cell transfection

The short hairpin RNA vectors targeting USP2 (sh-USP2#1, sh-USP2#2), the small interfering RNA (siRNA) against EZH2, or the corresponding negative controls were obtained from GenePharma (Shanghai, China). The complementary DNA (cDNA) of EZH2 was cloned into the pcDNA3.1 vector (Invitrogen). The full length USP2 cDNA was inserted into the pcDNA™6/myc-His A, B, and C to overexpress USP2 (His-USP2) in BLCA cells. Transfection was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocols. After 48 h, cells were collected for subsequent experiments.

Cell counting Kit-8 (CCK-8) assay

Cells (2 × 103 cells/well) were seeded onto 96-well plates and incubated at 37 °C. Then, 10 μL of CCK-8 solution (Sigma) was added to each well at 0, 24, 48, and 72 h. The plates were incubated for an additional 2 h. Cell proliferation capacity was assayed by recording the absorbance at 450 nm with a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

EdU assay

Cells were seeded on glass coverslips in 24-well plates (5 × 104 cells/well). Cell proliferation was evaluated using a 5-Ethynyl-2′-deoxyuridine (EdU) Kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. Briefly, cells were cultured in 50 μmol/L EdU medium for 2 h at 37 °C, then fixed with 4 % paraformaldehyde and stained with Apollo fluorochrome and hoechst. After mounting on glass slides, images were collected using a fluorescence microscope (Olympus, Tokyo, Japan).

Wound healing assay

Approximately 5 × 105 cells were seeded in 6-well plates. Once the cells reached 90 % confluence, a 200 μl pipette tip was used to create a linear wound. Images of five optical fields were randomly captured after 24 h incubation in serum-free medium using a microscope (Olympus). Wound closure was calculated with ImageJ software (National Institutes of Health, NIH, NY, USA).

Cell invasion assay

Cells (1 × 105) in serum-free medium were seeded in the upper chamber of transwell pre-covered with Matrigel (BD Biosciences, San Jose, CA, USA). The lower chamber was filled with medium plus 10 % FBS. After culturing for 24 h, a cotton swab was used to wipe cells on the upper membrane. Afterward, 4 % paraformaldehyde was utilized to fix cells on the lower membrane, and cells were then stained with 0.1 % crystal violet. The number of invaded cells was recorded in five random fields under a light microscope (Olympus).

Real-time quantitative PCR (RT-qPCR)

Total RNA from tissue samples or cells was extracted with TRIzol reagent (Invitrogen). The extracted RNA was reverse transcribed into cDNA using the PrimeScript RT reagent kit (Takara, Dalian, China). RT-qPCR was performed using the SYBR Green PCR kit (Toyobo, Osaka, Japan) on an ABI 7300 Sequence Detection System (Nanjing, Jiangsu, China). The designed primers are listed as follows: USP2 F: 5′-CCGCGCTTTGTTGGCTATAA-3′, R: 5′-CCCGATCCTACTGTCTTCCC-3′; EZH2 F: 5′-TTCATGCAACACCCAACACTT-3′, R: 5′-GGTGGGGTCTTTATCCGCTC-3′; SOX1 F: 5′-AGACCTAGATGCCAACAATTGG-3′, R: 5′-GCACCACTACGACTTAGTCCG-3′. The results were analyzed using the 2−∆∆Ct method and normalized against GAPDH.

Western blot analysis

Cells and tissues were lysed by adding RIPA lysis buffer (Sigma) to extract total protein. Protein concentration was determined by the BCA method (Sigma), and then 30 μg of protein per lane was used for separating on 10 % SDS-PAGE gels. After electrophoresis, the protein was wet transferred to a PVDF membrane (Millipore, Boston, MA, USA). Subsequently, the membranes were blocked with 5 % nonfat milk for 2 h. Membranes were then exposed to primary antibodies overnight at 4 °C: USP2 (15,404–1-AP, 1:500, Proteintech, USA), EZH2 (ab227648, 1:1000, Abcam), SOX1 (ab109290, 1:1000, Abcam), H3K27me3 (#720,069, 1:1000, Thermo Fisher Scientific, Waltham, MA, USA). After washing with TBST, the membranes were incubated with HRP-labeled secondary antibody (#7074, 1:1000, Cell Signaling Technology, Danvers, MA, USA) for 1 h. Subsequently, the membranes were washed with TBST again, and an ECL detection kit (Millipore) was utilized for developing in the dark. The gray value of protein bands was analyzed by ImageJ software (NIH). Tubulin was used as the control protein.

Co-immunoprecipitation (Co-IP)

Cells were lysed with RIPA buffer, and then centrifuged at 12,000 rpm at 4 °C for 20 min. Cell lysates were incubated with anti-IgG, anti-USP2 or anti-EZH2 antibodies overnight at 4 °C. Samples were then precipitated with Protein A G-1 agarose beads (Roche, Mannheim, Germany) for 30 min. The eluted proteins were subjected to western blot analysis using anti-EZH2 (ab227648, Abcam) and anti-USP2 (15,404–1-AP, Proteintech) antibodies. For cells transfected with His-USP2 or HA-EZH2, the cell lysates were incubated with anti-His antibody overnight at 4 °C, followed by the addition of Protein A G-1 agarose beads slurry. After washing, the samples were subjected to western blot analysis to detect potential interacting proteins.

Chromatin immunoprecipitation (ChIP) assay

The ChIP assay was performed with the EZ-ChIP kit (Millipore). Transfected cells were crosslinked with formaldehyde. The cross-linked chromatin was immunoprecipitated using anti-EZH2 (ab191250, Abcam), anti-H3K27me3 (ab192985, Abcam) or IgG (#2729, Cell Signaling Technology; used as a negative control) antibodies after sonication. Thereafter, the chromatin was incubated with protein A/G-Sepharose beads (Roche). Finally, the precipitated chromatin DNA was analyzed by PCR using SOX1 binding sites with its specific primers (F: 5′-GAGATTCATCTCAGGATTGAGATTCTA-3′, R: 5′-GGCCTACTGTAATCTTTTCTCCACT-3′).

Xenograft model

5637 cells (6 × 106) stably transfected with sh-NC+vector, sh-USP2+vector, or sh-USP2+EZH2 were subcutaneously injected into the athymic BALB/c nude mice (4–6 weeks old, 14–16 g, n = 5 for each group), which were purchased from Beijing Laboratory Animal Research Center (Beijing, China). The tumor size was monitored every 5 days. After five weeks, the mice were sacrificed, and the tumor weight was measured. Tumor tissues were collected for IHC assay. All mouse experiments were approved by the Animal Care Committee of The Second Affiliated Hospital of Nanchang University (2023R015-KS02).

Immunohistochemistry (IHC)

Tumor sections (4 μm) were deparaffinized and rehydrated using graded alcohol. Antigen retrieval was conducted in citrate buffer (pH 6.0) at 120 °C for 5 min. The tissue sections were then exposed to SOX1 antibody (ab109290, Abcam) overnight at 4 °C, followed by IgG H&L (HRP) secondary antibody incubation for 1 h at room temperature. Staining was performed in diaminobenzidine (DAB, R&D Systems, Minneapolis, MN, USA)). Images were observed using an Olympus microscope and analyzed using Image-Pro Plus software (Media Cybernetics, Carlsbad, CA, USA).

The tissue microarrays consisting of 120 cases of tumor tissues and 25 cases of normal tissues collected from The Second Affiliated Hospital of Nanchang University were analyzed by IHC. The tissues were stained with USP2 (ab195289, 1:500, Abcam) and EZH2 (ab227648, 1:100, Abcam) antibodies, respectively. Semiquantitative analysis of USP2 and EZH2 protein levels was conducted using ImageScope software (Aperio Technologies).

Statistical analysis

The results are expressed as mean ± standard deviation (SD) from three independent experiments. SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) was applied for all analyses. Student's t-test was utilized to compare two groups. Statistical differences among multiple groups were processed using one-way ANOVA. Spearman correlation analysis was conducted to estimate the relationship between USP2 and EZH2. The comparison between survival curves of BLCA patients were estimated and analyzed using the Kaplan-Meier method with the log-rank test. Statistics with P-value < 0.05 were deemed statistically significant.

Results

Abnormally elevated expression of USP2 in BLCA tissues and cell lines

To investigate the precise role of USP2 in BLCA, we initially examined its differential expression patterns in 46 paired BLCA specimens. As illustrated in Fig. 1A, we discovered that USP2 expression in BLCA tissues was significantly elevated, compared with adjacent normal tissues, as detected by RT-qPCR. This observation was consistent with the abnormal increase in USP2 protein levels, as confirmed through Western blot analysis of randomly selected four BLCA tissue samples (Fig. 1B). Besides, RT-qPCR analysis further clarified that a higher expression level of USP2 was detected in 27 out of the total 46 tumor tissues (Fig. 1C). Accordingly, Kaplan-Meier analysis demonstrated that these 27 cases of BLCA patients with elevated USP2 levels exhibited a reduced overall survival rate (Fig. 1D). These findings lend support to the notion that USP2 could serve as a potential biomarker for BLCA.Fig. 1 Abnormally elevated expression of USP2 in BLCA tissues and cell lines. (A) RT-qPCR analysis of USP2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) The protein level of USP2 was examined in 4 BLCA patients. (C) RT-qPCR was used to test USP2 expression in BLCA tissues. ΔCTN represented comparative Ct in normal tissues. ΔCTC was comparative Ct in tumor tissues. (D) Kaplan-Meier survival analysis of the relationship between USP2 level and overall survival of BLCA patients. (E, F) RT-qPCR and western blot analysis of USP2 level in BLCA cell lines and normal bladder epithelial cells SV-HUC-1. Values are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 1

In addition, at the cellular level, both mRNA and protein levels of USP2 were also expressed at a generally high level in several BLCA cell lines (especially in 5637 and J82 cells), when compared with normal bladder epithelial cells SV-HUC-1 (Fig. 1, Fig. 1). Thus, we speculated that USP2 may play a functional role in BLCA cell processes.

Interestingly, the TCGA database revealed that the expression level of USP2 was generally low in most tumors. Specifically, when compared with normal controls (n = 19), the expression of USP2 in BLCA tumor tissues was decreased (n = 408) (Figure S1A and S1B). Additionally, there was no statistical difference between the expression level of USP2 and the survival rate of BLCA patients (Figure S1C). Given this inconsistency, further validation is urgently required to determine the precise role of USP2 in BLCA progression.

Depletion of USP2 weakened cell proliferation, migration and invasion of BLCA cells

In order to explore the effect of USP2 on the biological functions of BLCA cells, we knocked down USP2 level using two specific shRNA duplexes. As expected, USP2 was extremely decreased in 5637 and J82 cells transfected with sh-USP2#1 or sh-USP2#2, compared to negative control (Fig. 2A and 2B), as determined by using RT-qPCR and western blot analysis. Data from CCK-8 assay showed that compared with sh-NC group, the proliferative capacity of BLCA cells was remarkably impaired after USP2 silencing (Fig. 2C). Similarly, the number of EdU-positive cells was notably reduced in USP2-depleted group (Fig. 2D). Moreover, BLCA cells with USP2 knockdown exhibited impaired migration and invasion abilities, when compared with their control cells, as assessed by wound healing and transwell assays (Fig. 2E-2F). In summary, there experimental data suggest that USP2 acts as an oncogenic driver, promoting BLCA cell proliferation, migration and invasion in vitro.Fig. 2 Depletion of USP2 weakened cell proliferation, migration and invasion of BLCA cells. (A, B) USP2 expression was extremely decreased in 5637 and J82 cells transfected with sh-USP2#1 or sh-USP2#2. (C) CCK-8 assay displayed cell growth curves in 5637 and J82 cells. (D) Representative images and quantification of EdU assay were performed in BLCA cells. (E) The migration of BLCA cells was assessed using the wound healing assay. (F) Transwell assay was used to test the capacity of cell invasion in BLCA cells. Results expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 2

EZH2 was increased in BLCA and correlated with USP2 level

Subsequently, we aimed to uncover the potential downstream mechanism of USP2 in BLCA development. As previously described, EZH2 is known to undergo proteasome-mediated ubiquitination degradation, and several E3 ligases have been identified as crucial regulators in mediating EZH2 degradation. Based on this premise, we proposed that there might be an intrinsic connection between USP2 and EZH2 in BLCA tumorigenesis. Therefore, we next measured the exact expression level of EZH2 in BLCA tissues and the results demonstrated that EZH2 was generally expressed at higher levels in BLCA tissues compared to their matched adjacent normal tissues (Fig. 3A). Specifically, EZH2 levels were elevated in 56.52 % (30 out of 46) of BLCA tissues (Fig. 3B). Additionally, Kaplan-Meier analysis revealed that BLCA patients with relatively high expression of EZH2 (30 cases) suffered from reduced overall survival (Fig. 3C). Notably, Spearman analysis confirmed a positive correlation between EZH2 and USP2 expression in BLCA tissues (Fig. 3D). Furthermore, we observed a generally high level of EZH2 expression in BLCA cells (Fig. 3E). Interestingly, USP2 depletion did not significantly change the EZH2 mRNA level (Fig. 3F). After USP2 silencing, EZH2 protein level and its related H3K27me3 expression were remarkably reduced (Fig. 3G). These results suggest a positive correlation between USP2 and EZH2 in BLCA.Fig. 3 EZH2 was increased in BLCA and correlated with USP2 level. (A) RT-qPCR analysis of EZH2 level in tumor tissues and adjacent normal tissues of 46 BLCA patients. (B) RT-qPCR was used to test EZH2 expression in BLCA tissues. (C) Kaplan-Meier survival analysis of the relationship between EZH2 level and overall survival of BLCA patients. (D) The correlation between USP2 and EZH2 levels in BLCA tissues was assessed using Spearman analysis. (E) RT-qPCR analysis of EZH2 level in BLCA cell lines and normal bladder epithelial cells. (F) RT-qPCR detection of EZH2 expression in 5637 and J82 cells transfected with sh-USP2. (G) Western blot analysis of EZH2 and H3K27me3 levels in 5637 and J82 cells transfected with sh-USP2. Data are represented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3

USP2 interacted with and stabilized EZH2 by inhibiting its ubiquitination and degradation in BLCA cells

We further explored the specific molecular mechanism of USP2 regulation and its interaction with EZH2 in BLCA cells. Co-IP assay demonstrated that endogenous USP2 and EZH2 interacted with each other in BLCA cells (Fig. 4A). Additionally, USP2 was co-immunoprecipitated with EZH2 in BLCA cells ectopically expressing both proteins (Fig. 4B). Notably, USP2 overexpression significantly inhibited EZH2 degradation in BLCA cells (Fig. 4C). Western blot assay further confirmed that the protein level of EZH2 was restored in USP2-silenced cells after treatment with proteasome inhibitor MG132 (20 μM) (Fig. 4D). Western blot assay next showed that overexpression of USP2 significantly inhibited the ubiquitination level of EZH2, while USP2 silencing promoted the ubiquitination modification of EZH2 (Fig. 4E). Therefore, these findings suggest that USP2 stabilizes EZH2 by suppressing its ubiquitination and degradation.Fig. 4 USP2 stabilized EZH2 by inhibiting its ubiquitination and degradation in BLCA cells. (A) 5637 and J82 cell lysates were immunoprecipitated using antibodies against USP2 or EZH2, followed by immunoblotting with the indicated antibodies. (B) BLCA cells were transfected with His-USP2 and HA-EZH2. Cell lysates were immunoprecipitated using anti-His magnetic beads, and immunoblotted with His and HA antibodies. (C) Cells overexpressing USP2 were treated with 50 ng/ml Cycloheximide (CHX) for 3, 6, and 9 h. Western blot was used to detect EZH2 expression. (D) Western blot was used to detect the expressions of USP2 and EZH2 in cells transfected with sh-NC or sh-USP2 with/without proteasomal inhibitor MG132 (20 μM) for 5 h. (E) EZH2 in BLCA cells transfected with His-USP2 or sh-USP2 was immunoprecipitated to detect ubiquitylation. Data are represented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Fig 4

Reinforced EZH2 abolished USP2 silencing-mediated biological functions on BLCA cells

Furthermore, we investigated whether EZH2, as a downstream signaling molecule of USP2, participated in the regulation of biological functions of BLCA cells. 5637 cells transfected with USP2 silencing plasmids were treated with or without ectopic reinforced EZH2. Compared with the control group, USP2 downregulation significantly suppressed cell proliferative activity, while EZH2 upregulation promoted cell proliferation in sh-USP2 cells (Fig. 5A and 5B). In addition, the number of invaded cells in the sh-USP2 group was greatly reduced compared to the control group. However, upon overexpression of EZH2, the invasive ability in cells with sh-USP2 was obviously induced (Fig. 5C). Western blot analysis showed that USP2 knockdown decreased the protein levels of USP2 and EZH2, and EZH2 upregulation further increased EZH2 expression (Fig. 5D). Taken together, USP2 silencing inhibited BLCA cell proliferation and invasion through EZH2.Fig. 5 Overexpressed EZH2 abolished USP2 silencing-mediated biological functions on BLCA cells. We knocked down the level of USP2 and overexpressed EZH2 expression in 5637 cells. (A) The CCK-8 assay displayed cell growth curves in 5637 cells. (B) Representative images and quantification of EdU assay in 5637 cells. (C) Transwell detection of invasion in 5637 cells. (D) Western blot analysis of USP2 and EZH2 protein levels. Values were expressed as mean ± SD of three separate determinations. **P < 0.01, ***P < 0.001.

Fig 5

USP2-mediated EZH2 stabilization was responsible for epigenetic repression of SOX1

To identify the underlying mechanism of EZH2 in BLCA, firstly, the transfection efficiency of si-EZH2 was evaluated by RT-qPCR and WB analysis. As shown in Figure S2A and S2B, the expression of EZH2 mRNA and protein levels were extremely decreased in both 5637 and J82 cells transfected with si-EZH2. Subsequently, we explored the molecular mechanism by which EZH2 can regulate biological functions of BLCA cells. We selected seven genes (OTX1, SOX1, VIM, OSTM1, MEIS1, RUNX3, ZNF154) that have been reported as targets of EZH2 in human tumors as candidates [[20], [21], [22]]. Among these, SOX1 was discovered to be markedly increased in BLCA cells upon depletion of USP2 or EZH2 (Fig. 6A and 6B). Additionally, ChIP assay demonstrated that USP2 silencing significantly reduced the recruitment of EZH2 and H3K27me3 to the SOX1 promoter region (Fig. 6, Fig. 6). Furthermore, USP2 silencing greatly promoted SOX1 expression, whereas this effect was abolished by EZH2 upregulation (Fig. 6, Fig. 6). Besides, compared to control cells, EZH2 level was remarkably decreased in cells with sh-USP2. After overexpressing EZH2, opposite result was observed (Fig. 6F). These results implied that USP2 epigenetically inhibited SOX1 by interacting with EZH2.Fig. 6 USP2 epigenetically repressed SOX1 expression via EZH2 stabilization. RT-qPCR was used to detect the levels of candidate gene in BLCA cells transfected with sh-USP2 (A) or si-EZH2 (B). (C, D) ChIP assay was performed to assess the occupancy of EZH2 and H3K27me3 in the SOX1 promoter region in cells with sh-NC or sh-USP2. (E, F) RT-qPCR and western blot analysis of EZH2 and SOX1 expression in BLCA cells transfected with sh-USP2 and/or pcDNA3.1-EZH2. Error bars stand for the mean ± SD of at least triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 6

USP2 downregulation suppressed tumorigenicity of BLCA in vivo

We next evaluated the contribution of USP2 to BLCA growth in vivo. 5637 cells stably expressing sh-USP2 or EZH2 were separately injected into the flanks of BALB/c nude mice to establish a xenograft tumor model. As shown in Fig. 7A-7C, tumors with repressed USP2 expression exhibited significantly smaller sizes and lower weights compared to the control group. However, these suppressive effects were overturned by EZH2 overexpression. Meanwhile, IHC staining revealed that USP2 depletion greatly elevated SOX1 expression in tumor tissues, while EZH2 upregulation led to a decrease in the level of SOX1 in tumors derived from USP2 shRNA-transfected BLCA cells (Fig. 7D). The above data indicated USP2 silencing played a crucial role in inhibiting BLCA growth in vivo. Taken together, USP2 suppressed the ubiquitination of EZH2 to trigger the epigenetic suppression of SOX1, thereby accounting for BLCA progression (Fig. 7E).Fig. 7 The USP2-EZH2-SOX1 axis in tumor growth of BLCA in vivo. The 5637 cells stably expressing sh-USP2 or EZH2 were separately injected into the flanks of BALB/c nude mice to establish a xenograft tumor model. (A) Representative photographs of tumors from the xenograft nude mice. (B) Tumor weight in the three groups. (C) Tumor volumes were determined on the indicated days. (D) IHC analysis detected the expression of SOX1 in tumor tissues. (E) Schematic graph illustrating the role of USP2 in regulating the progression of BLCA. The results are representative of three independent experiments. All data are represented as mean ± SD. **P < 0.01, ***P < 0.001.

Fig 7

USP2 positively correlated with EZH2 in clinical samples of BLCA

Having established that USP2 regulates the expression of SOX1 through EZH2 in BLCA progression, we proceeded to assess the expression of USP2 and EZH2 in situ in patient samples of BLCA. In our follow-up study, we collected 120 cases of tumor tissues and 25 cases of normal tissues. As illustrated in Figure S3A by IHC analysis, USP2 staining was weakly positive in normal tissues, while USP2 expression was remarkably stronger in BLCA tissues, especially in high grade of BLCA. More precisely, the positive staining rate of USP2 was 94.17 % in BLCA tissues, but only 5.83 % in normal tissues (Figure S3B). In addition, the data from Figure S3B further validated the prognostic importance of USP2 in BLCA, as higher levels of USP2 were observed in BLCA patients with histological grade III-IV. Similarly, the positive expression of EZH2 exhibited an analogous tendency, while the difference was that the positive EZH2 was not observed in normal tissues (Figure S3C and S3D). Furthermore, the original advanced tissue microarray data for examining USP2 and EZH2 in BLCA tissues has been shown in Figure S4A and S4B. Moreover, the reciprocal increased in USP2 and EZH2 expression may also imply their positive correlation in clinical samples of BLCA. These findings supplied more convincing evidence supporting the oncogenic role of USP2 and EZH2 in BLCA progression.

Discussion

BLCA is a common malignant tumor of the urinary system, accounting for 5 % of all cancer-related deaths [23]. In recent years, the incidence and mortality rates of BLCA have been increasing year by year [24]. The growth and metastasis of BLCA are significant contributors to patient mortality [25]. In this study, we identified USP2 as a key regulator that deubiquitinates and stabilizes EZH2, thus suppressing SOX1 expression and promoting BLCA progression.

The USP family is extensively studied and recognized as the most widely investigated group of deubiquitinating enzymes [26]. These enzymes are capable of catalyzing the cleavage of the isopeptide bond between the ubiquitin molecule and the lysine residue of the target protein, thereby playing a crucial role in deubiquitination processes [27]. USP2 is a special member of the USP family [6]. Studies have demonstrated that USP2 is aberrantly expressed in a variety of malignant tumors and exerts oncogenic effects. For example, the combination of USP2 and HSP90 inhibitors effectively restrained the growth of ErbB2-positive breast cancer xenograft [28]. USP2 depletion enhanced the responsiveness of triple-negative breast cancer to chemotherapy [29]. In BLCA, USP2a promotes tumor progression via binding to cyclin A1 [30]. USP2a mRNA can serve as a diagnostic marker for BLCA [31]. The results of this study discovered that USP2 was highly expressed in BLCA tissues and cells, consistent with previous literature reports. High expression of USP2 was significantly associated with poor prognosis in BLCA patients. In order to explore the effect of USP2 on BLCA progression, we constructed a cell line with silenced USP2, and observed that downregulation of USP2 reduced the proliferation, migration and invasion abilities of BLCA cells. Additionally, USP2 downregulation suppressed the tumorigenicity of BLCA in vivo.

Through its deubiquitination activity, USP2 can regulate the stability of its substrates and play an important role in DNA damage repair, epigenetic regulation and immune response [32]. Results from this study identified EZH2 as a novel substrate of USP2. EZH2 is an epigenetic modulator and is often overexpressed in many types of cancers [10]. Early studies have demonstrated that EZH2 can be degraded by multiple E3 ubiquitin ligases [33]. Additionally, USP21 was found to deubiquitinate EZH2, promoting BLCA cell proliferation and metastasis [14]. In breast cancer, ZRANB1 functions as a deubiquitinase to regulate the stabilization of EZH2[34]. Our study identified that EZH2 was increased in BLCA and positively correlated with USP2 level. Importantly, USP2 regulated BLCA cell growth and invasion through the stabilization of EZH2 and prevention of EZH2 ubiquitination. These results align with recent studies that have demonstrated the growth-promoting effects of EZH2 in BLCA. For instance, lncRNA CASC9 induced BLCA progression by interacting with EZH2 [35]. LncRNA SNHG1 promoted BLCA cell proliferation and invasion through the modulation of EZH2 [36].

Studies have shown that EZH2 plays a role in promoting H3K27me3 and epigenetically regulating the expression of target genes during cancer occurrence and development [37]. As an example, EZH2-mediated enrichment of H3K27me3 on the promoter of lncRNA MEG3 was found to induce glioma cell growth and metastasis [38]. LncRNA BLACAT1 was found to enhance pancreatic cancer cell proliferation and migration through EZH2-induced H3K27me3 [39]. SOX1 encodes a member belonging to the SOX (SRY-related high mobility group box) family of transcription factors that participate in governing embryonic development and determining cellular destiny [40]. As a tumor suppressor, SOX1 inhibits cell proliferation and reduces migration and invasion ability in various carcinomas, including lung cancer, breast cancer and nasopharyngeal carcinoma [[41], [42], [43]]. In BLCA, a correlation has been discovered between SOX1 expression and muscle invasiveness [20]. In this study, we showed that USP2 regulated SOX1 level via interacting with EZH2 in BLCA cells. Mechanically, EHZ2 could bind to the SOX1 promoter and epigenetically suppress SOX1 expression in BLCA cells. Moreover, USP2 depletion significantly suppressed the recruitment of EZH2 and H3K27me3 to the SOX1 promoter region, leading to a substantial induction of SOX1 expression. Therefore, the USP2/EHZ2/SOX1 axis forms a regulatory network that modulates BLCA progression.

In conclusion, our study demonstrated that USP2 was highly expressed in BLCA, and knockdown of USP2 inhibited BLCA cell proliferation, migration and invasion. Additionally, as a deubiquitinase, USP2 interacted with EZH2 to downregulate SOX1 expression, thereby promoting BLCA tumorigenesis. Therefore, USP2 is expected to become a novel therapeutic target for BLCA patients. Nevertheless, it is likely that several other signaling pathways may also be involved. Further studies are needed to investigate a deeper characterization of possible mechanisms associated with USP2-mediated BLCA progression. In addition, the prediction made based on TCGA database does not reflect the actual expression level of this gene in tumor tissue. It is important to consider certain factors such as regional variability in sample collection, limited sample sizes, individual patient differences, and the time elapsed since the prediction analysis was conducted. However, despite these limitations, we remain confident in the significance of the findings from this study. Additionally, increasing evidence indicates that USP2 plays an oncogenic role in many tumors, including choroidal melanoma [44], triple negative breast cancer [8], and prostate cancer [45]. Notably, a recent study reported that tight junction protein 1 promotes tumor angiogenesis in BLCA via recruiting USP2, which in turn deubiquitinates TWIST1, thereby protecting TWIST1 from proteasome-mediated protein degradation [9]. These studies imply that the results of database predictions should be used as a reference and should not be solely relied upon. The data from TCGA predictions should be approached with caution, as there are still questions that warrant further consideration and exploration.

Ethics approval

This study has obtained approval of the Ethics Committee of The Second Affiliated Hospital of Nanchang University and the patient's written informed consent.

CRediT authorship contribution statement

Fanghua Xu: Writing – original draft, Visualization, Validation, Formal analysis, Data curation. Xiangda Xu: Data curation. Huanhuan Deng: Data curation. Zhaojun Yu: Formal analysis. Jianbiao Huang: Formal analysis. Leihong Deng: Writing – review & editing. Haichao Chao: Writing – review & editing, Methodology, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Figure S1. (A) TCGA database was used to analyze the expression profile of USP2 in common human tumor types. (B) Differential expression of USP2 in BLCA tissues (n = 408) and normal tissues (n = 19) in TCGA database. (C) Prediction in TCGA database revealed the correlation between USP2 level in BLCA tissues and patient survival prognosis.

Image, image 1

Figure S2. Determination of the transfection efficiency of EZH2 siRNA. (A, B) RT-qPCR and Western blot showed that EZH2 was extremely decreased in 5637 and J82 cells transfected with si-EZH2. Results expressed as mean ± SD of three independent experiments. **P < 0.01.

Image, image 2

Figure S3. USP2 positively correlates with EZH2 in BLCA clinical samples. (A) IHC staining of USP2 in normal and tumor tissues of BLCA patients. (B) Quantification of IHC analysis showing differential expression of USP2 in normal tissues (n = 25) and tumor tissues (n = 120, including 60 cases of high grade and 60 cases of low grade). (C) IHC staining of EZH2 in normal and tumor tissues of BLCA patients. (D) Quantification of IHC analysis showing differential expression of EZH2 in normal tissues (n = 25) and tumor tissues (n = 120, including 60 cases of high grade and 60 cases of low grade). All data are represented as mean ± SD. ***P < 0.001.

Image, image 3

Figure S4. (A, B) The original advanced tissue microarray data for examining USP2 and EZH2 in BLCA tissues.

Image, image 4

Acknowledgments

None.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102104.
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