Introduction
Bladder cancer (BC), a carcinoma of the urothelial system, is one of the most common malignancies worldwide, with over 550,000 patients diagnosed and 190,000 succumbed to the disease worldwide every year [
1]. Cigarette smoking, male sex, and advanced age contribute to the development of bladder cancer. Bladder cancer is classified into two subclasses: non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Tumours restricted to the urothelium and the lamina propria are classified as NMIBC, which is not life-threatening. NMIBCs account for approximately 70% of newly diagnosed BC cases, and the five-year survival rate of NMIBC is above 90% [
2]. Tumours that invade the detrusor muscle are classified as MIBC. MIBC is accompanied by invasion and metastasis, which have increased the death rate annually. The five-year survival rate of MIBC is 50% [
3]. NMIBCs are treated with endoscopic resection and adjuvant intravesical therapy. For patients with MIBC, radical cystectomy and urinary diversion or trimodal therapy with maximal endoscopic resection, radiosensitizing chemotherapy, and radiation is warranted [
4]. The advent of checkpoint inhibitors, targeted therapies, and antibody-drug conjugates for immunotherapy has greatly improved the treatment of bladder cancer. Improved understanding of the molecular biology of bladder cancer is important for the development of diagnosis and treatment.
The c-Jun N-terminal kinase (JNK) pathway is a mitogen-activated protein kinase (MAPK) pathway [
5]. JNK signalling is involved in many physiological processes and pathological conditions, including inflammation, neurodegenerative diseases and multiple tumorigenic processes [
6‐
10]. JNK plays pivotal roles in aspects related to bladder cancer, such as tumorigenesis [
11,
12], apoptosis [
13], the chemotherapy response [
14] and metastasis [
15]. The transcription factor activator protein 1 (AP-1) may be activated by MAPKs, particularly JNK. c-Jun is a member of the Jun family and is a component of AP-1 complexes [
16]. c-Jun was the first purely oncogenic transcription factor discovered. It is important in processes related to cellular homeostasis, including proliferation, apoptosis and survival. The relationship between c-Jun and tumorigenesis has been widely investigated [
17,
18]. Usually, c-Jun is activated by JNK, which binds to the c-Jun transactivation domain and phosphorylates it at Ser63 and Ser73 [
19]. c-Jun is degraded through a ubiquitination mechanism. Several studies have revealed that c-Jun is ubiquitinated by several E3 ubiquitin ligases [
20,
21]. However, the regulation of the c-Jun by deubiquitinating enzymes (DUBs) requires further study.
The ubiquitin‒proteasome system is responsible for protein stability. DUBs remove ubiquitin moieties from substrates. DUBs can be classified into five families based on their sequence and structural homology [
22]: Otubain proteases (OTUs), ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), Machado-Joseph disease proteases (MJDs), and JAB1/MPN/Mov34 metalloenzymes (JAMMs). USP5 is a cysteine deubiquitinating enzyme belonging to the USP family. The
USP5 gene is located on chromosome 12p13 and encodes the 93.3-kDa protein USP5 [
23]. Analysis of data in the Human Protein Atlas (
https://www.proteinatlas.org/) revealed that USP5 is highly expressed in testicular cancer, prostate cancer, breast cancer and urothelial cancer. Several studies have demonstrated that USP5 plays an important role in cancers by targeting its substrates. In hepatocellular carcinoma (HCC) and colorectal cancer (CRC), USP5 is highly expressed and closely associated with malignancy and pathological progression [
24]. In pancreatic ductal adenocarcinoma (PDAC), USP5 stabilizes FoxM1 to promote tumour growth [
25]. In non-small cell lung cancer, USP5 promotes cell proliferation, colony formation and migration [
26]. However, the role of USP5 in BC needs to be explored.
We found that USP5 was overexpressed in bladder cancer samples and that patients with high USP5 expression had an unfavourable prognosis. In vitro, USP5 overexpression promotes the proliferation and migration of EJ bladder cancer cells. Consistent with these results, USP5 deficiency inhibits the proliferation and migration of T24 bladder cancer cells. Through RNA sequencing (RNA-seq) and luciferase assays, we found that USP5 activates the JNK pathway. We showed that USP5 binds to and stabilizes c-Jun by mediating its deubiquitination, thereby promoting the JNK signalling cascade. Finally, we revealed a novel mechanism of USP5 in bladder cancer development and progression.
Materials and methods
Antibodies and plasmids
The following antibodies were used: rabbit polyclonal anti-USP5 (10473-1-AP, Proteintech, Wuhan, China); rabbit monoclonal anti-c-Jun (ab40766, Abcam, Waltham, USA, 1:2000 dilution); mouse anti-HA (M180-3, MBL, Japan, 1:5000 dilution); mouse anti-Flag (M185-11R, MBL, Japan, 1:5000 dilution); mouse anti-Myc (M192-3, MBL, Japan,1:5000 dilution); mouse anti-GAPDH (ANT011, AntGene, Wuhan, China, 1:5000 dilution); HRP-labelled goat anti-mouse IgG (H + L) (A0216, Beyotime, Shanghai, China, 1:5000 dilution); and HRP goat anti-rabbit IgG (H + L) (ANT020, AntGene, Wuhan, China, 1:5000 dilution).
The plasmids PHAGE-3×Flag-USP5(FLAG-USP5), pHAGE-3×HA-USP5 and pHAGE-3×HA-USP5 C335A (HA-USP5 C335A) were constructed according to the methods in the “Molecular Cloning Experiment Guide”. The plasmid pcDNA3.1-3xFlag-c-Jun (FLAG-c-Jun) was purchased from Youbio (F118284).
Xenografts
USP5−/− and parental T24 cells were collected and washed twice with PBS. A total of 5 × 106 cells were resuspended in 0.2 mL of PBS and inoculated into the flanks of 5 4-week-old female BALB/c nude mice. Tumours were measured every other day after the appearance of subcutaneous tumours. The tumour volume was calculated as follows: volume = (length×width2) × 0.5. 30 days after inoculation, the mice will be deeply anesthetized with isoflurane (5%) for approximately 3 min. Then, the mice were killed by cervical dislocation. All animal studies were conducted in accordance with the Guidelines of the China Animal Welfare Legislation and were approved by the Committee on Ethics in the Care and Use of Laboratory Animals of Hunan Normal University (permit number: 2,021,286). All efforts were made to minimize animal suffering.
Cell culture and cell lines
All cell lines were purchased from ATCC. HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, USA). T24 cells were maintained in McCoy’s 5a medium (BasalMedia, Shanghai, China). EJ cells were maintained in RPMI 1640 medium (Gibco, Carlsbad, USA). The media were supplemented with 10% foetal bovine serum (FBS, Gibco, Carlsbad, USA) and 1% penicillin/streptomycin (Gibco, Carlsbad, USA). All cells were cultured at 37 °C in a 5% CO2 incubator. No mycoplasma contamination was detected.
Cell proliferation, colony formation, transwell and scratch assays
A Cell Counting Kit-8 (CCK8, BS350B, Biosharp, China) was used for cell proliferation assays. A total of 1 × 103 cells/well were seeded into 96-well plates. CCK8 solution (10 µL in 100 µL medium) was added to each well and incubated at 37 °C for 1 h. The optical density was measured at a wavelength of 450 nm. Colony formation was performed as we previously described. Briefly, 4 × 102 cells were cultured in 6-well plates for 14 days. Colonies were stained with 0.025% crystal violet, and images were acquired using a scanner. For transwell assays, medium with 40% FBS was added to the lower chamber, and serum-free medium was added to the upper chamber. Cells were seeded into the upper chamber. After 36–48 h, cells that migrated into the lower chamber were stained with 0.02% crystal violet. For the scratch assays, cells subjected to different treatments were seeded in 6-well plates. When the cells formed a confluent monolayer, a cell-free area was artificially created in the centre of each well. Images of the wounds were acquired every 12 h using a light microscope.
Western blot analysis
Cells were lysed with SDS lysis buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol) at 95 °C for 10 min. Total protein was separated by 10% SDS‒PAGE and transferred to PVDF membranes (IPVH00010; Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 h. The membranes were incubated with primary antibodies overnight at 4 °C. The next day, the membranes were washed in TBST and then incubated with HRP-labelled secondary antibodies at room temperature for 1 h. A Tanon 5500 chemiluminescence image analysis system (Tanon, Shanghai, China) was used to evaluate the chemiluminescence of the protein bands. GAPDH was used as the internal control.
Coimmunoprecipitation
Cells were lysed using NP-40 lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1% NP-40) in the presence of protease inhibitor cocktails. The lysates were centrifuged at 12,000 rpm for 10 min at 4 °C and then incubated with the approprioat antibody and rProtein A/G Magarose Beads (SM005002, SMART Lifesciences, China) overnight at 4 °C. The next day, the magarose beads were washed with lysis buffer 3 times. The immunoprecipitated proteins were boiled in 2 × SDS‒PAGE loading buffer for 10 min at 95 °C and separated using SDS‒PAGE.
Luciferase assay
HEK293T cells (30% confluence) were seeded into 24-well plates. The reporter plasmid (100 ng, contains 34 signaling pathways), pRL-CMV (5 ng) with the indicated gene-expressing plasmids (Flag-USP5, 500 ng) or empty vector were transient transfected into the HEK293T cells in each well. were transfected into the cells. After 48 h, luciferase reporter assays were performed with a dual luciferase assay kit (E1960, Promega, Beijing).
Immunofluorescence
Cells were fixed with 4% paraformaldehyde for 15 min, blocked with 0.1% Triton X-100 and then washed with PBS. The samples were then stained with a primary antibody and the corresponding secondary antibody. Finally, the slides were observed and digitally photographed using a confocal microscope (Leica TCS SP8 SR. Leica, Germany).
Immunohistochemistry and H&E staining
Bladder cancer tissue microarrays were purchased from Wuhan Shuangxuan Biotechnology Co., Ltd. (Cat. No. IWLT-N-140BL61, contains 64 bladder cancer tissues and 48 normal tissues). Immunohistochemistry examination with USP5 antibody at WuHan Servicebio Technology Co., Ltd. Tumors of each mouse were prepared for histopathological sections, and subjected to HE staining at WuHan Servicebio Technology Co., Ltd.
Obtaining USP5-knockout cell lines using CRISPR-CAS9
USP5-knockout cells were obtained by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing [
27]. Briefly, sgRNAs targeting USP5 exon 6 were designed via the CRISPR Design Tool (sgRNA-1: TGTGGGCGACGCTACTTCGA, sgRNA-2: TACCCGTTAGCTGTCAAGCT). The annealed sgRNA oligos were inserted into the lentiCRISPRv2 vector (Addgene plasmid 52,961) [
28] to generate the USP5 knockout plasmids. Transfection and infection were performed using PEI MAX transfection reagent (Polysciences) according to the manufacturer’s instructions as previously described [
29]. Positive cells were selected by puromycin (1 ug/ml). The western blot analysis was used for cell line identification.
Statistical analysis
All experiments were performed 3 times. Two-tailed unpaired Student’s t test was used to compare two groups of data. One-way ANOVA was used to compare multiple groups of data. A P value of less than 0.05 was considered significant. KEGG pathway enrichment analysis was performed using the BGI Dr.Tom.
Discussion
DUBs are essential for maintaining ubiquitin homeostasis and are required for diverse cellular functions. There are more than 100 DUBs encoded in the human genome. USP5, also called ubiquitin isopeptidase (ISOT), has been reported to be involved in multiple cellular processes, including stress responses, DNA repair and inflammatory responses [
30]. USP5 has also been found to be associated with cancers, including breast, prostate, testicular and urothelial cancers. Several studies have revealed the role of USP5 in HCC, PDAC and CRC. However, the mechanism of USP5 in bladder cancer needs to be determined. First, we analysed the expression of USP5 in bladder cancer in the online GEPIA database, and the results suggested that USP5 is upregulated in bladder cancer patients. The results of IHC staining experiments with a tissue microarray were revealed that USP5 was highly expressed in bladder cancer compared with normal tissue. To determine the function of USP5 in bladder cancer, we constructed USP5-overexpressing and USP5-deficient cancer cell lines. The phenotype results suggested that USP5 promotes the development and progression of bladder cancer. Next, we performed RNA-seq analysis and luciferase pathway screening to explore the mechanism of USP5 in bladder cancer. The results pointed to the involvement of the JNK pathway. Previous studies have revealed that the JNK pathway is strongly associated with bladder cancer [
31]. To explore how USP5 activates the JNK signalling pathway, we performed coimmunoprecipitation experiments to determine whether USP5 can interact with important molecules in the JNK signalling pathway. We verified the physical association between USP5 and c-Jun. We detected USP5 and c-Jun colocalization in the nucleus.
Posttranslational modifications, including phosphorylation, ubiquitination, and acetylation, are important to the function of c-Jun. Ubiquitination is responsible primarily posttranslational modification for the stability of c-Jun. Several studies have revealed that c-Jun is ubiquitinated by several E3 ubiquitin ligases [
20,
32]. In 2018, Lin et al. published a paper in which they described that USP6 regulates the stability of the c-Jun protein [
33]. We sought to determine whether USP5 could regulate the stability of the c-Jun protein. We further verified that overexpression of USP5 could stabilize the c-Jun protein. It has been reported that USP5 cleaves K6-linked, K29-linked, K48-linked, K63-linked and linear ubiquitin chains, especially Lys48-linked polyubiquitin chains [
34]. To determine whether USP5 stabilizes the c-Jun protein by deubiquitination, we constructed a plasmid expressing the catalytically inactive mutant USP5 C335A. CHX chase assays and deubiquitination assays were performed, and the results suggested that USP5 stabilizes the c-Jun protein by inhibiting its ubiquitination. Considering the phosphorylation of c-Jun is closely related to its stability, further studies need to be performed to evaluate whether USP5 affects c-Jun phosphorylation. Finally, an in vivo xenograft mouse model was used to study the role of USP5 in bladder cancer. The results demonstrated that USP5 potentially promotes bladder tumour growth.
USPs involves in a wide range of pathological processes of malignancy, they have been considered targets for drug development. The development of USP inhibitors has also become a perspective and possibility for cancer therapy. Several USP5 inhibitors have been developed to treat human cancers. Such as WP1130 [
35], PYR-41 [
36] formononetin [
24]. However, there was few findings suggest that USP5 could be a potential target for bladder cancer therapy. Our experimental results showed that USP5 is associated with poor prognosis in bladder cancer, Further studies need to be performed to evaluate whether this treatment strategy works against bladder cancer development and progression through specific inhibitors selectively targeting USP5.
In conclusion, our experimental results showed that USP5 is associated with poor prognosis in bladder cancer. USP5 plays an oncogenic role through deubiquitination of c-Jun, which is an important downstream target of the JNK pathway in bladder cancer. Our study reveals a new potential therapeutic target for bladder cancer.
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