Ovarian cancer is one of the most severe malignancies in women and the 5th leading cause of malignancy-related mortality. There are three major types of ovarian tumors: epithelial, germ cell, and sex cord stromal tumors. The first accounts for approximately 95% of all ovarian cancer cases.[2–5] Recent progress in the screening and prevention of ovarian cancer has resulted from the combined use of genetic and epidemiological factors to predict the risk of ovarian cancer in different individuals. However, the overall survival rate of ovarian cancer remains low because of the rapid metastasis and the lack of ideal biomarkers. Therefore, it is important to investigate the pathogenesis of ovarian cancer and identify new biomarkers to improve prevention and treatment.
Ubiquitin-specific peptidase 7 (USP7), also known as herpesvirus-associated USP, is a deubiquitinating enzyme widely overexpressed in human cancers and contributing to cancer progression. Zhang et al. found that USP7 was robustly upregulated in ovarian cancer tumor tissues, which correlated with poor patient prognosis. USP7 inhibition induces cancer cell death through a p53-dependent mechanism. Hence, several small, selective inhibitors targeting USP7 have been developed for cancer treatment. Moreover, USP7 appears to facilitate colorectal progression cancer via β-catenin deubiquitination and Wnt signaling. However, the functional role of USP7 in ovarian cancer is unclear.
Tumor necrosis factor receptor associated factor 4 (TRAF4) is a member of the TRAF family that regulates multiple cellular processes including embryogenesis and development. As an E3 ligase for protein ubiquitination, TRAF4 is involved in the pathogenesis of multiple cancers. TRAF4 reportedly promotes endometrial cancer growth by activating PI3K/AKT signaling,[13,14] and mediates chemoresistance in breast cancer cells by upregulating β-catenin. Furthermore, Shi et al. reported TRAF4 upregulation in ovarian tumor tissues, having an oncogenic effect in the malignant phenotype of ovarian cancer. Interestingly, USP7 may regulate TRAF4 expression via deubiquitination. Hence, we hypothesized that USP7 may regulate ovarian cancer via TRAF4.
Ribosomal protein S6 kinase 4 (RSK4) is a tumor suppressor downregulated in many types of cancers. RSK4 overexpression was found to attenuate the metastasis of human breast cancer and colorectal cancer. Mechanically, RSK4 inactivates PI3K/AKT signaling. Particularly in breast cancer, RSK4 is negatively regulated by TRAF4. Moreover, low RSK4 correlates with advanced stages of ovarian cancer. Accordingly, TRAF4 might activate PI3K/AKT signaling by downregulating RSK4 in ovarian cancer.
In the present study, we demonstrated USP7 upregulation in ovarian cancer cells; USP7 inhibition decreased the proliferation, migration, and invasion of ovarian cancer cells. Mechanically, USP7 inhibition increased TRAF4 ubiquitination, promoting TRAF4 degradation. Decreased TRAF4 upregulated RSK4 expression and inactivated PI3K/AKT signaling. Our findings may provide novel insights into the tumor-suppressive role of USP7 in ovarian cancer.
MATERIALS AND METHODS
Database analysis and clinical tissues samples
The gene expression profiles of patients with ovarian cancer were obtained from the GEPIA and UALCAN databases. We compared USP7, TRAF4, and RSK4 expression between ovarian tumor tissues and normal tissues.
The normal ovarian epithelial cell line IOSE80 and the ovarian cancer cell lines CAOV-3, SW626, and SKOV3 were obtained from ATCC (American Type Culture Collection). In addition, the ovarian cancer cell line Anglne was purchased from Procell Life Science & Technology (Wuhan, China), and the ovarian cancer cell line IGROV1 was obtained from Cobioer Biosciences (Nanjing, China). All cells were confirmed by STR profiling. Primary fallopian tube epithelial cells (FTECs) were isolated from disease-free fallopian tubes of women undergoing partial salpingectomy as previously reported. All cells were maintained in high glucose Dulbecco’s modified Eagle’s medium (Invitrogen, BRL, USA) supplemented with 10% FBS, 1% penicillin, and 1% streptomycin (Sigma, ST, USA) in an incubator with 5% CO2 at 37°C. Cells subjected to MG132 treatment were incubated with 20 mM of MG132 (Sigma) for 3 h.
RSK4 full-length cDNA and shRNAs targeting USP7, TRAF4, RSK were purchased from GenePharma (Shanghai, China) and subcloned into pLKO (TRC Cloning Vector-protocol) plasmids. Plasmids were then transfected to HEK293T cells with a psPAX2 packaging plasmid and a pMD2.G envelope plasmid using Lipofectamine 3000. The obtained lentiviral vectors were then used to transfect cells.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay
The proliferation of ovarian cancer cells following transfection was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates (5 × 103 cell per well) and transfected for 48 h. Then, 10 mL MTT reagent (YEASEN, Shanghai, China) was added into each well and incubated for 2 h at 37°C. Afterwards, the mixture in the well was replaced by 100 mL DMSO. After incubation, the OD value was measured at 490 nm by a microplate reader.
Transwell migration assay
Transwell chambers with 8-mm porous membranes (BD Biosciences, USA) were used for a migration assay. First, migration chambers were inserted in 24-well plates filled with 750 mL culture medium with serum. Secondly, 200 mL of cell suspension in serum-free medium (a density of 5 × 104) was added to each upper chamber. After overnight incubation at 37°C, the medium was discarded and the inner surface of the upper chambers was scraped with cotton swabs and washed twice with PBS. Then, the migrating cells adhered to the chambers were fixed in 4% paraformaldehyde for 3 min at room temperature. Cells were stained with crystal violet for 10 min. The remaining dying solution was washed with PBS. The migrating cells were observed and counted under a microscope.
Transwell invasion assay
Melted Matrigel (BD Biosciences) was diluted and mixed with serum-free medium at 1:8. Then, 100 mL matrix gum was added to each upper chamber and incubated for 2 h at 37°C. The remaining experimental procedures were the same as the Transwell migration assay. Non-invading cells were scraped from the inner surface. The invading cells were observed and counted under a microscope.
Cells transfected with sh-USP7 or sh-NC were harvested and lysed in lysate buffer containing protease inhibitors (CST, USA). After 30 min, the mixture was centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was used for immunoprecipitation with anti-TRAF4 antibody (CST) for 4 h at 4°C. Then, protein A/G beads (Thermo Fisher Scientific) were added and incubated for 12 h at 4°C. Protein complexes were sequentially eluted from the beads with lysis buffer. The mixture was centrifuged, and the precipitates were analyzed by immunoblotting.
CAOV-3 and SKOV3 cells (1 × 106) were cultured in a 15-cm Petri dish until ~80% confluence and transfected with either tagged USP7 or TRAF4 or both for 48 h. Subsequently, cells were lysed with 2 mL lysis buffer (Sigma), centrifuged at 12,000 rpm for 10 min, and incubated with 35 mL of 50% suspension containing antibodies against FLAG tag or HA tag (Sigma) for 3 h. Then, 40 mL of sample buffer was added to the immunoprecipitates. Finally, the immunoprecipitated proteins were analyzed by Western blot.
A Pierce Desthiobiotinylation Kit (Thermo Fisher Scientific) was used for USP7 desthiobiotin labeling. Biotin-labeled USP7 was kept at –80°C. Streptavidin magnetic beads (Thermo Fisher Scientific) were incubated with biotin-labeled USP7 in binding buffer for 12 h. Then, the beads were incubated with the cell lysates, and the complex was examined by Western blot.
Thirty female, 6-week-old BALB/c nude mice were purchased from Laboratory Animal Center (Shanghai, China) and housed in a controlled environment (22 ± 2°C, 50% humidity, 12-h light/dark cycle) with free access to water and food. CAOV-3 and SKOV3 cells were transfected with sh-USP7 or sh-NC as previously mentioned. After one-week acclimation, mice were randomly divided into three groups: control, sh-NC, and sh-USP7 (n = 5 per group). Mice assigned to the sh-NC and sh-USP7 groups were subcutaneously injected with 250 mL of cell suspension (~1 × 107 cells) into the right flank. The control group received the same volume of normal saline. Tumor volume was measured at 0, 5, 10, 15, 20, and 25 days after inoculation using the following formula: tumor volume (cm3) = (p × length × width2)/6. Then, all mice were euthanized and tumor xenografts removed. Tumor tissue homogenates were prepared for Western blot and quantitative real-time PCR (qRT-PCR). All animal experiments were approved by the Animal Care and Use Committee of the local hospital and performed following the Guide for the Care and Use of Laboratory Animals.
USP7, TRAF4, RSK4, PI3K, and AKT protein levels were detected by Western blot. The total protein was extracted from cell lysates or tissue homogenates using RIPA buffer with PMSF. Protein concentration was measured by a BCA kit and equal amounts of total protein were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transferring the proteins onto the PVDF membrane, nonspecific binding was minimized by incubation with blocking reagent. Protein expression was detected by incubation with primary antibodies against USP7 (Abcam), TRAF4 (Abcam), RSK4 (14813S, CST), PI3K (4249S, CST), p-PI3K (17366S, CST), AKT (4691S, CST), p-AKT (4060S, CST), and GAPDH (3683S, CST) overnight at 4°C. Then, the membranes were incubated with a goat anti-mouse or goat anti-rabbit secondary antibody. The signal intensity of protein bands was quantified by ImageJ software.
Quantitative real-time PCR
Total RNA was extracted from the cells using TRIzol (Takara, Japan), and its concentration measured. RNA (1 mg) was reverse transcribed to cDNA in 20 mL of reverse transcriptase reagent (Takara) using the following parameters: 42°C for 2 min and 37°C for 15 min. mRNA levels were determined by qRT-PCR using SYBR Green (Invitrogen). cDNA (1 mL) was amplified in a reaction system consisting of 5 mL SYBR Primix Ex Taq II, 0.2 mL ROX II, and 0.2 mL primer. The qRT-PCR conditions were as follows: pre-denaturation for 30 s at 95°C, followed by 40 amplification cycles (5 s at 95°C and 34 s at 60°C). Target RNA expression was analyzed using the 2-ΔΔCt method. The primer sequences used were as follows:
USP7 Forward 5´-TCGTCGCACATTGAGACGG-3´
USP7 Reverse 5´-CTTGTCGGCATGGTTGGGAAT-3´
TRAF4 Forward 5´-ACTTCAGTGGGGAGGCCTAT-3´
TRAF4 Reverse 5´-ACTGGTACTGGTGGCTCTGG-3´
RSK4 Forward 5´-AATACTACCATTCGCTCCTCA-3´
RSK4 Reverse 5´-TTACAGGCCAGTTGATGTTCGC-3´
GADPH Forward 5´-TGCTGAGTATGTCGTGGAG-3´
GADPH Reverse 5´-GCATCAAAGGTGGAAGAAT-3´
Statistical analyses were performed using the GraphPad Prism software (version 8.0). One-way analysis of variance (ANOVA) and t-tests were used to compare the differences between groups. Data are shown as mean ± standard deviation. Differences were considered statistically significant at P < 0.05.
USP7 and TRAF4 were upregulated in ovarian cancer
The gene expression profiles obtained from the GEPIA and UALCAN databases showed USP7 and TRAF4 upregulation and RSK4 downregulation in ovarian tumor tissues compared to normal tissues [Figure 1a-c]. We also evaluated the mRNA levels of USP7, TRAF4, and RSK4 in ovarian cell lines. USP7 and TRAF4 were upregulated in all ovarian cancer cell lines (Anglne, IGROV1, CAOV-3, SW626, and SKOV3) compared to IOSE80 and FTECs [Figure 1d]. Similarly, RSK4 was downregulated in comparison to IOSE80 and FTECs [Figure 1d]. Taken together, these findings indicate that USP7 and TRAF4 are upregulated and RSK4 downregulated in ovarian cancer.
USP7 inhibition attenuated the proliferation, migration, and invasion of ovarian cancer cells
To identify the functional role of USP7 in ovarian cancer cells, shRNA was used to generate two knockdown USP7 ovarian cancer cell lines (CAOV-3 and SKOV3). The efficiency of USP7 knockdown was evaluated using qRT-PCR and Western blotting [Figure 2a and 2b]. The MTT assay showed impaired cell proliferation upon USP7 knockdown at 72 h in both cell lines [Figure 2c]. Moreover, Transwell assays revealed that knocking down USP7 decreased the number of migrating CAOV-3 and SKOV3 cells in the upper chamber [Figure 2d]. Similarly, the invasive capacity of CAOV-3 and SKOV3 cells was suppressed by the USP7 knockdown [Figure 2e]. Accordingly, USP7 inhibition attenuated the proliferation, migration, and invasion of ovarian cancer cells.
USP7 deubiquitinated TRAF4, promoting the proliferation, migration, and invasion of ovarian cancer cells
A previous study indicated that TRAF4 competitively binds to USP7 and induces the ubiquitination and degradation of p53 (12). We explored whether TRAF4 expression and ubiquitination were regulated by USP7 in ovarian cancer cells. qRT-PCR and Western blotting showed that USP7 inhibition decreased TRAF4 expression at both mRNA and protein levels [Figure 3a and 3b]. TRAF4 ubiquitination increased with USP7 knockdown, as shown in Figure 3c. However, the addition of MG132, a ubiquitin inhibitor, eliminated the effect of the USP7 knockdown on TRAF4 ubiquitination. These data indicated that USP7 deubiquitinates TRAF4. We further demonstrated a direct interaction between USP7 and TRAF4 [Figure 3d and 3e]. We further verified the oncogenic role of TRAF4 in ovarian cancer cells. TRAF4 expression was successfully decreased by TRAF shRNA at both the mRNA and protein levels [Figure 3f and 3g]. The MTT assay showed that cell proliferation was inhibited by the TRAF4 knockdown at 72 h in both cell lines [Figure 3h]. Moreover, the Transwell assay suggested that the TRAF4 knockdown caused a notable decrease in the number of migrated CAOV-3 and SKOV3 cells [Figure 3i]. Similarly, the invasive capacity of CAOV-3 and SKOV3 cells was suppressed after knocking down TRAF4 [Figure 3j]. Taken together, these results demonstrate that USP7 deubiquitinates TRAF4, promoting the proliferation, migration, and invasion of ovarian cancer cells.
TRAF4 negatively regulated RSK4, suppressing the proliferation, migration, and invasion of ovarian cancer cells
A previous study revealed that RSK4 was negatively regulated by TRAF4 in breast cancer (16). Next, we investigated whether RSK4 is regulated by TRAF4 in ovarian cancer. qRT-PCR showed increased RSK4 expression upon TRAF4 downregulation [Figure 4a]. Moreover, the RSK4 protein level was upregulated, whereas phosphorylated PI3K and AKT levels decreased upon TRAF4 knockdown [Figure 4b]. To verify the functional significance of RSK4 in ovarian cancer, we overexpressed RSK4 in CAOV-3 and SKOV3 cells. Transfection with overexpression plasmids successfully upregulated RSK4 protein levels, while decreasing the phosphorylated PI3K and AKT levels, indicating the suppression of PI3K/AKT signaling [Figure 4c]. In addition, RSK4 overexpression inhibited proliferation of CAOV-3 and SKOV3 cells, as indicated by the MTT assay [Figure 4d]. Transwell assays also showed impaired migratory and invasive cell capacity with RSK4 overexpression [Figure 4e and 4f]. Collectively, our results showed that TRAF4 negatively regulates RSK4, suppressing the proliferation, migration, and invasion of ovarian cancer cells.
RSK4 was involved in USP7’s regulatory network in ovarian cancer cells
To investigate whether USP7 affects ovarian cancer cells via RSK4 and PI3K/AKT signaling, we simultaneously knocked down USP7 and RSK4 in said cells. qRT-PCR showed that transfection with shRNA targeting RSK4 effectively inhibited the USP7 knockdown-induced RSK4 upregulation in ovarian cancer cells [Figure 5a]. Figure 5B shows that USP7 inhibition increased RSK4 expression and inhibited the activation of PI3K/AKT signaling. However, the simultaneous inhibition of RSK4 compromised the effect of the USP7 knockdown by reactivating PI3K/AKT signaling. Moreover, the viability of CAOV-3 and SKOV3 cells, decreased by knocking down USP7, was recovered after suppressing RSK4 [Figure 5c]. The migratory and invasive capacities of cells were repressed by the USP7 knockdown, whereas the simultaneous RSK4 downregulation restored the number of migratory and invasive cells [Figure 5d and 5e]. These results collectively demonstrate that RSK4 is involved in USP7’s regulatory network in ovarian cancer cells.
Knocking down USP7 suppressed ovarian tumor growth by regulating RSK4 and PI3K/AKT signaling
To validate the effects of USP7 downregulation on ovarian tumor growth, we inoculated mice with CAOV-3 and SKOV3 cells transfected with sh-NC or sh-USP7. The USP7 knockdown significantly suppressed ovarian tumor growth in vivo [Figure 6a]. In addition, mice inoculated with sh-USP7-transfected cells displayed significantly decreased TRAF4 levels and upregulated RSK4 levels in tumor tissues [Figure 6b]. Moreover, Western blotting revealed that USP7 knockdown downregulated TRAF4 protein expression and reduced PI3K and AKT phosphorylation but increased RSK4 levels in tumor tissues [Figure 6c]. Collectively, these data confirmed that knocking down USP7 suppressed ovarian tumor growth in mouse xenografts by regulating RSK4 and PI3K/AKT signaling.
The present study revealed the functional significance of USP7 in ovarian cancer. We first showed that USP7 was upregulated in ovarian tumor tissues and cell lines and that knocking it down notably inhibited their proliferation, migration, and invasion. Further research demonstrated that USP7 inhibition increased TRAF4 ubiquitination, promoting TRAF4 degradation and upregulating RSK4 expression. Moreover, TRAF4 knockdown and RSK4 overexpression lines had similar effects to the USP7 knockdown on ovarian cancer cell proliferation, migration, and invasion. In vivo data confirmed that the USP7 knockdown suppressed ovarian tumor growth by regulating RSK4 and PI3K/AKT signaling.
Ubiquitination and deubiquitination play a vital role in cancer progression. USP10, another member of the USP family, exhibits a tumor suppressive effect in lung cancer via PTEN deubiquitination and stabilization. Moreover, USP10 was reported to play an oncogenic role in colon cancer by stabilizing the expression of Musashi-2. Meanwhile, USP4 promotes metastasis and the epithelial-to-mesenchymal transition of cancer cells by deubiquitinating the TGF-b type I receptor, and USP5 facilitates pancreatic tumor growth by stabilizing FoxM1. Similarly, USP7 protects MDM2 and MDMX from ubiquitination and degradation, negatively regulating the tumor suppressor p53.[7,9,28] USP7 also promotes breast cancer tumorigenesis by deubiquitination of histone demethylase PHF8. In the present study, we illustrated for the first time, that USP7 regulated the proliferation and migration of ovarian cancer cells via a novel deubiquitination target, TRAF4. This finding hints at a potential regulatory mechanism of USP7 in cancer via deubiquitination.
TRAF4 is an oncogenic regulator in many types of cancers via ubiquitination-mediated activation. For instance, TRAF4 is upregulated in metastatic prostate cancer, playing a vital role in RTK-mediated cancer metastasis via ubiquitination at the kinase domain of TrkA. In addition, TRAF4 promotes CHK1 ubiquitination and activation in colorectal cancer cells. Moreover, TRAF4 facilitates TGF-b-induced migration of breast cancer cells via ubiquitinating and activating TGF-b-activated kinase (TAK) 1. In addition, TRAF4 can activate PI3K/AKT signaling, a major survival and oncogenic signaling in cancer cells.[13,33] However, the mechanism by which TRAF4 activates the PI3K/AKT signaling remains unknown. In the present study, we identified RSK4 as a critical mediator between TRAF4 and PI3K/AKT signaling, consistent with a previous study showing that TRAF4 mediates the progression of breast cancer via negative regulation of RSK4 and AKT signaling. However, the detailed mechanisms by which TRAF4 regulates RSK4 expression warrant further investigation.
In conclusion, our study demonstrated that USP7 inhibition suppressed proliferation, migration, and invasion of ovarian tumor cells as well as tumor growth in vivo. Mechanically, USP7 inhibition increased TRAF4 ubiquitination, thus promoting TRAF4 degradation. Decreased TRAF4 upregulated RSK4 expression and inactivated PI3K/AKT signaling, leading to a tumor-suppressive effect in ovarian cancer cells. Our findings may provide novel insights into the functional role of USP7 in ovarian cancer.
USP7, ubiquitin specific peptidase 7; TRAF4, TNF receptor associated factor 4; RSK4, ribosomal protein S6 kinase 4; PI3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative real-time PCR; TGF-b, transforming growth factor beta.
Financial support and sponsorship
This work was supported by the Scientific Research Project of Hunan Provincial Health Commission (No. 202105011313), National Natural Scientific Foundation of China (No. 81603207), Natural Science Foundation of Hunan Province (No. 2017JJ3189), Hunan Key Laboratory of Cancer Metabolism (No. 2020TP1018), National Natural Science Foundation of China (No. 82003050), China Postdoctoral Science Foundation (No. 2022M711126) and the Natural Science Foundation of Hunan Province (No. 2020JJ5338).
Conflicts of interest
There are no conflicts of interest.
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