Downregulated expression levels of USP46 promote the resistance of ovarian cancer to cisplatin and are regulated by PUM2.

Ovarian cancer (OC) is a major contributor to cancer‑related mortality in women. Despite numerous drugs being available for the treatment and improving the prognosis of OC, resistance to clinical chemotherapy remains a major obstacle for the treatment of advanced OC. Therefore, determining how to reverse the chemoresistance of OC has become a research hotspot in recent years. The present study aimed to reveal the potential mechanism of OC chemoresistance. Reverse transcription‑quantitative PCR and western blot analysis were performed to detect the expression levels of Ubiquitin‑specific peptidase 46 (USP46) and Pumilio 2 (PUM2) in OC. Cell viability and apoptosis were evaluated by Cell Counting Kit‑8 assay and flow cytometry, respectively. The association between USP46 and PUM2 was assessed by RNA immunoprecipitation. The results of the present study revealed that the expression levels of USP46 which is associated with tumor progression, was downregulated, while PUM2 expression levels were upregulated in cisplatin (DDP)‑resistant OC cells and patient tissues. The downregulation of USP46 expression levels in SKOV3 cells significantly inhibited cell apoptosis and increased cell viability. In SKOV3/DDP cells, the upregulation of USP46 expression levels notably suppressed cell viability and increased cell apoptosis. The results of the RNA immunoprecipitation chip assay demonstrated that PUM2 bound to USP46 and regulated its expression. Furthermore, following the knockdown of USP46 expression, the mRNA and protein expression levels of the cell apoptosis‑related protein, Bcl‑2, were upregulated, whereas the expression levels of caspase‑3, caspase‑9 and Bax were significantly downregulated. In addition, phosphorylated AKT expression levels were notably upregulated. Following the overexpression of USP46 in SKOV3/DDP cells, the opposite trends were observed. In SKOV3 cells, the knockdown of PUM2 could reverse the DDP resistance induced by small interfering RNA‑USP46 as the expression levels of Bcl‑2 were downregulated whereas those of caspase‑3, caspase‑9 and Bax were upregulated compared with the small interfering‑USP46 group. Similarly, in SKOV3/DDP cells, the overexpression of PUM2 could reverse DDP sensitivity induced by the overexpression of USP46. In conclusion, the findings of the present study suggested that the downregulation of USP46 expression levels may promote DDP resistance in OC, which may be regulated by PUM2. Therefore, targeting PUM2/USP46 may be an effective way to reverse DDP resistance in OC.

Introduction

Ovarian cancer (OC), as a common type of gynecological malignant cancer, accounts for high incidence and high mortality rates worldwide, with a 5-year survival rate of ~47%. Early diagnosis improves survival, but only 15% of OC cases are diagnosed at an early or localized stage. OC therefore presents a significant threat to the health of women worldwide (1,2). Although there are numerous chemotherapeutics available to treat OC and improve patient prognosis, the chemoresistance of OC remains a major obstacle for the successful treatment of OC (3). At present, platinum drugs, such as cisplatin (DDP), combined with paclitaxel represent the main chemotherapeutic regimen for OC, and overall, ~80–90% patients respond well to initial chemotherapy (4). However, numerous patients eventually develop chemoresistance towards DDP, which significantly impedes the prognosis of patients with OC (5). Therefore, it is vital to understand the mechanisms of resistance to DDP in OC to determine novel chemoresistance-related molecular markers for reversing the chemoresistance of OC.

Ubiquitin-specific proteases (USPs) are a group of cysteine protease isomers that primarily act on ubiquitinated protein substrates in the ubiquitin-proteasome system (6). USPs, a subfamily of the deubiquitinase family and deubiquitinated proteolytic enzymes, serve roles in cell proliferation, genetic transcription, mitosis, DNA damage repair and other physiological and pathological processes (7–9). In addition, USPs have been reported to exert important roles in the occurrence and development of malignant cancer types, such as triple-negative breast cancer (10) and hepatic carcinoma (11) by suppressing tumor activity, thus serving antitumor roles (12). For example, the aberrant expression of USP11 was found to interact with nuclear factor 90 and promote its deubiquitination, to promote the proliferation and metastasis of hepatocellular carcinoma (13). In addition, USP46 was discovered to serve as a deubiquitinating enzyme (DUB) within processes regulating the circadian clock and depressive behavior disorders (14,15). Furthermore, USP46 has also been found to be involved in the metastasis and growth in numerous types of cancer, including colon cancer, similar to most other DUB family members (16,17). However, to the best of our knowledge, no previous study has reported a role for USP46 in OC. Another member of the USP family, USP39, was reported to promote OC malignant phenotypes and carboplatin chemoresistance (18). USP11 was discovered to be a predictive and prognostic factor post-neoadjuvant therapy in female patients with breast cancer (19). Therefore, it was hypothesized that USP46 may also be associated with DDP resistance in OC.

Pumilio 2 (PUM2) is an RNA binding protein belonging to the PUF family, and a positive regulator of cell proliferation. PUM2 can bind to the 3′ untranslated region (UTR) of specific target mRNAs to block the formation of the translation initiation complex and inhibit the expression of target genes (20,21). Therefore, PUM2 is considered to be a transcription inhibitor. In addition, previous studies have confirmed that PUM2 is associated with the pathogenesis of solid tumors, such as bladder (22) and colon cancer (23). However, little is known about the role of PUM2 in DDP resistance in OC.

The aim of the present study was to investigate the role of USP46 in DDP resistance in OC and the potential mechanism. This may help provide an enhanced understanding of the roles of USP46 in DDP resistance in OC.

Materials and methods

Cell lines

The human ovarian cancer cell line SKOV3 was purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The SKOV3/DDP cell line, which was DDP-resistant, was obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. SKOV3 and SKOV3/DDP cells were cultured in DMEM and Iscove's modified Dulbecco's medium (Invitrogen; Thermo Fisher Scientific, Inc.), respectively. Both cells were supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution, and maintained in a humidified incubator with 5% CO2 at 37°C. To maintain DDP resistance, the SKOV3/DDP cell culture solution was incubated with 1 µg/ml DDP. Cells in the logarithmic growth phase were used in subsequent experiments.

Patient studies

Fresh frozen tissue samples were randomly collected from female chemosensitive (n=48; age, 40–56 years) and chemoresistant patients (n=48; age, 42–58 years) who underwent surgery for OC at the People's Hospital of Qingdao West Coast New Area (Qingdao, China) between March 2017 and October 2019, and were stored in a refrigerator at −80°C until required for subsequent analysis. A total of 40 paraffin-embedded tissue sections were harvested from each group due to the other 16 tissues were improperly stored. All patients had received ovarian cytoreductive surgery and 6–8 months of postoperative chemotherapy of DDP combined with paclitaxel, without preoperative chemotherapy or radiotherapy. According to the National Comprehensive Cancer Network guidelines (24) inclusion criteria, the chemoresistant group generated some response to initial chemotherapy but recurred during a later course of chemotherapy or six months post-chemotherapy. The patients in the chemosensitive group recurred 12 months post-chemotherapy or did not recur, and other patients were excluded. Written consent was acquired from all patients in the study. The present study was reviewed and approved by the Ethics Committee of the People's Hospital of Qingdao West Coast New Area and was performed according to the principles of the Declaration of Helsinki.

Cell transfection

Geneseed Biotech, Inc. designed and synthesized the small interfering (si)RNAs targeting USP46 (si-USP46, 5′-TGATGGTTGGTCTCTAATA-3′) and PUM2 (si-PUM2, 5′-GCTCCCAGAGTAGTTCTTTATTTCAAGAGAATAAAGAACTACTCTGGGAGCTTTTTT-3′), or overexpression (OE)-USP46 and OE-PUM2 plasmids (pcDNA3.1), as well as the corresponding nonspecific negative controls (NCs), si-NC (GCAAGCTGACCCTGAAGTT) or OE-NC (empty plasmid). When SKOV3 and SKOV3/DDP cell confluence reached 80%, cells were transfected with each plasmid (4 µg) or oligonucleotide (60 µM) using 8 ng/ml polybrene (8 µl) with Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 20 min at 25°C and 24–48 h later, cells were harvested for subsequent experiments according to the manufacturer's protocol.

Reverse transcription-quantitative (RT-q)PCR

Total RNA was extracted from tissues and cultured cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR was subsequently performed using both TaqMan™ Universal Master Mix II (cat. no. 4440043; Applied Biosystems; Thermo Fisher Scientific, Inc.) and SYBR® Premix Ex Taq™ II (cat. no. RR420L; Takara Bio, Inc.), according to the manufacturers' protocols, on a LightCycler 480 instrument (Roche Diagnostics). RT-qPCR reaction conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec, 60°C for 10 sec and 72°C for 30 sec. The following primer sequences were used for the qPCR: GAPDH forward, 5′-CAGGAGGCATTGCTGATGAT-3′ and reverse, 5′-GAAGGCTGGGGCTCATTT-3′; USP46 forward, 5′-AGAAGAAGGTTGGCGTCATCC-3′ and reverse, 5′-TGTCCGCAATAGTGTTTAGCAAA-3′; PUM2 forward, 5′-TTCTCAGCAGGCCTTGCTC-3′ and reverse, 5′-GGTGGAACCACTGCTGGAC-3′; Bcl-2 forward, 5′-GGGCTACGAGTGGGATACTGGAG-3′ and reverse, 5′-CGGGCGTTCGGTTGCTCT-3′; Bax forward, 5′-GGTGGTTGCCCTTTTCTACTTTGC-3′ and reverse, 5′-GCTCCCGGAGGAAGTCCAGTG-3′; Caspase-3 forward, 5′-ACTGGAAAGCCGAAACTCTTCATCAC-3′ and reverse, 5′-GGAAGTCGGCCTCCACTGGTA-3′; and Caspase-9 forward, 5′-GGCTGTCTACGGCACAGATGCA-3′ and reverse, 5′-CTGGCTCGGGGTTACTGCCAG-3′. The expression levels were quantified using the 2−ΔΔCq method and normalized to GAPDH (25).

Western blotting

Total protein was extracted from cells and tissues. Cell lysis was performed in RIPA buffer containing protease and phosphatase inhibitors (Beyotime Institute of Biotechnology). Total protein was quantified using a BCA assay (Beyotime Institute of Biotechnology), according to the manufacturer's protocol, and proteins (40 µg protein per lane) were separated via 10% SDS-PAGE. The separated proteins were transferred onto nitrocellulose membranes and blocked with 5% skimmed milk powder in TBS-Tween-20 (5% Tween-20) for 1 h at 25°C. The membranes were incubated with the following primary antibodies overnight at 4°C: Anti-USP46 (1:1,000; cat. no. ab244215; Abcam), anti-PUM2 (1:1,000; cat. no. ab92390; Abcam), anti-Bcl-2 (1:1,000; cat. no. ab32124; Abcam), anti-Bax (1:1,000; cat. no. ab32503; Abcam), anti-caspase-3 (1:1,000; cat. no. ab13847; Abcam), anti-caspase-9 (1:1,000; cat. no. ab32539; Abcam), anti-AKT (1:1,2000; cat. no. ab179463; Abcam), anti-phosphorylated (p)-AKT (1:1,000; cat. no. ab38449; Abcam), anti-mTOR (1:2,000; cat. no. ab134903; Abcam), anti-p-mTOR (1:1,000; cat. no. ab109268; Abcam) and anti-GAPDH (1:1,000; cat. no. 5174S; Cell Signaling Technology, Inc.). Following the primary antibody incubation, the membranes were washed with TBST and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (cat. nos. A0208 and A0216; both Beyotime Institute of Biotechnology) for 1 h at 25°C. Protein bands were visualized using an ECL Plus Western Blotting substrate (cat. no. 32134; Pierce; Thermo Fisher Scientific, Inc.). ImageJ (1.8.0; National Institutes of Health) was applied for semi-quantification.

Cell Counting Kit-8 (CCK-8) assay

Cells were seeded into 96-well plates (1×104 cells/well) and incubated for 24 h at 37°C, prior to the treatment with different concentrations (SKOV3, 0, 1, 2, 4, 8, 12 and 16; SKOV3/DDP, 0, 3, 6, 9, 15, 20 and 30 µg/ml) of DDP for 24 h at 37°C. Subsequently, cell viability was conducted using a CCK-8 assay (Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol. Cells were cultured for 48 h. After 24 h, CCK-8 reagent (10%) was added and cells were incubated for 1–2 h at 37°C away from light. The absorbance was measured using a microplate reader (Model 680; Bio-Rad Laboratories, Inc.) at a wavelength of 450 nm. All data are generated from three independent experiments.

RNA immunoprecipitation (RIP) assay

In order to detect the regulatory mechanism of USP46, bioinformatics analysis was applied (starbase.sysu.edu.cn/starbase2/browseRbpMrna.php). For the detection of RNA abundance using anti-PUM2 antibody, RIP analysis was performed using an EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (cat. no. 17–701; Merck KGaA). Briefly, ~2×107 cells were harvested and centrifugated at 1,000 × g, 4°C, washed twice with PBS, and then 10 ml PBS and 0.01% formaldehyde were added and incubated at 4°C for 15 min for crosslinking. Cells were subsequently centrifuged with 1.4 ml glycine (2 mol/l) at 1,000 × g for 5 min at 4°C following 5 min of mixing. The supernatant was then discarded, the pellets were washed twice with PBS, followed by cell lysis using RIPA lysis buffer (Beyotime Institute of Biotechnology). Cell lysates were split into two groups: The first group was incubated with 4 µg anti-PUM2 primary antibody (1:200) as the experimental group, while the other group was incubated with normal rabbit anti-IgG antibody (1:200; cat. no. ab172730; Abcam), as the control group, overnight at 4°C. Another group was incubated with anti-AGO2 antibody (1:50; cat. no. ab186733; Abcam) as a positive control. The supernatant was subsequently discarded, the protein A resin was washed off with PBS for four runs and then 50 µl PBS was added to the pellet for suspension. After mixing, the lysates were split in two parts: One part was used for western blotting analysis and the other part for RNA extraction and analysis via RT-qPCR to analyze the abundance of USP46 mRNA.

Flow cytometric analysis of apoptosis

Cells were plated in DMEM and Iscove's Modified Dulbecco's Medium at a density of 1×105 cells/ml into a 6-well plate for 24 h at 37°C. Then, cells were treated with 4 µg/ml DDP at 24 h post-transfection at 37°C for a further 24 h. Cells were collected at 1,000 × g, 4°C for 10 min and washed twice using cold PBS after 48 h of treatment. An Annexin V-FITC Apoptosis Detection kit (US Everbright, Inc.) was used for the detection of cell apoptosis. Briefly, cells were incubated with 5 µl Annexin V and 2 µl propidium iodide in binding buffer for 15 min in the dark at 25°C. Apoptotic cells were analyzed using a FACSAria fusion flow cytometer (BD Biosciences). CELL QUEST analysis software (BD Biosciences) was used to analyze cell apoptosis. The apoptotic rate was calculated via early apoptotic + late apoptotic cells (Q1-2+Q1-4).

Immunohistochemistry (IHC)

Briefly, 5-µm tumor sections were collected and deparaffinized at 60°C for 10 min followed by 15 min immersion in xylene. The sections were rehydrated via sequential incubation in 100, 90 and 70% ethanol. Samples were rinsed with PBS followed by distilled water and incubated for 30 min in H2O2. Antigen retrieval was performed via microwave irradiation for 10 min. The sections were incubated with an anti-USP46 antibody (1:100; cat. no. ab244215; Abcam) overnight at 4°C. Following primary antibody incubation, the sections were conjugated by SignalStain® Boost IHC Detection reagent (cat. no. 31926S; Cell Signaling Technology, Inc.) at 25°C for 2 h. The sections were then stained with DAB (cat. no. SK-4100; Vector Laboratories, Inc.; Maravai LifeSciences) and mounted with VECTASHIELD® PLUS Antifade mounting medium (cat. no. H-1900; Vector Laboratories, Inc.; Maravai LifeSciences). Sections were visualized using an Olympus 600 auto-biochemical analyzer and a light microscope (Olympus Corporation) at 40× magnification. The percentage of cells with positive staining was determined using ImageJ software (v1.8.0; National Institutes of Health).

Statistical analysis

All statistical analyses were performed using SPSS version 22 software (IBM Corp.). Statistical differences between 2 groups were determined using Student's t-test. One-way ANOVA with Tukey's post hoc test was used to analyze the statistical differences between multiple groups. Pearson's correlation analysis was performed to determine the correlation between USP46 and PUM2 expression levels. Receiver operator characteristic curve (ROC) analysis was used to detect the accuracy of USP46 to distinguish chemoresistant patients with OC from chemosensitive patients. All data are presented as the mean ± SD of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

Results

USP46 expression levels in OC tissues

As indicated by RT-qPCR analysis, USP46 expression levels were significantly downregulated in OC chemoresistant tissues compared with chemosensitive tissues (Fig. 1A). These data suggested that USP46 may be a potential target gene of DDP resistance. ROC analysis was conducted to detect the specificity of USP46 to distinguish chemoresistant patients with OC from those who were chemosensitive indicating USP46 could be used as a predictor of drug resistance in patients with OC. The results demonstrated a cutoff value of 3.501 and an area under the curve of 0.8676 (Fig. 1B). The results of western blotting and IHC experiments also revealed that the expression levels of USP46 were significantly downregulated in OC chemoresistant tissues compared with chemosensitive tissues (Fig. 1C and D). These results suggested that the downregulated expression levels of USP46 in patients with OC may represent a potential biomarker to determine chemoresistant characteristics.

Figure 1.

USP46 expression levels in OC tissues. (A) mRNA expression levels of USP46 in OC chemoresistant and chemosensitive tissues. (B) Receiver operating characteristic curve analysis was performed to assess the specificity of USP46 in predicting whether patients with OC were chemoresistant. (C) Western blotting and (D) immunohistochemistry (scale bar, 100 µm) were used to analyze the protein expression levels of USP46 in OC chemosensitive and chemoresistant tissues. *P<0.05. USP46, ubiquitin-specific protease 46; OC, ovarian cancer; AUC, area under the curve.

USP46 regulates OC cell proliferation and apoptosis

The expression levels of USP46 in SKOV3/DDP cells were significantly downregulated compared with SKOV3 cells (Fig. 2A). Therefore, to further investigate the effects of USP46 on cell proliferation and DDP resistance, numerous si-USP46s were transfected into SKOV3 cells and an OE-USP46 vector into SKOV3/DDP cells; the transfection efficiencies are shown in Fig. 2B and C. si-USP46-1 was selected for use in following experiments as it downregulated USP46 expression levels to the greatest extent. Cells were treated with DDP at different concentrations for 24 h and cell viability was subsequently analyzed. Compared with the si-NC group, SKOV3 cell viability was significantly increased in the si-USP46 group in SKOV3 cells treated with DDP in a dose-dependent manner. (Fig. 2D). Conversely, transfection of SKOV3/DDP cells with the OE-USP46 plasmid significantly decreased cell viability compared with the NC group treated with DDP in a dose-dependent manner. In addition, following the treatment of SKOV3 cells with DDP post-transfection with si-USP46, the cell apoptotic rate was significantly reduced compared with the si-NC-transfected cells. In contrast, following the overexpression of USP46, the apoptotic rate of SKOV3/DDP cells was significantly increased compared with the OE-NC group (Fig. 2E). These results suggested that USP46 may be a key factor involved in the DDP resistance of SKOV3/DDP cells.

Figure 2.

USP46 regulates ovarian cancer cell proliferation and apoptosis. (A) Protein expression levels of USP46 in SKOV3 and SKOV3/DDP cell lines were analyzed via western blotting. (B) Transfection efficiencies of siRNAs targeting USP46 in SKOV3 cells were analyzed using western blotting. (C) Transfection efficiency of OE-USP46 plasmid in SKOV3/DDP cells was analyzed using western blotting. (D) Following DDP treatment, the cell viability of SKOV3 or SKOV/DDP cells post-transfection with si-USP46 or OE-USP46 plasmid, respectively, was analyzed using a Cell Counting Kit-8 assay. (E) Following the treatment with DDP of SKOV3 or SKOV3/DDP cells transfected with si-USP46 or OE-USP46, respectively, the apoptotic rate was analyzed. *P<0.05. USP46, ubiquitin-specific protease 46; DDP, cisplatin; siRNA/si, small interfering RNA; NC, negative control; OE, overexpression.

Relationship between PUM2 and USP46

In order to detect the regulatory mechanism of USP46, bioinformatic analysis was applied and PUM2 was found to interact with USP46. In OC chemosensitive tissues, the mRNA and protein expression levels of PUM2 were significantly downregulated compared with the chemoresistant tissues (Fig. 3A and B). Furthermore, PUM2 expression levels were revealed to be negatively correlated with USP46 expression levels (r=−0.4208; P=0.0029; Fig. 3C). Notably, SKOV3/DDP cells exhibited significantly upregulated PUM2 expression levels compared with SKOV3 cells (Fig. 3D). As shown in Fig. 3E, the results of the RIP experiments revealed that PUM2 could bind to USP46 in SKOV3 and SKOV3/DDP cells. Furthermore, following overexpression of PUM2 in SKOV3 and SKOV3/DDP cells, the expression levels of USP46 were significantly downregulated compared with the OE-NC group (Fig. 3F and G). Following treatment with DDP, the transfection of si-PUM2 was able to partially reverse the si-USP46-induced increase in cell viability and decrease in apoptosis of SKOV3 cells (Fig. 3H and I). However, in SKOV3/DDP cells, the OE-USP46-induced suppression of cell viability and increase in apoptosis was partially reversed by the co-transfection with OE-PUM2 (Fig. 3J and K). The transfection efficiency of the OE-PUM2 vector (in both cell lines) and si-PUM2 vector (in SKOV3 cells) is shown in Fig. S1. These data suggested that USP46 may be a downstream gene of PUM2, and its function may be regulated by PUM2.

Figure 3.

Association between PUM2 and USP46. (A) mRNA and (B) protein expression levels of PUM2 in OC chemosensitive and chemoresistant tissues. (C) mRNA expression levels of USP46 and PUM2 in OC chemosensitive tissues were negatively correlated (r=0.4208, P=0.0029). (D) SKOV3/DDP cells exhibited significantly upregulated PUM2 expression levels compared with SKOV3 cells. (E) PUM2 could bind to USP46 in both SKOV3 and SKOV3/DDP cells, as confirmed using an RNA immunoprecipitation assay. Protein expression levels of USP46 were significantly downregulated in (F) SKOV3 and (G) SKOV3/DDP cells overexpressing PUM2. (H) Cell viability of SKOV3 cells transfected with si-USP46 with or without si-PUM2. (I) Apoptotic rate of SKOV3 cells following transfection with si-USP46 with or without si-PUM2. (J) Cell viability of SKOV3/DDP cells transfected with OE-USP46 plasmid with or without OE-PUM2 plasmid. (K) Apoptotic rate of SKOV3/DDP cells following the transfection with OE-USP46 plasmid with or without OE-PUM2 plasmid. *P<0.05. PUM2, pumillo RNA binding family member 2; USP46, ubiquitin-specific protease 46; DDP, cisplatin; OC, ovarian cancer; NC, negative control; si, small interfering RNA; OE, overexpression; FITC-A, Annexin V-FITC.

USP46 activates the Bcl-2/caspase-3 apoptotic signaling pathway and inactivates AKT activity

To determine the signaling pathway through which USP46 may regulate cell viability and apoptosis, the expression levels of the apoptosis-related genes, caspase-3, caspase-9, Bcl-2 and Bax, and the phosphorylation levels of AKT were analyzed using RT-qPCR and western blotting following the transfection of cells with si-USP46 or the OE-USP46 plasmid. After the transfection of SKOV3 cells with si-USP46, the expression levels of Bcl-2 were upregulated, while the expression levels of caspase-3, caspase-9 and Bax were downregulated, compared with the transfection with si-NC (Fig. 4A and B). Following the transfection of OE-USP46 in SKOV3/DDP cells, the reverse trend was observed (Fig. 4C and D). In addition, the levels of p-AKT were significantly increased following the knockdown of USP46 and reduced following USP46 overexpression compared with the respective negative controls in SKOV3 and SKOV3/DPP cells, respectively (Fig. 4E and F).

Figure 4.

USP46 activates the Bcl-2/caspase-3 apoptotic signaling pathway and inactivates AKT activity. (A) mRNA and (B) protein expression levels of Bcl-2, Bax, caspase-3 and caspase-9 in SKOV3 cells transfected with si-USP46. (C) mRNA and (D) protein expression levels of Bcl-2, Bax, caspase-3 and caspase-9 in SKOV3/DDP cells transfected with OE-USP46 plasmid. Expression levels of p-AKT in (E) SKOV3 cells following the knockdown of USP46 and (F) SKOV3/DDP cells following the overexpression of USP46. *P<0.05. USP46, ubiquitin-specific protease 46; si, small interfering RNA; NC, negative control; OE, overexpression; p-, phosphorylated; DDP, cisplatin.

Effects of USP46 on apoptosis and AKT signaling pathway activity are regulated by PUM2

The findings of the present study suggested that USP46 may be regulated by PUM2. Therefore, it was further determined whether the effects of USP46 on apoptosis and the AKT signaling pathway were also regulated by PUM2. In SKOV3 cells, knockdown of PUM2 expression could partially recover the effects of knockdown of USP46 expression on the mRNA and protein expression levels of Bcl-2, Bax, caspase-3 and caspase-9 (Fig. 5A and B). Similarly, in SKOV3/DDP cells, transfection with OE-PUM2 could partially reverse the OE-USP46-induced downregulation of Bcl-2 expression levels and upregulation of Bax, caspase-3 and caspase-9 expression levels (Fig. 5C and D). In addition, knockdown of PUM2 expression in SKOV3 cells partially recovered the si-USP46-induced increase in p-AKT and downstream p-mTOR levels (Fig. 5E). Conversely, transfection with OE-PUM2 in SKOV3/DDP cells partially recovered the OE-USP46-induced suppressive effects on p-AKT and p-mTOR expression levels (Fig. 5F).

Figure 5.

Effects of USP46 on apoptosis and the AKT/mTOR signaling pathway are regulated by PUM2. (A) mRNA and (B) protein expression levels of Bcl-2, Bax, caspase-3 and caspase-9 in SKOV3 cells transfected with si-USP46 with or without si-PUM2. (C) mRNA and (D) protein expression levels of Bcl-2, Bax, caspase-3 and caspase-9 in SKOV3/DDP cells transfected with OE-USP46 plasmid with or without OE-PUM2 plasmid. Expression levels of p-mTOR, mTOR, p-AKT and AKT in (E) SKOV3 cells following the transfection with si-USP46 with or without si-PUM2 and (F) SKOV3/DDP cells following the transfection with OE-USP46 plasmid with or without OE-PUM2 plasmid. *P<0.05. USP46, ubiquitin-specific protease 46; si, small interfering RNA; PUM2, pumillo RNA binding family member 2; p-, phosphorylated; OE, overexpression.

Discussion

Due to the ovaries being located deep in the pelvic cavity, the poor specificity of clinical manifestations and the lack of available biomarkers for the early diagnosis of OC, >70% of patients with OC are diagnosed with advanced tumors upon the first diagnosis and therefore, cannot be effectively treated (26,27), which results in an unfavorable prognosis. The main therapeutic regimen for OC is currently surgery-based with the addition of adjuvant chemotherapy. It is estimated that 70–80% of patients develop chemoresistance following initial surgery and standardized chemotherapy, although through standardized treatment, patients with OC exhibit favorable perioperative efficacy and prognosis. The development of OC chemoresistance is a complex process involving multiple factors, genes and stages, such as BRAF inhibitors and CD44 (28,29), and severely limits the quality of life and prognosis of patients with OC (6,30). The results of the present study revealed that USP46 expression levels were downregulated in OC chemoresistant tissues compared with chemosensitive tissues, suggesting that abnormal expression of USP46 may be crucial for the development of resistance in OC.

USP46 expression levels in DDP-resistant OC were also analyzed in vitro using the OC cell lines, SKOV3 and SKOV3/DDP. The findings revealed that SKOV3/DDP cells became sensitive to DDP and DDP-mediated apoptosis was enhanced through the overexpression of USP46. In contrast, SKOV3 cells became less responsive to chemotherapy treatment following the knockdown of USP46 expression. These findings suggested that USP46 may represent a promising marker to help identify chemoresistant and chemosensitive patients with OC who are receiving platinum-based chemotherapy.

Previous studies have reported that the majority of chemotherapeutic drugs kill cancer cells by promoting cell apoptosis, the levels of which are often notably decreased in drug-resistant cancer cells. Bcl-2, which is mainly found distributed in the mitochondria and rough endoplasmic reticulum, is involved in the intrinsic pathway of cell apoptosis by suppressing the oligomerization of Bax, thereby lengthening the life of the cell cycle (31). Bcl-2 was discovered to promote tumorigenesis by inducing the immortalization of injured cells, promoting cell proliferation and suppressing cell apoptosis (32). Bax induces the permeabilization of mitochondrial outer membranes, activates members of the caspase family and participates in the transduction of cell apoptosis signals (33,34). The present experimental results demonstrated that the knockdown of USP46 upregulated Bcl-2 expression levels and downregulated the expression levels of Bax, caspase-3 and caspase-9. Conversely, the overexpression of USP46 induced opposing effects on the expression levels of these apoptotic-related factors. These findings suggested that USP46 may affect the chemoresistance of OC cells via the Bcl-2/caspase-3 signaling pathway.

USP46 was previously reported to serve as a tumor suppressor by deubiquitinating PH domain leucine-rich-repeats protein phosphatase (PHLPP) and decreasing its protein degradation (17). PHLPP is a serine/threonine protein phosphatase that is crucial for maintaining cell homeostasis and signaling networks such as PI3K/AKT and MAPK signaling pathway by regulating several key protein kinases, including AKT and MAPK (16). The PI3K/AKT/mTOR signaling pathway is pivotal for cell survival and proliferation via its regulation of downstream signaling pathways, and is disturbed in the majority of human cancer types (35,36). In the present study, it was revealed that downregulation of USP46 expression levels could also promote the phosphorylation of AKT and mTOR, while the overexpression of USP46 could inhibit the phosphorylation levels. The aforementioned findings suggested that the proliferative ability of OC cells was affected by regulating AKT/mTOR phosphorylation, thereby regulating the chemoresistance of OC.

PUM2 is a RNA-binding protein and a positive regulator of cell proliferation (37). PUM2 has been found to block the formation of the translation initiation complex by binding to the 3′-UTR of specific target mRNAs, thereby suppressing the expression levels of target genes (20,21). Therefore, PUM2 is considered as a transcriptional inhibitor. Currently, >1,000 PUM2 binding motif-carrying mRNAs have been identified, including mRNAs involved in cell signaling pathways associated with cell proliferation and differentiation, indicating that PUM2 may be a post-transcriptional regulator of these genes, such as PNRC2 and LBA1 (38). PUM2 has been shown to be crucial for the development of mammalian neural stem cells (39), epilepsy (40) and the development of human germ cells (41), and was found to be associated with cell adhesion, cell migration, synaptic function, and the differentiation and development of neurons (42). Previous studies have also identified that PUM2 may be important in tumor occurrence and development. For example, PUM2 promoted the proliferation and migration of glioblastoma cells by repressing BTG anti-proliferation factor 1 expression (43). PUM2 inhibited osteosarcoma progression by the partial and competitive binding to the 3′-UTR of StAR related lipid transfer domain containing 13 with miRNAs (such as miR-590-3p and miR-9) (44). However, the roles of PUM2 in the process of resistance in OC and its targets remain unclear.

By using bioinformatics prediction analysis in the present study, PUM2 was predicted to bind to USP46; however, whether USP46 is regulated by PUM2 during the drug resistance process of OC is currently unknown. Therefore, the expression levels of PUM2 were first detected in OC tissues; the results revealed that PUM2 expression levels were significantly upregulated in chemoresistant tissues, and were negatively correlated with USP46 expression levels. In addition, following the overexpression of PUM2 in OC cells, USP46 expression levels were discovered to be downregulated. Further experiments revealed that PUM2 attenuated the effects of USP46 on the expression levels of apoptosis-related proteins and p-AKT/mTOR expression. Recovery experiments also revealed that the concurrent downregulation of PUM2 expression in SKOV3 cells was able to partially recover the USP46 knockdown-induced increase in proliferation and decrease in apoptosis. Furthermore, the overexpression of PUM2 in SKOV3/DDP cells partially recovered the suppressive effect over proliferation and the increased levels of apoptosis induced by the overexpression of USP46. Taken together, these findings suggested that USP46-mediated OC resistance may be under the regulation of PUM2.

In conclusion, the findings of the present study suggested that chemoresistant OC tissues had significantly downregulated USP46 expression levels compared with chemosensitive tissues. Furthermore, the results indicated that USP46 influenced cell proliferation and apoptosis by regulating the Bcl-2/caspase-3 signaling pathway and the phosphorylation levels of AKT, and these functions may be regulated by PUM2. Thus, USP46 may be a candidate target for the treatment of DDP-resistant OC.