& Peter Chang-Whan Lee1,2,3,* 
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.Despite recent advances in treatment strategies, gastric cancer is the fifth most common cancer and the third leading cause of cancer-related deaths (1). Surgery and systemic therapy are mainstream treatments for gastric cancer, and curative treatment with surgical resection is possible for patients with localized disease (2). However, for the remaining patients with inoperable or metastatic gastric cancer, combination chemotherapy (including 5-fluorouracil (5-FU)-based regimens) has become the first line of systemic treatment, based on the findings of randomized phase III trials (3-5). Unfortunately, most advanced-stage patients are resistant to this conventional chemotherapy, and their prognosis remains poor, with a 5-year relative survival rate of 6% (6). Therefore, the development of novel targeted drugs capable of suppressing chemoresistance in advanced gastric cancer is essential, as these therapies have the potential to prolong patient survival periods.
The ubiquitin-proteasome system (UPS) is a crucial regulator of various functions in cells; it determines the fate of most proteins in eukaryotic cells (7, 8). Ubiquitin (Ub) ligase action can be counteracted by deubiquitinases (DUBs) that cleave a single ubiquitin or entire ubiquitin chains from target proteins (9, 10). DUBs are involved in various physiological processes, such as apoptosis, autophagy, and the cell cycle, as well as pathological processes, including neurodegenerative diseases (7, 11-14). Currently, approximately 100 DUBs have been identified, some of which are implicated in the development of various types of cancer (15-17). Among them, USP14 has been linked to the progression of various cancers, including gastric cancer (18). The inhibition of USP14 activity triggers antiproliferative effects and apoptosis, which result in cell death (15, 19, 20). Although the precise function of USP14 in gastric cancer remains unclear, its expression was found to be elevated in gastric cancer tissues; thus, USP14 may serve as an independent marker for disease-free survival in gastric cancer patients (21, 22).
We investigated the mechanisms underlying the anticancer effects of the IU1-induced inhibition of USP14 activity. Our results indicated that IU1 successfully inhibited the activity of USP14 and markedly reduced the growth of gastric cancer cells. Furthermore, IU1 treatment enhanced the sensitivity of gastric cancer cells to 5-FU chemotherapy and suppressed 5-FU resistance in these cells. These results indicate that USP14 may serve as a novel target for the treatment or diagnosis of gastric cancer.
To investigate the USP14 expression in gastric cancer, we analyzed the protein level of USP14 in 48 pairs of patient-derived gastric cancer tissues and adjacent non-tumor tissues using IHC analysis and western blotting. The USP14 expression was found to be significantly higher in tumor tissues than in the adjacent non-tumor tissues (Fig. 1A, B and Supplementary Fig. 1). Additionally, we performed the RT-PCR in 76 pairs of patients-derived gastric cancer tissues to evaluate the mRNA level of USP14 and significantly elevated USP14 mRNA in gastric cancer tissues were observed (Fig. 1C). Furthermore, we conducted a survival analysis based on the USP14 expression level in patients with gastric cancer using Kaplan-Meier plotter (http://kmplot.com/analysis/) and observed a positive correlation between the USP14 expression level and poor prognosis (Fig. 1D). These results suggest that USP14 overexpression is strongly associated with tumor formation in gastric cancer and the prognosis of gastric cancer patients.
To confirm the role of USP14 in tumorigenesis in gastric cancer, we evaluated the effects of the inhibition of USP14 activity using IU1 or the inhibition of USP14 expression using USP14-specific siRNA on the proliferation of MKN74 and SNU216 gastric cancer cells. Treatment with IU1 or USP14-specific siRNA resulted in a significant dose-dependent reduction in cell proliferation compared to the proliferation of the control cells treated with dimethyl sulfoxide only (Fig. 2A and Supplementary Fig. 2A). We further investigated the effects of the inhibition of USP14 activity using IU1 (100 μM) or the inhibition of USP14 expression using USP14-specific RNA on the migration and wound-healing abilities of MKN74 and SNU216 cells using colony formation, Transwell migration, and wound healing assays. The results showed that cells treated with IU1 and USP14-specific siRNA exhibited markedly lower proliferation and migration abilities and slower wound healing than the control cells (Fig. 2B-D and Supplementary Fig. 2B-D). Therefore, the decrease in the activity of USP14 after treatment with the USP14 inhibitor IU1 and the decrease in the expression of USP14 after the treatment with the USP14-specific siRNA effectively suppressed the proliferation, migration, and wound-healing abilities of gastric cancer cells.
As previously shown in the present study, the inhibition of USP14 activity suppressed the proliferation of gastric cancer cells. To validate the downstream mechanisms underlying the effects of IU1 and USP14 knockdown in the inhibition of gastric cancer cell growth, we evaluated the expression levels of apoptosis- and autophagy-related proteins in MKN74 and SNU216 cells subjected to IU1 treatment and USP14 knockdown. Apoptosis that occurred via apoptosis-related proteins, such as PARP, Caspase3, and p21, was induced in the IU1-treated and cells showing USP14 knockdown (Fig. 3A, C). In addition, IU1 treatment enhanced the induction of apoptosis mediated by the cleaved PARP protein in a time-dependent manner (Fig. 3B). The expression levels of the autophagy-related proteins LC3 I and II and Beclin-1 remained unchanged after IU1 treatment and USP14 knockdown (Fig. 3A, C).
Subsequently, we detected apoptotic MKN74 and SNU216 cells, which showed positive staining with annexin-V, to demonstrate the apoptosis mechanism associated with the role of the USP14 protein in gastric cancer. The FACS analysis further confirmed that apoptosis was induced in IU1- and siRNA-treated cells (Fig. 3D, MKN74-IU, P = 0.008; MKN74-siRNA, P = 0.041; SNU216-IU, P = 0.024; SNU216-siRNA, P = 0.024). Additionally, we evaluated the expression level of PARP after overexpression of USP14 with or without IU1 to prove the USP14-mediated apoptotic regulation of IU1 in gastric cancer. As expected, decreased cleaved form of PARP and caspase3 with USP14 overexpression was reversed after IU1 treatment (Fig. 3E). Collectively, these results indicate that the inhibition of USP14 activity using IU1 and USP14 knockdown reduce gastric cancer growth via apoptotic mechanisms.
To evaluate the therapeutic potential of the inhibition of USP14 activity against drug-resistant gastric cancer, 5-FU-resistant SNU620 cells were treated with IU1. The results showed that the combined application of IU1 and 5-FU in SNU620 cells enhanced the apoptosis of SNU620 cells compared to the case after the treatment with 5-FU alone (Fig. 4A). Additionally, the combined treatment exhibited a similar effect on cell growth inhibition, migration and invasion (Fig. 4B and Supplementary Fig. 3). In 5-FU-resistant SNU620 cells, apoptosis did not occur after treatment with 5-FU alone, but the degree of apoptosis increased after the combined treatment with IU1 and 5-FU (Fig. 4C); the same effects were observed regarding cell growth inhibition, migration and invasion (Fig. 4D and Supplementary Fig. 3).
These results show that the USP14 inhibitor IU1 is effective in suppressing 5-FU resistance. Thus, the inhibition of USP14 activity suppresses 5-FU resistance in gastric cancer cells.
Gastric cancer has a high mortality rate, and targeted therapies are limited in patients with advanced disease (23, 24). Although chemotherapy, including 5-FU treatment, is a standard treatment approach for advanced gastric cancer, its effectiveness is often limited due to the development of drug resistance in patients (25, 26). This study aimed to investigate the role of USP14 in gastric cancer tumorigenesis and demonstrate the feasibility of using USP14 as a novel target for the treatment of advanced gastric cancer.
Our results indicated that the expression of USP14 increased in gastric cancer cells; the inhibition of its expression reduced gastric cancer cell growth via the induction of apoptosis. In addition, the inhibition of USP14 activity using IU1 effectively suppressed 5-FU resistance.
Among the various DUBs, USP14 is extensively engaged in various canonical cellular signaling pathways, including the nuclear factor kappa B (NF-κB) and Wnt/β catenin signaling pathways. Dysregulation of USP14 activity has been associated with multiple pathological conditions, and its overexpression has been observed in various cancers, such as head and neck, breast, lung, and gastric cancers; high levels of USP14 have been associated with a poor prognosis among cancer patients (27). Therefore, an inhibitor targeting USP14, IU1, has been developed but only a few studies have investigated the effects of IU1 on cancer progression and downstream signaling pathways in cancer cells (28).
We observed an increase in USP14 expression in gastric cancer; the inhibition of its activity using the USP14 inhibitor IU1 and the inhibition of USP14 expression using USP14-specific siRNA reduced gastric cancer cell growth. These results suggest that the USP14 has the potential to serve as a prognostic biomarker and a treatment target for gastric cancer.
The mechanisms underlying the anti-tumor effects of the inhibition of USP14 activity are diverse, and discordant results have been reported for different types of cancers. Cell cycle arrest, apoptotic pathway, and autophagic pathway have emerged as regulatory mechanisms underlying the role of USP14 in tumorigenesis. Further, in our previous study, we reported that USP14 regulates the proliferation of lung cancer cells, but it does so via the modulation of autophagy and not apoptosis (19). Although the apoptotic mechanism by inhibition of USP14 is not clear yet, the inhibition of Notch1 and Wnt/β-catenin signlaing lead to apoptosis of breast and hepatocellular carcinoma cell (29). In gastric cancer, inhibition of USP14 activity has been found to induce apoptosis through the inactivation of the Akt and ERK signaling pathways (18, 22, 30). Interestingly, in the present study, USP14 was found to regulate gastric cancer cell growth via induction of the apoptotic pathway.
As previously mentioned, the response rate of 5-FU-based chemotherapy in advanced gastric cancer is lower than 32% due to 5-FU resistance (31, 32); thus, circumventing 5-FU resistance represents a challenge for treating this disease. Several factors, including the degradation of 5-FU by dihydropyrimidine dehydrogenase, increased deoxyuridine triphosphatase activity, and the overexpression of thymidylate synthase, B-cell lymphoma 2 (BCL2), BCL-XL, and the MCL1 apoptosis regulator BCL2 family member protein have been investigated as mechanisms involved in 5-FU resistance (33, 34). Surprisingly, in the present study, the inhibition of USP14 activity using IU1 was found to suppress 5-FU resistance by enhancing the apoptotic pathway in 5-FU-resistant gastric cancer cells. These results show that USP14 may serve as a novel target for treating 5-FU-resistant gastric cancer.
In conclusion, USP14 regulates gastric cancer cell growth via the apoptotic pathway. Inhibition of USP14 activity suppresses 5-FU resistance in gastric cancer cells; however, the specific mechanisms underlying this process remain unclear. Our research suggests that USP14 is a useful prognostic biomarker and a valuable treatment target for advanced gastric cancer. However, further studies are urgently required to identify the precise mechanism whereby USP14 inhibitors, such as IU1, suppress 5-FU resistance and to validate the effectiveness of using IU1 for treating patients with advanced gastric cancer.
Written informed consent was obtained from all the gastric cancer patients who participated in this study. All experimental protocols were approved by the Institutional Review Board of the Asan Medical Center and the University of Ulsan College of Medicine (2016-1275). Human gastric tissues were obtained from the Asan Bio-Resource Center (2016-24(136).
All materials and methods of cells culture and transfection were referred to previous study (19). MKN74 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM). The SNU216, SNU620, and 5FU resistance SNU620 cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and cultured in Roswell Park Memorial Institute (RPMI) supplemented with 10% FBS. The MKN74, SNU216 and SNU 620 cells were maintained in a 5% CO2 incubator at 37°C. The cells were transfected with a plasmid using iNfect (Intron, Seoungnam, South Korea) according to the manufacturer’s instructions, and the transfected cells were harvested for analysis after 48 h. Lipofectamine RNAiMax (Invitrogen) was used for USP14 knockdown using USP14-specific siRNA, according to the manufacturer’s guidelines. The sequences of USP14 specific siRNA -1 and -2 were 5’-GUU GAG AGC UUC AGG AGA A-3’ and 5’-GGA UAC AAA UGA UGA GAG U-3’, respectively.
The cells were lysed with 1% SDS-lysis buffer (40 mM Tris pH 8.0, 150 mM NaCl, 1% SDS, and 1 mM EDTA); the lysates were collected and the protein levels were measured using a BCA protein assay kit (Pierce, Thermo Fisher, Rockford, IL, USA). Subsequently, western blotting was performed to analyze the expression of various proteins using primary antibodies against USP14 (Novus, Centennial, CO, USA), LC3 (Cell Signaling, Danvers, MA, USA), Beclin-1 (Cell Signaling, Danvers, MA, USA), PARP (Cell Signaling), Bcl-2 (Santa Cruz, Dallas, TX, USA), Caspase-3 (Cell signaling), cleaved Caspase-3 (Cell Signaling), HA (Covance, Biolegend, San Diego, CA, USA), and β-actin (Santa Cruz). A secondary anti-mouse antibody (BETHYL) was used for the western blotting analysis; the protein bands were detected using an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA).
RNA was extracted from chopped tissue samples using the easy-BLUETM kit, and the total RNA concentration was determined by measuring the optical density of the samples at 260 nm. A260/A280 ratios greater than 1.8 were considered as a criterion for assessing the purity of the RNA samples. A total of 1 μg of RNA was reverse-transcribed into cDNA using a cDNA Synthesis Kit (iNTRON). Real-Time PCR 2x Master Mix (SYBR green, EBT-1801, Elpis Biotech) was utilized for the amplification of cDNA using the following primer pairs spanning various exons: human USP14 and GAPDH. The PCR thermocycling conditions for the duplication of USP14 and GAPDH cDNA were as follows: 1 cycle of denaturing at 95°C for 5 min, and 30 cycles at 95°C for 30 s/58°C for 30 s/72°C for 90 s, followed by final extension at 72°C for 5 min. The forward and reverse sequences of USP14 and GAPDH were 5’-GCAGAGAAGGGCGAACAAG-3’, 5’-TGTTGCAGGACTCTCATCATTTG-3’ and 5’-ATCACTGCCACCCAGAAGACT-3’, 5’-CATGCCAGTGAGCTTCCCGTT-3’, respectively.
Immunohistochemical analysis was performed using formalin fixed, paraffin-embedded samples. After deparaffinization with xylene (10 min, twice), the sections were rehydrated using 100% ethanol (twice) and subsequently, using a graded series of ethanol solutions with decreasing concentrations (95%, 85%, and 70%), followed by washing for 5 min. Two sections were obtained from each sample. The samples were stained using anti-USP14 antibodies (Novus, Centennial, CO, USA).
To investigate the degree of cell death, an FITC annexin-V apoptosis detection kit (BD Bioscience) was used in accordance with the manufacturer’s guidelines; the stained cells were analyzed using FACS.
All materials and methods of cell viability, wound healing, colony formation, migration and invasion assays refer to previous study (19). To evaluate cell viability, the cells were seeded at a density of 1 × 103 cells/well in a 96-well cell culture plate and observed on days 0-3. At each analysis time point, the Cell Titer-Glo reagent (Promega, Madison, WI, USA) was added to the wells and the plate was incubated for 5 min on a shaker. For the wound-healing assay, the cells were seeded at a density of 8 × 105 cells/well in a 6-well cell culture plate. Next, the cell monolayer was scratched using a 200-μl pipette tip, followed by washing twice with PBS; the degree of wound closure was observed under a microscope on days 0-2. For the colony-formation assay, the cells were seeded at a density of 1 × 103 cells/well in a 6-well cell culture plate, followed by incubation for 1-2 weeks. Next, the cells were washed with PBS, fixed using 4% paraformaldehyde for 20 min, washed twice with PBS, and stained with 0.5% crystal violet for 20 min. For the Transwell migration assay, the cells were seeded at a density of 3 × 105 cells in the upper chamber of an 8-μm Transwell insert that was plated in a 24-well cell culture plate. The lower chamber was filled with medium supplemented with 10% FBS and 1% penicillin-streptomycin and incubated for 24 h. The cells that had migrated into the Transwell inserts were washed with PBS, fixed using 4% paraformaldehyde for 20 min, washed with PBS, and stained with 0.5% crystal violet for 20 min. The cells remaining in the upper chamber of the Transwell insert were removed with a swab; the stained migrated cells were analyzed under a microscope. For the Transwell invasion assay, the cells were seeded at a density of 3 × 105 cells in the upper chamber of an 8 μm transwell insert coated with 1 mg of matrigel (BD Bioscience). The process after this is the same as Transwell migration assay.
The authors have no conflicting interests.
Fig. 1. USP14 expression level in gastric cancer. (A) Representative immunohistochemistry (IHC) for USP14 in paired tumor and adjacent non-tumor tissues. Scale bar, 50 μm. N: Non-tumor tissue, T: Tumor tissue. (B) Pie chart representing the number of patients stratified based on the expression level of USP14 protein. (C) mRNA expression of USP14 in 76 gastric cancer cases. The P-values represent the probabilities of USP14 mRNA levels between variable subgroups, as determined using the paired t-test (P = 0.0008). (D) Kaplan-Meier representation of the overall survival between two groups of patients with high (red line) and low (black line) USP14 protein expression. All data shown were collected from three independent experiments (P < 0.0001).
Fig. 2. Inhibition of USP14 activity using the USP14 inhibitor IU1 or the inhibition of USP14 expression using USP14-specific siRNA decreases gastric cancer cell growth. (A) Effects of the inhibition of USP14 activity using IU1 (50 μM and 100 μM, left) or USP14 knockdown (right) on the viability of SNU216 cells (n = 3, *P < 0.05, **P < 0.01). (B) Effects of the inhibition of USP14 activity using IU1 (left) or USP14 knockdown (right) on the colony-forming ability of MKN74 and SNU216 cells (n = 3). (C) Effects of the inhibition of USP14 activity using IU1 (left) or USP14 knockdown (right) on the migration ability of MKN74 and SNU216 cells. Scale bar = 100 μm. (D) Effects of the inhibition of USP14 activity with IU1 (left) or USP14 knockdown (right) on wound healing ability of SNU216 cells (n = 3, *P < 0.05, **P < 0.01).
Fig. 3. Inhibition of USP14 activity induces apoptosis in gastric cancer cells. (A) MKN74 and SNU216 cells were treated with IU1 and lysed; they were then subjected to immunoblotting analysis. (B) MKN74 and SNU216 cells were treated with IU1 for 48 h and subjected to immunoblotting analysis. (C) MKN74 and SNU216 cells were subjected to USP14 knockdown, followed by immunoblotting analysis. (D) FACS analysis, along with the annexin-V apoptosis assay, of MKN74 and SNU216 cells after IU1 treatment and USP14 knockdown. (E) MKN74 cells were overexpressed with USP14 plasmid and treated with or without IU1; then, they were subjected to immunoblotting analysis.
Fig. 4. Inhibition of USP14 activity suppressed 5-FU resistance in gastric cancer cells. (A) Immunoblotting analysis for assessing the levels of USP14 and PARP levels in SNU620 cells after treatment with DMSO, 5-FU (100 μM), and IU1 (100 μM) and co-treatment with 5-FU/IU1. (B) Effects of treatment with 5-FU (100 μM) and IU1 (100 μM) and co-treatment with 5-FU/IU1 on the viability of SNU620 cells. (n = 5, *P < 0.05, **P < 0.01). (C) Immunoblotting analysis for assessing the levels of USP14 and PARP after treatment with DMSO, 5-FU (100 μM), and IU1 (100 μM) and co-treatment with 5-FU/IU1 in 5-FU-resistance SNU620 cells. (D) Effects of treatment with 5-FU (100 μM) and IU1 (100 μM) and co-treatment with 5-FU/IU1 on the viability of 5-FU-resistant SNU620 cells (n = 5, **P < 0.01, ***P < 0.001).
Author ORCID Information
Jun-O Jin Peter Chang-Whan Lee
Funding Information National Research Foundation of Korea Ministry of Science and ICT, South Korea |
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