Knockdown of ubiquitin‑specific protease 51 attenuates cisplatin resistance in lung cancer through ubiquitination of zinc‑finger E‑box binding homeobox 1.

Lung cancer is a devastating cancer with high morbidity and mortality. Ubiquitin‑specific protease (USP) is a type of deubiquitinating enzyme (DUB) that has been implicated in numerous cancers, including colorectal, myeloma and breast. In the present study, the expression of USP51 was determined in the lung cancer cell line A549 and cisplatin (also known as DDP)‑resistant lung cancer strain A549/DDP. The expression of zinc‑finger E‑box binding homeobox 1 (ZEB1), a transcriptional repressor, was also examined. The effects of USP51 knockdown or overexpression on proliferation and apoptosis, as well as the impact of ZEB1 overexpression and USP51 interference on apoptosis and ubiquitination were then assessed. Notably, increased expression of USP51 and ZEB1 in A549/DDP cells was observed, and treatment with DDP significantly inhibited proliferation in A549/DDP cells. In addition, knockdown of USP51 in A549/DDP cells significantly induced apoptosis, decreased ZEB1 expression and increased cleaved poly ADP‑ribose polymerase 1 (PARP1) and cleaved caspase‑3 levels. Consistently, USP51 overexpression in A549 cells displayed the opposite effects and potently attenuated DDP‑induced apoptosis. Notably, overexpression of ZEB1 in A549/DDP cells potently attenuated the effects of USP51 knockdown on apoptosis, and co‑IP experiments further demonstrated interaction between USP51 and ZEB. Lastly, knockdown of USP51 promoted ZEB1 ubiquitination, leading to ZEB1 degradation. Collectively, the present findings demonstrated that USP51 inhibition attenuated DDP resistance in A549/DDP cells via ubiquitin‑mediated degradation of ZEB1. Hence, targeting USP51 may serve as a novel therapeutic target for DDP resistance in lung cancer.

Introduction

Lung cancer is among the most malignant of human cancers, with escalating growth in morbidity and mortality. In the past 50 years, lung cancer incidence and mortality have increased worldwide, ranking first and second as the most malignant cancer in men and women, respectively (1–3). At present, the pathogenesis of lung cancer remains elusive. Past research has associated lung cancer occurrence to long-term, large-scale smoking, and smokers are 10 to 20 times more likely to develop lung cancer than non-smokers (4–6).

Lung cancer mortality is mostly attributed to tumor invasion and metastasis (7,8). Studies have revealed that epithelial-mesenchymal transition (EMT) serves an essential role in tumor metastasis (9–12). Zinc-finger E-box binding homeobox 1 (ZEB1), a transcriptional repressor, is a crucial inducer of EMT in a variety of human cancers, such as colorectal and breast (13,14). ZEB1 contains two zinc finger clusters on the N-terminal and C-terminal regions, which bind to the E-Box sequence (CACCT) or similar sequence (CACCG), thereby regulating downstream target gene expression. ZEB1 has been revealed to promote tumor cell metastasis, invasion and therapy resistance (15–20). Studies have revealed that decreased expression of the miR-200 family of microRNAs, including miR-200a, miR-200b and miR-200c, is often accompanied with increased ZEB1 expression, which is known to downregulate the CDH1 gene, thus suppressing EMT (21–24). This regulatory pathway has been confirmed in other cancers, including colon cancer and head and neck squamous cell carcinoma (21,25). ZEB1 expression has been associated with treatment resistance in multiple cancers (9,16,18,26), and inhibition of ZEB1 was revealed to reverse chemoresistance in docetaxel-resistant human lung cancer cells (27).

Ubiquitin-specific protease (USP) is a type of deubiquitinating enzyme (DUB). DUBs are known to regulate both proteolytic degradation and non-proteolytic processes, including kinase activation, gene transcription and cell cycle progression. USP51 is a ZEB1-binding DUB that promotes ZEB1 deubiquitination and stabilization (28). USP51 can deubiquitinate histones to prevent aberrant DNA repair and can also regulate tumor growth (29,30). However, the functions of USP51 and ZEB, and whether they are associated, in lung cancer drug resistance have not been elucidated.

In the present study, it was revealed that USP51 and ZEB1 expression was increased in cisplatin (also known as DDP)-resistant lung cancer strain A549/DDP, and A549/DDP cell proliferation was inhibited by treatment with 100 µmol/l DDP. Knockdown of USP51 in A549/DDP cells significantly promoted apoptosis, decreased ZEB1 expression, and increased cleaved poly ADP-ribose polymerase 1 (PARP1) and cleaved caspase-3 protein levels, while USP51 overexpression displayed the opposite outcomes and potently attenuated the effects induced by DDP. Furthermore, overexpression of ZEB1 in A549/DDP cells weakened the effects of USP51 knockdown. Lastly, USP51 and ZEB1 were revealed to interact by co-IP experiments, and USP51 knockdown promoted ZEB1 ubiquitination and degradation. Collectively, these findings indicated that USP51 and ZEB1 may serve crucial roles in DDP resistance in lung cancer.

Materials and methods

Cell culture

Cisplatin (also known as DDP)-resistant lung cancer strain A549/DDP, parental A549 cell line, and normal lung bronchial epithelial 16HBE cell line were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 medium (product no. SH30809.01B; Logan; GE Healthcare Life Sciences) containing 10% FBS (cat. no. 16000-044; Gibco; Thermo Fisher Scientific, Inc.) and 1% double antibody (penicillin and streptomycin; cat. no. P1400-100; Beijing Solarbio Science & Technology Co., Ltd.) at 37°C in a 5% CO2 humidified-incubator (Thermo Forma 3111; Thermo Fisher Scientific, Inc.).

Construction of lentiviral constructs

Targeting different sites of the USP51 gene (NM_201286.3), three short hairpin RNA (shRNA) sequences were synthesized (Table I) and double-strand annealed to form three shRNA constructs which were then inserted into the pLKO.1-puro vector (Addgene, Inc.) at AgelI/EcoRI restriction sites. The coding DNA sequence (CDS) region of USP51, full-length of 2,136 bp, as well as ZEB1, were respectively synthesized (cat. no. 10878; Genewiz, Inc.) and inserted into the EcoRI/BamHI restriction sites of the pLVX-Puro (Clontech Laboratories, Inc.) vector. After confirmation of DNA sequencing (Shanghai Meiji Biomedical Technology Co., Ltd.), Lipofectamine® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.; according to the manufacturer's protocol) was used to transfect 3 µg pLKO.1-shUSP51, 4 µg pLVX-Puro-USP51 or 4 µg pLVX-Puro-ZEB1 into 2×105 293T cells/well in 6-well plates along with two viral packaging plasmids, psPAX2 and pMD2G (Addgene, Inc.). The virus particles in the medium were collected by ultracentrifugation (8,000 × g; 4°C; 2 h) following 48 h of transfection at 37°C.

Table I.

Short hairpin RNA sequences for ubiquitin-specific protease 51.

Gene	Sequence (5′→3′)
USP51 target site 1 (895–913)	CCATTTAGCTGTAGACCTT
USP51 target site 2 (1803–1821)	GCTACCAGGAGTCTACTAA
USP51 target site 3 (2152–2170)	GGACTTACTCTACAGTGAA
shNC	UUCUCCGAACGUGUCACGU

sh/shRNA, short hairpin RNA; NC, negative control.

Experimental grouping

A549 cells were infected with USP51 overexpression (USP51) or control vector (empty plasmid). A549/DDP cells were infected with USP51 interference (shUSP51-1/-2/-3) or negative control (shNC) vector. A549 or A549/DDP cells treated with RMPI-1640 medium were used as controls. Efficiency of shUSP51 and USP51 lentiviruses was determined by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Following treatment with gradient concentrations of DDP (0, 50, 100, 200, 400, 800 µmol/l), cell proliferation was assessed.

Next, A549/DDP cells were divided into seven groups to receive treatment as follows: i) shNC group which received shNC + 100 µmol/l DDP; ii) shUSP51-1 group which received shUSP51-1 + 100 µmol/l DDP; iii) shUSP51-2 group which received shUSP51-2 + 100 µmol/l DDP; iv) Vector group which received Vector + 5 µmol/l DDP; v) USP51 group which received USP51 + 5 µmol/l DDP; vi) DDP + Vector + shNC group which received DDP + Vector + sh-NC; and vii) DDP + ZEB1 + shNC which received DDP + ZEB1 + sh-NC. Apoptosis and expression of related-genes were then examined. A co-immunoprecipitation (CO-IP) assay was performed to determine interaction between USP51 and ZEB1. After USP51 interference, ZEB1 ubiquitination was detected.

Cell proliferation assay

A549 and A549/DDP cells in logarithmic growth phase were trypsinized and resuspended in fresh medium. Cell suspension (3,000 cells/well) was added into 96-well plates and cultured overnight in a 5% CO2 incubator at 37°C. The following day, the cells were cultured with RPMI-1640 media containing gradient concentrations of DDP (0, 50, 100, 200, 400, 800 µmol/l). After 0, 24, 48, 72 h of culture, 100 µl Cell Counting Kit-8 (CCK-8; cat. no. CP002; Signalway Antibody LLC) solution (CCK-8 to serum-free medium, 1:10) was added, according to the manufacturer's protocol, and cells were incubated for 1 h. Cell proliferation was assessed by measuring the absorbance value (OD) at 450 nm using a microplate reader (DNM-9602; Perlong Medical Equipment Co., Ltd.).

RT-qPCR

Total RNA from A549 or A549/DDP cells with the indicated treatments was extracted using TRIzol® reagent (cat. no. 1596-026; Invitrogen; Thermo Fisher Scientific, Inc.). After quantification and confirmation of RNA integrity, extracted 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. Using cDNA as templates, qPCR was conducted on an ABI 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with a SYBR® Green PCR kit (Thermo Fisher Scientific, Inc.). The following thermocycling conditions were used: 95°C for 10 min; 40 cycles of 95°C for 15 sec and 60°C for 45 sec (31). Thereafter, the mRNA expression of USP51 and ZEB1, relative to GAPDH, was analyzed by 2−ΔΔCq method (32). The primers were as follows: USP51 forward, 5′-CCTCAGACACGGAGAAGC-3′ and reverse, 5′-GGACCCTGACCAAACTCG-3′; ZEB1 forward, 5′-AATGTACTTAAAGTGGCGGTAG-3′ and reverse, 5′-ATGGCTGAAATAACAGAATGG-3′; GAPDH forward, 5′-AATCCCATCACCATCTTC-3′ and reverse, 5′-AGGCTGTTGTCATACTTC-3′.

Western blot analysis

Using RIPA buffer containing protease and phosphatase inhibitors (cat. no. R0010, Beijing Solarbio Science & Technology Co., Ltd.), total protein from A549 or A549/DDP cells with the indicated treatments was isolated. After quantification by a BCA kit (cat. no. PICPI23223; Thermo Fisher Scientific, Inc.), proteins (~25 µg) were subjected to 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (cat. no. HATF00010; EMD Millipore) by a semi-dry transfer. Following 1 h of blocking in 5% skimmed milk (cat. no. BYL40422; BD Biosciences) at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: USP51 (1:1,000; cat. no. PA5-68358; Invitrogen; Thermo Fisher Scientific, Inc.), ZEB1 (1:1,000; cat. no. ab124512; Abcam), cleaved PARP1 (1:3,000; cat. no. ab32064; Abcam), cleaved caspase-3 (1:3,000; cat. no. ab32351; Abcam) and GAPDH (1:2,000; cat. no. 5174; Cell Signaling Technology, Inc.). The membranes were washed six times with TBS-0.1% Tween 20 (TBST) and subsequently incubated with goat anti-rabbit (cat. no. A0208) secondary antibodies labeled with horseradish peroxidase (HRP; 1:1,000; Beyotime Institute of Biotechnology) at room temperature for 1 h. The membranes were washed again with TBST and subsequently developed using a chemiluminescent reagent (cat. no. WBKLS0100; EMD Millipore) and exposed on an ECL imaging system (Tanon-5200; Tanon Science and Technology Co., Ltd.). Relative protein expression was normalized to GAPDH and calculated using ImageJ version 1.47v software (National Institutes of Health).

Flow cytometric analysis of apoptosis

Flow cytometric analysis was employed to evaluate apoptosis in A549 or A549/DDP cells. After treatment, according to the experimental grouping, A549 or A549/DDP cells were subjected to Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining (cat. no. C1063; Beyotime Institute of Biotechnology) assay according to the manufacturer's protocol. Briefly, 5×105−1×106 cells were resuspended in 195 µl Annexin V-FITC binding buffer and then incubated with 5 µl Annexin V-FITC for 15 min, followed by 5 min of incubation in 5 µl PI at 4°C in the dark. Cells without Annexin V-FITC and PI were used as a negative control. Percentages of apoptotic cells were analyzed by flow cytometry and evaluated by the BD Accuri™ C6 Software (version 1.0.264.21; BD Biosciences).

CO-IP and ubiquitination detection

Proteins [protein was isolated using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing protease and phosphatase inhibitors] isolated from A549 or A549/DDP cells with treatments according to experimental grouping were incubated with rabbit-IgG (1:400; 1 µg; cat. no. sc-2027; Santa Cruz Biotechnology, Inc.) or IP-indicated antibody (1 µg) overnight at 4°C. Appropriate amounts of extracted proteins served as input controls. Then, 30 µl of Protein A/G PLUS-Agarose was respectively added to aforementioned two tubes and incubated at 4°C for 2 h to form an immune complex. The solution was centrifuged at 2,500 × g for 4 min in 4°C, and the Protein A/G Plus-Agarose beads were washed four times with 1 ml lysate. Appropriate volumes of SDS-PAGE sample loading buffer (cat. no. P1015; Beijing Solarbio Science & Technology Co., Ltd.) were added and samples were boiled for 5 min, followed by 1 min of centrifugation at 2,500 × g at 4°C. The supernatants were collected for western blot analysis. Anti-ZEB1 antibody (1:500; cat. no. 21544-1-AP; ProteinTech Group, Inc.) and an anti-USP51 antibody (1:500; cat. no. orb181545; Biorbyt Ltd.) were used for IP. Anti-ZEB1 antibody (1:100; cat. no. ab124512; Abcam), anti-USP51 antibody (1:1,000; cat. no. PA5-68358; Thermo Fisher Scientific, Inc.), anti-Ubiquitin antibody (1:2,000; cat. no. ab7780; Abcam) and goat anti-rabbit HRP-labeled secondary antibody (1:2,000; cat. no. A0208; Beyotime Institute of Biotechnology) were used for western blotting, which was performed as previously described.

Statistical analysis

GraphPad prism 7.0 software (GraphPad Software, Inc.) was applied for statistical analysis. One-way ANOVA followed by Tukey's post hoc test was used to determine significance among multiple comparisons. All data were presented as the mean ± SD of three repeated experiments. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression of USP51 and ZEB1 is significantly increased in A549 or A549/DDP cells

It was previously reported that A549/DDP cells acquired an EMT phenotype, with morphological changes including acquisition of a spindle-like fibroblastic phenotype, downregulation of E-cadherin and upregulation of mesenchymal markers (33). After treatment with gradient concentrations of DDP (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 µmol/l), cell proliferation was detected to determine drug resistance of A549/DDP cells to DDP. As revealed in Fig. 1A, the half-maximal inhibitory concentration (IC50) of A549/DDP cells was significantly higher than that of A549 cells, which confirmed that A549/DDP cells were DDP resistant. Consistent with previous studies (34,35), 100 µmol/l of DDP was used for subsequent experiments. To determine the expression of USP51 and ZEB1 in A549 or A549/DDP cells, RT-qPCR and western blotting were conducted. It was revealed that both the mRNA expression and protein levels of USP51 (Fig. 1B and D) and ZEB1 (Fig. 1C and D) were higher in A549 cells than in 16HBE cells. Moreover, when compared with A549 cells, the expression of both USP51 and ZEB1 in A549/DDP cells was significantly increased.

Figure 1.

Expression of USP51 and ZEB1 is significantly increased in A549 or A549/DDP cells. Total RNA and protein were extracted from A549, A549/DDP and 16HBE cells. (A) After treatment with gradient concentrations of DDP (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 µmol/l), cell proliferation was assessed to determine drug resistance in the indicated cells (A549 and A549/DDP) to DDP. The mRNA expression of (B) USP51 and (C) ZEB1 was detected by reverse transcription-quantitative PCR. (D) Protein expression levels of USP51 and ZEB1 were detected by western blotting. **P<0.01 and ***P<0.001 vs. 16HBE, and ##P<0.01 and ###P<0.001 vs. A549. A549, lung cancer cells; A549/DDP, DDP-resistant A549 cells; 16HBE, normal lung cells. USP, ubiquitin-specific protease; ZEB1, zinc-finger E-box binding homeobox 1.

Knockdown or overexpression by lentivirus infection efficiently alters USP51 expression in A549 or A549/DDP cells

A549 and A549/DDP cells were infected with lentiviruses of USP51/vector and shUSP51/shNC, respectively. As revealed in Fig. 2A, USP51 overexpression by USP51 lentivirus in A549 cells resulted in USP51 upregulation, both at the mRNA and protein levels (Fig. 2A), whereas knockdown of USP51 in A549/DDP cells by shUSP51 lentivirus (Fig. 2B) resulted in USP51 downregulation. Among the lentiviral constructs for knockdown, shUSP51-1 and −2 exhibited higher efficiency and therefore were used for follow-up experiments.

Figure 2.

Knockdown or overexpression of USP51 in A549 or A549/DDP cells by lentivirus infection. A549 cells were infected with USP51/vector while A549/DDP cells were infected with shUSP51/shNC. (A and B) USP51 mRNA expression (upper) and protein levels (lower) in (A) A549 or (B) A549/DDP cells were determined by reverse transcription-quantitative PCR and western blotting, respectively. ***P<0.001 vs. vector or shNC. A549, lung cancer cells; A549/DDP, DDP-resistant A549 cells. USP, ubiquitin-specific protease; sh, short hairpin RNA; NC, negative control.

Knockdown of USP51 significantly decreases cisplatin resistance in A549/DDP cells by promoting apoptosis

Likewise, after treatment with gradient concentrations of DDP (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 µmol/l) and shUSP51 lentivirus, cell proliferation was detected to determine drug resistance of A549/DDP cells infected with shUSP51 to DDP. As revealed in Fig. 3A, the shUSP51 group was more sensitive to DDP than the shNC group. Moreover, flow cytometric analysis indicated that knockdown of USP51 (7.4% increase of apoptosis) or treatment with 100 µmol/l DDP (2.9% increase of apoptosis) in A549/DDP cells significantly promoted apoptosis. Furthermore, knockdown of USP51 potently enhanced the effects of DDP in A549/DDP cells (5.5% increase of apoptosis) (Fig. 3B). Conversely, overexpression of USP51 potently attenuated DDP-induced apoptosis in A549 cells (Fig. 3C). Concurrently, decreased ZEB1 protein and increased levels of cleaved PARP1 and cleaved caspase-3 were observed in USP51-silenced A549/DDP cells, while USP51-overexpressing A549 cells displayed increased ZEB1 protein (Fig. 3D). Given the roles of PARP as a DNA repair enzyme and cleavage substrate for caspases (36), and of caspase-3 as a major apoptosis-executing enzyme cleaving PARP (37), these results indicated that USP51 may be an oncogene in lung cancer and that USP51 may play a role in DDP resistance in lung cancer by regulating ZEB1.

Figure 3.

Knockdown of USP51 significantly decreases DDP resistance in A549/DDP cells by promoting apoptosis. (A) After treatment with gradient concentrations of DDP (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 µmol/l), cell proliferation was assessed to determine drug resistance in the indicated cells (A549/DDP + shNC, A549/DDP + shUSP51) to DDP. (B) After treatment with shUSP51 lentivirus and 100 µmol/l DDP, the percentage of apoptotic cells in A549/DDP cells was detected by flow cytometry. (C) After treatment with USP51 lentivirus and 5 µmol/l DDP, apoptotic cells in A549 cells were detected. Knockdown of USP51 significantly decreases DDP resistance in A549/DDP cells by promoting apoptosis. (D) Protein levels of USP51, ZEB1, PARP and caspase-3 in USP51-silenced A549/DDP cells, as well as USP51 and ZEB1 in USP-overexpressing A549 cells, were determined by western blotting. **P<0.01 and ***P<0.001 vs 0 µmol/l DDP, shNC or vector; and #P<0.05 and ###P<0.001 vs shUSP51-1 or USP51; and ++P<0.01 vs. shUSP51-2. A549/DDP, DDP-resistant A549 cells. USP, ubiquitin-specific protease; ZEB1, zinc-finger E-box binding homeobox 1; sh, short hairpin RNA; NC, negative control.

Knockdown of USP51 decreases DDP resistance in A549/DDP cells by modulating ZEB1 ubiquitination

To determine the relationship and mechanism of USP51 and ZEB1 in regulating DDP resistance in lung cancer, ZEB1 was overexpressed in A549/DDP cells by lentivirus infection (Fig. 4A) and apoptosis was analyzed. It was revealed that overexpression of ZEB1 significantly suppressed DDP-induced apoptosis in A549/DDP cells (1.2% decrease of apoptosis), and the effects of USP51 knockdown on apoptosis of A549/DDP cells were potently attenuated by ZEB1 overexpression (2.7% decrease of apoptosis) (Fig. 4B). Notably, ZEB1 mRNA expression was unaltered in USP51-silenced A549/DDP cells (Fig. 4C). Rather, the co-IP assay demonstrated that USP51 interacted with ZEB1 in A549/DDP cells (Fig. 4D), and that USP51 knockdown promoted the ubiquitination and degradation of the ZEB1 protein (Fig. 4E). Collectively, the present data indicated that knockdown of USP51 decreased DDP resistance in A549/DDP cells likely via ZEB1 ubiquitination and degradation.

Figure 4.

Knockdown of USP51 decreases DDP resistance in A549/DDP cells by modulating ZEB1 ubiquitination. (A) After infection of ZEB1/vector lentivirus in A549/DDP cells, ZEB1 mRNA and protein levels were detected. (B) A549/DDP cells were infected with shUSP51 and ZEB1 lentiviruses and treated with 100 µmol/l DDP, and apoptotic cells were detected. (C) ZEB1 mRNA expression in USP51-silenced A549/DDP cells was detected. (D) Interaction between USP51 and ZEB1 was determined by Co-Immunoprecipitation experiment. (E) Ubiquitin-mediated degradation of ZEB1 in USP51-silenced A549/DDP cells, as well as Zeb1 and USP51 protein, was examined by western blotting. *P<0.05 and ***P<0.001 vs. vector or DDP + vector + shNC, and ##P<0.01 vs. DDP + vector + shUSP51; and +++P<0.001 vs. DDP + ZEB1 + shNC. A549/DDP, DDP-resistant A549 cells. USP, ubiquitin-specific protease; ZEB1, zinc-finger E-box binding homeobox 1; sh, short hairpin RNA; NC, negative control.

Discussion

Increasing evidence in recent years has suggested USP as an attractive therapeutic focus and target for cancer treatment. For instance, in early-stage non-small cell lung cancer, overexpression of USP22 can predict poor survival of patients (38). Likewise, by directly targeting USP25, miR-200c can inhibit tumor cell invasion and metastasis (39). Furthermore, USP14 was revealed to participate in cell adhesion-mediated drug resistance of multiple myeloma cells (40). On a related note, upregulation of ZEB1 is involved in DDP resistance of multiple cancers, such as osteosarcoma and epithelial ovarian cancer (41,42). In the present study, increased expression of USP51 and ZEB1 in A549/DDP cells was observed, indicating that their expression may be associated with DDP resistance of lung cancer cells. Furthermore, cell proliferation in A549/DDP was significantly inhibited by 100 µmol/l of DDP. Similar to DDP treatment, knockdown of USP51 in A549/DDP cells, strongly induced apoptosis, which was potently attenuated by ZEB1 overexpression. Moreover, USP51 overexpression potently attenuated DDP-induced apoptosis. These results indicated that knockdown of USP51 could reverse the resistance of A549/DDP cells to DDP, likely through regulation of ZEB1.

The mechanism between USP51 and ZEB1 in regulating resistance of A549/DDP cells to DDP was also investigated in this study. DDP is known to bind the N7 reactive center on purine residues and as such can cause DNA damage in cancer cells, blocking cell division and resulting in apoptotic cell death. Several molecular mechanisms of action have been described, including induction of oxidative stress through reactive oxygen species production and lipid peroxidation, induction of p53 signaling and cell cycle arrest, downregulation of proto-oncogenes and anti-apoptotic proteins, and activation of both intrinsic and extrinsic pathways of apoptosis (43,44). Studies have revealed that ZEB1 serves a critical role in cancer cell plasticity, tumor recurrence and therapy resistance (9,16). ZEB1 protein is subjected to proteolytic ubiquitination and, in certain conditions, can be stabilized (28). It has been revealed recently that Siah1/2 and Skp1-Pam-Fbxo45 complex, the ubiquitin ligase, promote ubiquitination and degradation of ZEB1 (45,46). In the present study, decreased expression of ZEB1 and increased expression of cleaved PARP1 and cleaved caspase-3 was revealed in USP51-silenced A549/DDP cells. Moreover, the effects of USP51 knockdown in A549/DDP cells were potently attenuated by ZEB1 overexpression. USP51 interacted with ZEB1, and knockdown of USP51 markedly induced ubiquitin-mediated degradation of ZEB1. These results indicated that knockdown of USP51 may reverse the resistance of A549/DDP cells to DDP through ZEB1 ubiquitination and degradation, thus activating apoptosis. This is consistent with a study that reported that USP51 may act as a ZEB1 deubiquitinase and possibly act as an alternative pathway for targeting ZEB1 (28). In addition, multiple anti-cancer agents have been revealed to be used in combination with DDP to enhance treatment. For example, retigeric acid B, a topoisomerase II inhibitor, can enhance the cytotoxicity of DDP in prostate cancer (47), while ursane triterpenoid can be combined with DDP in bladder cancer (48). Consistent with the studies that revealed that USP7 inhibitor can overcome bortezomib resistance in multiple myeloma cells (49), the present findings indicated the pharmacological potential of USP51 inhibitors in the treatment of lung cancer. However, at the current medical level, there is a lack of research on USP51 inhibitors. Thus, development of USP51 inhibitors, used in combination with DDP, may offer a better therapy for lung cancer.

In conclusion, the present study demonstrated the inhibitory effects of USP51 knockdown on DDP resistance in lung cancer via induction of apoptosis, likely through ubiquitination of ZEB1. Targeting USP51 is likely to be an alternative pathway for targeting ZEB1, thus providing a novel therapeutic target for DDP resistance in lung cancer.