USP51, an oncogene for pancreatic cancer (PC) cell growth via Wnt/β-catenin pathway

Background: Currently, targeting ubiquitin-specic protease (USP) is popular in cancer treatment. Aim: To study the roles of USP51, a member of USPs, in human pancreatic cancer (PC) and the underlying mechanism. Method: Expression of USP51 in tumor pancreatic tissue and PC cell lines were assessed. Overall survival rate of PC patients was measured by Kaplan-Meier method. The association between USP51 and β-catenin in PC cells was determined by Co-Immunoprecipitation (Co-IP) and ubiquitination assay. XAV939 was used to inhibit β-catenin. Protein levels of β-catenin and Cyclin D1 were used to assess the mechanism. Result: Herein, we evidenced high levels of USP51 in tumor tissues of PC patients, which predicted a poor survival. Knockdown of USP51 in MiaPaca2 and SW1990 (two PC cells) inhibited proliferation, arrested MiaPaca2 and SW1990 cells at G0/G1 phases, and impaired tumorigenicity of MiaPaca2 in nude mice, demonstrating the anti-cancer effect of siUSP51 in PC. Moreover, Knockdown of USP51 prevented β-catenin and its downstream Cyclin D1, suggesting the blockade of β-catenin pathway in this process. On the contrary, overexpression of USP51 in ASPC1 (one PC cell) resulted in the opposite effect, facilitating the tumorigenesis of PC and the activation of β-catenin pathway, which were signi�cantly reversed by XAV939. Interestingly, USP51 bound to β-catenin and knockdown of USP51 increased β-catenin ubiquitination. Conclusion: USP51 is an oncogene that is upregulated in PC. USP51 acted as a deubiquitinase that favored β-catenin accumulation, then activated Wnt/β-catenin pathway and exerted carcinogenesis in PC.


Background
Increased activity of β-catenin, a key transcriptional activator of canonical Wnt pathway, is an essential cause in human pancreatic cancer (PC), and a promising therapeutic target to block this disease [4].
Mechanically, once Wnt signal is stimulated at cell plasma membrane, β-catenin is accumulated in the nucleus to assemble into a complex with lymphoid enhancer binding factor (LEF)/T-cell factor (TCF) to stimulate its downstream oncogenes, such as Cyclin D1, and ultimately contributes to cancer cell proproliferation, more aggressive phenotype and chemo-resistance [8].Usually, level of β-catenin can be determined by the following steps: APC/Axin1 destruction complex-mediated-phosphorylation, E3 ligase (β-TrCP) mediated-ubiquitination and proteasome-dependent degradation [5].However, the speci c regulator in controlling β-catenin levels in PC is not fully understood.
Ubiquitin speci c proteases (USPs) functioned as de-ubiquitinating enzymes to favor protein recycle from proteasome degradation, and play a role in protein localization or activation [3].Recently, UPS appears as an rich source for drug targets in cancer treatment [15].Evidence suggests that USPs member USP51 is necessary for cell viability, and hindering USP51 expression represses tumor growth [1].Expression of USP51 is enhanced and accompanied with a poor survival in human gastric cancer (GC) [17].However, the roles of USP51 in PC remained largely unknown.
In human cancers, USPs (such as USP4, USP7 and USP47) retarded β-catenin degradation through a deubiquitination process [9,12,16].However, in PC, whether USP51 functioned as a de-ubiquitinating enzyme to control β-catenin expression remained largely unknown.Herein, we found evaluated levels of USP51 in human PC, which correlated with a poor survival.Our data disclosed that knockdown of USP51 inhibited proliferation of PC cells and induced cell cycle arrest.However, overexpression of USP51 resulted in the opposite.We con rmed USP51 as a deubiquitination enzyme to enhanced β-catenin expression and the subsequent accumulation of Cyclin D1 (β-catenin downstream oncogene), which ultimately induced the tumorigenesis of PC cells.

Tissue samples
In RT-PCR method, 25 pairs of tumor and adjacent non-tumor tissues in PC patients were acquired from Fudan University Shanghai Cancer Center.In IHC analysis, human PC tissue microarrays (n = 67) and corresponding control healthy (n = 25) were prepared by Shanghai Outdo Biotech Co., Ltd.(Shanghai, China).According to IHC results, USP51 expression in PC tissue were divided into USP51 high (n = 37) and USP51 low (n = 30) group, then the overall survival rate for PC patients were assessed.

Immunohistochemistry (IHC) assay
Expression of USP51 in human PC tissue microarrays, as well as expression of Ki67 in tumor of a Xenograft model was assessed, using IHC method.Para nembedded samples (cut into 4 mm thick) from human pancreatic or mice tumor tissue were routinely dewaxing, rehydration, and then blocked using 3% H 2 O 2 .IHC was conducted using DAB substrate (Long Island Biotech) and hematoxylin (714094, BASO) following manufacturer's instructions.The primary antibody for the IHC was rabbit Ki67 polyclonal antibody (ab16667, abcam) and rabbit USP51 polyclonal antibody (NBP1-86167, NOVUS).
To study the effect of USP51 on the levels of β-catenin ubiquitination (Ub-β-catenin), Ub-β-catenin, precipitated in the immune-complexes, was determined using Anti-ubiquitin antibody (ab7780), according the western blot.

Xenograft model
Nude mice with 4-6-week-old were provided by Shanghai Laboratory Animal Company.MiaPaca2 cells transfected with siNC/siUSP51 (7 × 10 6 cells , 100 μl) were subcutaneously injected into nude mice (n = 6 in each group), and then fed separately.Tumor formation in siNC and siUSP51 groups was monitored from 12 th day to 33 th day.On 33 th day, all mice were killed.Tumor weight (g) was recorded.Histological changes in pancreatic tumor tissue were recorded using Hematoxylin-eosin (H&E) staining.IHC was conducted to evaluate the expression of Ki67, using antibody against Rb Ki67 (ab16667, abcam).

Statistics and data analysis
Data was showed as mean ± standard error of the mean (SEM), calculated from at least 3 parallels.Comparison between two groups was evaluated using Student's test with P < 0.05 being statistical signi cance.Overall survival of PC patients was analyzed by Kaplan-Meier method using medcalc sofeware.

Results
Expression of USP51 was enhanced in PC and predicted a poor survival Expression of USP51 in PC tissues was detected.Figure 1A showed that expression of USP51, assessed by RT-PCR, was signi cantly enhanced in tumor tissues (n = 25) when compared with adjacent non-tumor tissue (n = 25), suggesting the involvement of USP51 in human PC, which was further con rmed using an IHC method (Figure 1B).Besides, similar change tread was also observed in human PC cell lines (ASPC1, BXPC3, MiaPaca2, PANC1 and SW1990) when compared with normal pancreatic HPC-Y5 cells, con rming the up-regulation of USP51 in human PC in vitro (Figure 1C).We then investigated whether enhanced USP51 levels were correlated with the survival of PC patients.Kaplan-Meier analysis showed that PC patients with lower USP51 levels were more likely to survive than that with the higher USP51 expression (Figure 1D, P = 0.0083).
Inhibitory effect of siUSP51 on PC cell proliferation, cycle and the activation of β-catenin pathway Among PC cell lines, USP51 was highly expressed in MiaPaca2 and SW1990 cells while lowly expressed in ASPC1 cells (Figure 1C), therefore, we selected MiaPaca2, SW1990 and ASPC1 cells in the subsequent study.
Proliferation and cell cycle of PC cells transfected with siUSP51 was examined.Our data noted that siUSP51-1 and siUSP51-2 time-dependently reduced PC cell proliferation (Figure 2C, D), and arrested MiaPaca2 and SW1990 in G0/G1 phase (Figure 2E, F).
During cycle progression, the transition from G0/G1 to S phase is modulated by Cyclin D1, which is target gene of β-catenin [2].siUSP51-1 and siUSP51-2 exerted inhibitory effect on protein levels of β-catenin and Cyclin D1 (Figure 2G, H), as well as mRNA expression of Cyclin D1 (data not shown) in MiaPaca2 and SW1990 cells, suggesting that knockdown of USP51 inhibited PC cell growth likely via blocking βcatenin/Cyclin D1 pathway.
USP51 acted on β-catenin to in uence cell growth cells were transfected with oeUSP51 to overexpress USP51.oeUSP51 enhanced USP51 mRNA and protein expression, in comparison to vector (all P < 0.01), indicating the successful construction of USP51 overexpression in ASPC1 cells (Figure 3A).ASPC1 cells with oeUSP51 were treated with XAV939 (a speci c β-catenin inhibitor).Our data showed that oeUSP51 time-dependently enhanced cell proliferation (Figure 3B), promoted the transition of ASPC1 from G0/G1 to S phase (Figure 3C), and up-regulated β-catenin and Cyclin D1 expression (Figure 3D) when compared with vector.However, oeUSP51-induced those changes were reversed by XAV939 (P < 0.01).

Knockdown of USP51 suppressed tumorigenicity of cells in nude mice
Ki67, a nuclear associated antigen, indicates the nuclei of proliferating, and can be used to label the proliferative activity of pancreatic tumors.Evidence suggests that Ki67 is the most reliable factor in the prognosis of PC [6].In the present study, after tumor formation using MiaPaca2 cells transfected with siUSP51 in nude mice, tumor growth and its pathological observation, as well as the expression levels of Ki67 were measured.Our data showed that siUSP51 remarkably inhibited tumor growth and weight (Figure 5A, B), obviously reduced cell in ltration and viability, as evidenced by relatively sparse cytoplasm and dispersed chromatin around cell membrane (Figure 5C), and reduced the proportion of Ki67-labeled cells (Figure 5D), suggesting the inhibitory effect of siUSP51 on tumor growth in PC in vivo.

Discussion
Currently, more than 30 USPs have been implicated in cancer, considering the diversity roles of different USPs in regulating cellular functions, and various substrates involved and modulated by them [10].Evidence suggests that USP51 is overexpressed and indicate a poor survival in GC [17], which was further con rmed in PC, substantiating the involvement of USP51 in human cancers.
Cancer cells are bene ts from USPs for their sustained growth and progression of cell cycle [13].USP51 is implicated in cell proliferation and cycle progression, and also involved in tumor growth modulation [1].Presently, we rstly proposed USP51 as an oncogene in PC.Knockdown of USP51 suppressed PC cell proliferation and tumor growth, and overexpression of USP51 resulted in the opposite.Besides, we also evidenced the inhibited effect of USP51 knockdown on tumorigenicity of PC cells in nude mice, further con rming the anti-cancer effect of siUSP51 in PC. 89-90% of intracellular proteins can be degraded by an important process, named deubiquitination [10].Evidence suggested that USPs, as deubiquitinating enzymes, regulates β-catenin ubiquitin, and is emerging as an activator for β-catenin [9,12,16].In this study, we con rmed an up-regulation of β-catenin in PC tumorous tissue.Interference experiment using XAV939 suggested that USP51 regulated β-catenin to determin PC cell growth.Besides, our data further substantiated that USP51 deubiquitinates β-catenin, thereby leading to accumulation of β-catenin protein.Furthermore, oncogene Cyclin D1, a key regulator for transition from G1 to S phase and a downstream of β-catenin, is overexpressed in many types of cancers [14].Cyclin D1 is regulated by USP2 in a deubiquitination process [11].Our data suggested that down-regulation of cyclin D1 is implicated in the inhibitory effect of siUSP51 on PC cells growth, however, whether USP51 was a speci c deubiquitinase for cyclin D1, and whether there was a USP51/βcatenin/cyclin D1 axis that contributes to PC cell growth needed further con rmed.

In Conclusion
USP51 was up-regulated in PC and associated with a poor survival.USP51 enhanced β-catenin accumulation via deubiquitination process, and then favoring the tumor progression of PC both in vivo and in vitro.Targeting USP51 may represent a therapeutic strategy in PC treatment.

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