PCNP promotes ovarian cancer progression by accelerating β‐catenin nuclear accumulation and triggering EMT transition

Abstract Ever reports showed that PCNP is associated with human cancers including neuroblastoma and lung cancer. However, the role and underlying molecular mechanism of PCNP in ovarian cancer have not been plenty elucidated. Herein, we first investigated the expression of PCNP in ovarian cancer tissues and cells, the effects of PCNP in ovarian cancer proliferation, apoptosis, migration and invasion, and determined the molecular mechanism of PCNP in ovarian cancer progression. The results indicated that PCNP was significantly overexpressed in human ovarian cancer tissues and cells, and related to poor prognosis in ovarian cancer patients. In addition, we also detected that PCNP promoted ovarian cancer cells growth, migration and invasion, as well as inhibited ovarian cancer cells apoptosis. Mechanistically, PCNP binding to β‐catenin promoted β‐catenin nuclear translocation and further activated Wnt/β‐catenin signalling pathway. Moreover, PCNP regulated the expression of genes involved in EMT and further triggered EMT occurrence. Conclusionally, PCNP may promote ovarian cancer progression through activating Wnt/β‐catenin signalling pathway and EMT, acting as a novel and promising target for treating ovarian cancer.


| INTRODUC TI ON
Ovarian cancer (OC) is the leading cause of death in female reproductive system malignancies and has a 5-year survival rate of ~47%. 1,2 There are approximately 239 000 new cases and 152 000 deaths worldwide annually with regard to this disease. [3][4][5] Only 15% of ovarian cancer are diagnosed at clinically early stage, and most patients carry metastatic tumours when they are diagnosed as ovarian cancer patients, further acquire poor prognosis and high mortality rate. So far, the treatment for most ovarian cancers may be surgery and cytoreduction, 6 repeated treatment and drug-resistance also lead to a low 5-year survival. Thus, it is imperative to explore novel and effective biomarker, and underlying molecular mechanism of ovarian cancer metastasis, further supplying new therapeutic interventions for ovarian cancer. 7 PEST-containing nuclear protein (PCNP) is firstly identified in the nucleus through database mining. 8  Nervous system (CNS) cancer compared with non-tumour normal, without any biological function exploration for PCNP in cancers. 9,10 Recently, Wang DY et al summarized that PCNP promotes cell proliferation, migration and invasion in lung adenocarcinoma; however, Wu DD et al considered that PCNP inhibited human neuroblastoma progression. 11,12 However, the role of PCNP in OC tumorigenesis and progression remains to be elucidated. Herein, we investigated the effects and the underlying mechanism of PCNP in ovarian cancer.
Wnt/β-catenin signalling pathway, which is critically involved in both development and homeostasis of tissues, is indispensable in regulating a variety of cellular biological activities. 13 Abnormal activation of this signal pathway has been implicated in various tumours including ovarian cancer, which can drive a variety of tumorigenic functions including regulation of transformation, cell proliferation and invasion. [13][14][15] In the absence of WNT ligand stimulation, β-catenin is constitutively phosphorylated via interaction with glycogen synthase kinase 3β (GSK3β) in a destruction complex composed of scaffold proteins Axin and adenomatous polyposis coli (APC), leading to ubiquitin-mediated degradation of β-catenin and inactivation of the signalling. 16 Recently, studies exhibit that mRNA of PCNP has been detected in some cancer cells, including U-937 myeloid leukaemia cells, HepG2 hepatoma cells and HT-1080 fibrosarcoma cells, 17 suggesting the potential and promising relation of PCNP to other cancer cells including ovarian cancer cells. The tumour-related characteristic of PCNP and the vital role of Wnt/β-catenin in ovarian cancer prompt us to be interested in potential functional connections between PCNP and β-catenin.
In ovarian cancer, the dysregulation of Wnt/β-catenin can induce the epithelial-mesenchymal transition (EMT). 18 EMT, plays a critical role in embryonic development and also involved in cancer progression and metastasis, 19,20 contributes to new tumour cell properties required for invasiveness and vascular intravasation during metastasis. 21,22 It is a changeable process during which the epithelial cells lose epithelial properties and acquire mesenchymal characteristics by disassembly of cell-cell junctions, loss of cell polarity, and reorganization of the cytoskeleton, thereby promoting cells to acquire increased motility. 23 EMT can be defined according to EMT-associated markers, such as mesenchymal-specific markers, epithelial-specific markers and transcription factors. 19 In this study, we examined the effect of PCNP on OC cells prolifer-

| Construction of cell models
PCNP expression plasmid and empty vector, the PCNP shRNA (sh-PCNP group) and scramble shRNA (sh-Scb group) were purchased from Genechem (Shanghai, China) and were transfected into OC cells with Lipofectamine 3000 Transfection Reagent (Invitrogen, Shanghai, China) to construct stable cell lines. They were screened, respectively, by G418 (Solarbio, Shanghai, China) at a concentration of 800 μg/mL and puromycin (Solarbio, Shanghai, China) at a concentration of 2 μg/mL. The un-transfected tumour cells were used as controls. Seventy-two hours post-transfection, the localization of PCNP within tumour cells was detected under a fluorescent microscope (Eclipse Ti, Nikon, Melville, NY, USA).

| Real-time fluorescence quantitative PCR (qPCR)
The relative expression level of PCNP was measured by RT-PCR. The total RNA was extracted using the Trizol reagent The reaction procedure is as follows: denaturation at 95°C for 5 min; followed by 35 cycles of 95°C for 30 second, annealing at 60°C for 30 second, and a final extension at 72°C for 30 second, and then at 72°C for 10 min. Data were analysed using the 2 −ΔΔCt method, and the relative mRNA expression levels were normalized against GAPDH.

| Cell proliferation assay
The 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay was performed using the Cell-Light EdU Apollo 567 In Vitro Imaging Kit

| Colony formation assay
Cells were introduced into a 6-well plate at 5 × 10 2 cells/well and incubated for a week, after washing by phosphate-buffered saline (PBS) for 3 times, the colonies were subjected into methanol at room temperature for 15 minutes. Then, 0.1% crystal violet was added into each well and incubated for 30 minutes at room temperature.
After that, wash the plate by water gently and air-dried at room temperature. The plate was photographed under an Olympus CKX41 microscope and then measured using Image J software.

| Soft agar assay
Cells were suspended in 0.6% agarose and medium supplemented with 20% FBS at 1 × 10 4 cells/well, and the mixture was seeded in 6-well plates containing a basal layer of 1.2% agarose. The top layer is added with medium for twice per week to prevent drying. After 2 weeks of routine culture, colonies were photographed under an Olympus CKX41 microscope. Only colonies larger than 0.1 mm in diameter were counted.

| Migration and invasion assays
Cell migration and invasion were evaluated using transwell chambers with 8μm pores. 5 × 10 4 cells/well in serum-free medium were

| Immunoblottingting analysis
Cells were collected and added appropriate amount of IP lysate

| Immunohistochemistry and HE assay
Four μm thick tissue sections were cut, dried, dewaxed and hydrated according to standard procedures. An EDTA (Solarbio, BeiJing, China) heat-induced antigen retrieval process is then performed. Staining was optimized using markers (PCNP, CD31, Ki67, N-cadherin and E-cadherin) with Universal two-step assay kit (mouse/rabbit reinforced polymer assay system) and DAB (Zhong Shan Jin Qiao, BeiJing, China).
CHX interferes with the translocation step in protein synthesis and is widely used in biomedical research to inhibit protein synthesis in vitro. Samples were collected at 0, 1, 2, 3, 4 hour and stored for Immunoblottingting to detect β-catenin degradation.

| Co-immunoprecipitation (Co-IP) assay
A pierce Co-Immunoprecipitation (Co-IP) Kit was purchased from Thermo Scientific (Shanghai, China). Then, all the procedures were followed by this kit. The bound proteins were analysed with Western blottingting using relevant antibodies.

| Statistical analysis
All values were expressed as the mean ± standard deviation (SD).

| PCNP was overexpressed and related to overall survival in ovarian cancer
To preliminary detect the association of PCNP with OC, its transcriptome data in the Cancer Genome Atlas (TCGA) was collected and identified, discovering that PCNP was significantly higher in OC than normal tissues (P < .001; Figure 1A). Moreover, immunohistochemical analysis of microarray chip including normal and OC patient samples showed the protein expression of PCNP in OC tissues was significantly increased, which is consistent to the identification using TCGA data ( Figure 1B-C). During the analysis of clinical and transcriptional data of OC in four GEO datasets, it was illustrated that patients with high PCNP level had a worse overall survival (OS) F I G U R E 3 The effects of PCNP on apoptosis of OC cells. A, Apoptosis level of SK-OV-3 and A2780 cells was detected by Tunel staining; original magnification 200×. B, The calculation and analysis of apoptosis rate of SK-OV-3 and A2780. C. Western blotting analysis for the expression of cleaved-caspas8, cleaved-caspase9, cleaved-PARP, bad, bcl-2 and bcl-xl in PCNP over-or down-expressed group for SK-OV-3 and A2780 cells. D, The analysis of proteins mentioned in (C) was performed, normalized to the corresponding GAPDH level. Data are presented as mean ± SEM of three independent experiments; * P < .05, ** P < .01 compared with the Mock group; # P < .05, ## P < .01 compared with the sh-Scb group

| PCNP promoted ovarian cancer cells proliferation and suppressed its apoptosis
To evaluate the role of PCNP in OC cells proliferation, we conducted EDU assay and detected that the number of cells in the proliferative phase for overexpressed PCNP group was significantly higher than that of mock group, meanwhile, the PCNP knock-down group was significantly lower than that of sh-scb group ( Figure 2D-E), showing the proliferative capacity of PCNP in OC cells.
To further and visually clarify the function of PCNP to promote growth, we also conducted colony formation, and verified that PCNP promoted OC cells proliferation and PCNP knock-down played the opposite role ( Figure 2F-G). Multi-cellular organisms maintain tissue homeostasis by balancing proliferation and removal of cells including cancer cells via apoptosis. Here, TUNEL assays were used to investigate whether PCNP promotes OC cells proliferation was accompanied by the suppression of cells apoptosis. As a result, in PCNP overexpressed group, the percentage of apoptotic cells was significantly lower than that in mock group, while knock-down PCNP group was higher than sh-scb group ( Figure 3A-B). Results of Western blotting presented that PCNP up-regulated Bcl-2 and Bcl-xl, which inhibited cell apoptosis, meanwhile, down-regulated cleaved-caspase8, 9, PARP, Bad, which promoted cell apoptosis, yet PCNP knock-down had an opposite effect ( Figure 3C-D). Together, PCNP may possess the proliferative and anti-apoptotic capacity in ovarian cancer.

| PCNP promoted ovarian cancer migration, invasion and EMT
Furthermore, to reveal the biological function of PCNP in OC, we investigate the effect of PCNP on OC cells migration and invasion.
Results showed that the migration rate in PCNP overexpressed group for both SK-OV-3 and A2780 cells was significantly increased compared with corresponding mock group. However, PCNP knock-down obviously lowered the migration rate ( Figure 4A-B). Consistently, soft agar colony formation assays and transwell assays were identified to present the same trend, together, demonstrating that invasion and migration of OC cells in vitro could be promoted by PCNP overexpression and inhibited by PCNP knock-down ( Figure 4C-H).
To visually investigate the morphological changes for OC cells with the conversion of the metastatic ability, observation was conducted using microscope and morphological changes named slender shape was detected, which was beneficial to cell metastasis and invasion and was typically characteristic of EMT ( Figure 4I). To further identify the association of PCNP with EMT in OC cells, the protein expression of EMT-related markers such as N-cadherin, E-cadherin and ZEB1 were checked, results in the Figure 4J-K showed that PCNP significantly increased protein expression of mesenchymal markers ZEB1, N-cadherin and decreased it of epithelial marker E-cadherin, which were just also features of EMT. Summatively, PCNP could promote OC cells migration and invasion and EMT in vitro.

| PCNP interacted to β-catenin and increased nuclear accumulation of β-catenin
Wnt/β-catenin and PI3K/Akt signalling pathways appear to play an important role in ovarian cancer. To expound the precisely potential mechanism of PCNP in OC progression, Western blotting was performed and resulted in that PCNP facilitated the up-regulation of β-catenin ( Figure 5A-B). However, total proteins of PI3K, Akt and GSK3β were not affected by PCNP, moreover, PCNP also scarcely regulated phosphorylated Akt, phosphorylated PI3K and phosphorylated GSK3β. To further confirm the association of β-catenin with PCNP, we conducted Co-IP assay and indicated PCNP could combine to β-catenin ( Figure 5C). Consistently and interestingly, the positive correlation of PCNP and Wnt/β-catenin up-regulated genes listed in File1 according to NetPath-Signal Transduction Pathways database was also obtained from GEPIA database ( Figure 5D). In Wnt/β-catenin signal, the accumulation of β-catenin promotes its nuclear translocation and further activates the downstream genes.
As shown in Figure 5E-F, results demonstrated that PCNP overexpression increased the accumulation of β-catenin in the nucleus in both SK-OV-3 and A2780 cells. To explore the potential mechanism of PCNP-mediated regulation of β-catenin expression, we performed protein stability assays. As CHX is widely used to inhibit protein F I G U R E 5 The identification of relation of PCNP with β-catenin. A, Western blottingting analysis for the expression of AKT, P-AKT, PI3K, P-PI3K, GSK-3β, P-GSK-3β, β-catenin. B, The densitometry analysis of proteins shown in (A) was performed, normalized to the corresponding GAPDH level. C, Co-IP assays reveal the association between PCNP and β-catenin in A2780 cells. Inputs were used as control. CO-IP was performed using antibodies as indicated. D, The correlation analysis of PCNP with Wnt/β-catenin pathway up-regulated genes according to GEPIA database. E, Expression of β-catenin in the nucleus and cytoplasm of each group of SK-OV-3 and A2780. F, The densitometry analysis of β-catenin was performed, normalized to the corresponding LaminB1 (nucleus) and β-Tubulin (cytoplasm) level. G, β-catenin degradation was investigated by cycloheximide (CHX). H. The densitometry analysis of β-catenin in 0, 1, 2, 3, 4 h was performed, normalized to the corresponding GAPDH. Data are presented as mean ± SEM of three independent experiments; * P < .05, ** P < .01 compared with the Mock group; # P < .05, ## P < .01 compared with the sh-Scb group synthesis in eukaryotic cells, so we use it to detect the stability of β-catenin. The half-life of β-catenin was significantly increased in the PCNP overexpression group compared with the mock group within 0-4h after CHX treatment ( Figure 5G-H), synthetically, suggesting that PCNP could interact to β-catenin and obstruct its degradation, further increase β-catenin nuclear accumulation.

| D ISCUSS I ON
In our prior studies, PCNP was reported to play a vital role in growth, migration and invasion in lung cancer and neuroblastoma, 11,12 despite acting as tumour-promoter or -suppressor. Ovarian cancer whose little progress has been made to improve treatment 24 is regarded as a disease with a poor prognosis and high mortality. In this study, we expounded the role of PCNP and its potential mechanism in ovarian cancer progression.
To preliminary investigate the role of PCNP in ovarian cancer, According to recent reports, Wnt/β-catenin pathway has been the most well depicted in various cancers, specifically in ovarian cancer. 25 The increased nuclear translocation of β-catenin regarded as critical protein in Wnt pathway contributes to abnormal activation of Wnt/βcatenin pathway. 26 In this study, the up-regultion of total and nuclear β-catenin in overexpressed-PCNP group hinted that PCNP may activate Wnt/β-catenin pathway by fortifying β-catenin nuclear accumulation. Using GEPIA which can employ gene expression profiling to analysis correlation of genes, 27  Together, the increase of β-catenin and the stability of its upstream F I G U R E 6 Effects of PCNP on the growth of SK-OV-3 and A2780 cells in vivo. A, Representative xenograft tumours taken from different groups of nude mice. B, C, The tumour volume of each group was measured every day, and the TVDT was calculated by the graph shown above. D, E, The tumours were weighed and the inhibition rates of tumour growth were calculated by the formula shown above. F, G, The bodyweight change curve of each group during the experiment and the bodyweight of each group on the first day (day 0) and the last day (day 21). H, Western blottingting analysis for the expression of PCNP and β-catenin in tumours in nude mice of SK-OV-3 and A2780. I, The densitomsis of PCNP and β-catenin was performed, normalized to the corresponding GAPDH level. Data are presented as mean ± SEM of three independent experiments; * P < .05, ** P < .01 compared with the Mock group; # P < .05, ## P < .01 compared with the sh-Scb group signal PI3K/Akt/GSK3β manifested that the effect of PCNP on β-catenin may be direct interaction instead of the upstream regulation. To validate the hypothesis, we conducted Co-IP assays and clarified the interaction of PCNP to β-catenin.
EMT is a reversible process which is tightly regulated by various transcription factors such as ZEB1. 32 Wnt/β-catenin signalling mediates ZEB1 expression, which induces the phenotype and cellular plasticity of EMT. 33,34 EMT is characterized by loss of the epithelial marker E-cadherin and increased expression of mesenchymal markers, such as Vimentin. 35 In our results, the mediated protein levels of ZEB1, E-cadherin and Vimentin because of PCNP reflected that PCNP may regulate EMT. The morphological change of A2780 cells because of PCNP further clarified the conclusion.
Current studies suggest that the up-regulated expression of β-catenin in the nucleus is associated with poor prognosis and metastasis. 36 Hence, the PCNP/β-catenin axis promotes ovarian cancer progression.
In summary, the present study here signifies that PCNP is overexpressed in OC and positively correlated with poor survival. PCNP enhances OC cell proliferation, migration and invasion by interacting to β-catenin and prompting β-catenin nuclear translocation, providing the first evidence for PCNP/β-catenin network in OC ( Figure 7D).
Together, these data indicate that PCNP/β-catenin signalling may be an effective and significant therapeutic target for OC treatment.
However, further studies should be performed to identify the precise sites regulating the association between PCNP and β-catenin, which will be a key to developing specific inhibitors targeting PCNP/ β-catenin signalling.

ACK N OWLED G EM ENTS
The research was supported by two National Natural Science Foundation of China (81670088, 81602708).

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Xin-Ying Ji: Conceptualization (supporting); Funding acquisition (lead).

E TH I C A L A PPROVA L S TATE M E NT
This study was reviewed and approved by the Ethical Board at Henan University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.