Liver cancer: WISP3 suppresses hepatocellular carcinoma progression by negative regulation of β‐catenin/TCF/LEF signalling

Abstract Objectives Wnt1‐inducible signalling pathway protein 3 (WISP3/CCN6) belongs to the CCN (CYR61/CTGF/NOV) family of proteins, dysregulation of this family contributed to the tumorigenicity of various tumours. In this study, we need to explore its role in hepatocellular carcinoma that remains largely elusive. Materials and Methods The expression of WISP3/CCN6 was analysed by qRT‐PCR and Western blotting. Effects of WISP3 on proliferation and metastasis of HCC cells were examined, respectively, by MTT assay and Boyden Chamber. Roles of WISP3 on HCC tumour growth and metastatic ability in vivo were detected in nude mice. Related mechanism study was confirmed by immunofluorescence and Western blotting. Results The expression of WISP3 was significantly downregulated in HCC clinical samples and cell lines, and reversely correlated with the tumour size. Forced expression of WISP3 in HCC cells significantly suppressed cell growth and migration in vitro as well as tumour growth and metastatic seeding in vivo. In contrast, downregulation of WISP3 accelerated cell proliferation and migration, and promoted in vivo metastasis. Further study revealed that WISP3 inhibited the translocation of β‐catenin to the nucleus by activating glycogen synthase kinase‐3β (GSK3β). Moreover, constitutively active β‐catenin blocked the suppressive effects of WISP3 on HCC. Conclusions Our study showed that WISP3 suppressed the progression of HCC by negative regulation of β‐catenin/TCF/LEF signalling, providing WISP3 as a potential therapeutic candidate for HCC.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is the most common malignant liver cancer, ranking the sixth most common cancer and the second leading cause of cancer-related death worldwide. 1 Major risk factors for HCC include chronic hepatitis, alcohol consumption and exposure to other known mutagens. 2 The incidence and mortality of HCC is higher in China than other countries, due to the infection of hepatitis B virus. Moreover, the prognosis of HCC is very poor, and the 5-year survival rate worldwide is <5%. 2 β-catenin, the central component of the canonical Wnt pathway, plays an important role in cell proliferation, growth, epithelial-mesenchymal transitions (EMT), cell death and liver regeneration. [3][4][5] Various molecular and genetic factors, which participated in the aberrant activation of Wnt/β-catenin pathway, were implicated in tumorigenesis. 4,5 It has been reported that mutations activating the Wnt/β-catenin pathway were the most frequent genetic event in human HCC. 6 Meanwhile, inhibition of β-catenin signalling exerted anti-tumour effects on HCC cell lines. 7,8 Considering that aberrant activation of the β-catenin signal pathway leads to the tumorigenesis and progression of HCC, 4 some of the endogenous inhibitors of β-catenin signal pathway may have potential therapeutic effects.
Wnt1-inducible signalling pathway protein 3 (WISP3/CCN6) belongs to the CCN (named after CYR61, CTGF and NOV) family of secreted, extracellular matrix (ECM)-associated signalling matricellular proteins. 9 Members of CCN family are involved in many vital biological functions, including cell proliferation, adhesion, angiogenesis, ECM production, migration, tumour growth and wound healing, 10 and dysregulation of this family contributed to the tumorigenicity of various tumours. [11][12][13] WISP3 has previously been implicated in tumourigenesis and metaplasia in a variety of tissues. [14][15][16] Moreover, it was reported that β-catenin dysregulation was partially correlated with inactivating mutations in WISP3 gene in metaplastic carcinomas of the breast. 17 In microarray-based screening of differentially expressed genes in HCC, we found that the expression of WISP3 was significantly decreased in HCC samples compared with the adjacent normal tissues (data not shown). In this study, we reported that the expression of WISP3 was significantly decreased in HCC samples and reversely correlated with clinical features of HCC patients, such as AFP level and tumour size. WISP3 suppressed tumour growth, migration and metastasis by negatively regulating β-catenin/TCF/LEF signalling. Our study has revealed the significance of loss of WISP3 in the progression of HCC and therefore provides a potential drug candidate in HCC therapy.

| RNA extraction and real-time polymerase chain reaction analysis
Total RNA was isolated from cell lines and tissues using Trizol reagent Amplification reactions were performed in a 15 µL volume of the metastasis. Further study revealed that WISP3 inhibited the translocation of βcatenin to the nucleus by activating glycogen synthase kinase-3β (GSK3β). Moreover, constitutively active β-catenin blocked the suppressive effects of WISP3 on HCC.

Conclusions:
Our study showed that WISP3 suppressed the progression of HCC by negative regulation of β-catenin/TCF/LEF signalling, providing WISP3 as a potential therapeutic candidate for HCC.
LightCycler-DNA Master SYBR Green I mix (Roche Applied Science, Penzberg, Germany) with 10 pmol/L of primer, 2 mmol/L MgCl 2 , 200 µmol/L deoxynucleotide triphosphate mixture, 0.5 units of Taq DNA polymerase and universal buffer. All of the reactions were performed in triplicates in Mx3000 system (Stratagene), and the thermal cycling conditions were as follows: 95°C for 3 minutes; 40 cycles of 95°C for 30 seconds, 60°C for 20 seconds and 72°C for 20 seconds; 72°C for 10 minutes. The relative mRNA level of WISP3 was normalized to that of β-actin.

| Construction of plasmids and transfection
The coding sequence for the full length of WISP3 was cloned into the pcDNA3.

| Construction of shRNA targeted WISP3
ShRNA specially targeting WISP3 was constructed as follows: the complete coding sequence of human WISP3 was retrieved from the NCBI GenBank database, and two paired insert DNA fragments according to the coding regions of WISP3, starting at 232 and 331, were designed using software offered by Qiagen (Valencia, CA, USA).
Transfected cells were selected with puromycin, and the expression of WISP3 in the resistant clones was further confirmed by Western blot.

| MTT assay
The in vitro cell growth was measured by MTT assay. Cells were plated in 96-well plates with a concentration of 1000 cells per well. To measure cell growth, 20 µL 5 mg/mL MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) was added into the medium and cultured at 37°C for 4 hours. Then, the medium was removed, 200 µL DMSO was added to dissolve the generated formazan, and the OD550 value of the solvent was measured by an automatic microplate reader. The measurement process was performed every 24 hours for 7 days to generate a cell growth curve.
For experiments with recombinant human WISP3 (rhWISP3), Huh7 and PVTT-1 cells were plated in 96-well plates with a concentration of 1500 cells per well for 24 hours followed by serum starvation for 16 hours. Cells were treated with 400 ng/mL of rhWISP3 (Peprotech, Rocky Hill, NJ, USA) or with diluent in 10% FBS-DMEM medium for 6 days. The cell growth curve was generated using the MTT assay.

| In vivo tumorigenicity assay
All procedures were performed in agreement with SIBS Guide for the Care and Use of Laboratory Animals and approved by Animal Care and Use Committee, Shanghai Institutes for Biological Sciences.
Male nude mice were housed under standard conditions. Cell suspensions (2 × 10 5 ) of Huh7/vec or Huh7/WISP3 cells in a total volume of 100 µL were injected subcutaneously into the 4-week-old nude mice (four mice for each group, randomly assigned). On the 28th day after inoculation, the tumours were harvested, photographed and weighted.

| In vivo metastasis assay
Cells (5 × 10 5 for PVTT-1 and 5 × 10 6 for QSG-7701) labelled with luciferase were injected into the left ventricle of 6-week-old nude mice (five mice for each group, randomly assigned). The metastatic lesions were monitored weekly by luciferase expression, which was determined by an in vivo Living Image System (Xenogen, Alameda, CA, USA). Before mice were anesthetized with isoflurane (Abbott, Abbott Park, IL, USA), an aqueous solution of luciferin (Xenogen, 150 mg/kg intraperitoneally) was injected 10 minutes prior to imaging. The mice were placed into a light-tight chamber of the CCD camera system (Xenogen), and the photons emitted from the luciferase-expressing cells within the animal were quantified for 1 minutes using the software program Living Image as an overlay on Igor (Wavemetrics, Seattle, WA, USA).

| Cell migration assay
The migration assay was carried out using a 10-well Boyden Chamber (Neuro Probe, Gaithersburg, MD, USA) with a polycarbonate membrane of 8 µm pore size. Briefly, the underside of the filter was coated with 50 µg/mL rat tail type I collagen at 4°C overnight. Cells (2 × 10 5 ) in 1% foetal bovine serum were plated in the upper wells. Medium with 10% FBS was used as a chemoattractant in the bottom wells.
The chamber was then incubated at 37°C for 6-8 hours. Cells that did not migrate through the pores of the filter were removed. Cells that migrated to the underside of the filter were stained with eosin and photographed using an inverted microscope. Six fields of each well were counted, and the experiments were repeated at least three times.
For experiments with recombinant human WISP3 (rhWISP3), Huh7 and PVTT-1 cells (2 × 10 5 ) with 400 ng/mL of rhWISP3 (Peprotech, Rocky Hill, NJ, USA) or with diluent in 1% foetal bovine serum were plated in the upper wells. The other steps were the same with above description of cell migration assay.

| Western blotting
Cells or HCC samples were lysed in RIPA buffer for 30 minutes on ice, and the lysates were centrifuged at 10 000 g for 15 minutes at immunoblotted with specific antibodies. The immunoreactive protein bands were visualized using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA). ImageJ software was used for quantification of Western blots.

| Immunofluorescence
Cells were digested with trypsin and plated on the slides. Twenty-four hours later, cells were washed thrice with phosphate-buffered saline (PBS) at room temperature, fixed in methanol at −20°C for 5 minutes, blocked with 1% bovine serum albumin at room temperature for 1 hour and then incubated with the primary antibody overnight at 4°C.
After washing three times in PBS, cells were incubated with the secondary antibody (1:1000) at room temperature for 1 hour. Cells were washed three times with PBS for 5 minutes each in darkness, and the nuclei were stained with Hoechst. Fluorescence was monitored by an inverted confocal laser microscopy (Carl Zeiss, New York, NY, USA).

| Nuclear protein extraction
The cytoplasmic and nuclear proteins were prepared as described previously. 20

| Luciferase reporter assay
Cells were plated at a subconfluent density and cotransfected with 0.05 µg of the reporter plasmid, 0.5 µg of expression vectors and 0.02 µg of Renilla luciferase pRL-TK (internal control for transfection efficiency). Cell lysates were prepared 24 hours after transfection, and the reporter activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Transfections were performed in triplicate and repeated three times to ensure reproducibility.

| Preparation of conditioned medium and cell treatment
Conditioned medium (CM) from confluent cultures of QSG-7701 cells stably transfected with either the control (WISP3 abundant) or shRNA targeted WISP3 vector (WISP3 scarcity) was collected, centrifuged at 600 g for 10 minutes, filtered, sterilized and stored at −70°C for use. Huh7 cells were treated with the CM 1:1 diluted with the fresh medium. After incubation for 24 hours, cell migration assay was performed as described above.

| Statistical analysis
All data are presented as mean ± SE. Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Statistically significant differences were determined by Student's t test, and oneway ANOVA test. Differences between groups were considered statistically significant at P < 0.05.

| WISP3 is downregulated in HCC cell lines and clinical samples, and correlates with the clinical features
To explore the potential roles of WISP3 in HCC, we first examined its expression in normal hepatic cell lines (QSG-7701 and LO2) and  Figure 1B). Results of Western blotting further confirmed that the protein level of WISP3 also significantly decreased in HCC samples ( Figure 1C). In addition, downregulation of WISP3 in liver cancer samples compared with normal liver samples was further confirmed by an online Bioinformatics tool (GEPIA, Figure 1D). The correlation between WISP3 mRNA levels and pathological characteristics was analysed, and we found that the decreased level of WISP3 mRNA was correlated with larger tumour size and higher AFP level (Table S1). These results suggested that WISP3 was dysregulated in clinical HCC patients and indicated its suppressive role in the progression of HCC.

| Overexpression of WISP3 in HCC cells suppresses cell growth and migration in vitro as well as tumorigenesis and metastasis in vivo
To investigate the function of WISP3 in HCC, WISP3 was stably overexpressed in PVTT-1 and Huh7 cells, and the expression was confirmed by Western blot (Figure 2A). Since the expression of WISP3 was reversely correlated with tumour size, we first examined the effect of WISP3 on cell growth by MTT assay. As shown in Figure 2B, overexpression of WISP3 remarkably decreased cell growth in both PVTT-1 and Huh7 cells. In addition, forced expression of WISP3 significantly suppressed cell migration examined by Boyden Chamber assay, an important process involved in tumour metastasis ( Figure 2C). Since WISP3 is an ECM proteins, we examined the effects of recombinant human WISP3 on HCC cell growth and migration. As shown in Figure S1A,B, rhWISP3 (400 ng/mL) significantly inhibited the growth and migration of Huh7 and PVTT-1.

| Knockdown of WISP3 in HCC cells promotes cell growth and migration in vitro as well as tumorigenesis and metastasis in vivo
To further elucidate the role of WISP3 in HCC cell growth and migration, we employed siRNA (shWISP3 6# and shWISP3 8#) to knock down the expression of endogenous WISP3 in QSG-7701 and LO2 cells ( Figure 3A). Silencing the expression of WISP3 in QSG-7701 and LO2 cells dramatically stimulated cell proliferation and migration ( Figure 3B,C). Furthermore, knockdown of WISP3 facilitated metastasis of QSG-7701 cells to distant organs ( Figure 3D). Taken together, these results suggested that WISP3 acted as a negative regulator in the progression of HCC.

| WISP3 inhibits cell growth and migration by negatively regulating Akt/GSK3β/β-catenin/ TCF signalling
To identify the underlying molecular mechanism for the suppressive effects of WISP3 in HCC, we firstly screened several signalling pathways with luciferase reporter assays. We found that WISP3 remarkably inhibited the activity of TOPflash, a classic Wnt-responsive reporter in HEK293T, as well as HCC cell Huh7 and PVTT-1. Figure 3A and Figure Figure S2E,F). It was known that β-catenin was regulated at multiple levels including stabilization/translocation. 21 Then, we examined the localization of β-catenin. As shown in Figure 3B Glycogen synthase kinase 3β is a key negative regulator of β-catenin. 3-5 PI3K/Akt was reported to act upstream of GSK3β. Therefore, we examined whether WISP3 affected the activation of Akt and GSK3β. As shown in Figure 3D, forced expression of WISP3 dramatically abrogated phosphorylation of Akt (Thr308 and Ser473) and GSK3β. As expected, downregulation of WISP3 enhanced the phosphorylation of Akt and increased the inhibition-associated phosphorylation of GSK3β on Ser-9. Taken together, the phosphorylation of Akt/GSK3β may involve in inhibition of nuclear accumulation of β-catenin by WISP3.

As shown in
We next examined whether the effects of WISP3 on the progression of HCC were β-catenin/TCF-dependent. Constitutively active β-catenin (CA β-catenin) was introduced into Huh7 cells ( Figure 4A). Figure 4B,C, WISP3-induced inhibitory effects on cell growth and migration were rescued by CA β-catenin. These observations indicated that the suppressive effects of WISP3 were β-catenin/TCF signalling-dependent.

| D ISCUSS I ON
CCN proteins influence multiple physiological and pathological processes, mounting evidence indicated that abnormal levels of CCN proteins played different roles in tumorigenesis, either enhance or inhibit cancer progression. 13,28,29 WISP3 was frequently studied in breast cancer. It was reported that WISP3 was downregulated in 80% of the aggressive inflammatory breast cancers with high metastatic potential. 15 Low CCN6 protein levels were associated with F I G U R E 2 Forced expression of WISP3 inhibited cell growth, migration and tumorigenesis. A, Overexpression of WISP3 in PVTT-1 and Huh7 cells. Cells were infected with lentivirus expressing WISP3 or vector control, followed by selection with puromycin. Lysate was examined by Western blot. α-tubulin was used as a loading control. B, The effects of WISP3 on the proliferation of PVTT-1 and Huh7 cells were measured by MTT assay. C, The effects of WISP3 on the migration of PVTT-1 and Huh7 cells were measured by Boyden Chamber assay. Left photograph: Boyden Chamber assay of PVTT-1 and Huh7 cell lines after forced expression of WISP3. Right photograph: calculation of cells that migrated through the filter following eosin staining. Scale bar = 200 μm. D, The effects of WISP3 on growth of Huh7 in vivo. Left photograph: representative photograph of the xenografts. Right photograph: tumour weight. E, Overexpression of WISP3 inhibited HCC cell growth in distant organs. WISP3 overexpressing cells and control cells labelled with luciferase were injected into the left ventricle of nude mice. The metastatic lesions were measured by bioluminescent signals on days 0 and 28 (five mice for each group). Results were presented as mean ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs Lenti-vector cells Among the mechanisms leading to the malignant transformation of liver cells, hyperactivation of β-catenin/TCF signalling was very common. 35 WISP genes were first described as downstream targets of the Wnt signalling pathway, 9 whereas the β-catenin/TCF/ LEF signalling activity was regulated by the WISP family. It has been reported that WISP1 auto-regulated its expression through regulation of β-catenin phosphorylation and nuclear translocation. 11 Also, our results suggested WISP3 could affect the subcellular localization of β-catenin. In the unstimulated cells, Serine-45 in the β-catenin protein was constitutively phosphorylated by CK1 and GSK3β, preventing β-catenin accumulation in the cytoplasm and the nucleus.
During the inhibition of GSK3β induced by Wnt activation, β-catenin was accumulated and translocated to the nucleus to regulate the transcription of Wnt target genes. [3][4][5] We demonstrated that the overexpression of WISP3 was associated with decreased phosphorylation of Akt and GSK3β as well as the reduced expression of β-catenin in the nucleus. In contrast, knockdown of endogeneous WISP3 enhanced phosphorylation of Akt, leading to increased phosphorylation of GSK3β and free cytoplasmic pool of β-catenin, which facilitated its nuclear translocalization and transcriptional function.
The nuclear β-catenin induced a gene expression pattern favouring tumour metastasis, including cyclin D1 and MMP7, 23  E-cadherin andβ-catenin are important epithelial adhesion molecules in normal epithelium, 36 and β-catenin plays a crucial role in cellcell adhesion mediated by E-cadherin. 21 A disturbance in epithelial cell adhesion, which leads to a more invasive and metastatic phenotype, is a hallmark of tumour progression. 21 Our result also indicated that overexpression of WISP3 induced mesenchymal-epithelial transition ( Figure S6A), and enhanced the expression of E-cadherin in Huh7 and PVTT-1 ( Figure S6B). Consistently, it has been reported that WISP3 knockdown activates growth factor-independent survival that requires activation of the PI3K/Akt signalling pathway 37 and regulation of E-cadherin. 38 Loss of E-cadherin liberated β-catenin from the membrane, which resulted in accumulation of β-catenin in the nuclear and made cells more motile and invasive. 39 Our results suggested that WISP3 might alternatively influence the subcellular localization of β-catenin by regulating the expression of E-cadherin which recruited β-catenin to adherens junctions and then inhibited β-catenin/TCF signalling. Taken together, reduction of WISP3 in HCC contributed to downregulation of E-cadherin and inhibition of GSK3β, both of which lead to the accumulation and activation of β-catenin in nucleus.
In summary, our study has shown that the expression of WISP3 was significantly decreased in clinical samples of HCC. Silencing the expression of WISP3 accelerated cell growth and migration, whereas forced expression of WISP3 suppressed tumour growth and metastasis seeding. All of these findings provide the first evidence that WISP3 exerts a strong inhibitory effect in human HCC. The aberrant activation of β-catenin signal pathway occurs frequently in HCC and many other types of solid tumour. Wnt inhibitors in the extracellular compartment prevent the aberrant βcatenin signalling by regulating the accessibility of the Wnt ligands to Frizzled receptors. 35 In this context, WISP3, an ECM protein, might be a novel therapy candidate to retard the development and metastasis of HCC by inhibiting β-catenin signalling.
F I G U R E 3 Knockdown of WISP3 promoted cell growth and migration, as well as metastasis in vivo. A, Silencing the expression of WISP3 in QSG-7701 and LO2 cells. Left panel: Result of Western blot. Right panel: Grey analysis results of the Western blot bands. B, Knockdown the expression of WISP3 promoted the growth of QSG-7701 and LO2 cells by MTT assay. C, Knockdown the expression of WISP3 promoted the migration of QSG-7701 and LO2 cells by Boyden Chamber assay. Left photograph: Boyden Chamber assay of QSG-7701 and LO2 cell lines after knockdown the expression of WISP3. Right photograph: calculation of cells that migrated through the filter following eosin staining. Scale bar = 200 μm. D, Knockdown of WISP3 promoted cell growth in distant organs. The metastatic lesions were measured by bioluminescent signals on days 0 and 63 (five mice for each group). Results were presented as mean ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs Lenti-vector cells | 11 of 12 GAO et Al.

ACK N OWLED G EM ENTS
We thank the New World Group for their Charitable Foundation to establish the Institute for Nutritional Sciences, SIBS, CAS-New World Joint laboratory, which have given full support to this study.
The ATF2 luciferase reporter was a gift from Dr. Shi De-Li.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S' CO NTR I B UTI O N S
Hong Gao and Fen-Fen Yin contributed to the conception and design, collection and assembly of data, and wrote the manuscript; all authors were involved in final approval of the submitted and published versions.