Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53‐dependent apoptosis and anticancer effect

Abstract This study investigated the efficacy of Toxoplasma GRA16, which binds to herpes virus‐associated ubiquitin‐specific protease (HAUSP), in anticancer treatment, and whether the expression of GRA16 in genetically modified hepatocellular carcinoma (HCC) cells (GRA16‐p53‐wild HepG2 and GRA16‐p53‐null Hep3B) regulates PTEN because alterations in phosphatase and tensin homologue (PTEN) and p53 are vital in liver carcinogenesis and the abnormal p53 gene appears in HCC. For this purpose, we established the GRA16 cell lines using the pBABE retrovirus system, assessed the detailed mechanism of PTEN regulation in vitro and established the anticancer effect in xenograft mice. Our study showed that cell proliferation, antiapoptotic factors, p‐AKT/AKT ratio, cell migration and invasive activity were decreased in GRA16‐stable HepG2 cells. Conversely, the apoptotic factors PTEN and p53 and apoptotic cells were elevated in GRA16‐stable HepG2 cells but not in Hep3B cells. The change in MDM2 was inconspicuous in both HepG2 and Hep3B; however, the PTEN level was remarkably elevated in HepG2 but not in Hep3B. HAUSP‐bound GRA16 preferentially increased p53 stabilization by the nuclear localization of PTEN rather than MDM2‐dependent mechanisms. These molecular changes appeared to correlate with the decreased tumour mass in GRA16‐stable‐HepG2 cell‐xenograft nude mice. This study establishes that GRA16 is a HAUSP inhibitor that targets the nuclear localization of PTEN and induces the anticancer effect in a p53‐dependent manner. The efficacy of GRA16 could be newly highlighted in HCC treatment in a p53‐dependent manner.

Toxoplasma gondii (T gondii) is an intracellular parasite that infects multiple organs and tissues in acute infection and the brain in chronic infection 9,10 and regulates the host immunity for its survival during infection. 10,11 Briefly, the immunomodulatory activities of T gondii are mediated by infection as well as several T gondii-specific molecules such as rhoptry proteins (ROP), dense granule proteins (GRA), T gondii profilin-like protein (TgPLP) and the lysate antigenic proteins. 9,10 Thus, our objective was to determine the intermediate events between HAUSP inhibition and p53 stabilization and also the anticancer effect. In particular, p53 transcriptional activity is often disrupted in HCC by highly expressed HAUSP; moreover, the expression of nuclear PTEN decreases in patients with advancedstage HCC. 5,7,17,18 Thus, HCC forms an appropriate model for our study; indeed, it has been known that HCC is one of the 10 most common cancer types worldwide with no ideal treatment. 17 Thus, this study aimed to investigate transcriptional gene expressions associated with PTEN and subsequent apoptosis after HAUSP inhibition by GRA16. Furthermore, it investigated the characteristics of molecular networks primarily associated with nuclear PTEN changes between HAUSP and p53 in GRA16-stable cells.

| Plasmid construction for preparing GRA16gene stable cell line
The T gondii-derived dense granule protein 16 (TgGRA16) was prepared by gene cloning for the preparation of a stable cell line that continuously expressed the GRA16 gene. Furthermore, the GRA16 gene was amplified by PCR with a pair of specific prim-

| Co-immunoprecipitation for binding between GRA16 and USP7/HAUSP
For the co-immunoprecipitation (co-IP) assay, total proteins in 5 × 10 6 HepG2 or Hep3B cells were extracted by incubating for 15 minutes at room temperature (RT) using 100-μL mammalian protein extraction reagent (M-PER; Pierce Biotechnology, Inc, Rockford, IL, USA). Protein A/G-plus agarose beads previously reacted with HA-Tag Ab were added in 0.5 mg of each extracted protein (HepG2 or Hep3B) and incubated for 4 hours at 4°C. The supernatant of each sample was analysed by Western blotting with the HAUSP Ab (Cell Signaling Technology, Danvers, MA, USA).

| Cell proliferation
A total of 5 × 10 3 cells were seeded on a 96-well plate and incubated for 2, 4 and 6 days to assess the cytotoxicity of cells that were divided into experimental groups (control, vector, GRA16) in HepG2 and Hep3B cells. Cell viability at each incubation time was assessed using the Cell Counting Kit (CCK-8; Dojindo, Kumamoto, Japan) and measured in terms of optical density at 451 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). For the cell proliferation analysis, HepG2 and Hep3B cells were seeded into 24-well plates at 3 × 10 4 cells/ well and cultured for 6 days. The degree of cell proliferation was monitored by the Trypan blue exclusion test using a haemocytometer on days 2, 4 and 6 after cell seeding. All experiments were performed in triplicates, and results were obtained through three independent experiments for each study.

| Real-time PCR
Real-time PCR was performed to target genes using the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) and SYBR Green I detection chemistry (Bio-Rad Laboratories). Primer sequences are presented in Table 1. The data were analysed using the Bio-Rad CFX manager software ver 3.1 (Bio-Rad Laboratories).
All cDNA samples were assessed by the fold change in the target gene expression compared with the control and, then, calculated for the fold change in the gene expression for vector (transfected by empty vector) or GRA16 (stable cells with GRA16) compared with the control in each target gene.

| Western blotting
Cells were cultured in a 6-well plate with complete DMEM medium for 6 days, and total proteins were extracted from cells using the M-PER Mammalian Protein Extraction Kit (Pierce Biotechnology, Inc). Proteins

| Immunofluorescence for the GRA16 expression and PTEN nuclear localization
Immunofluorescence images of cells were obtained after immunostaining with HA-Tag Ab (Elabscience) and anti-PTEN Ab (Santa Cruz Biotechnology) to investigate GRA16 expression in HepG2and Hep3B-GRA16 cells and the nuclear localization of PTEN. The immunostained cells were observed using fluorescent microscopy (Leica DMI6000 B).

| Cell cycle analysis
HepG2 and Hep3B cells with and without GRA16 were seeded at

| Wound healing assay
HepG2 and Hep3B cells with and without GRA16 were seeded at and a microscope (BX-51; Olympus Corporation) with the AF6000 Leica Las-X software (Leica Microsystems). Based on the wound width, data were evaluated by the migration distance using the ImageJ program.

| Transwell migration/invasion assay
The migration and invasion ability of cells were assessed using the

| Statistical analysis
All statistical analyses were performed using the GraphPad Prism 5 software (GraphPad, La Jolla, CA, USA). Data are presented as mean ± standard deviation (SD) of three independent experiments, each performed in triplicates. One-way analysis of variance (ANOVA) was performed followed by the Tukey's multiple comparison test to assess the differences between the experimental groups. Two-way ANOVA followed by the Bonferroni's post hoc comparisons test was used to assess differences between the experimental groups.
P < 0.05 was considered statistically significant.

| Changes in the cell proliferation and total cell counts after establishing a GRA16-expressing stable cell line for HepG2 and Hep3B
A stable cell line expressing the GRA16 protein for HepG2 and Hep3B, which are p53-wild-and p53-null type, respectively, as human HCC cells was established to determine the role of GRA16 as an HAUSP inhibitor in cancer cells ( Figure 1). All experimental groups were divided into control (no transfection), vector (transfected with pBABE vector) and GRA16 (transfected with pBABE-GRA16 vector).
We confirmed GRA16 expression in the stable cell line by intracellular GRA16 expression using green fluorescent protein (GFP)-immunostaining ( Figure 1A) and co-immunoprecipitation (co-IP) assay results between GRA16 and HAUSP ( Figure 1B). As shown in

| Protein expressions of PTEN and p53 as well as AKT phosphorylation in GRA16-stable-HepG2 and -Hep3B cells
p53 protein expression was observed in p53-wild HepG2 cells but not in p53-null Hep3B cells (Figure 2A-a and B-a). Although PTEN expression was significantly increased, AKT phosphorylation was significantly decreased in GRA16-stable HepG2 cells in comparison to the control and vector groups (P < 0.05; Figure 2A As anticipated, the relative intensity of the p53 protein normalized by β-actin was significantly increased in GRA-stable HepG2 cells in comparison to the control and vector groups (P < 0.05; Figure 2C-b). However, because the p53-null-type Hep3B is absent in p53 expression, further regulation of PTEN expression and AKT phosphorylation was not observed in this study ( Figure 2B-b and C-b). Accordingly, these findings suggested that in the presence of endogenous p53, GRA16 as a HAUSP inhibitor increases PTEN expression and simultaneously decreases AKT phosphorylation, subsequently increasing p53 expression. In the absence of p53, the role of GRA16 is so limited that it cannot increase PTEN expression and p53 stabilization.  and Hep3B. *The significant difference between the control and GRA16 groups (P < 0.05). †The significant difference between the vector and GRA groups (P < 0.05)

| The retained cell cycle of the G 2 -M phase in GRA16-stable-HepG2
The results presented above, which demonstrate a decrease in the total cell number and AKT phosphorylation as well as an increase in cell apoptotic factors and nuclear PTEN in GRA16-stable-HepG2 cells, implied subsequent changes in cell cycles ( Figure 4). Thus, we quantified the percentage of cells occupied during each phase of the cell cycle using the FACS assay. Figure Table 2 presents these findings in detail.

| Differences in inductions of apoptosis and p53-related apoptotic factors between GRA16stable-HepG2 and -Hep3B cells
To investigate p53-dependent cell apoptosis in GRA16-stable For the apoptosis related with p53 in GRA16-stable HepG2 cell, we validated mRNA expressions for apoptotic (Foxo3a, BAX and P21) and antiapoptotic factors (Bcl-2, Survivin and MDM2) associated with the p53 function ( Figure 5C). As anticipated, the mRNA levels of apoptotic factors (Foxo3a, BAX and P21) in GRA16-stable HepG2 cells were significantly increased in comparison to both control and vector groups (P < 0.05; Figure 5C). In contrast, the mRNA levels of antiapoptotic factors (Bcl-2 and Survivin) were decreased in GRA16-stable HepG2 cells (P < 0.05; Figure 5C).
However, the increase in apoptotic factors was not observed in Hep3B cells ( Figure 5C). Meanwhile, antiapoptotic factors exhibited no change in GRA16-stable Hep3B cells as well as the control and vector groups ( Figure 5C). These findings implied that the role of GRA16 as a HASUP inhibitor was exhibited in p53-wild-type cells.

| Effects of GRA16 on the mobility and invasive activity of GRA16-stable-HepG2 and -Hep3B cancer cells
We investigated the changes in the cell mobility (wound closure effect) and invasive activity (Transwell cell migration activity) of cancer cells in HepG2 and Hep3B ( Figure 6A-a and b, B-a and b and C-a). In addition, we assessed the mRNA levels of MMP2 and PTK2 (important cell migration factors), which can be increased by AKT phosphorylation and cell mobility factor, which is inhibited by PTEN respectively (Figure 6A-c and B-c). In HepG2 cells, the presence of GRA16 significantly reduced cell mobility at 24 and 48 hours after incubation in comparison to the control and vector groups (P < 0.05; Figure 6A-b). In addition, the decreased cell mobility suggests that the wounded area was not reduced ( Figure 6A-a). However, in Hep3B, the wounded area (%) after scratching was covered in the same degree among the control, vector and GRA16 groups, indicating no effect of GRA16 ( Figure 6B Figure 6A-c), but they were not reduced in GRA16-stable Hep3B cells ( Figure 6B-c). Moreover, the Transwell invasion assay exhibited similar results as follows ( Figure 6C-a). When the result was calculated by the relative migration capability (%) compared with the control (100%), it was also consistent with that of the wound healing assay ( Figure 6C-b). Hence, we are convinced that the effect of GRA16 as a HAUSP inhibitor could induce the anticancer effect in p53-wild HCC cells.

| Tumour reduction in xenograft mice
Nude mice xenograft with control-and vector-HepG2 or Hep3B cells exhibited a gradual increase in the tumour size and mass ( Figure 7A-a and d and B-a and d). However, the tumour size and mass in nude mice xenograft with GRA-stable HepG2 cells were significantly lower than that in the control and vector group (P < 0.05; Figure 7A-a and A-b). The tumour weights in nude mice xenograft with GRA16-stable HepG2 cells were significantly smaller than those in the control and vector group (P < 0.05; Figure 7A-d). However, the tumour mass and tumour weights in nude mice xenograft with GRA-stable Hep3B cells were continuously increased without differences in comparison to the control and vector group ( Figure 7B-a and B-d). Hence, we are convinced that GRA16 in nude mice xenograft enhanced anticancer effects in the presence of p53.

| D ISCUSS I ON
The inhibition of USP (or HAUSP) has been reported as an anticancer drug target. 1 The action of HAUSP is highly specific for substrates  is worth investigating HCC suppression using a HAUSP inhibitor. [1][2][3]6 Typically, GRA proteins of T gondii secreted from parasites reside in the parasitophorous vacuole and play a role in the intracellular survival and replication of parasites. 13 Of these, GRA16 migrates to the nucleus and participates in the regulation of the p53 oncogene signalling pathway. 13 We assessed whether an anticancer effect could be induced by using the HAUSP-binding effect of GRA16 in HCC, and, moreover, the underlying mechanisms inducing p53 stabilization after HAUSP inhibition. As some human cancer types, including HCC, exhibit an abnormal p53 gene or have disrupted p53 gene activation pathways, the effect of GRA16 should be evaluated in conditions with and without the p53 gene. 17 Thus, in our study, we developed genetically modified GRA16-stable cancer cells for p53-wild-type HepG2 and p53-null-type Hep3B, and examined the binding between GRA16 and HAUSP within cells using the co-IP.
However, Hep3B cells did not exhibit any changes in the levels of were under the following situation by which p21, p27, p73 and Bax/ Bcl-2 were increased by the action of an MDM2 antagonist or by which AKT inhibition was observed by notch1 overexpression, 22,23 indicating that the precise mechanisms should be determined by the type of HAUSP inhibitor and the target substrate for the HAUSP inhibitor.
The p53 protein cooperates with PTEN and could be an essential blockage in the development of mammary tumours 6 ; thus, we can comprehend that the therapeutic efficiency of anticancer agents strongly depends on their detailed mechanisms to trigger apoptosis in targeted cancer cells. 24 As p53 plays a pivotal role in regulating tumour apoptosis, our study elucidated the prominence of the p53dependent mechanism on the anti-HCC effect of GRA16. In HCC tumours, down-regulation of the nuclear PTEN is an essential step in hepatocarcinogenesis. 5,19,20 As revealed in this study, PTEN levels tics. Furthermore, this study newly proves that the major role of GRA16 is to inhibit PTEN translocation from the nucleus to the cytoplasm through HAUSP inhibition and suggests that GRA16 can be applied as an alternative treatment for HCC.

CO N FLI C T O F I NTE R E S T
The authors have no competing interests to declare.

AUTH O R CO NTR I B UTI O N S
SGK, SHS and EHS conceived and designed the experiments. SGK and SHS prepared the GRA16 stable cell line and performed the experiments. JHS, JPY and EHS analysed the data. SHL and EHS contributed reagents/materials/analysis tools. EHS supported the idea and was responsible for overall project administration, acquiring financial support and writing the paper.