A super gene expression system enhances the anti-glioma effects of adenovirus-mediated REIC/Dkk-3 gene therapy

Reduced expression in immortalized cells/Dickkopf-3 (REIC/Dkk-3) is a tumor suppressor and therapeutic gene in many human cancers. Recently, an adenovirus REIC vector with the super gene expression system (Ad-SGE-REIC) was developed to increase REIC/Dkk-3 expression and enhance therapeutic effects compared with the conventional adenoviral vector (Ad-CAG-REIC). In this study, we investigated the in vitro and in vivo effects of Ad-SGE-REIC on malignant glioma. In U87ΔEGFR and GL261 glioma cells, western blotting confirmed that robust upregulation of REIC/Dkk-3 expression occurred in Ad-SGE-REIC-transduced cells, most notably after transduction at a multiplicity of infection of 10. Cytotoxicity assays showed that Ad-SGE-REIC resulted in a time-dependent and significant reduction in the number of malignant glioma cells attaching to the bottom of culture wells. Xenograft and syngeneic mouse intracranial glioma models treated with Ad-SGE-REIC had significantly longer survival than those treated with the control vector Ad-LacZ or with Ad-CAG-REIC. This study demonstrated the anti-glioma effect of Ad-SGE-REIC, which may represent a promising strategy for the treatment of malignant glioma.


Cytotoxicity of Ad-SGE-REIC against normal human astrocytes. The in vitro cytotoxic effect of
Ad-REIC on normal human astrocyte (NHA) cells was investigated. Incubation with Ad-LacZ, Ad-CAG-REIC, or Ad-SGE-REIC at an MOI of 10 for the indicated time did not alter the proliferation rate of NHA cells (Fig. 3).
The Wnt signaling pathway additionally regulates cell survival by inhibition of proteasome-dependent proteolysis of β -catenin. Therefore, we evaluated the impact of Ad-LacZ, Ad-CAG-REIC, and Ad-SGE-REIC treatment on β -catenin expression in malignant glioma cells. β -catenin protein levels were more potently reduced by Ad-SGE-REIC treatment than by Ad-CAG-REIC treatment.
Moreover, the activity of caspase-9 was evaluated in U87Δ EGFR cells. The cleaved form of caspase-9 expression was also increased in cells treated with Ad-SGE-REIC compared with those treated with Ad-CAG-REIC or Ad-LacZ (Fig. 5).
Therapeutic efficacy of Ad-SGE-REIC in xenograft mouse models and a syngeneic model. The anti-tumor effect of Ad-CAG-REIC and Ad-SGE-REIC was tested in mice bearing intracerebral glioma (U87Δ EGFR or GL261) and a syngeneic model (GL261). In all experiments, mice were injected with 3.6 × 10 7 plaque-forming units (pfu) of adenovirus. Kaplan-Meier curves were used to analyze the survival time of the U87Δ EGFR mouse glioma model after treatment with Ad-LacZ, Ad-CAG-REIC, or Ad-SGE-REIC. The median survival time was longer in mice treated with Ad-SGE-REIC than with Ad-LacZ (22 and 18 days, respectively; P = 0.0038; Fig. 6A). Median survival was also longer in mice treated with Ad-SGE-REIC than with Ad-CAG-REIC (22 and 19 days, respectively; P = 0.0107; Fig. 6A). In the GL261 mouse glioma model, the median survival time was also significantly longer in mice treated with Ad-SGE-REIC than with Ad-LacZ (41 and 33 days, respectively; P = 0.0257; Fig. 6B). In cytotoxicity assays, the proliferation rate of malignant glioma cells was reduced in a time-dependent manner after treatment with Ad-SGE-REIC and the effect was stronger compared with that of Ad-CAG-REIC (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001).
In the GL261 syngeneic model, the median survival time of mice treated with Ad-CAG-REIC was significantly longer than that of those treated with Ad-LacZ (47 and 36 days, respectively; P = 0.024; Fig. 6C). The median survival time of mice treated with Ad-SGE-REIC was also significantly longer than that of those treated with Ad-LacZ (103 and 36 days, respectively; P = 0.004; Fig. 6C).

Infection with Ad-REIC induces lymphocyte and dendritic cell infiltration into glioma.
Immunological reactions to Ad-REIC were investigated by histological evaluation of GL261 gliomas at 21 days after viral infection. Few CD8-and CD11c-positive cells had infiltrated the tumor specimens treated with Ad-LacZ (Figs 7 and 8). In stark contrast, obvious infiltration of both CD8-and CD11c-positive cells was detected in tumors treated with Ad-SGE-REIC or Ad-CAG-REIC (Figs 7 and 8). Infiltration of CD8-and CD11c-positive cells was significantly greater in tumors treated with Ad-SGE-REIC relative to those treated with Ad-CAG-REIC (P < 0.0001).

Discussion
Summary of results. This study focused on the anti-glioma activity of Ad-SGE-REIC, a novel adenoviral vector that produces higher protein expression and a superior therapeutic effect compared with the conventional system (Ad-CAG-REIC). REIC/Dkk-3 expression was upregulated in Ad-SGE-REIC-transduced glioma cells, and the most prominent effect was obtained after transduction at 10 MOI. In cytotoxicity assays, Ad-SGE-REIC time-dependently reduced the number of viable malignant glioma cells. In xenograft and syngeneic intracranial glioma models, Ad-SGE-REIC was associated with significantly longer survival than Ad-LacZ or Ad-CAG-REIC. Infiltration of CD8-and CD11c-positive cells was significantly greater in syngeneic gliomas treated with Ad-SGE-REIC than in those treated with Ad-CAG-REIC.  9 . Similarly, we previously found that enhancing REIC/Dkk-3 expression with an adenoviral vector led to a marked increase in the number of TUNEL-positive cells. Our data indicated that levels of the activated form of caspase-9 were significantly higher in glioma cells treated with Ad-SGE-REIC than in those treated with Ad-CAG-REIC and control.

Effects of Ad-REIC on glioma. Expression levels of REIC/Dkk
Moreover, the expressions of Bip, phosphorylated IRE1α , and phosphorylated SAPK/JNK were increased in Ad-SGE-REIC-infected cells compared with Ad-CAG-REIC-and Ad-LacZ-infected cells. This result indicated that ER stress was strongly evoked by Ad-SGE-REIC. ER stress was also found to be evoked by enhanced REIC/Dkk-3 expression in malignant mesothelioma and in prostate and testicular cancer cells 6,19 . Additionally, expression levels of β -catenin, a key element of the Wnt signaling pathway, declined in parallel with the increase in REIC/Dkk-3 expression. Wnt signaling inhibits the release of cytochrome C and the subsequent activation of caspase-9 induced by apoptotic stimuli 20 .
Ad-SGE-REIC. Watanabe et al. found that insertion of the triple translational enhancer sequences of hTERT, SV40, and CMV downstream of the BGH polyA sequence yielded the most potent gene expression 18 . The hTERT promoter/enhancer is well-characterized and has been frequently used for cancer-specific gene expression [21][22][23][24] . Several studies have demonstrated increased gene expression by insertion of the SV40 enhancer downstream of polyA sequences [15][16][17] . The CMV enhancer is used in the CMV early enhancer/chicken β -actin promoter (CAG promoter), which is known to improve gene expression in various cell types and tissues 16 . Because this novel gene expression system using triple enhancers significantly increases the expression of the gene(s) of interest in comparison with conventional systems using the strong CMV promoter, we termed this novel gene expression cassette, the SGE system.

Efficacy of Ad-SGE-REIC.
In various types of human cancer cell, the induction of apoptosis is significantly increased by transduction of Ad-SGE-REIC compared with conventional Ad-REIC vectors. Furthermore, the inhibitory effects of Ad-REIC treatment on tumor growth have been analyzed in xenograft models. In both mouse renal cell carcinoma and human prostate cancer models, strong suppression of tumor growth was observed in the Ad-SGE-REIC-treated groups relative to the other treatment groups 18 . Thus, the novel SGE system significantly augments the anti-tumor effects of Ad-REIC in mouse xenograft models, and the Ad-SGE-REIC vector was superior to the conventional Ad-CMV-REIC and Ad-CAG-REIC vectors in terms of the efficacy of in vivo intratumoral gene therapy. The present findings demonstrated that in xenograft models the survival time of mice treated with Ad-SGE-REIC was significantly longer than that of those treated with Ad-LacZ or Ad-CAG-REIC. Furthermore, in a syngeneic model, the survival time of mice treated with Ad-SGE-REIC was vastly longer than that of those treated with conventional Ad-REIC.
Anti-tumor effect of Ad-SGE-REIC in the syngeneic model. In the GL261 syngeneic mouse glioma model, mice treated with Ad-CAG-REIC survived significantly longer than those treated with Ad-LacZ. Infiltration of CD8-and CD11c-positive cells was significantly greater in tumors treated with Ad-CAG-REIC than in those treated with Ad-LacZ. In another study, intratumoral administration of REIC/Dkk-3 protein also significantly suppressed tumor growth, which was linked to accumulation of CD8-and CD11c-postiive cells (killer T marker and dendritic cells, respectively), and enhanced the anti-cancer cytolytic activity of splenocytes 11 . Furthermore, the survival time of mice treated with Ad-SGE-REIC was significantly longer than that of those Future direction. Ad-REIC is being developed for evaluation in clinical trials. At the time of publication, a first-in-human, phase I/IIa clinical trial of in situ Ad-REIC gene therapy for prostate cancer was done at Okayama University Hospital 25,26 . In addition, a phase I clinical trial of Ad-SGE-REIC for malignant mesothelioma was initiated in September 2015. According to the findings of these trials, a clinical trial of Ad-SGE-REIC for the treatment of glioma will be planned. Moreover, we showed that integrin antagonist cilengitide augmented the therapeutic effect of Ad-REIC gene therapy for malignant glioma 10 . Several preclinical studies have shown that cilengitide has an enhanced antitumor effect when administered in combinatorial therapeutic regimens [27][28][29][30][31][32][33] . Furthermore, combination therapy of Ad-REIC with chemotherapy, molecular targeted therapy, and immunotherapy should also be evaluated.
In conclusion, we demonstrated the anti-glioma effect of the Ad-SGE-REIC. Our results indicated that Ad-SGE-REIC has potential as a strategy for the treatment of malignant glioma.

Materials and Methods
Cell lines. The glioma cell lines U87Δ EGFR and GL261 were seeded on tissue culture dishes (BD Falcon, Franklin Lakes, NJ, USA) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U penicillin, and 0.1 mg/ml of streptomycin. GL261 cells were provided by Dr. A. Natsume, Nagoya University (Nagoya, Japan). NHA cells were purchased from Takara Bio Inc. (Shiga, Japan).

Adenovirus vector carrying SGE-REIC/Dkk-3.
For Ad-REIC under the control of the CAG promoter, the full-length human REIC/Dkk-3 gene was inserted into the cosmid vector pAxCAwt and then transferred into an adenoviral vector using the COS-TPC method (Takara Bio). The SGE system was made by inserting the triple translational enhancer sequences of human telomerase reverse transcriptase (hTERT), Simian virus 40 (SV40),

Western blot analysis.
After cell culture in flat-bottomed six-well dishes, the media were aspirated, the dishes were washed twice in phosphate-buffered saline, and the cells were lysed in 1% sodium dodecyl sulfate. The lysates were sonicated for analyzing whole-cell proteins. Nuclear proteins were isolated using an NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Extracted protein samples were separated by gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking in 5% skim milk, the membranes were incubated overnight with primary antibodies at 4 °C. The membranes were washed with Tris-buffered saline-Tween 20 (TBST), incubated with secondary antibodies at room temperature for 1 h, and rinsed with TBST. Signals were visualized using the ECL Prime Western Blotting Detection System (GE Healthcare Life Sciences, Buckinghamshire, UK). The primary antibodies, mouse anti-human REIC/DKK-3 (provided by the Department of Urology, Okayama University, Okayama, Japan), rabbit anti-human Bip, mouse anti-human β -catenin, rabbit anti-human caspase-9, rabbit anti-human SAPK/JNK, mouse anti-human phosphorylated SAPK/JNK, TATA-binding protein (TBP) (Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-human phosphorylated IRE1α (Novus Biochemicals, Littleton, CO, USA) were diluted 1:1000 in TBST, and β -actin antibody (Sigma, St Louis, MO, USA) was diluted Ethics and animal use statement. This study was conducted in strict accordance to the recommendations in the Guide for the Care and Use of Laboratory Animals in Japan. Animals were housed at 25 °C with 12-h light/dark cycles and free access to water and standard rodent chow in the Department of Animal Resources of Okayama University. All procedures and animal protocols were approved by the Committee on the Ethics of Animal Experimentation at Okayama University (Permit No. OKU-2014264). All surgery was performed under general anesthesia with ketamine/pentobarbital, and all efforts were made to minimize animal suffering.
In vivo experiments. Before implantation, 85 to 90% confluent U87Δ EGFR cells or GL261 cells were trypsinized and centrifuged at 100 g for 5 min; the cell pellet was resuspended in phosphate-buffered saline, and the cell concentration was adjusted to 1.0 × 10 5 cells/μ l. For the xenograft models, U87Δ EGFR or GL261 cells (2 μ l) were injected into 6-week-old female BALB/c nude mice (CLEA Japan Inc., Tokyo, Japan). For the syngeneic model, GL261 cells (2 μ l) were injected into 6-week-old female C57BL/6N mice. The mice were anesthetized and placed in stereotactic frames (Narishige, Tokyo, Japan) with their skulls exposed. Tumor cells were injected with a Hamilton syringe (Hamilton, Reno, NV, USA) into the right frontal lobe (3 mm lateral to the midline, 1 mm posterior to the coronal suture, 3 mm depth from the dura), and the syringe was withdrawn slowly after 5 min to prevent reflux. The skulls were then cleaned, and the incision was sutured. At 7 days after tumor inoculation, all mice bearing brain tumors were reanesthetized and stereotactically injected with Ad-SGE-REIC, Ad-CAG-REIC, or Ad-LacZ at the tumor inoculation site using the same coordinates.
Histological procedures. For the detection of CD8-or CD11c-positive cell infiltration into gliomas after Ad-REIC treatment, GL261 glioma cells were implanted, then 3.6 × 10 7 pfu of Ad-SGE-REIC, Ad-CAG-REIC, or Ad-LacZ was injected intratumorally 7 days after tumor inoculation. Mice were sacrificed, and their excised brains were embedded in paraffin at 28 days after tumor inoculation. Immunohistochemical staining was performed after samples were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Sections with a thickness of 4 μ m were incubated in 0.3% H 2 O 2 (30 min) and then autoclaved for 15 min at 121 °C in 10 mM sodium citrate buffer, pH 6.0. Immunohistochemical staining for CD8 was performed with mouse monoclonal CD8 antibody (1:50 dilution, no. 550281, BD Pharmingen, San Diego, CA, USA). The Dako Cytomation Envision+ System-HRP Kit was then applied according to the manufacturer's protocol (Dako Cytomation, Carpentaria, CA, USA). After washing in PBS, the sections were counterstained with hematoxylin. Immunohistochemical staining for CD11c was performed with mouse monoclonal anti-CD11c antibody (no. 550375, BD Pharmingen) using the same method.
Statistical analyses. Data on protein expression obtained by western blotting were analyzed using Student's t-test. The proliferation rates obtained from cytotoxicity assays were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Kaplan-Meier survival curves were compared using the log-rank test. The number of CD8-and CD11c-positive cells/field was analyzed using one-way ANOVA followed by Tukey's post hoc test. Statistical analyses were performed using SPSS statistical software (version 20; SPSS, Inc., Chicago, IL, USA). P-values < 0.05 were considered statistically significant.