ANTICANCER RESEARCH 37 : 301-308 (2017) doi:10.21873/anticanres.11321

Distant Bystander Effect of REIC/DKK3 Therapy Through Immune System Stimulation in Thoracic Malignancies KEN SUZAWA 1* , KAZUHIKO SHIEN 1,2* , HUANG PENG 3, MASAKIYO SAKAGUCHI 4, MASAMI WATANABE 5,6,7 , SHINSUKE HASHIDA 1,2 , YUHO MAKI 1, HIROMASA YAMAMOTO 1, SHUTA TOMIDA 8, JUNICHI SOH 1, HIROAKI ASANO 1, KAZUNORI TSUKUDA 1, YASUTOMO NASU 5,6,7 , HIROMI KUMON 3,5,7 , SHINICHIRO MIYOSHI 1 and SHINICHI TOYOOKA 1,2

Departments of 1Thoracic Surgery, 2Clinical Genomic Medicine and 4Cell Biology, 5Urology, 3Collaborative Research Center for Okayama Medical Innovation Center, and 8Biobank, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 6Center for Innovative Clinical Medicine, Okayama University Hospital, Okayama, Japan; 7Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University, Okayama, Japan

Abstract. Background: Reduced expression in immortalized Both advanced-stage non-small cell lung cancer (NSCLC) cell (REIC)/Dickkoph-3 (DKK3) is a tumor-suppressor gene, and malignant mesothelioma (MM) are aggressive tumors and and its overexpression by adenovirus vector (Ad-REIC) present a dismal prognosis. Despite advances in treatment exhibits a remarkable therapeutic effect on various human regimens for both diseases, such as surgical resection, cancer types through a mechanism triggered by endoplasmic chemotherapy, molecular-targeted therapy and radiotherapy, reticulum stress. Materials and Methods: We examined the treatment outcome is still unsatisfactory. Gene therapy for direct anti-tumor effect of Ad-REIC gene therapy on lung thoracic malignancies represents a novel therapeutic approach cancer and malignant mesothelioma cell lines in vitro, and and has been evaluated in a number of clinical trials over the the distant bystander effect using immunocompetent mouse last two decades (1). Previously, we identified expression of allograft models with bilateral flank tumors. Results: Ad- the tumor-suppressor gene Reduced expression in REIC treatment showed antitumor effect in many lung cancer immortalized cell ( REIC )/Dickkoph-3 ( DKK3 ) as being and malignant mesothelioma cell lines in vitro. In an in vivo reduced in many human cancer types (2-6). However, the model, Ad-REIC treatment inhibited the growth not only of impact of REIC/DKK3 in thoracic malignancies, such as the directly treated tumors but also of distant untreated tumors. frequency of its decreased expression among the subtypes of By immunohistochemical analysis, infiltration of T-cells and lung cancer, and the relationship between its loss and other natural killer (NK) cells and expression of the major oncogenic driver mutations, is yet to be investigated. histocompatibility complex (MHC) class I molecules were We demonstrated that overexpression of REIC/DKK3 by observed in bilateral tumors. Conclusion: Ad-REIC treatment adenovirus (Ad-REIC) led to remarkable therapeutic effects not only had a direct antitumor effect but also an indirect on several human cancer types, but not on normal cells (3, bystander effect through stimulation of the immune system. 4, 7-9). As the REIC/DKK3 gene expression is absent from cancer cells, the REIC/DKK3 folding system in cancer cells does not function well when the protein is overexpressed by Ad-REIC, which leads to endoplasmic reticulum (ER) stress-induced apoptosis. The activation of c- *These Authors contributed equally to this study. JUN N-terminal kinase (JNK) pathway occurs downstream of ER stress signaling, which is a critical event in apoptosis Correspondence to: Shinich Toyooka, MD, Ph.D., Department of (3, 4, 7-11). In addition to this direct antitumor effect, we Clinical Genomic Medicine, Okayama University Graduate School also showed Ad-REIC to have a host-mediated bystander of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, effect on human prostate cancer and scirrhous gastric cancer Japan. Tel: +81 862357436, Fax: +81 862357437, e-mail: [email protected] via the induction of anticancer immunity (12-14). Secreted REIC/DKK3 protein differentiates monocyte into the Key Words: REIC/DKK3 , gene therapy, lung cancer, mesothelioma, dendritic cells-like phenotype and then activates cytotoxic T- bystander effect. lymphocytes (CTLs) (12). Furthermore, in normal

0250-7005/2017 $2.00+.40 301 ANTICANCER RESEARCH 37 : 301-308 (2017) fibroblasts, Ad-REIC treatment does not cause apoptosis, but a modified 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]- induces interleukin 7 (IL7) secretion. This enhanced IL7 2-[4-sulfophenyl]-2H-tetrazolium, inner salt (MTS) assay with secretion activates natural killer (NK) cells (13, 14). These CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA) according to the manufacturer’s instruction. mechanisms up-regulate systemic antitumor immunity. Given this evidence, a clinical trial of REIC gene therapy Western blotting. The total cell lysate was extracted from cell lines for prostatic cancer is ongoing (UMIN000004929; with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor NCT01931046). In a first study in Man (a phase I/IIa study cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA), and of in situ Ad-REIC therapy for prostate cancer), direct and Complete Mini (Roche, Basel, Switzerland). Western blot analysis systemic antitumor effects were clearly detected in a patient was performed by conventional methods using the following with metastatic castration-resistant prostate cancer following primary antibodies: rabbit anti-human REIC/DKK3 antibody raised chemotherapy (15). in our laboratory, rabbit anti- stress-activated protein kinases (SAPK)/JNK, phospho-SAPK/JNK (Thr183/Tyr185), cleaved poly In this study, we examined potential direct and indirect (ADP-ribose) polymerase (PARP) (Cell Signaling Technology, bystander effects of Ad-REIC via activation of systemic Danvers, MA, USA), and mouse anti-actin (Merck Millipore, antitumor immunity in lung cancer and mesothelioma cells. Billerica, MA, USA). The following secondary antibodies were In order to examine the immune-mediated response in vivo , used: goat anti-rabbit or anti-mouse IgG-conjugated horseradish we used murine lines and an immunocompetent peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). To detect mouse model. specific signals, membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, Materials and Methods UK) and LAS-3000 (Fujifilm, Tokyo, Japan).

Gene expression analysis of clinical lung cancer tissues. Messenger Animal models. This study was carried out in accordance with the RNA (mRNA) expression, histological subtype, and genetic mutation Guidelines of the Okayama University Animal Committee. profiling for 158 lung carcinomas and five normal lung tissues (16) Pathogen-free immunocompetent female BALB/c mice aged 6 to 8 were obtained from Gene Expression Omnibus public database weeks were purchased from CLEA Japan (Tokyo, Japan). AB12 cells (1×10 6) were suspended in 100 μl of serum-free DMEM, and (GEO; http://www.ncbi.nlm.nih.gov/geo/; accession number 6 GSE11969). We assessed the association between mRNA expression KLN205 cells (1×10 ) were suspended in 200 μl DMEM with of REIC/DKK3 and the histological subtype or mutation profile. Matrigel, and each cell line was injected subcutaneously into the bilateral flanks of mice (n=3 per group and n=4 per group, 3 Cell lines. Four human lung cancer cell lines (A549, HCC827, respectively). When the tumors reached a volume of 200-250 mm , H1975, and PC9), three murine lung cancer cell lines (CMT64, the mice received intratumoral injection of Ad-SGE-REIC at a dose KLN205, and LL/2), four human MM cell lines (H28, H290, of 109 plaque-forming units (pfu) into the right-flank tumor on days 1 and 8. Control mice were treated in an analogous manner with H2052, and MSTO-211H), and three murine MM cell lines (AB1, 9 AB12, and AC29) were used in this study. A549 and LL/2 were injection of either 10 pfu Ad-LacZ or 100 μl of PBS. During the purchased from American Type Culture Collection (Manassas, VA, course of the study, mice were monitored for signs of pain or USA). HCC827, H1975, and four human MM cell lines were kindly distress, and loss of body weight twice a week. Tumor growth was provided by Dr. Adi F. Gazdar (University of Texas Southwestern monitored twice a week, and individual tumor volumes were measured using a digital caliper and approximated according to the Medical Center, Dallas, TX, USA). PC9 was purchased from 2 Immuno-Biological Laboratories (Takasaki, Japan). CMT64 was formula V=1/2 ab (a being the long diameter and b being the short obtained from the European Collection of Cell Cultures (Porton diameter of the tumor). No mouse encountered difficulty moving or Down, UK). KLN205 was obtained from RIKEN BioResource accessing feed and water due to tumor size, had to be euthanized Center (Tsukuba, Japan). AB1, AB12, and AC29 were were kindly prior to the experimental endpoint for the health reasons, nor provided by Steven M. Albelda (University of Pennsylvania, became severely ill or died. All mice were sacrificed on day 22 by Philadelphia, PA, USA). All human cells were cultured in RPMI- cervical dislocation under ketamine and xylazine anesthesia. After 1640 medium supplemented with 10% fetal bovine serum (FBS) sacrifice, tumors were subsequently harvested, measured, and and all murine cells were cultured in Dulbecco’s modified Eagle's pathologically examined. medium (DMEM) supplemented with 10% FBS. Immunohistochemical analysis. Dissected bilateral flank tumors Adenovirus vectors. REIC/DKK3 was overexpressed using an were fixed in 10% formaldehyde, embedded in paraffin, and cut into adenovirus (Ad-REIC). We modified the Ad-REIC adenovirus vector 4 μm-thick sections. The sections were deparaffinized and previously generated (first-generation Ad-REIC) (3) and named it rehydrated; endogenous peroxidase was inhibited by a 10-min Ad-SGE-REIC (second-generation Ad-REIC) (17). An adenovirus incubation with 3.0% H 2O2 solution. Following a blocking step with vector carrying the LacZ gene (Ad-LacZ) was used as control. normal horse serum, the sections were incubated overnight with the primary antibodies at 4˚C, except for anti-REIC/DKK3, in which Cell viability assay. Cells were plated in 96-well plates at a density sections were incubated for 1 h at room temperature. The primary of 1×10 3 cells/well. Twenty-four hours later, cells were infected with antibodies consisted of anti-REIC/DKK3, anti-histocompatibility 60 Ad-LacZ, or Ad-SGE-REIC was infected at a multiplicity of infection (H60) (Santa Cruz Biotechnology), anti-retinoic acid early inducible (MOI) of 200 or with phosphate-buffered saline solution (PBS) as 1 (RAE1) (Santa Cruz Biotechnology), anti-cluster of differentiation control. Cell viability after treatment was evaluated 72 h later using (CD) 49b (pan-NK cells) (BioLegend, San Diego, CA, USA), anti-

302 Suzawa et al : Distant Bystander Effect of REIC/DKK-3 Gene Therapy

Figure 2. Overexpression of reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) induces expression of apoptosis-rebuked in non-small cell lung cancer cells and malignant mesothelioma cells. Human non-small cell lung cancer and malignant mesothelioma cells were transiently infected with either adenoviral vectors containing LacZ gene (Ad-LacZ) or REIC/DKK3 gene (Ad-SGE- REIC) at a multiplicity of infection of 200 for 72 h. Lysates were then subjected to western blot analysis for the indicated proteins. JNK: c- JUN N-terminal kinase; PARP: poly (ADP-ribose) polymerase.

DAB Peroxidase Substrate Kit (Vector Laboratories), Mayer’s hematoxylin was used for counterstaining, and the protein expression was analyzed by standard light microscopy.

Statistical analysis. All statistical analyses were performed with GraphPad Prism, version 6.0.3, J (GraphPad Software, San Diego, CA, USA). Group differences were compared using t-test or ANOVA for repeated measurements. Values of p< 0.05 were considered statistically significant.

Figure 1. Comparison of expression of reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) among different Results histological subtypes and genetic profiles of clinical lung cancer tissues. A: The mRNA expression level of REIC/DKK3 was obtained from the Characteristics of REIC/DKK3 expression in lung cancer . microarray data of five normal lung tissues, 158 lung carcinomas, The microarray data of 149 NSCLCs, nine small-cell lung including 90 adenocarcinomas (Ad), 35 squamous cell carcinomas (Sq), four adenosquamous cell carcinomas (AdSq), 18 large cell lung cancers cancers (SCLC) and five normal lung tissue showed that (LCLC), two large neuroendocrine caricinomas (LCNEC), and nine REIC/DKK3 expression was significantly reduced in lung small cell lung cancers (SCLC). Data represent means±SD. B: The cancer samples, including adenocarcinomas, squamous cell mRNA expression level of REIC/DKK3 of 149 non-small cell lung carcinomas, large cell carcinomas, large cell neuroendocrine cancers (NSCLC), including 34 epidermal growth factor receptor carcinomas, and SCLCs, compared with normal lung tissues (EGFR)-mutant (MT) NSCLCs, 12 Kirsten ras (KRAS)-MT NSCLCs and 103 both EGFR and KRAS wild-type (WT) NSCLCs, was obtained from (Figure 1A). In addition, no relationship was identified the same database. Sixty-eight samples were NSCLCs with p53 between REIC/DKK3 expression and other typical oncogenic mutation. *Significantly different at p<0.05. driver mutations of epidermal growth factor receptor ( EGFR ) or Kirsten ras ( KRAS ), or tumor-suppressor gene p53 mutational status (Figure 1B).

CD8a (BD Pharmingen, San Diego, CA, USA), and anti-cleaved caspase-3 (Cell Signaling Technology). After a brief wash, sections Induction of apoptosis in NSCLC and MM cells by were incubated with the appropriate second antibody (Vector overexpression of REIC/DKK3 using Ad-SGE-REIC. We first Laboratories, Burlingame, CA, USA) for 30 min at room examined the ability of Ad-SGE-REIC treatment to induce temperature. Antibody binding was detected using the ImmPACT apoptosis of human NSCLC and MM cells. NSCLC and MM

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Figure 3. In vitro antitumor effect and gene transduction efficiency of adenoviral vector containing reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) gene (Ad-SGE-REIC) treatment in 14 non-small cell lung cancer and malignant mesothelioma cell lines. Human and murine non-small cell lung cancer cells and malignant mesothelioma cells were treated with adenoviral vector containing LacZ gene (Ad-LacZ) or Ad-SGE-REIC at a multiplicity of infection of 200, and cell viability after 72-h of treatment was compared with control (non-treatment) cell viability using an MTS assay. Error bars indicate standard deviations. *Significantly different at p<0.05.

cells were infected with either Ad-LacZ or Ad-SGE-REIC at assessed the relationship with the response to Ad-SGE-REIC 200 MOI for 72 h. We then analyzed the expression of treatment. No endogenous REIC/DKK3 expression was REIC/DKK3, JNK, and cleaved PARP, as an apoptosis observed in any of the cell lines, and the expression level of marker, by western blot analysis. As shown in Figure 2, Ad- ectopic REIC/DKK3 in Ad-SGE-REIC treated cells differed SGE-REIC treatment induced a significant expression of among cell lines. Cells which were resistant to Ad-SGE- REIC/DKK3 and up-regulation of phospho-JNK, which REIC treatment generally expressed only a low level of resulted in subsequent expression of apoptosis-related REIC/DKK3 when treated with Ad-SGE-REIC, and the proteins in both NSCLC and MM cells. ectopic expression of REIC/DKK3 positively correlated with antitumor effect of Ad-SGE-REIC treatment. These results Association between antitumor effect and gene transduction indicate that the effect of Ad-SGE-REIC treatment depends efficiency of Ad-SGE-REIC treatment. We then investigated on gene transduction efficiency. the association between anti-tumor effect and gene transduction efficiency of Ad-SGE-REIC treatment in several In vivo antitumor effect of Ad-SGE-REIC. A host-mediated NSCLC and MM cell lines. Cell viability inhibition rates bystander effect was investigated in vivo using murine cancer with Ad-SGE-REIC treatment differed between each cell cell lines and an immunocompetent mouse model. The line, ranging from 5.6% to 75.2% (Figure 3). Next, we experimental set-up is shown in Figure 4A. Mice received examined the expression of REIC/DKK3 by western blot an intratumoral injection of PBS, Ad-LacZ, or Ad-SGE- analysis to evaluate gene transduction efficiency and REIC twice at a dose of 10 9 pfu into the right-flank tumor

304 Suzawa et al : Distant Bystander Effect of REIC/DKK-3 Gene Therapy

Figure 4. Adenoviral vector containing reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) gene (Ad-REIC) treatment had an antitumor effect on tumor growth of distant untreated tumors as well as directly treated tumor. A: Experimental scheme. Mice received an intratumoral injection of either phosphate-buffered saline solution (PBS), adenoviral vector containing LacZ gene (Ad-LacZ), or Ad-SGE-REIC into only the right-flank (ipsilateral) tumor on days 1 and 8. B: The growth curve of subcutaneous AB12 tumors is shown. Three mice were analyzed in each group. Data represent means±SD. C: The growth curve of subcutaneous KLN205 tumors is shown. Four mice were analyzed for each group. Data represent means±SD. D: Dissected bilateral AB12 tumors were stained with the antibodies to the indicated proteins. HE, Hematoxylin–eosin staining. H60 and RAE1 are mouse major histocompatibility complex class I homologs. Infiltration by natural killer cells was detected using antibody to cluster of differentiation (CD) 49b. Infiltration by CD8-positive T-cells was detected using antibody to CD8a. Cleaved caspase-3 was also examined as an apoptosis marker. The scale bar in each photomicrograph is 100 μm. *Significantly different at p<0.05.

on days 1 and 8 (Figure 4A). As shown in Figure 4B and C, In order to assess the mechanism of the indirect bystander Ad-SGE-REIC treatment significantly inhibited the growth effects mediated by Ad-REIC, immunohistochemical analysis of tumor compared with the controls (PBS or Ad-LacZ) in of tumors was performed. To confirm the expression of REIC both murine lung cancer (KLN205) and MM (AB12) in tumors treated with Ad-SGE-REIC, mice with AB12 allograft models. Furthermore, in the Ad-SGE-REIC-treated tumors were sacrificed on day 14, and the tumors were group, the growth of untreated tumors of the contralateral stained immunohistochemically using anti-REIC/DKK3 side was also strikingly suppressed, whereas this bystander (Figure 4D). REIC was detected only in the tumors into effect was not observed in control groups. which Ad-SGE-REIC had been directly injected. Next, we

305 ANTICANCER RESEARCH 37 : 301-308 (2017) examined ligands related to NK cell-activating receptor Furthermore, overproduction of IL7 from co-infected normal (NKG2D), and NK cell and CD8-positive T-cell infiltration. fibroblasts activates NK cells (13), and secreted REIC/DKK3 Dissected bilateral flank AB12 tumors upon sacrifice were protein induces differentiation of monocytes into the dendritic stained with antibodies recognizing H60, RAE1, CD49b and cell phenotype and then activates CTLs (12). These CD8a in order to investigate activation of systemic antitumor mechanisms up-regulate systemic antitumor immunity and immunity. Immunohistochemical assessment revealed marked lead to antitumor effects at both treated and distant tumor expression of H60 and RAE1 (mouse MHC class I homologs sites. Thus, the indirect antitumor effect generated by the functioning as NKG2D ligands) in both treated and untreated immune system is an important factor in the treatment of tumors in the Ad-REIC-treated group. NK cells with CD49b malignant tumors, which frequently develop metastasis. expression also infiltrated into untreated as well as treated In addition to previous reports, in this study, we tumors in the Ad-REIC treated group. These findings suggest demonstrated that Ad-SGE-REIC treatment is highly that NKG2D ligands are expressed by tissues in response to effective against both lung cancer and MM cells. Indeed, Ad- stress and may induce NK-cell activation and provide T-cell SGE-REIC not only exhibited a direct antitumor effect but co-stimulation (18). On the other hand, H60 and RAE1 were also a distant bystander effect, considered as an indirect not expressed in the tumors of the control group; moreover, antitumor effect. Ad-SGE-REIC efficiently inhibited nor were infiltrating NK cells detected. Furthermore, CD8- proliferation of lung cancer and MM cells, which expressed positive T-cells infiltrated into both treated and untreated REIC/DKK3 protein after REIC gene transduction in vitro , tumors in the Ad-REIC-treated group, while they were rarely whereas some cell lines were resistant to Ad-SGE-REIC detected in the tumors of LacZ control group. Consistent with treatment because of inefficient REIC gene transduction. On these results, cleaved caspase-3 was detected not only in Ad- the other hand, in murine tumor allograft models using REIC-injected tumors but also in non-injected tumors in the immunocompetent mice, we showed that Ad-SGE-REIC Ad-REIC-treated group, whereas the expression of cleaved treatment inhibited growth not only of the tumor directly- caspase-3 was low in both sides in the control group. These injected with Ad-REIC but also of the distant, non-injected results suggest that Ad-SGE-REIC treatment exhibits a tumor. With regard to the precise mechanism of this indirect bystander antitumor effect through immune system-mediated effect, in addition to our previous reports of its indirect effect apoptosis and CD8-positive T-cell activation, and that protein through Ad-REIC-mediated cancer vaccination and expression of MHC class I molecules induces NK-cell production of IL7, we newly demonstrate here that Ad-SGE- infiltration of the tumor. REIC treatment induces expression of the MHC class I molecules H60 and RAE1 both in treated tumors and in Discussion distant untreated tumors, resulting in the presentation of targets for NK cells. Although we have not elucidated Despite advances in treatment approaches including whether this induction of MHC class I protein expression is chemotherapy, radiation therapy, molecular-target therapy, caused directly or indirectly by Ad-REIC treatment, and surgery, the overall 5-year survival rate of patients with infiltration by CD49b-positive NK cells was detected not NSCLC remains lower than 15% (19, 20). Lung cancer is a only in the treated tumor but also in the untreated tumor, potentially good disease target for gene therapy using with induction of apoptosis-related protein expression only adenovirus-vector because lung cancer cells typically exhibit in the Ad-REIC-treated group. Although it is still unknown high expression of coxsackievirus and adenovirus receptor what causes MHC class I protein expression in the distant (CAR), facilitating transfection of vectors to target cells (9). tumor site, three possibilities should be considered: Firstly, On the other hand, MM is an aggressive tumor of the serosa REIC protein, which was produced in the treated site, might and pleura. Current therapies for MM are only marginally act on distant cancer cells via an as-yet-unidentified receptor. effective, and mortality remains high, with a median survival Secondly, some damage-associated molecules released from of approximately 12 months, except for rare cases where the treated site might bind to cell-surface receptors of the complete resection is possible (21). MM is also a potentially tumor at the distant site. Thirdly, tumor-associated antigen good disease target for gene therapy because the large released from the treated site may activate T-cells, and stress surface area of the thoracic cavity where malignant cells from stimulation of activated T-cells might induce expression exist is expected to offer efficient, rapid, and diffuse gene of MHC class I molecules at the distant tumor site. Further transfer (1). Indeed, REIC gene therapy for MM is also studies are necessary to uncover the mechanism of up- ongoing (UMIN000013568; JapicCTI-152998). regulation of expression of MHC class I molecules at distant We previously showed that Ad-REIC gene therapy exhibits tumor sites. a potent antitumor effect in various types of cancer cells (3, In conclusion, Ad-SGE-REIC treatment not only had a 4, 6-9). The direct antitumor effect is due to cancer-selective direct antitumor effect, but also an indirect bystander effect apoptosis as a result of ER stress in cancer cells (3, 9, 13). through immune system-mediated NK cell stimulation. Our

306 Suzawa et al : Distant Bystander Effect of REIC/DKK-3 Gene Therapy results support the therapeutic potential of Ad-SGE-REIC for 9 Shien K, Tanaka N, Watanabe M, Soh J, Sakaguchi M, Matsuo K, advanced lung cancer and MM. On the basis of these Yamamoto H, Furukawa M, Asano H, Tsukuda K, Nasu Y, Huh findings, we have initiated a clinical trial of Ad-REIC NH, Miyoshi S, Kumon H and Toyooka S: Anti-Cancer Effects of REIC/DKK-3-encoding adenoviral vector for the treatment of treatment of MM. non-small cell lung cancer. PLoS One 9: e87900, 2014. 10 Watanabe M, Nasu Y and Kumon H: Adenovirus-mediated Conflicts of Interest REIC/DKK3 gene therapy: Development of an autologous cancer vaccination therapy (Review). Oncol Lett 7: 595-601, 2014. Okayama University and Momotaro-Gene Inc., a start-up biotech 11 Sakaguchi M, Huh NH and Namba M: A novel tumor suppressor, company originating from Okayama University, hold the patents on REIC/DKK3 gene identified by our in vitro transformation model the Ad-REIC agent and are developing the agent as a cancer of normal human fibroblasts works as a potent therapeutic therapeutic medicine. Drs. M.S., M.W., Y.N., and H.K. own stocks antitumor agent. Adv Exp Med Biol 720 : 209-215, 2011. in Momotaro-Gene Inc. 12 Watanabe M, Kashiwakura Y, Huang P, Ochiai K, Futami J, Li SA, Takaoka M, Nasu Y, Sakaguchi M, Huh NH and Kumon H: Acknowledgements Immunological aspects of REIC/DKK3 in monocyte differentiation and tumor regression. Int J Oncol 34 : 657-663, 2009. The Authors thank Ms. Fumiko Isobe for her technical assistance. 13 Sakaguchi M, Kataoka K, Abarzua F, Tanimoto R, Watanabe M, This study was supported by a Grant-in-Aid for Scientific Research Murata H, Than SS, Kurose K, Kashiwakura Y, Ochiai K, Nasu from the Japan Society for the Promotion of Science (Grant number Y, Kumon H and Huh NH: Overexpression of REIC/DKK-3 in 26670627 to T. Shi). normal fibroblasts suppresses tumor growth via induction of interleukin-7. J Biol Chem 284 : 14236-14244, 2009. 14 Than SS, Kataoka K, Sakaguchi M, Murata H, Abarzua F, Taketa References C, Du G, Yashiro M, Yanagihara K, Nasu Y, Kumon H and Huh NH: Intraperitoneal administration of an adenovirus vector 1 Vachani A, Moon E, Wakeam E, Haas AR, Sterman DH and carrying REIC/DKK3 suppresses peritoneal dissemination of Albelda SM: Gene therapy for lung neoplasms. Clin Chest Med scirrhous gastric carcinoma. Oncol Rep 25 : 989-995, 2011. 32 : 865-885, 2011. 15 Kumon H, Sasaki K, Ariyoshi Y, Sadahira T, Ebara S, Hiraki T, 2 Tsuji T, Nozaki I, Miyazaki M, Sakaguchi M, Pu H, Hamazaki Y, Kanazawa S, Yanai H, Watanabe M and Nasu Y: Ad-REIC gene Iijima O and Namba M: Antiproliferative activity of REIC/DKK- therapy: promising results in a patient with metastatic CRPC 3 and its significant down-regulation in non-small-cell lung following chemotherapy. Clin Med Insights Oncol 9: 31-38, 2015. carcinomas. Biochem Biophys Res Commun 289 : 257-263, 2001. 16 Takeuchi T, Tomida S, Yatabe Y, Kosaka T, Osada H, 3 Abarzua F, Sakaguchi M, Takaishi M, Nasu Y, Kurose K, Ebara Yanagisawa K, Mitsudomi T and Takahashi T: Expression S, Miyazaki M, Namba M, Kumon H and Huh NH: Adenovirus- profile-defined classification of lung adenocarcinoma shows mediated overexpression of REIC/Dkk-3 selectively induces close relationship with underlying major genetic changes and apoptosis in human prostate cancer cells through activation of clinicopathologic behaviors. J Clin Oncol 24 : 1679-1688, 2006. c-JUN-NH 2-kinase. Cancer Res 65 : 9617-9622, 2005. 17 Watanabe M, Sakaguchi M, Kinoshita R, Kaku H, Ariyoshi Y, 4 Kashiwakura Y, Ochiai K, Watanabe M, Abarzua F, Sakaguchi M, Ueki H, Tanimoto R, Ebara S, Ochiai K, Futami J, Li SA, Huang Takaoka M, Tanimoto R, Nasu Y, Huh NH and Kumon H: Down- P, Nasu Y, Huh NH and Kumon H: A novel gene expression regulation of inhibition of differentiation-1 via activation of system strongly enhances the anticancer effects of a REIC/ DKK3- activating transcription factor 3 and SMAD regulates REIC/ encoding adenoviral vector. Oncol Rep 31 : 1089-1095, 2014. -3-induced apoptosis. Cancer Res 68 : 8333-8341, 2008. 18 Diefenbach A, Jamieson AM, Liu SD, Shastri N and Raulet DH: 5 Nozaki I, Tsuji T, Iijima O, Ohmura Y, Andou A, Miyazaki M, Ligands for the murine NKG2D receptor: expression by tumor Shimizu N and Namba M: Reduced expression of REIC/DKK-3 cells and activation of NK cells and macrophages. Nat Immunol gene in non-small cell lung cancer. Int J Oncol 19 : 117-121, 2001. 1: 119-126, 2000. 6 Tsuji T, Miyazaki M, Sakaguchi M, Inoue Y and Namba M: A 19 Molina JR, Yang P, Cassivi SD, Schild SE and Adjei AA: Non- REIC gene shows down-regulation in human immortalized cells small cell lung cancer: epidemiology, risk factors, treatment, and and human tumor-derived cell lines. Biochem Biophys Res survivorship. Mayo Clin Proc 83 : 584-594, 2008. Commun 268 : 20-24, 2000. 20 Yang P: Epidemiology of lung cancer prognosis: quantity and 7 Kawasaki K, Watanabe M, Sakaguchi M, Ogasawara Y, Ochiai quality of life. Methods Mol Biol 471 : 469-486, 2009. K, Nasu Y, Doihara H, Kashiwakura Y, Huh NH, Kumon H and 21 Robinson BW and Lake RA: Advances in malignant Date H: REIC/DKK-3 overexpression down-regulates P- mesothelioma. N Engl J Med 353 : 1591-1603, 2005. glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer. Cancer Gene Ther 16 : 65-72, 2009. 8 Tanimoto R, Abarzua F, Sakaguchi M, Takaishi M, Nasu Y, Kumon H and Huh NH: REIC/DKK-3 as a potential gene Received October 19, 2016 therapeutic agent against human testicular cancer. Int J Mol Med Revised November 15, 2016 19 : 363-368, 2007. Accepted November 22, 2016

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