Research ArticleClinical Studies

Distant Bystander Effect of REIC/DKK3 Gene Therapy Through Immune System Stimulation in Thoracic Malignancies

KEN SUZAWA, KAZUHIKO SHIEN, HUANG PENG, MASAKIYO SAKAGUCHI, MASAMI WATANABE, SHINSUKE HASHIDA, YUHO MAKI, HIROMASA YAMAMOTO, SHUTA TOMIDA, JUNICHI SOH, HIROAKI ASANO, KAZUNORI TSUKUDA, YASUTOMO NASU, HIROMI KUMON, SHINICHIRO MIYOSHI and SHINICHI TOYOOKA
Anticancer Research January 2017, 37 (1) 301-307;

Abstract

Background: Reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) is a tumor-suppressor gene, and its overexpression by adenovirus vector (Ad-REIC) exhibits a remarkable therapeutic effect on various human cancer types through a mechanism triggered by endoplasmic reticulum stress. Materials and Methods: We examined the direct anti-tumor effect of Ad-REIC gene therapy on lung cancer and malignant mesothelioma cell lines in vitro, and the distant bystander effect using immunocompetent mouse allograft models with bilateral flank tumors. Results: Ad-REIC treatment showed antitumor effect in many lung cancer and malignant mesothelioma cell lines in vitro. In an in vivo model, Ad-REIC treatment inhibited the growth not only of directly treated tumors but also of distant untreated tumors. By immunohistochemical analysis, infiltration of T-cells and natural killer (NK) cells and expression of the major histocompatibility complex (MHC) class I molecules were observed in bilateral tumors. Conclusion: Ad-REIC treatment not only had a direct antitumor effect but also an indirect bystander effect through stimulation of the immune system.

Both advanced-stage non-small cell lung cancer (NSCLC) and malignant mesothelioma (MM) are aggressive tumors and present a dismal prognosis. Despite advances in treatment regimens for both diseases, such as surgical resection, chemotherapy, molecular-targeted therapy and radiotherapy, treatment outcome is still unsatisfactory. Gene therapy for thoracic malignancies represents a novel therapeutic approach and has been evaluated in a number of clinical trials over the last two decades (1). Previously, we identified expression of the tumor-suppressor gene Reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) as being reduced in many human cancer types (2-6). However, the impact of REIC/DKK3 in thoracic malignancies, such as the frequency of its decreased expression among the subtypes of lung cancer, and the relationship between its loss and other oncogenic driver mutations, is yet to be investigated.

We demonstrated that overexpression of REIC/DKK3 by adenovirus (Ad-REIC) led to remarkable therapeutic effects on several human cancer types, but not on normal cells (3, 4, 7-9). As the REIC/DKK3 gene expression is absent from cancer cells, the REIC/DKK3 protein 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-JUN N-terminal kinase (JNK) pathway occurs downstream of ER stress signaling, which is a critical event in apoptosis (3, 4, 7-11). In addition to this direct antitumor effect, we also showed Ad-REIC to have a host-mediated bystander effect on human prostate cancer and scirrhous gastric cancer via the induction of anticancer immunity (12-14). Secreted REIC/DKK3 protein differentiates monocyte into the dendritic cells-like phenotype and then activates cytotoxic T-lymphocytes (CTLs) (12). Furthermore, in normal fibroblasts, Ad-REIC treatment does not cause apoptosis, but induces interleukin 7 (IL7) secretion. This enhanced IL7 secretion activates natural killer (NK) cells (13, 14). These mechanisms up-regulate systemic antitumor immunity.

Given this evidence, a clinical trial of REIC gene therapy for prostatic cancer is ongoing (UMIN000004929; NCT01931046). In a first study in Man (a phase I/IIa study of in situ Ad-REIC therapy for prostate cancer), direct and systemic antitumor effects were clearly detected in a patient with metastatic castration-resistant prostate cancer following chemotherapy (15).

In this study, we examined potential direct and indirect bystander effects of Ad-REIC via activation of systemic antitumor immunity in lung cancer and mesothelioma cells. In order to examine the immune-mediated response in vivo, we used murine cancer cell lines and an immunocompetent mouse model.

Materials and Methods

Gene expression analysis of clinical lung cancer tissues. Messenger RNA (mRNA) expression, histological subtype, and genetic mutation profiling for 158 lung carcinomas and five normal lung tissues (16) were obtained from Gene Expression Omnibus public database (GEO; http://www.ncbi.nlm.nih.gov/geo/; accession number GSE11969). We assessed the association between mRNA expression of REIC/DKK3 and the histological subtype or mutation profile.

Cell lines. Four human lung cancer cell lines (A549, HCC827, H1975, and PC9), three murine lung cancer cell lines (CMT64, KLN205, and LL/2), four human MM cell lines (H28, H290, H2052, and MSTO-211H), and three murine MM cell lines (AB1, AB12, and AC29) were used in this study. A549 and LL/2 were purchased from American Type Culture Collection (Manassas, VA, USA). HCC827, H1975, and four human MM cell lines were kindly provided by Dr. Adi F. Gazdar (University of Texas Southwestern Medical Center, Dallas, TX, USA). PC9 was purchased from Immuno-Biological Laboratories (Takasaki, Japan). CMT64 was obtained from the European Collection of Cell Cultures (Porton Down, UK). KLN205 was obtained from RIKEN BioResource Center (Tsukuba, Japan). AB1, AB12, and AC29 were were kindly provided by Steven M. Albelda (University of Pennsylvania, Philadelphia, PA, USA). All human cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and all murine cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS.

Adenovirus vectors. REIC/DKK3 was overexpressed using an adenovirus (Ad-REIC). We modified the Ad-REIC adenovirus vector previously generated (first-generation Ad-REIC) (3) and named it Ad-SGE-REIC (second-generation Ad-REIC) (17). An adenovirus vector carrying the LacZ gene (Ad-LacZ) was used as control.

Cell viability assay. Cells were plated in 96-well plates at a density of 1×103 cells/well. Twenty-four hours later, cells were infected with Ad-LacZ, or Ad-SGE-REIC was infected at a multiplicity of infection (MOI) of 200 or with phosphate-buffered saline solution (PBS) as control. Cell viability after treatment was evaluated 72 h later using a modified 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt (MTS) assay with CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA) according to the manufacturer's instruction.

Western blotting. The total cell lysate was extracted from cell lines with lysis buffer, a mixture of RIPA buffer, phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, St. Louis, MO, USA), and Complete Mini (Roche, Basel, Switzerland). Western blot analysis was performed by conventional methods using the following primary antibodies: rabbit anti-human REIC/DKK3 antibody raised in our laboratory, rabbit anti- stress-activated protein kinases (SAPK)/JNK, phospho-SAPK/JNK (Thr183/Tyr185), cleaved poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Danvers, MA, USA), and mouse anti-actin (Merck Millipore, Billerica, MA, USA). The following secondary antibodies were used: goat anti-rabbit or anti-mouse IgG-conjugated horseradish peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). To detect specific signals, membranes were examined using the ECL Prime Western Blotting Detection System (GE Healthcare, Amersham, UK) and LAS-3000 (Fujifilm, Tokyo, Japan).

Animal models. This study was carried out in accordance with the Guidelines of the Okayama University Animal Committee. Pathogen-free immunocompetent female BALB/c mice aged 6 to 8 weeks were purchased from CLEA Japan (Tokyo, Japan). AB12 cells (1×106) were suspended in 100 μl of serum-free DMEM, and KLN205 cells (1×106) were suspended in 200 μl DMEM with Matrigel, and each cell line was injected subcutaneously into the bilateral flanks of mice (n=3 per group and n=4 per group, respectively). When the tumors reached a volume of 200-250 mm3, the mice received intratumoral injection of Ad-SGE-REIC at a dose 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 injection of either 109 pfu Ad-LacZ or 100 μl of PBS. During the course of the study, mice were monitored for signs of pain or distress, and loss of body weight twice a week. Tumor growth was monitored twice a week, and individual tumor volumes were measured using a digital caliper and approximated according to the formula V=1/2ab2 (a being the long diameter and b being the short diameter of the tumor). No mouse encountered difficulty moving or accessing feed and water due to tumor size, had to be euthanized prior to the experimental endpoint for the health reasons, nor became severely ill or died. All mice were sacrificed on day 22 by cervical dislocation under ketamine and xylazine anesthesia. After sacrifice, tumors were subsequently harvested, measured, and pathologically examined.

Immunohistochemical analysis. Dissected bilateral flank tumors were fixed in 10% formaldehyde, embedded in paraffin, and cut into 4 μm-thick sections. The sections were deparaffinized and rehydrated; endogenous peroxidase was inhibited by a 10-min incubation with 3.0% H2O2 solution. Following a blocking step with normal horse serum, the sections were incubated overnight with the primary antibodies at 4°C, except for anti-REIC/DKK3, in which sections were incubated for 1 h at room temperature. The primary antibodies consisted of anti-REIC/DKK3, anti-histocompatibility 60 (H60) (Santa Cruz Biotechnology), anti-retinoic acid early inducible 1 (RAE1) (Santa Cruz Biotechnology), anti-cluster of differentiation (CD) 49b (pan-NK cells) (BioLegend, San Diego, CA, USA), anti-CD8a (BD Pharmingen, San Diego, CA, USA), and anti-cleaved caspase-3 (Cell Signaling Technology). After a brief wash, sections were incubated with the appropriate second antibody (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Antibody binding was detected using the ImmPACT DAB Peroxidase Substrate Kit (Vector Laboratories), Mayer's hematoxylin was used for counterstaining, and the protein expression was analyzed by standard light microscopy.

Figure 1.
Figure 1.

Comparison of expression of reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) among different histological subtypes and genetic profiles of clinical lung cancer tissues. A: The mRNA expression level of REIC/DKK3 was obtained from the microarray data of five normal lung tissues, 158 lung carcinomas, including 90 adenocarcinomas (Ad), 35 squamous cell carcinomas (Sq), four adenosquamous cell carcinomas (AdSq), 18 large cell lung cancers (LCLC), two large neuroendocrine caricinomas (LCNEC), and nine small cell lung cancers (SCLC). Data represent means±SD. B: The mRNA expression level of REIC/DKK3 of 149 non-small cell lung cancers (NSCLC), including 34 epidermal growth factor receptor (EGFR)-mutant (MT) NSCLCs, 12 Kirsten ras (KRAS)-MT NSCLCs and 103 both EGFR and KRAS wild-type (WT) NSCLCs, was obtained from the same database. Sixty-eight samples were NSCLCs with p53 mutation. *Significantly different at p<0.05.

Figure 2.
Figure 2.

Overexpression of reduced expression in immortalized cell (REIC)/Dickkoph-3 (DKK3) induces expression of apoptosis-rebuked proteins 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.

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.

Results

Characteristics of REIC/DKK3 expression in lung cancer. The microarray data of 149 NSCLCs, nine small-cell lung cancers (SCLC) and five normal lung tissue showed that REIC/DKK3 expression was significantly reduced in lung cancer samples, including adenocarcinomas, squamous cell carcinomas, large cell carcinomas, large cell neuroendocrine carcinomas, and SCLCs, compared with normal lung tissues (Figure 1A). In addition, no relationship was identified between REIC/DKK3 expression and other typical oncogenic driver mutations of epidermal growth factor receptor (EGFR) or Kirsten ras (KRAS), or tumor-suppressor gene p53 mutational status (Figure 1B).

Induction of apoptosis in NSCLC and MM cells by overexpression of REIC/DKK3 using Ad-SGE-REIC. We first examined the ability of Ad-SGE-REIC treatment to induce apoptosis of human NSCLC and MM cells. NSCLC and MM cells were infected with either Ad-LacZ or Ad-SGE-REIC at 200 MOI for 72 h. We then analyzed the expression of REIC/DKK3, JNK, and cleaved PARP, as an apoptosis marker, by western blot analysis. As shown in Figure 2, Ad-SGE-REIC treatment induced a significant expression of REIC/DKK3 and up-regulation of phospho-JNK, which resulted in subsequent expression of apoptosis-related proteins in both NSCLC and MM cells.

Figure 3.
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.

Association between antitumor effect and gene transduction efficiency of Ad-SGE-REIC treatment. We then investigated the association between anti-tumor effect and gene transduction efficiency of Ad-SGE-REIC treatment in several NSCLC and MM cell lines. Cell viability inhibition rates with Ad-SGE-REIC treatment differed between each cell line, ranging from 5.6% to 75.2% (Figure 3). Next, we examined the expression of REIC/DKK3 by western blot analysis to evaluate gene transduction efficiency and assessed the relationship with the response to Ad-SGE-REIC treatment. No endogenous REIC/DKK3 expression was observed in any of the cell lines, and the expression level of ectopic REIC/DKK3 in Ad-SGE-REIC treated cells differed among cell lines. Cells which were resistant to Ad-SGE-REIC treatment generally expressed only a low level of REIC/DKK3 when treated with Ad-SGE-REIC, and the ectopic expression of REIC/DKK3 positively correlated with antitumor effect of Ad-SGE-REIC treatment. These results indicate that the effect of Ad-SGE-REIC treatment depends on gene transduction efficiency.

In vivo antitumor effect of Ad-SGE-REIC. A host-mediated bystander effect was investigated in vivo using murine cancer cell lines and an immunocompetent mouse model. The experimental set-up is shown in Figure 4A. Mice received an intratumoral injection of PBS, Ad-LacZ, or Ad-SGE-REIC twice at a dose of 109 pfu into the right-flank tumor on days 1 and 8 (Figure 4A). As shown in Figure 4B and C, Ad-SGE-REIC treatment significantly inhibited the growth of tumor compared with the controls (PBS or Ad-LacZ) in both murine lung cancer (KLN205) and MM (AB12) allograft models. Furthermore, in the Ad-SGE-REIC-treated group, the growth of untreated tumors of the contralateral side was also strikingly suppressed, whereas this bystander effect was not observed in control groups.

Figure 4.
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.

In order to assess the mechanism of the indirect bystander effects mediated by Ad-REIC, immunohistochemical analysis of tumors was performed. To confirm the expression of REIC in tumors treated with Ad-SGE-REIC, mice with AB12 tumors were sacrificed on day 14, and the tumors were stained immunohistochemically using anti-REIC/DKK3 (Figure 4D). REIC was detected only in the tumors into which Ad-SGE-REIC had been directly injected. Next, we examined ligands related to NK cell-activating receptor (NKG2D), and NK cell and CD8-positive T-cell infiltration. Dissected bilateral flank AB12 tumors upon sacrifice were stained with antibodies recognizing H60, RAE1, CD49b and CD8a in order to investigate activation of systemic antitumor immunity. Immunohistochemical assessment revealed marked expression of H60 and RAE1 (mouse MHC class I homologs functioning as NKG2D ligands) in both treated and untreated tumors in the Ad-REIC-treated group. NK cells with CD49b expression also infiltrated into untreated as well as treated tumors in the Ad-REIC treated group. These findings suggest that NKG2D ligands are expressed by tissues in response to stress and may induce NK-cell activation and provide T-cell co-stimulation (18). On the other hand, H60 and RAE1 were not expressed in the tumors of the control group; moreover, nor were infiltrating NK cells detected. Furthermore, CD8-positive T-cells infiltrated into both treated and untreated tumors in the Ad-REIC-treated group, while they were rarely detected in the tumors of LacZ control group. Consistent with these results, cleaved caspase-3 was detected not only in Ad-REIC-injected tumors but also in non-injected tumors in the Ad-REIC-treated group, whereas the expression of cleaved caspase-3 was low in both sides in the control group. These results suggest that Ad-SGE-REIC treatment exhibits a bystander antitumor effect through immune system-mediated apoptosis and CD8-positive T-cell activation, and that protein expression of MHC class I molecules induces NK-cell infiltration of the tumor.

Discussion

Despite advances in treatment approaches including chemotherapy, radiation therapy, molecular-target therapy, and surgery, the overall 5-year survival rate of patients with NSCLC remains lower than 15% (19, 20). Lung cancer is a potentially good disease target for gene therapy using adenovirus-vector because lung cancer cells typically exhibit high expression of coxsackievirus and adenovirus receptor (CAR), facilitating transfection of vectors to target cells (9). On the other hand, MM is an aggressive tumor of the serosa and pleura. Current therapies for MM are only marginally effective, and mortality remains high, with a median survival of approximately 12 months, except for rare cases where complete resection is possible (21). MM is also a potentially good disease target for gene therapy because the large surface area of the thoracic cavity where malignant cells exist is expected to offer efficient, rapid, and diffuse gene transfer (1). Indeed, REIC gene therapy for MM is also ongoing (UMIN000013568; JapicCTI-152998).

We previously showed that Ad-REIC gene therapy exhibits a potent antitumor effect in various types of cancer cells (3, 4, 6-9). The direct antitumor effect is due to cancer-selective apoptosis as a result of ER stress in cancer cells (3, 9, 13). Furthermore, overproduction of IL7 from co-infected normal fibroblasts activates NK cells (13), and secreted REIC/DKK3 protein induces differentiation of monocytes into the dendritic cell phenotype and then activates CTLs (12). These mechanisms up-regulate systemic antitumor immunity and lead to antitumor effects at both treated and distant tumor sites. Thus, the indirect antitumor effect generated by the immune system is an important factor in the treatment of malignant tumors, which frequently develop metastasis.

In addition to previous reports, in this study, we demonstrated that Ad-SGE-REIC treatment is highly effective against both lung cancer and MM cells. Indeed, Ad-SGE-REIC not only exhibited a direct antitumor effect but also a distant bystander effect, considered as an indirect antitumor effect. Ad-SGE-REIC efficiently inhibited proliferation of lung cancer and MM cells, which expressed REIC/DKK3 protein after REIC gene transduction in vitro, whereas some cell lines were resistant to Ad-SGE-REIC treatment because of inefficient REIC gene transduction. On the other hand, in murine tumor allograft models using immunocompetent mice, we showed that Ad-SGE-REIC treatment inhibited growth not only of the tumor directly-injected with Ad-REIC but also of the distant, non-injected tumor. With regard to the precise mechanism of this indirect effect, in addition to our previous reports of its indirect effect through Ad-REIC-mediated cancer vaccination and production of IL7, we newly demonstrate here that Ad-SGE-REIC treatment induces expression of the MHC class I molecules H60 and RAE1 both in treated tumors and in distant untreated tumors, resulting in the presentation of targets for NK cells. Although we have not elucidated whether this induction of MHC class I protein expression is caused directly or indirectly by Ad-REIC treatment, infiltration by CD49b-positive NK cells was detected not only in the treated tumor but also in the untreated tumor, with induction of apoptosis-related protein expression only in the Ad-REIC-treated group. Although it is still unknown what causes MHC class I protein expression in the distant tumor site, three possibilities should be considered: Firstly, REIC protein, which was produced in the treated site, might act on distant cancer cells via an as-yet-unidentified receptor. Secondly, some damage-associated molecules released from the treated site might bind to cell-surface receptors of the tumor at the distant site. Thirdly, tumor-associated antigen released from the treated site may activate T-cells, and stress from stimulation of activated T-cells might induce expression of MHC class I molecules at the distant tumor site. Further studies are necessary to uncover the mechanism of up-regulation of expression of MHC class I molecules at distant tumor sites.

In conclusion, Ad-SGE-REIC treatment not only had a direct antitumor effect, but also an indirect bystander effect through immune system-mediated NK cell stimulation. Our results support the therapeutic potential of Ad-SGE-REIC for advanced lung cancer and MM. On the basis of these findings, we have initiated a clinical trial of Ad-REIC treatment of MM.

Acknowledgements

The Authors thank Ms. Fumiko Isobe for her technical assistance. This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Grant number 26670627 to T. Shi).

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Conflicts of Interest

    Okayama University and Momotaro-Gene Inc., a start-up biotech company originating from Okayama University, hold the patents on the Ad-REIC agent and are developing the agent as a cancer therapeutic medicine. Drs. M.S., M.W., Y.N., and H.K. own stocks in Momotaro-Gene Inc.

  • Received October 19, 2016.
  • Revision received November 15, 2016.
  • Accepted November 22, 2016.

References

  1. ↵
    1. Vachani A,
    2. Moon E,
    3. Wakeam E,
    4. Haas AR,
    5. Sterman DH,
    6. Albelda SM
    : Gene therapy for lung neoplasms. Clin Chest Med 32: 865-885, 2011.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Tsuji T,
    2. Nozaki I,
    3. Miyazaki M,
    4. Sakaguchi M,
    5. Pu H,
    6. Hamazaki Y,
    7. Iijima O,
    8. Namba M
    : Antiproliferative activity of REIC/DKK-3 and its significant down-regulation in non-small-cell lung carcinomas. Biochem Biophys Res Commun 289: 257-263, 2001.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Abarzua F,
    2. Sakaguchi M,
    3. Takaishi M,
    4. Nasu Y,
    5. Kurose K,
    6. Ebara S,
    7. Miyazaki M,
    8. Namba M,
    9. Kumon H,
    10. Huh NH
    : Adenovirus-mediated overexpression of REIC/Dkk-3 selectively induces apoptosis in human prostate cancer cells through activation of c-JUN-NH2-kinase. Cancer Res 65: 9617-9622, 2005.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Kashiwakura Y,
    2. Ochiai K,
    3. Watanabe M,
    4. Abarzua F,
    5. Sakaguchi M,
    6. Takaoka M,
    7. Tanimoto R,
    8. Nasu Y,
    9. Huh NH,
    10. Kumon H
    : Down-regulation of inhibition of differentiation-1 via activation of activating transcription factor 3 and SMAD regulates REIC/Dickkopf-3-induced apoptosis. Cancer Res 68: 8333-8341, 2008.
    OpenUrlAbstract/FREE Full Text
    1. Nozaki I,
    2. Tsuji T,
    3. Iijima O,
    4. Ohmura Y,
    5. Andou A,
    6. Miyazaki M,
    7. Shimizu N,
    8. Namba M
    : Reduced expression of REIC/DKK-3 gene in non-small cell lung cancer. Int J Oncol 19: 117-121, 2001.
    OpenUrlPubMed
  6. ↵
    1. Tsuji T,
    2. Miyazaki M,
    3. Sakaguchi M,
    4. Inoue Y,
    5. Namba M
    : A REIC gene shows down-regulation in human immortalized cells and human tumor-derived cell lines. Biochem Biophys Res Commun 268: 20-24, 2000.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Kawasaki K,
    2. Watanabe M,
    3. Sakaguchi M,
    4. Ogasawara Y,
    5. Ochiai K,
    6. Nasu Y,
    7. Doihara H,
    8. Kashiwakura Y,
    9. Huh NH,
    10. Kumon H,
    11. Date H
    : REIC/DKK-3 overexpression down-regulates P-glycoprotein in multidrug-resistant MCF7/ADR cells and induces apoptosis in breast cancer. Cancer Gene Ther 16: 65-72, 2009.
    OpenUrlCrossRefPubMed
    1. Tanimoto R,
    2. Abarzua F,
    3. Sakaguchi M,
    4. Takaishi M,
    5. Nasu Y,
    6. Kumon H,
    7. Huh NH
    : REIC/DKK-3 as a potential gene therapeutic agent against human testicular cancer. Int J Mol Med 19: 363-368, 2007.
    OpenUrlPubMed
  9. ↵
    1. Shien K,
    2. Tanaka N,
    3. Watanabe M,
    4. Soh J,
    5. Sakaguchi M,
    6. Matsuo K,
    7. Yamamoto H,
    8. Furukawa M,
    9. Asano H,
    10. Tsukuda K,
    11. Nasu Y,
    12. Huh NH,
    13. Miyoshi S,
    14. Kumon H,
    15. Toyooka S
    : Anti-Cancer Effects of REIC/DKK-3-encoding adenoviral vector for the treatment of non-small cell lung cancer. PLoS One 9: e87900, 2014.
    OpenUrl
    1. Watanabe M,
    2. Nasu Y,
    3. Kumon H
    : Adenovirus-mediated REIC/DKK3 gene therapy: Development of an autologous cancer vaccination therapy (Review). Oncol Lett 7: 595-601, 2014.
    OpenUrl
  11. ↵
    1. Sakaguchi M,
    2. Huh NH,
    3. Namba M
    : A novel tumor suppressor, REIC/DKK3 gene identified by our in vitro transformation model of normal human fibroblasts works as a potent therapeutic antitumor agent. Adv Exp Med Biol 720: 209-215, 2011.
    OpenUrlPubMed
  12. ↵
    1. Watanabe M,
    2. Kashiwakura Y,
    3. Huang P,
    4. Ochiai K,
    5. Futami J,
    6. Li SA,
    7. Takaoka M,
    8. Nasu Y,
    9. Sakaguchi M,
    10. Huh NH,
    11. Kumon H
    : Immunological aspects of REIC/DKK3 in monocyte differentiation and tumor regression. Int J Oncol 34: 657-663, 2009.
    OpenUrlPubMed
  13. ↵
    1. Sakaguchi M,
    2. Kataoka K,
    3. Abarzua F,
    4. Tanimoto R,
    5. Watanabe M,
    6. Murata H,
    7. Than SS,
    8. Kurose K,
    9. Kashiwakura Y,
    10. Ochiai K,
    11. Nasu Y,
    12. Kumon H,
    13. Huh NH
    : Overexpression of REIC/DKK-3 in normal fibroblasts suppresses tumor growth via induction of interleukin-7. J Biol Chem 284: 14236-14244, 2009.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Than SS,
    2. Kataoka K,
    3. Sakaguchi M,
    4. Murata H,
    5. Abarzua F,
    6. Taketa C,
    7. Du G,
    8. Yashiro M,
    9. Yanagihara K,
    10. Nasu Y,
    11. Kumon H,
    12. Huh NH
    : Intraperitoneal administration of an adenovirus vector carrying REIC/DKK3 suppresses peritoneal dissemination of scirrhous gastric carcinoma. Oncol Rep 25: 989-995, 2011.
    OpenUrlPubMed
  15. ↵
    1. Kumon H,
    2. Sasaki K,
    3. Ariyoshi Y,
    4. Sadahira T,
    5. Ebara S,
    6. Hiraki T,
    7. Kanazawa S,
    8. Yanai H,
    9. Watanabe M,
    10. Nasu Y
    : Ad-REIC gene therapy: promising results in a patient with metastatic CRPC following chemotherapy. Clin Med Insights Oncol 9: 31-38, 2015.
    OpenUrl
  16. ↵
    1. Takeuchi T,
    2. Tomida S,
    3. Yatabe Y,
    4. Kosaka T,
    5. Osada H,
    6. Yanagisawa K,
    7. Mitsudomi T,
    8. Takahashi T
    : Expression profile-defined classification of lung adenocarcinoma shows close relationship with underlying major genetic changes and clinicopathologic behaviors. J Clin Oncol 24: 1679-1688, 2006.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Watanabe M,
    2. Sakaguchi M,
    3. Kinoshita R,
    4. Kaku H,
    5. Ariyoshi Y,
    6. Ueki H,
    7. Tanimoto R,
    8. Ebara S,
    9. Ochiai K,
    10. Futami J,
    11. Li SA,
    12. Huang P,
    13. Nasu Y,
    14. Huh NH,
    15. Kumon H
    : A novel gene expression system strongly enhances the anticancer effects of a REIC/DKK3-encoding adenoviral vector. Oncol Rep 31: 1089-1095, 2014.
    OpenUrl
  18. ↵
    1. Diefenbach A,
    2. Jamieson AM,
    3. Liu SD,
    4. Shastri N,
    5. Raulet DH
    : Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat Immunol 1: 119-126, 2000.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Molina JR,
    2. Yang P,
    3. Cassivi SD,
    4. Schild SE,
    5. Adjei AA
    : Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 83: 584-594, 2008.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Yang P
    : Epidemiology of lung cancer prognosis: quantity and quality of life. Methods Mol Biol 471: 469-486, 2009.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Robinson BW,
    2. Lake RA
    : Advances in malignant mesothelioma. N Engl J Med 353: 1591-1603, 2005.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Distant Bystander Effect of REIC/DKK3 Gene Therapy Through Immune System Stimulation in Thoracic Malignancies
KEN SUZAWA, KAZUHIKO SHIEN, HUANG PENG, MASAKIYO SAKAGUCHI, MASAMI WATANABE, SHINSUKE HASHIDA, YUHO MAKI, HIROMASA YAMAMOTO, SHUTA TOMIDA, JUNICHI SOH, HIROAKI ASANO, KAZUNORI TSUKUDA, YASUTOMO NASU, HIROMI KUMON, SHINICHIRO MIYOSHI, SHINICHI TOYOOKA
Anticancer Research Jan 2017, 37 (1) 301-307;
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords