Division of Gene Therapy Science
Division of Gene Therapy Science Graduate School of Medicine, Osaka University.
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Research contents
I. Cancer research based on virus-cancer cell interaction and development of novel anti-cancer strategies.
1.

As a tool for cancer research, we have used HVJ which was isolated in Japan in the early 1950ís and is famous for inducing membrane fusion. It is a mouse parainfluenza virus belonging to the paramixoviridae genus containining negative strand RNA (appr. 15kb). Two glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN) protein, are present on the viral envelope.

We developed a novel drug delivery system based on inactivated HVJ particle (HVJ envelope vector; HVJ-E). The vector can directly introduce plasmid DNA, siRNA, proteins and anti-cancer compounds to various cells both in vitro and in vivo (Fig. 1).

However, without incorporating therapeutic materials, we discovered anti-tumor activities in HVJ-E itself which has lost the ability for viral genome replication and viral protein synthesis.

Those activities are the activation of anti-tumor immunity and the induction of cancer cell-selective death. We have been analyzing the mechanism for those activities as follows (Fig. 2);

1) CTL and NK cell against cancers are generated by the activation of type I interferon and several chemokines (Cancer Res, 2007, etc). Those factors are activated by the introduction of viral RNA fragment into immune cells, especially dendritic cells, independently of Toll-like receptor pathway. RIG-I, a cytoplasmic RNA receptor, activates the production of those factors by recognizing viral RNA fragments. We identified the structure of HVJ-E-derived to stimulate RIG-I signaling pathway (Mol. Therapy 2016).
2) Macrophages and neutrophils in tumor microenvironment generally are pro-tumorigenic. HVJ-E can covert the character of those immune cells to anti-tumorigenic. HVJ-E-stimulated macrophages and neutrophils enhance the cytotoxic function of effector T cells by up-regulating expression of Th1-related genes (Oncotarget 2016).
3) Regulatory T cells are suppressed by HVJ-E injection in cancer tissues and regional lymphnodes. It results from the suppression of FoxP3 by HVJ-E-induced IL-6 secretion from dendritic cells. We identified that F protein of HVJ is necessary for IL-6 production in dendritic cells (FEBS Lett. 2008). The mechanism is independent on either Toll-like receptor or cytoplasmic RNA receptor. A novel receptor for F protein may be present on dendritic cells.
4) Cancer-cell selective apoptosis has been investigated (Clin. Cancer Res. 2012). We conclude that the apoptosis is achieved by the introduction of viral RNA fragment. The signaling pathway is mediated by RIG-I/MAVS. Different expression of apoptotic genes results from the difference of epigenetic control of those gene loci in cancer cells and non-cancerous cells.
5) In caspase 8-defective neuroblastoma cells, HVJ-E induced programmed necrosis (necroptosis), not apoptosis, by increasing cytoplasmic calcium followed by the activation of CaM-kinase II (Cancer Res. 2014.
6) We are also analyzing other pathways for HVJ-induced cancer cell death.

These pathways described above provide us with the possible targets for cancer therapy. Based on our findings, we are also developing therapeutic molecules for cancers (Oncogene 2008, etc).

Clinical trials of melanoma and prostate cancer are ongoing in Japan. A new clinical trial to treat malignant mesothelioma will be started in 2015.

2. Cancer initiating cells and exosomes are also our research targets for cancer therapy.
1) A novel pathway for anti-cancer drug-resistance has been elucidated in prostate cancers (Mol. Cancer Res. 2013).
2) We focused on Nanog gene for conferring the property of cancer-stem cells and analyzed the finction of Nanog gene using next-generation sequencing technologies and CRISPR-Cas9-mediated genome editing technologies (Oncotarget 2015).
3) We developed technologies to convert cancer cell-derived exosomes to anti-cancer particles by modifying surface proteins.
II. Development of cancer-targeting vectors.
HVJ-E vector can deliver plasmid DNA, siRNA, proteins and several anti-cancer drugs. However, the vector is not available for either systemic injection or cancer- targeting. We have tried to solve these limitations by two approaches.
1) One approach involves modification of fusion proteins with targeting molecules by genetic engineering (Human Gene Ther. 2007 etc).
2) Using genetic modification, we succeeded in constriction of HN-defective HVJ-E with single chain IL-12 protein on the envelope. This IL-12/HVJ-E enhanced anti-tumor immunity in various cancer models compared with HVJ-E itself. The particle also suppressed lung metastatic foci of melanoma by systemic administration (Clin. Cancer Res. 2013) (Fig. 3).
3) Another is the incorporation of HVJ-E into biomaterials. We developed a platelet vector incorporating HVJ-E. The platelet vector dominantly accumulated in tumor tissues and was activated to release HVJ-E. By systemic injection of the vector, tumor regression and prolonged survival were achieved in mouse melanoma model (Mol. Therapy 2014) (Fig. 4).
Fig. 1. Development of HVJ envelope vector (HVJ-E). HVJ-E incorporates plasmid DNA, siRNA, proteins and anti-cancer compounds. Via membrane fusion, those therapeutic molecules are directly introduced into the cytoplasm of various cells both in vitro and in vivo.
Fig. 2. Anti-tumor activities of HVJ-E. Viral RNA fragments induces anti-tumor immunity and cancer-cell specific apoptosis via RIG-I/Mavs signaling pathway.
Fig. 3. Construction of IL-12-bound HVJ-E. Killer T ell activity is highly enhanced by IL-12-bound HVJ-E. By systemic injection, the vector accumulates in lung and reduces the number of metastatic foci of melanoma in mouse lung.
Fig. 4. Platelet vector containing HVJ-E. ①The vector reaches tumor tissues, is activated and releases HVJ-E. ②HVJ-E affects endothelial cells and tumor cells. ③Anti-tumor immunity is induced by HVJ-E. ④Cancer cell-specific death is induced by HVJ-E.
Recent publications related to cancers, vectors, therapeutic molecules and stem cells. (2012~2016)
  1. Chang CY, Tai JA, Li S, Nishikawa T, Kaneda Y. Virus-stimulated neutrophils in the tumor microenvironment enhance T cell-mediated anti-tumor immunity. Oncotarget in press.
  2. Jiang Y, Saga K, Miyamoto Y, Kaneda Y. Cytoplasmic calcium increase via fusion with inactivated Sendai virus induces apoptosis in human multiple myeloma cells by downregulation of c-Myc oncogene. Oncotarget in press.
  3. Li, Y-T., Nishikawa, T., and Kaneda, Y. Platelet-cytokine Complex Suppresses Tumour Growth by Exploiting Intratumoural Thrombin-dependent Platelet Aggregation. Scientific Reports, 2016 Apr 27;6:25077.
  4. Liu, L-W, Nishikawa, T. and Kaneda, Y. An RNA molecule derived from Sendai virus DI particles induces anti-tumor immunity and cancer-selective apoptosis. Mol. Therapy, 24(1):135-45, 2016.
  5. Kawamura, N., Nimura, K., Nagano, H., Yamaguchi, S., Nonomura, N. and Kaneda, Y. CRISPR/Cas9-mediated gene knockout of NANOG1 and NANOGP8 leads to low malignant potential in prostate cancer. Oncotarget, Sep 8;6(26):22361-74, 2015.
  6. Gerardo Rodriguez-Araujo, Hironori Nakagami, Yoichi Takami, Tomohiro Katsuya, Hiroshi Akasaka, Shigeyuki Saitoh, Kazuaki Shimamoto, Ryuichi Morishita, Hiromi Rakugi, and Yasufumi Kaneda "Low Alpha synuclein levels in the blood are associated with insulin resistance" Scientific Reports Jul 10;5:12081, 2015.
  7. Iinuma S, Aikawa E, Tamai K, Fujita R, Kikuchi Y, Chino T, Kikuta J, McGrath J, Uitto J, Ishi M, Iizuka H and Kaneda Y. Transplanted bone marrow-derived circulating PDGFRα+ cells restore type VII collagen in recessive dystrophic epidermolysis bullosa mouse skin graft. J. Immunology, 194, 1996-2003, 2015.
  8. Fujita R, Tamai K, Aikawa E, Nimura K, Ishino S, Kikuchi Y, Kaneda Y. Endogenous Mesenchymal Stromal Cells in Bone Marrow are Required to Preserve Muscle Function in Mdx Mice. Stem Cells. 33, 962-975, 2015.
  9. Fei, Y., Nimura, K., Lo, W-N, Saga, K., Kaneda, Y. Histone H3 lysine 36 methyltransferase Whsc1 promotes the association of Runx2 and p300 in the activation of bone-related genes. PLOS-ONE, Sep 4;9(9):e106661, 2014.
  10. Nishikawa, T., Tung L-Y, Kaneda, Y. Systemic administration of platelets incorporating inactivated Sendai virus eradicates melanoma in mice. Mol. Therapy, 22(12):2046-55, 2014.
  11. Nomura, M., Ueno, A., Saga, K., Fukuzawa, M. and Kaneda, Y. Accumulation of cytosolic calcium induces necroptotic cell death in human neuroblastoma. Cancer Res. 74, 1056-1066, 2014.
  12. Hatano, K., Yamaguchi, S., Nimura, K., Murakami, K., Nagahara, A., Fujita, K., Uemura, M., Nakai, Y., Tsuchiya, M., Nakayama, M., Nonomura, N., and Kaneda, Y. Residual prostate cancer cells after docetaxel therapy increase the tumourigenic potential via constitutive CXCR4, ERK1/2 and c-Myc signalling loop activation. Mol. Cancer Res., 11:1088-100. 2013.
  13. Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y. The Transcription Factors Tbx18 and Wt1 Control the Epicardial Epithelial-Mesenchymal Transition through Bi-Directional Regulation of Slug in Murine Primary Epicardial Cells. PLoS One. 2013;8(2):e57829.
  14. Saga, K., Tamai, K., Yamasaki, T., and Kaneda, Y. Systemic administration of a novel immune-stimulatory pseudovirion suppresses lung metastatic melanoma by regionally enhancing IFN-γ production. Clinical Cancer Research., 1;19(3):668-79, 2013.
  15. Nomura, M., Shimbo, T., Miyamoto, Y., Fukuzawa, M. and Kaneda, Y. 13-cis retinoic acid can enhance the anti-tumor activity of non-replicating Sendai virus particle against neuroblastoma. Cancer Science, 104(2):238-44, 2013.
  16. Gerardo Rodriguez-Araujo, G., Nakagami, H., Hayashi, H., Mori, M., Shiuchi, T., Minokoshi, Y., Nakaoka, Y., Takami, Y., Komuro, I., Morishita, R., and Kaneda, Y. Alpha-synuclein elicits glucose uptake and utilization in adipocytes through the Gab1/PI3K/Akt transduction pathway. J. Cellular and Molecular Life Sciences, 70(6):1123-33, 2013.
  17. Matsushima-Miyagi, T., Hatano, K., Nomura, M., Li-Wen, L., Nishikawa, T., Saga, K., Shimbo, T. and Kaneda, Y. TRAIL and Noxa are selectively up-regulated in prostate cancer cells downstream of the RIG-I/MAVS signaling pathway by non-replicating Sendai virus particles. Clinical Cancer Research, 18, 6271-83, 2012.
  18. Mori, M., Nakagami, H., Rodriguez-Araujo, G., Nimura, K. and Kaneda, Y. Essential role for miR-196a in brown adipogenesis of white fat progenitor cells. PLoS Biology, Apr;10(4):e1001314, 2012.
  19. Hayashi, H., Nakagami, H., Takeichi, M., Shimamura, M., Koibuchi, N., Oiki, E., Sato, N., Koriyama, H., Gerardo-Araujo, R., Maeda, A., Morishita, R., Tamai, K., and Kaneda, Y. HIG1, a novel regulator of mitochondrial γ-secretase, maintains normal mitochondrial function. The FASEB J. 26(6):2306-17, 2012.
  20. Hatano, K., Miyamoto, Y., Mori, M., Nimura, K., Nakai, Y., Nonomura, N., and Kaneda, Y. Androgen-Regulated Transcriptional Control of Sialyltransferases in Prostate Cancer Cells. PLoS ONE, 2012;7(2):e31234.
  21. Kiyohara, E., Tamai, K., Katayama, I., Kaneda, Y. The combination of chemotherapy with HVJ-E containing Rad51 siRNA elicited diverse anti-tumor effects and synergistically suppressed melanoma. Gene Therapy, 19, 731-41, 2012.
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Division of Gene Therapy Science, Graduate School of Medicine, Osaka University
2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
Phone: +81-6-6879-3901 Fax: +81-6-6879-3909
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