Details of a Researcher - Kawakami, Yutaka

Papers - Kawakami, Yutaka

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Downregulation of KIF23 suppresses glioma proliferation

Takahashi Satoshi, Fusaki Noemi, Ohta Shigeki, Iwahori Yoshihiro, Iizuka Yukihiko, Inagawa Kohei, Kawakami Yutaka, Yoshida Kazunari, Toda Masahiro

Journal of Neuro-Oncology 106 ( 3 ) 519 - 529 2012.02

ISSN 0167-594X

To identify therapeutic molecular targets for glioma, we performed modified serological identification of antigens by recombinant complementary DNA (cDNA) expression cloning using sera from a mouse glioma model. Two clones, kinesin family member 23 (Kif23) and structural maintenance of chromosomes 4 (Smc4), were identified as antigens through immunological reaction with sera from mice harboring synergic GL261 mouse glioma and intratumoral inoculation with a mutant herpes simplex virus. The human Kif23 homolog KIF23 is a nuclear protein that localizes to the interzone of mitotic spindles, acting as a plus-end-directed motor enzyme that moves antiparallel microtubules in vitro. Expression analysis revealed a higher level of KIF23 expression in glioma tissues than in normal brain tissue. The introduction of small interfering RNA (siRNA) targeting KIF23 into two different glioma cell lines, U87MG and SF126, downregulated KIF23 expression, which significantly suppressed glioma cell proliferation in vitro. KIF23 siRNA-treated glioma cells exhibited larger cell bodies with two or more nuclei compared with control cells. In vivo analysis using mouse xenograft showed that KIF23 siRNA/DNA chimeratreated tumors were significantly smaller than tumors treated with control siRNA/DNA chimera. Taken together, our results indicate that downregulation of KIF23 decreases proliferation of glioma cells and that KIF23 may be a novel therapeutic target in malignant glioma.

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Human tumor antigens recognized by T cells and their implications for cancer immunotherapy

Ueda Ryo, Yaguchi Tomonori, Kawakami Yutaka

335 - 345 2012.01

Recent clinical trials of immunotherapies indicate that tumor reactive autologous T cells are able to regress even advanced, large tumors in melanoma patients. For example, the adoptive transfer of CD8+ cytotoxic T lymphocytes (CTL) specifically targeted for identified tumor antigens following lymphodepletive treatment, such as fludarabine/cyclophosphamide administration and total body irradiation, led to objective tumor responses in more than 70% of patients with melanoma (Dudley et al. 2008). Immunological analyses on these tumor tissues demonstrated that administered T cells may eliminate tumor cells through direct killing and cytokine secretion. Therefore, CD8+ CTLs that recognize MHC class I positive cancer cells are important for in vivo tumor rejection.

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Defining the critical hurdles in cancer immunotherapy

Fox Bernard A., Schendel Dolores J., Butterfield Lisa H., Aamdal Steinar, Allison James P., Ascierto Paolo Antonio, Atkins Michael B., Bartunkova Jirina, Bergmann Lothar, Berinstein Neil, Bonorino Cristina C., Borden Ernest, Bramson Jonathan L., Britten Cedrik M., Cao Xuetao, Carson William E., Chang Alfred E., Characiejus Dainius, Raja Choudhury A., Coukos George, de Gruijl Tanja, Dillman Robert O., Dolstra Harry, Dranoff Glenn, Durrant Lindy G., Finke James H., Galon Jerome, Gollob Jared A., Gouttefangeas Cécile, Grizzi Fabio, Guida Michele, Håkansson Leif, Hege Kristen, Herberman Ronald B., Stephen Hodi F., Hoos Axel, Huber Christoph, Hwu Patrick, Imai Kohzoh, Jaffee Elizabeth M., Janetzki Sylvia, June Carl H., Kalinski Pawel, Kaufman Howard L., Kawakami Koji, Kawakami Yutaka, Keilholtz Ulrich, Khleif Samir N., Kiessling Rolf, Kotlan Beatrix, Kroemer Guido, Lapointe Rejean, Levitsky Hyam I., Lotze Michael T., Maccalli Cristina, Maio Michele, Marschner Jens Peter, Mastrangelo Michael J., Masucci Giuseppe, Melero Ignacio, Melief Cornelius, Murphy William J., Nelson Brad, Nicolini Andrea, Nishimura Michael I., Odunsi Kunle, Ohashi Pamela S., O'Donnell-Tormey Jill, Old Lloyd J., Ottensmeier Christian, Papamichail Michael, Parmiani Giorgio, Pawelec Graham, Proietti Enrico, Qin Shukui, Rees Robert, Ribas Antoni, Ridolfi Ruggero, Ritter Gerd, Rivoltini Licia, Romero Pedro J., Salem Mohamed L., Scheper Rik J., Seliger Barbara, Sharma Padmanee, Shiku Hiroshi, Singh-Jasuja Harpreet, Song Wenru, Straten Per Thor, Tahara Hideaki, Tian Zhigang, van der Burg Sjoerd H., von Hoegen Paul, Wang Ena, Welters Marij J P, Winter Hauke, Withington Tara, Wolchok Jedd D., Xiao Weihua, Zitvogel Laurence, Zwierzina Heinz, Marincola Francesco M., Gajewski Thomas F., Wigginton Jon M., Disis Mary L.

Journal of Translational Medicine 9 ( 1 ) 2011.12

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.

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Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors

Iwata-Kajihara Tomoko, Sumimoto Hidetoshi, Kawamura Naoshi, Ueda Ryo, Takahashi Tomomi, Mizuguchi Hiroyuki, Miyagishi Makoto, Takeda Kiyoshi, Kawakami Yutaka

Journal of Immunology 187 ( 1 ) 27 - 36 2011.07

ISSN 0022-1767

STAT3 signaling constitutes an important negative feedback mechanism for the maintenance of immune homeostasis, a suppressive signal for the Th1 immune response in murine macrophages, and a cancer immune evasion signal in various immune cells. The strategy for STAT3 signal inhibition should be considered, because these features could impede effective cancer immunotherapy. We have evaluated the effects of STAT3 inactivation in dendritic cells (DCs) on immune responses in mice and humans. DCs derived from LysMcre/STAT3^flox/flox mice displayed higher cytokine production in response to TLR stimulation, activated T cells more efficiently, and were more resistant to the suppression of cytokine production by cancer-derived immunosuppressive factors compared with DCs from control littermates. Antitumor activities of STAT3-depleted and control DCs were compared by intratumoral administration of gp70 Ag peptide-pulsed DCs in the therapeutic MC38 tumor model. Intratumoral administration of STAT3-depleted DCs significantly inhibited MC38 tumor growth of both injected and nontreated remote tumors. The inhibition was accompanied by an increase in gp70-specific T cell response as well as in systemic Th1 immune response. STAT3-depleted human DCs with adenoviral STAT3 short hairpin RNAwere also capable of producing more cytokines with TLR stimulation and more resistant to cancer-derived factors, and they induced tumor Ag-specific T cells more efficiently than control DCs. The identified role of DC STAT3 signaling in both in vivo therapeutic tumor models in mice and in vitro-specific T cell induction in humans indicates that STAT3-inactivated DCs may be a promising approach for cancer immunotherapy.

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Functional analysis of HOXD9 in human gliomas and glioma cancer stem cells

Tabuse Masanao, Ohta Shigeki, Ohashi Yohei, Fukaya Raita, Misawa Aya, Yoshida Kazunari, Kawase Takeshi, Saya Hideyuki, Thirant Cécile, Chneiweiss Hérve, Matsuzaki Yumi, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Molecular Cancer 10 2011.05

Background: HOX genes encode a family of homeodomain-containing transcription factors involved in the determination of cell fate and identity during embryonic development. They also behave as oncogenes in some malignancies.Results: In this study, we found high expression of the HOXD9 gene transcript in glioma cell lines and human glioma tissues by quantitative real-time PCR. Using immunohistochemistry, we observed HOXD9 protein expression in human brain tumor tissues, including astrocytomas and glioblastomas. To investigate the role of HOXD9 in gliomas, we silenced its expression in the glioma cell line U87 using HOXD9-specific siRNA, and observed decreased cell proliferation, cell cycle arrest, and induction of apoptosis. It was suggested that HOXD9 contributes to both cell proliferation and/or cell survival. The HOXD9 gene was highly expressed in a side population (SP) of SK-MG-1 cells that was previously identified as an enriched-cell fraction of glioma cancer stem-like cells. HOXD9 siRNA treatment of SK-MG-1 SP cells resulted in reduced cell proliferation. Finally, we cultured human glioma cancer stem cells (GCSCs) from patient specimens found with high expression of HOXD9 in GCSCs compared with normal astrocyte cells and neural stem/progenitor cells (NSPCs).Conclusions: Our results suggest that HOXD9 may be a novel marker of GCSCs and cell proliferation and/or survival factor in gliomas and glioma cancer stem-like cells, and a potential therapeutic target.

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Recommendations from the iSBTc-SITC/FDA/NCI workshop on immunotherapy biomarkers

Butterfield Lisa H., Palucka A. Karolina, Britten Cedrik M., Dhodapkar Madhav V., Håkansson Leif, Janetzki Sylvia, Kawakami Yutaka, Kleen Thomas Oliver, Lee Peter P., Maccalli Cristina, Maecker Holden T., Maino Vernon C., Maio Michele, Malyguine Anatoli, Masucci Giuseppe, Pawelec Graham, Potter Douglas M., Rivoltini Licia, Salazar Lupe G., Schendel Dolores J., Slingluff Craig L., Song Wenru, Stroncek David F., Tahara Hideaki, Thurin Magdalena, Trinchieri Giorgio, Van Der Burg Sjoerd H., Whiteside Theresa L., Wigginton Jon M., Marincola Francesco, Khleif Samir, Fox Bernard A., Disis Mary L.

Clinical Cancer Research 17 ( 10 ) 3064 - 3076 2011.05

ISSN 1078-0432

Purpose: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements. Experimental Design: The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations. Results and Conclusions: Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.

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AMP kinase-related kinase NUAK2 affects tumor growth, migration, and clinical outcome of human melanoma

Namiki Takeshi, Tanemura Atsushi, Valencia Julio C., Coelho Sergio G., Passeron Thierry, Kawaguchi Masakazu, Vieira Wilfred D., Ishikawa Masashi, Nishijima Wataru, Izumo Toshiyuki, Kaneko Yasuhiko, Katayama Ichiro, Yamaguchi Yuji, Yin Lanlan, Polley Eric C., Liu Hongfang, Kawakami Yutaka, Eishi Yoshinobu, Takahashi Eishi, Yokozeki Hiroo, Hearing Vincent J.

Proceedings of the National Academy of Sciences of the United States of America 108 ( 16 ) 6597 - 6602 2011.04

ISSN 0027-8424

The identification of genes that participate in melanomagenesis should suggest strategies for developing therapeutic modalities. We used a public array comparative genomic hybridization (CGH) database and real-time quantitative PCR (qPCR) analyses to identify the AMP kinase (AMPK)-related kinase NUAK2 as a candidate gene for melanomagenesis, and we analyzed its functions in melanoma cells. Our analyses had identified a locus at 1q32 where genomic gain is strongly associated with tumor thickness, and we used real-time qPCR analyses and regression analyses to identify NUAK2 as a candidate gene at that locus. Associations of relapse-free survival and overall survival of 92 primary melanoma patients with NUAK2 expression measured using immunohistochemistry were investigated using Kaplan-Meier curves, log rank tests, and Cox regression models. Knockdown of NUAK2 induces senescence and reduces S-phase, decreases migration, and down-regulates expression of mammalian target of rapamycin (mTOR). In vivo analysis demonstrated that knockdown of NUAK2 suppresses melanoma tumor growth in mice. Survival analysis showed that the risk of relapse is greater in acral melanoma patients with high levels of NUAK2 expression than in acral melanoma patients with low levels of NUAK2 expression (hazard ratio = 3.88; 95% confidence interval = 1.44-10.50; P = 0.0075). These data demonstrate that NUAK2 expression is significantly associated with the oncogenic features of melanoma cells and with the survival of acral melanoma patients. NUAK2 may provide a drug target to suppress melanoma progression. This study further supports the importance of NUAK2 in cancer development and tumor progression, while AMPK has antioncogenic properties.

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The mechanisms of cancer immunoescape and development of overcoming strategies

Yaguchi Tomonori, Sumimoto Hidetoshi, Kudo-Saito Chie, Tsukamoto Nobuo, Ueda Ryo, Iwata-Kajihara Tomoko, Nishio Hiroshi, Kawamura Naoshi, Kawakami Yutaka

International Journal of Hematology 93 ( 3 ) 294 - 300 2011.03

ISSN 0925-5710

Cancer-induced immunosuppression is a major problem as it reduces the anti-tumor effects of immunotherapies. In cancer tissues, cancer cells, immune cells, and other stromal cells interact and create an immunosuppressive microenvironment through a variety of immunosuppressive factors. Some cancer subpopulations such as cancer cells undergoing epithelial-mesenchymal transition and cancer stem-like cells have immunosuppressive and immunoresistant properties. The production of immunosuppressive factors by cancer cells is mechanistically attributed to oncogenic signals frequently activated in cancer cells, including the STAT3, MAPK, NF-κB, and Wnt/β-catenin signals, which are upstream events leading to immunosuppressive cascades. Moreover, some of these signals are also activated in immunosuppressive immune cells stimulated by cancer-derived factors and contribute to their immunosuppressive activities. Therefore, targeting these signals both in cancer cells and immunosuppressive immune cells may result in the restoration of immuno-competence in cancer patients and improve current immunotherapy.

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Adjuvant effects of formalin-inactivated HSV through activation of dendritic cells and inactivation of myeloid-derived suppressor cells in cancer immunotherapy

Ohkusu-Tsukada Kozo, Ohta Shigeki, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 128 ( 1 ) 119 - 131 2011.01

ISSN 0020-7136

Use of adequate adjuvant is necessary for induction of effective antitumor immune responses. To develop an effective adjuvant for cancer immunotherapy, we selected formalin-inactivated (f)-HSV as an adjuvant component, and analyzed the mechanisms underlying its adjuvant effects. First, we found that f-HSV can induce the tumor antigen-specific CTLs by enhancing antigen cross-presentation by dendritic cells (DCs), mainly through TLR2, but not TLR9. Next, f-HSV was also found to prevent the accumulation of myeloid-derived suppressor cells (MDSCs). We demonstrated that the expansion of MDSCs in the blood and spleen during tumor progression required B cells producing the inflammatory angiogenesis factors, vascular endothelial growth factor (VEGF)-A and neuropilin-1 (NRP-1), a co-receptor for VEGF receptor-2 (VEGFR-2). Interestingly, the transmembrane-type NRP-1 on B cells changed to soluble-type NRP-1 (sNRP-1) by f-HSV treatment. We further showed that the sNRP-1 and VEGF-A secreted from B cells by f-HSV treatment could abrogate the immunosuppressive ability of MDSCs. These results suggest that f-HSV can enhance antitumor immune responses as an adjuvant, not only through activation of DCs, but also inactivation of MDSCs via B cells.

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Generation of human melanocytes from induced pluripotent stem cells

Ohta Shigeki, Imaizumi Yoichi, Okada Yohei, Akamatsu Wado, Kuwahara Reiko, Ohyama Manabu, Amagai Masayuki, Matsuzaki Yumi, Yamanaka Shinya, Okano Hideyuki, Kawakami Yutaka

PLoS One 6 ( 1 ) 2011

Epidermal melanocytes play an important role in protecting the skin from UV rays, and their functional impairment results in pigment disorders. Additionally, melanomas are considered to arise from mutations that accumulate in melanocyte stem cells. The mechanisms underlying melanocyte differentiation and the defining characteristics of melanocyte stem cells in humans are, however, largely unknown. In the present study, we set out to generate melanocytes from human iPS cells in vitro, leading to a preliminary investigation of the mechanisms of human melanocyte differentiation. We generated iPS cell lines from human dermal fibroblasts using the Yamanaka factors (SOX2, OCT3/4, and KLF4, with or without c-MYC). These iPS cell lines were subsequently used to form embryoid bodies (EBs) and then differentiated into melanocytes via culture supplementation with Wnt3a, SCF, and ET-3. Seven weeks after inducing differentiation, pigmented cells expressing melanocyte markers such as MITF, tyrosinase, SILV, and TYRP1, were detected. Melanosomes were identified in these pigmented cells by electron microscopy, and global gene expression profiling of the pigmented cells showed a high similarity to that of human primary foreskin-derived melanocytes, suggesting the successful generation of melanocytes from iPS cells. This in vitro differentiation system should prove useful for understanding human melanocyte biology and revealing the mechanism of various pigment cell disorders, including melanoma.

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Phase I clinical trial of the vaccination for the patients with metastatic melanoma using gp100-derived epitope peptide restricted to HLA-A*2402

Baba Toshiyuki, Sato-Matsushita Marimo, Kanamoto Akira, Itoh Akihiko, Oyaizu Naoki, Inoue Yusuke, Kawakami Yutaka, Tahara Hideaki

Journal of Translational Medicine 8 2010.09

Background: The tumor associated antigen (TAA) gp100 was one of the first identified and has been used in clinical trials to treat melanoma patients. However, the gp100 epitope peptide restricted to HLA-A*2402 has not been extensively examined clinically due to the ethnic variations. Since it is the most common HLA Class I allele in the Japanese population, we performed a phase I clinical trial of cancer vaccination using the HLA-A*2402 gp100 peptide to treat patients with metastatic melanoma.Methods: The phase I clinical protocol to test a HLA-A*2402 gp100 peptide-based cancer vaccine was designed to evaluate safety as the primary endpoint and was approved by The University of Tokyo Institutional Review Board. Information related to the immunologic and antitumor responses were also collected as secondary endpoints. Patients that were HLA-A*2402 positive with stage IV melanoma were enrolled according to the criteria set by the protocol and immunized with a vaccine consisting of epitope peptide (VYFFLPDHL, gp100-in4) emulsified with incomplete Freund's adjuvant (IFA) for the total of 4 times with two week intervals. Prior to each vaccination, peripheral blood mononuclear cells (PBMCs) were separated from the blood and stored at -80°C. The stored PBMCs were thawed and examined for the frequency of the peptide specific T lymphocytes by IFN-γ- ELISPOT and MHC-Dextramer assays.Results: No related adverse events greater than grade I were observed in the six patients enrolled in this study. No clinical responses were observed in the enrolled patients although vitiligo was observed after the vaccination in two patients. Promotion of peptide specific immune responses was observed in four patients with ELISPOT assay. Furthermore, a significant increase of CD8⁺gp100-in4⁺CTLs was observed in all patients using the MHC-Dextramer assay. Cytotoxic T lymphocytes (CTLs) clones specific to gp100-in4 were successfully established from the PBMC of some patients and these CTL clones were capable of lysing the melanoma cell line, 888 mel, which endogenously expresses HLA-restricted gp100-in4.Conclusion: Our results suggest this HLA-restricted gp100-in4 peptide vaccination protocol was well-tolerated and can induce antigen-specific T-cell responses in multiple patients. Although no objective anti-tumor effects were observed, the effectiveness of this approach can be enhanced with the appropriate modifications.

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Cancer treatment by comprehensive regulation of anti-tumor immune network

Kawakami Yutaka

Nippon rinsho. Japanese journal of clinical medicine 68 ( 6 ) 1094 - 1099 2010.06

ISSN 0047-1852

Anti-tumor effects of current cancer vaccines are limited. However, detailed analyses of clinical trials revealed several important points to be improved towards immunological tumor rejection. These are, (1) identification of tumor antigens involved in tumor cell proliferation/survival and expressed in cancer stem cells, (2) development of in situ tumor destruction methods which release tumor antigens in an immunogenic manner to induce immune responses to multiple endogenous tumor antigens, (3) development of methods to augment antigen processing and presentation by dendritic cells, including effective adjuvants, (4) development of methods to expand CTL and helper T cells in vivo, (5) understanding of mechanisms underlying cancer induced immunosuppression and development of methods to overcome them. Comprehensive interventions on these points in the antitumor immune network will lead to effective cancer immunotherapy.

Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line, SK-MG-1

Fukaya Raita, Ohta Shigeki, Yamaguchi Masayuki, Fujii Hirofumi, Kawakami Yutaka, Kawase Takeshi, Toda Masahiro

Cancer Letters 291 ( 2 ) 150 - 157 2010.05

ISSN 0304-3835

Accumulating evidence suggests that in several types of brain tumors, including glioma, only a phenotypic subset of tumor cells called brain cancer stem cells (BCSCs) may be capable of initiating tumor growth. Recently, the isolation of side population (SP) cells using Hoechst dye has become a useful method for obtaining cancer stem cells in various tumors. In this study, we isolated cancer stem-like cells from human glioma cell lines using the SP technique. Flow cytometry analysis revealed that SK-MG-1, a human glioblastoma cell line, contained the largest number of SP cells among the five glioma cell lines that were analyzed. The SP cells had a self-renewal ability and were capable of forming spheres in a neurosphere culture medium containing EGF and FGF2. Spheres derived from the SP cells differentiated into three different lineage cells: neurons, astrocytes and oligodendrocytes. RT-PCR analysis revealed that the SP cells expressed a neural stem cell marker, Nestin. The SP cells generated tumors in the brains of NOD/SCID mice at 8 weeks after implantation, whereas the non-SP cells did not generate any tumors in the brain. These results indicate that SP cells isolated from SK-MG-1 possess the properties of cancer stem cells, including their self-renewal ability, multi-lineage differentiation, and tumorigenicity. Therefore, the SP cells from SK-MG-1 may be useful for analyzing BCSCs because of the ease with which they can be handled and their yield.

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Targeted inhibition of IL-10-secreting CD25^- Treg via p38 MAPK suppression in cancer immunotherapy

Ohkusu-Tsukada Kozo, Toda Masahiro, Udono Heiichiro, Kawakami Yutaka, Takahashi Kimimasa

European Journal of Immunology 40 ( 4 ) 1011 - 1021 2010.04

ISSN 0014-2980

Cancer-induced immunotolerance mediated by inducible Treg (iTreg) is a major obstacle to cancer immunotherapy. In a basic study of immunotolerance, injection of an endogenous superantigen, i.e. the minor lymphocyte stimulatory (Mls)-1^a, into specific TCR Vβ8.1-Tg mice enabled generation of anergic CD25^- iTreg, the immunosuppressive function of which was maintained by IL-10 production via p38-MAPK activation. Interestingly, although p38-chemical inhibitor (p38-inhibitor) is capable of breaking CD25^- iTreg-induced immunotolerance, the p38-inhibitor had hardly any immunotolerance breaking effect when CD25⁺ Treg were present, suggesting that depletion of CD25⁺ Treg is necessary for p38-inhibitor to be effective. Peptide OVA_323-339 iv.-injection into its specific TCR-Tg (OT-II) mice also induced adaptive tolerance by iTreg. Peptide immunotherapy with p38-inhibitor after CD25⁺ Treg-depletion was performed in an OVA-expressing lymphoma E.G7-bearing tolerant model established by adoptive transfer of OT-II CD25^- iTreg, which resulted in suppression of tumor growth. Similarly, the antitumor immunity induced by peptide immunotherapy in colon carcinoma CT26-bearingmice, in which the number of IL-10-secreting iTreg is increased, was augmented by treatment with p38-inhibitor after CD25 ⁺ Treg-depletion and resulted in inhibition of tumor progression. These results suggest that simultaneous inhibition of two distinct Treg-functions may be important to the success of cancer immunotherapy.

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Identification of HLA-A2- And A24-restricted T-cell epitopes derived from SOX6 expressed in glioma stem cells for immunotherapy

Ueda Ryo, Ohkusu-Tsukada Kozo, Fusaki Noemi, Soeda Akio, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 126 ( 4 ) 919 - 929 2010.02

ISSN 0020-7136

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma-bearing mice. In this study, we attempted to identify SOX6-derived peptides as specific targets for effective and safe T-cell-mediated immunotherapy targeting SOX6-positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)-A*2402 (A24)-restricted peptides, RFENLGPQL (SOX6₅₀₄) and PYYEEQARL (SOX6₆₂₈) or the HLA-A*0201 (A2)-restricted peptide, ALFGDQDTV (SOX6₄₄₇) was capable of inducing SOX6 peptide-specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I-restricted and an antigen-dependent manner. Furthermore, peptide vaccines of SOX6₆₂₈, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H-2^d, induced CTLs specific for SOX6₆₂₈ in H-2^d mice. Normal autologous cells from mice, in which SOX6-specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA-A24 or -A2 positive glioma patients and should be considered as a promising strategy for safe and effective T-cell-based immunotherapy of patients with gliomas.

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Aberrant fatty acid-binding protein-7 gene expression in cutaneous malignant melanoma

Goto Yasufumi, Koyanagi Kazuo, Narita Norihiko, Kawakami Yutaka, Takata Minoru, Uchiyama Aya, Nguyen Linhda, Nguyen Tung, Ye Xing, Morton Donald L., Hoon Dave S B

Journal of Investigative Dermatology 130 ( 1 ) 221 - 229 2010.01

ISSN 0022-202X

Fatty acid-binding protein-7 (FABP7) has been shown to be expressed in cutaneous melanoma; however, its role in tumor progression is unclear. Expression of FABP7 was assessed during melanoma progression through assessment of various clinicopathology stages of primary tumor progression and metastasis. FABP7 mRNA was highly expressed in 60 of 87 (69%) primary melanomas, compared with significant (P0.0001) reduction in 13 of 68 (19%) metastatic melanomas. Analysis of 37 paired primary and metastatic melanomas by immunohistochemistry with anti-FABP7 Ab showed 73 and 27% positivity, respectively (P0.001). FABP7 detection of metastatic tissues was inversely correlated with relapse-free (P0.0001) and overall (P0.0001) survival. To examine FABP7 expression loss in advanced melanomas, loss of heterozygosity (LOH) was assessed using microsatellite markers encompassing the FABP7 gene. LOH was identified in 10 of 20 (50%) metastatic melanomas at 6q22.31, compared with 0 of 14 primary melanomas (P0.0017). FABP7 as a surrogate biomarker for circulating tumor cells (CTCs) in the blood was assessed by quantitative real-time (qRT)-PCR from melanoma patients blood (n134). Assessment of patients blood showed that FABP7() CTC decreased with disease progression. FABP7 may function as a tumor progression gene and can be used as a potential diagnostic biomarker of early-stage melanoma systemic spreading in blood.

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The RNA silencing technology applied by lentiviral vectors in oncology.

Sumimoto Hidetoshi, Kawakami Yutaka

Methods in molecular biology (Clifton, N.J.) 614 187 - 199 2010

ISSN 1064-3745

Since the discovery of RNA interference (RNAi) in Caenorhabditis elegans in 1998, this mechanism has been found to be conserved in a wide variety of species, including insects, plants, and mammals. In mammals, small (or short) interfering RNA (siRNA) or short hairpin RNA (shRNA) can be expressed by using several expression vectors including lentiviral vectors. The lentiviral vector has several useful characteristics for RNAi experiments including broad host tropism and stable gene transduction to both dividing and nondividing cells, which permits stable depletion of target genes. This technology can be useful for several applications, including basic cancer research.

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Simultaneous suppression of MITF and BRAF^V600E enhanced inhibition of melanoma cell proliferation

Kido Kenji, Sumimoto Hidetoshi, Asada Sakiyo, Okada Starlyn M., Yaguchi Tomonori, Kawamura Naoshi, Miyagishi Makoto, Saida Toshiaki, Kawakami Yutaka

Cancer Science 100 ( 10 ) 1863 - 1869 2009.10

ISSN 1347-9032

Microphthalmia-associated transcription factor (MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth-associated molecules may be independent of MITF and dependent on BRAF^V600E. Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAF^V600E inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAFV600E significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment. (Cancer Sci 2009; 100: 1863-1869).

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Impairment of plasmacytoid dendritic cells for IFN production by the ligand for immunoglobulin-like transcript 7 expressed on human cancer cells

Tsukamoto Nobuo, Okada Starlyn, Onami Yoko, Sasaki Yusuke, Umezawa Kazuo, Kawakami Yutaka

Clinical Cancer Research 15 ( 18 ) 5733 - 5743 2009.09

ISSN 1078-0432

Purpose: Plasmacytoid dendritic cells (pDC) are specialized cells to produce type I IFN. Infiltration of pDCs in cancer tissues that have impaired ability to produce IFN-α has been suggested to play immunosuppressive roles in tumor immunity. To identify potential mechanisms causing pDC impairment in the cancer microenvironment, expression of immunoglobulin-like transcript 7 ligands (ILT7L), which inhibits pDC production of type I IFNs on the surface of various human cancer and noncancer cells, was examined. Experimental Design: To detect unidentified ILT7L, reporter cells, which express green fluorescent protein on interaction with ILT7L, were constructed. ILT7L expression on various human cancer cell lines as well as various noncancerous stromal cells and immune cells was examined. Cytokines and signals involved in the ILT7L expression were also investigated. Results: ILT7L was detected on all of the various types of human cancer cell lines tested. IFN-α, IFN-β, IFN-γ, tumor necrosis factor-α, interleukin-1β, transforming growth factor-β, lipopolysaccharide, and imiquimod induced ILT7L expression on cancer and noncancer cells. High ILT7L-expressing cancer cells inhibited production of IFN-α and tumor necrosis factor-α by pDC stimulated with CpG. ILT7L does not appear to be a member of classic or nonclassic HLAs. Additionally, NF-κB and mammalian target of rapamycin are involved in regulating ILT7L expression. Conclusions: ILT7L expression on cancer cells may be one of the mechanisms for impairment of pDCs in the cancer microenvironment. ILT7/ILT7L signaling may normally enable a negative immune response feedback following viral infection. Intervention of the ILT7L/ILT7 system may be useful for enhancing antitumor immunity as well as antiviral immunity.

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RNA helicase encoded by melanoma differentiation-associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis

Sato Shinji, Hoshino Kana, Satoh Takashi, Fujita Tomonobu, Kawakami Yutaka, Fujita Takashi, Kuwana Masataka

Arthritis and Rheumatism 60 ( 7 ) 2193 - 2200 2009.07

ISSN 0004-3591

Objective. To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods. An anti-CADM-140 antibody-positive prototype serum sample was used to screen a HeLa cell-derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro-transcribed and in vitro-translated products using anti-CADM-140 antibody-positive and anti-CADM-140 antibody-negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti-CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue Results. By cDNA library screening and immunoprecipitation of in vitro-transcribed and in vitro-translated products, we obtained a cDNA clone encoding melanoma differentiation-associated gene 5 (MDA-5). The anti-CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion. Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti-CADM-140 antibody routinely available.

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