Details of a Researcher - Kawakami, Yutaka

Papers - Kawakami, Yutaka

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Aberrant myosin 1b expression promotes cell migration and lymph node metastasis of HNSCC

Ohmura Gaku, Tsujikawa Takahiro, Yaguchi Tomonori, Kawamura Naoshi, Mikami Shuji, Sugiyama Juri, Nakamura Kenta, Kobayashi Asuka, Iwata Takashi, Nakano Hiroshi, Shimada Taketoshi, Hisa Yasuo, Kawakami Yutaka

Molecular Cancer Research 13 ( 4 ) 721 - 731 2015.04

Research paper (scientific journal), Joint Work, ISSN 1541-7786

Lymph node metastasis is the major clinicopathologic feature associated with poor prognosis in patients with head and neck squamous cell carcinoma (HNSCC). Here, web-based bioinformatics meta-analysis was performed to elucidate the molecular mechanism of lymph node metastasis of human HNSCC. Preferential upregulation of Myosin 1b (MYO1B) transcript in HNSCC datasets was identified. Myo1b mRNA was highly expressed in human HNSCC cells and patient tissue specimens compared with their normal counterparts as shown by quantitative PCR (qPCR) analyses. Immunohistochemistry (IHC)-detected Myo1b expression was significantly correlated with lymph node metastases in patients with oral cancer of the tongue. HNSCC with high expression of Myo1b and chemokine receptor 4 (CCR4), another metastasis-associated molecule, was strongly associated with lymph node metastasis. RNA interference (RNAi) of Myo1b in HNSCC cells, SAS and HSC4, significantly inhibited migratory and invasive abilities through decreased large protrusion formation of cell membranes. Finally, Myo1b knockdown in SAS cells significantly inhibited in vivo cervical lymph nodemetastases in a cervical lymph node metastatic mouse model system. Implications: Myo1b is functionally involved in lymph node metastasis of human HNSCC through enhanced cancer cell motility and is an attractive target for new diagnostic and therapeutic strategies for patients with HNSCC. Mol Cancer Res; 13(4); 721-31.

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Predominant development of mature and functional human NK cells in a novel human IL-2 - Producing transgenic NOG mouse

Katano Ikumi, Takahashi Takeshi, Ito Ryoji, Kamisako Tsutomu, Mizusawa Takuma, Ka Yuyo, Ogura Tomoyuki, Suemizu Hiroshi, Kawakami Yutaka, Ito Mamoru

Journal of Immunology 194 ( 7 ) 3513 - 3525 2015.04

Research paper (scientific journal), Joint Work, ISSN 0022-1767

We generated a severe immunodeficient NOD/Shi-scid-IL-2Rγnull (NOG) mouse substrain expressing the transgenic human IL-2 gene (NOG-IL-2 Tg). Upon transfer of human cord blood-derived hematopoietic stem cells (HSCs), CD3-CD56highCD16+/- cells developed unexpectedly, predominantly in the NOG-IL-2 Tg (hu-HSC NOG-IL-2 Tg). These cells expressed various NK receptors, including NKp30, NKp44, NKp46, NKG2D, and CD94, as well as a diverse set of killer cell Ig-like receptor molecules at levels comparable to normal human NK cells from the peripheral blood, which is evidence of their maturity. They produced levels of granzyme A as high as in human peripheral blood-derived NK cells, and a considerable amount of perforin protein was detected in the plasma. Human NK cells in hu-HSC NOG-IL-2 Tg produced IFN-γ upon stimulation, and IL-2, IL-15, or IL-12 treatment augmented the in vitro cytotoxicity. Inoculation of K562 leukemia cells into hu-HSC NOG-IL-2 Tg caused complete rejection of the tumor cells, whereas inoculation into hu-HSC NOG fully reconstituted with human B, T, and some NK cells did not. Moreover, when a CCR4+ Hodgkin's lymphoma cell line was inoculated s.c. into hu-HSC NOG-IL-2 Tg, the tumor growth was significantly suppressed by treatment with a therapeutic humanized anti-CCR4 Ab (mogamulizumab), suggesting that the human NK cells in the mice exerted active Ab-dependent cellular cytotoxicity in vivo. Taken together, these data suggest that the new NOG-IL-2 Tg strain is a unique model that can be used to investigate the biological and pathological functions of human NK cells in vivo.

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Cytokine profile in cervical mucosa of Japanese patients with cervical intraepithelial neoplasia

Iwata Takashi, Fujii Takuma, Morii Kenji, Saito Miyuki, Sugiyama Juri, Nishio Hiroshi, Morisada Tohru, Tanaka Kyoko, Yaguchi Tomonori, Kawakami Yutaka, Aoki Daisuke

International Journal of Clinical Oncology 20 ( 1 ) 126 - 133 2015.02

Research paper (scientific journal), Joint Work, ISSN 1341-9625

BACKGROUND: Immune responses in the uterine cervix are considered to play an important role in persistent human papillomavirus (HPV) infection and carcinogenesis, but many aspects of the mechanism are still unclear. The goal of this study was to measure cytokines to analyze immune responses in patients with cervical intraepithelial neoplasia (CIN).MATERIALS AND METHODS: The levels of 17 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, INF-γ, MCP-1, MIP-1β, and TNFα) in cervical mucus were simultaneously measured using a multiplex immunoassay in 52 high-grade squamous intraepithelial lesion (HSIL) cases and overproduction of IL-1β, IL-8, and MIP-1β was identified. The levels of these 3 cytokines were measured in 130 patients with or without CIN lesions using enzyme-linked immunosorbent assay. The associations of the cytokine levels with the cytology, infecting HPV type, and status of cigarette smoking were investigated.RESULTS: IL-1β and IL-8 levels were associated with the cytology, and these levels were higher in HSIL cases than in NILM (negative for intraepithelial lesion and malignancy) and LSIL (low-grade squamous intraepithelial lesion) cases (P = 0.005, P = 0.001, respectively). The MIP-1β level was significantly lower in smokers (P = 0.018) and high-risk (HR)-HPV-infected patients (P = 0.021).CONCLUSIONS: Enhanced expression of IL-1β and IL-8 indicates that Th2 inflammatory responses become stronger in the local uterine cervical region with the progression of CIN lesions, and a decrease in the MIP-1β level may be advantageous for immunoescape of HPV. Cigarette smoking may further facilitate persistent HPV infection.

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Prognostic significance of interleukin-8 and CD163-positive cell-infiltration in tumor tissues in patients with oral squamous cell carcinoma

Fujita Yohei, Okamoto Masato, Goda Hiroyuki, Tano Tomoyuki, Nakashiro Koh Ichi, Sugita Atsuro, Fujita Tomonobu, Koido Shigeo, Homma Sadamu, Kawakami Yutaka, Hamakawa Hiroyuki

PLoS One 9 ( 12 ) e110378 2014.12

Research paper (scientific journal), Joint Work

Purpose: We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC). Experimental Design: Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens. Results: We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p=0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p =0.033 and p=0.038, respectively), and they were associated with poor clinical outcome (p= 0.007 and p=0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10. Conclusions: These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.

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Functional analysis of a novel glioma antigen, EFTUD1

Saito Katsuya, Iizuka Yukihiko, Ohta Shigeki, Takahashi Satoshi, Nakamura Kenta, Saya Hideyuki, Yoshida Kazunari, Kawakami Yutaka, Toda Masahiro

Neuro-Oncology 16 ( 12 ) 1618 - 1629 2014.12

Research paper (scientific journal), Joint Work, ISSN 1522-8517

BACKGROUND: A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.METHODS: EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.RESULTS: EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.CONCLUSION: These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.

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Predictive biomarkers for the outcome of vaccination of five therapeutic epitope peptides for colorectal cancer

Hazama Shoichi, Takenouchi Hiroko, Tsunedomi Ryouichi, Iida Michihisa, Suzuki Nobuaki, Iizuka Norio, Inoue Yuka, Sakamoto Kazuhiko, Nakao Mitsuhiro, Shindo Yoshitaro, Kanekiyo Shinsuke, Tokumitsu Yukio, Yoshimura Kiyoshi, Maeda Noriko, Maeda Kazunari, Maeda Yoshinari, Matsui Hiroto, Yoshino Shigefumi, Nakamura Yusuke, Fujita Yusuke, Hamamoto Yoshihiko, Okamoto Masato, Fujita Tomonobu, Kawakami Yutaka, Oka Masaaki

Anticancer Research 34 ( 8 ) 4201 - 4206 2014.08

Research paper (scientific journal), Joint Work, ISSN 0250-7005

Background: We previously reported a phase I study of a cancer vaccine using five novel HLA-A∗2402-restricted peptides, and demonstrated the safety and the promising potential of our five-peptide cocktail for advanced colorectal cancer. The objective of this analysis was to investigate predictive biomarkers for the prior selection of patients who are likely to have clinical benefit from such therapy. Patients and Methods: Seventeen patients with colorectal cancer who were treated with the five peptides underwent a complete blood count, serum chemistry tests and enzyme-linked ImmunoSpot assay before the treatment as predictive markers of high reactivity to the peptides. Results: Interleukin-6 level was a significant predictor for overall survival of patients treated with the peptide cocktail (p=0.017). A high neutrophil/lymphocyte ratio was likely to have some association with the poor induction of peptide-specific immune reaction. Conclusion: Interleukin-6 level might be a good predictive biomarker for clinical benefit of patients treated with this peptide vaccine.

Absence of recognition of common melanocytic antigens by T cells isolated from the cerebrospinal fluid of a Vogt-Koyanagi-Harada patient

Abad Sébastien, Wieërs Grégoire, Colau Didier, Wildmann Claude, Delair Emmanuelle, Dhote Robin, Brézin Antoine P., Kawakami Yukata, Coulie Pierre G., van der Bruggen Pierre

Molecular Vision 20 956 - 969 2014.07

ISSN 1090-0535

Purpose: Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune disease characterized by inaugural uveomeningitidis and hearing loss and at late stages a depigmentation in eyes and skin. Melanocytes are the cells common to the four affected tissues, namely eye, brain, inner ear, and skin. Melanocytes are therefore considered as the source of self-antigens. The melanocytic proteins tyrosinase-related protein-1 (TRP1), TRP2, tyrosinase, and gp100 have been proposed as the proteins targeted by autoreactive T cells from VKH patients bearing human leukocyte antigen (HLA)-DRB1*04:05, the HLA allele classically associated with VKH disease. The objective of this work was to determine the antigens recognized by a large number of potentially autoreactive CD4 T lymphocytes obtained from the cerebrospinal fluid of one VKH patient who did not express HLA-DRB1*04:05. Methods: T cells were isolated from the cerebrospinal fluid of a newly diagnosed HLA-DRB1*14:01,*15:03;-DPB1*01:01,*04:02 patient in the acute phase of the VKH disease and cloned by limiting dilution. Each of the 107 T cell clones, of which 90% were CD4+, was tested for its ability to secrete cytokines upon contact with autologous antigen-presenting cells loaded with either of the melanocytic proteins TRP1, TRP2, tyrosinase, gp100, Melan-A and KU-MEL-1. The sensitivity of our recombinant bacteria-based approach was validated with a CD4 T cell clone with known antigen specificity. The ability of each of the 107 clones to secrete cytokines upon nonspecific stimulation was verified. Results: None of the 107 T cell clones was able to secrete tumor necrosis factor-α, interferon-γ, interleukin (IL)-5, or IL-17 upon contact with autologous B cells loaded with any of the six common melanocytic proteins. Nine clones secreted high-level IL-17 upon stimulation with beads coated with antibodies. Conclusions: The self-antigens that triggered the VKH disease in this patient probably derive from proteins other than the six melanocytic proteins mentioned above. Further study of antigens that are recognized by potential autoreactive T cells from VKH patients is likely to benefit from testing a broader set of melanocytic proteins.

NOD-Rag2^null IL-2Rγ^null mice an alternative to NOG mice for generation of humanized mice.

Katano Ikumi, Ito Ryoji, Kamisako Tsutomu, Eto Tomoo, Ogura Tomoyuki, Kawai Kenji, Suemizu Hiroshi, Takahashi Takeshi, Kawakami Yutaka, Ito Mamoru

Experimental Animals 63 ( 3 ) 321 - 330 2014.07

Research paper (scientific journal), Joint Work, ISSN 1341-1357

We have developed NOD-Rag2^null IL-2Rγ^null (NR2G) mice similar to NOD-scid IL-2Rγ^null (NOG) mice that are known as an excellent host to generate humanized mice. To evaluate the usefulness of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of human cells after human hematopoietic stem cell (HSC) transplantation were compared among NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate irradiation doses to expand the niche for human stem cells in the bone marrow were first determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG mice, respectively, were found to be appropriate. Next, 5 × 10⁴ human umbilical cord blood CD34⁺ cells were intravenously inoculated into irradiated adult or newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated that both NR2G and NOG mice showed high engraftment rates compared with NOD-scid mice, although NOG mice showed a slightly higher engraftment rate than that for NR2G mice. However, no difference was found in the human cell populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation experiments to adults and newborns of two immunodeficient mice also revealed that the HSC transplantation into newborn mice resulted in higher engraftment rate than those into adults. These results showed that NR2G mice could be used as an alternative host to NOG mice to generate humanized mice.

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Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor

Nishio H., Yaguchi T., Sugiyama J., Sumimoto H., Umezawa K., Iwata T., Susumu N., Fujii T., Kawamura N., Kobayashi A., Park J., Aoki D., Kawakami Y.

British Journal of Cancer 110 ( 12 ) 2965 - 2974 2014.06

ISSN 0007-0920

Background:Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated.Methods:The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ.Results:In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells.Conclusions:NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.

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A phase ΙI study of five peptides combination with oxaliplatin-based chemotherapy as a first-line therapy for advanced colorectal cancer (FXV study)

Hazama Shoichi, Nakamura Yusuke, Tanaka Hiroaki, Hirakawa Kosei, Tahara Ko, Shimizu Ryoichi, Ozasa Hiroaki, Etoh Ryuichi, Sugiura Fumiaki, Okuno Kiyotaka, Furuya Takumi, Nishimura Taku, Sakata Koichiro, Yoshimatsu Kazuhiko, Takenouchi Hiroko, Tsunedomi Ryouichi, Inoue Yuka, Kanekiyo Shinsuke, Shindo Yoshitaro, Suzuki Nobuaki, Yoshino Shigefumi, Shinozaki Hirokazu, Kamiya Akira, Furukawa Hiroyuki, Yamanaka Takeharu, Fujita Tomonobu, Kawakami Yutaka, Oka Masaaki

Journal of Translational Medicine 12 ( 1 ) 2014.04

Background: We previously conducted a phase I trial for advanced colorectal cancer (CRC) using five HLA-A*2402-restricted peptides, three derived from oncoantigens and two from vascular endothelial growth factor (VEGF) receptors, and confirmed safety and immunological responses. To evaluate clinical benefits of cancer vaccination treatment, we conducted a phase II trial using the same peptides in combination with oxaliplatin-based chemotherapy as a first-line therapy.Methods: The primary objective of the study was the response rates (RR). Progression free survival (PFS), overall survival (OS), and immunological parameters were evaluated as secondary objective. The planned sample size was more than 40 patients for both HLA2402-matched and -unmatched groups. All patients received a cocktail of five peptides (3 mg each) mixed with 1.5 ml of IFA which was subcutaneously administered weekly for the first 12 weeks followed by biweekly administration. Presence or absence of the HLA-A*2402 genotype were used for classification of patients into two groups.Results: Between February 2009 and November 2012, ninety-six chemotherapy naïve CRC patients were enrolled under the masking of their HLA-A status. Ninety-three patients received mFOLFOX6 and three received XELOX. Bevacizumab was added in five patients. RR was 62.0% and 60.9% in the HLA-A*2402-matched and -unmatched groups, respectively (p = 0.910). The median OS was 20.7 months in the HLA-A*2402-matched group and 24.0 months in the unmatched group (log-rank, p = 0.489). In subgroup with a neutrophil/lymphocyte ratio (NLR) of < 3.0, patients in the HLA-matched group did not survive significantly longer than those in the unmatched group (log-rank, p = 0.289) but showed a delayed response.Conclusions: Although no significance was observed for planned statistical efficacy endpoints, a delayed response was observed in subgroup with a NLR of < 3.0. Biomarkers such as NLR might be useful for selecting patients with a better treatment outcome by the vaccination.Trial registration: Trial registration: UMIN000001791.

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Induction of immunoregulatory CD271⁺ cells by metastatic tumor cells that express human endogenous retrovirus H

Kudo-Saito Chie, Yura Masahiro, Yamamoto Ryusuke, Kawakami Yutaka

Cancer Research 74 ( 5 ) 1361 - 1370 2014.03

ISSN 0008-5472

Human endogenous retroviruses (HERV) are associated with many diseases such as autoimmune diseases and cancer. Although the frequent expression of a variety of HERVs in tumor cells has been demonstrated, their functional contributions in cancer are as yet unclear. Intriguingly, HERVs and other retroviruses include an immunosuppressive domain in their transmembrane envelope proteins, but its mechanism of action and cancer relevance are obscure. In this study, we demonstrate that the human endogenous retrovirus HERV-H has a critical role in tumor metastasis and immune escape. We found that expression of herv-h mRNA was elevated in metastatic tumor cells undergoing epithelial-to-mesenchymal transition (EMT) and in primary tumor tissues from advanced colon cancer. The immunosuppressive peptide H17 derived from HERV-H was sufficient to induce EMT in tumor cells that expressed low levels of HERV-H, and it amplified this event within the tumor microenvironment. H17 also stimulated CCL19 expression in tumor cells, which in turn recruited and expanded a population of pluripotent immunoregulatory CD271⁺ cells, which included mesenchymal stem cells and myeloid-derived suppressor cells. In tumor tissues from patients with advanced colon cancer, we confirmed that CD271 ⁺ cells were increased in HERV-H⁺CCL19⁺ tumor tissues. Notably, RNAi-mediated change of HERVH or CCL19, or depletion of CD271⁺ cells, improved immune responses in vitro and in vivo accompanied by tumor regression. Together, our results argued that HERV-H is a critical determinant of immune escape in cancer, suggesting its candidacy as a promising therapeutic target to treat patients with advanced cancer.

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TGF-β1 in tumor microenvironments induces immunosuppression in the tumors and sentinel lymph nodes and promotes tumor progression

Nakamura Shoko, Yaguchi Tomonori, Kawamura Naoshi, Kobayashi Asuka, Sakurai Toshiharu, Higuchi Hajime, Takaishi Hiromasa, Hibi Toshifumi, Kawakami Yutaka

Journal of Immunotherapy 37 ( 2 ) 63 - 72 2014.02

ISSN 1524-9557

In cancer patients, sentinel lymph nodes (SLNs) are crucial in the induction of antitumor T cells. However, in many cases, SLNs and tumors appear to be in immunosuppressive condition through mechanisms yet to be elucidated. In this study, the role of tumor-derived TGF-β1 in the generation of immunosuppressive microenvironments of tumors and SLNs was investigated. Murine colorectal carcinoma CT26 transduced with TGF-β1 cDNA (CT26-TGF-β1) showed enhanced tumor growth compared with mock-transduced CT26 (CT26-Mock) when implanted in syngeneic Balb/c mice, even though CT26-TGF-β1 shows slower growth in vitro. This enhanced growth was not observed when implanted in immunodeficient mice, suggesting that TGF-β1 enhanced tumor growth by suppressing antitumor T-cell responses. Analysis of immune cells in CT26-TGF-β1-implanted mice revealed impairment of dendritic cells (DCs), decrease of CD8 T cells, and increase of MDSCs and Tregs in the tumors. Similarly, the SLNs of these mice showed an increase of MDSCs, Tregs, and PD-L1 DCs, and decrease of T-cell stimulatory activity of DCs accompanied by decreased CD80 expression and TNF-α production. In addition, induction of tumor antigen-specific T cells from SLNs of the CT26-TGF-β1-implanted mice was significantly reduced. These results demonstrate that overproduction of TGF-β1 is critical for the generation of immunosuppressive microenvironments in both tumors and SLNs, which may result in suppression of spontaneous antitumor CD8 T-cell responses. Therefore, TGF-β1 is an attractive target for restoration of immunosuppressive condition in cancer patients.

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Roles of signaling pathways in cancer cells and immune cells in generation of immunosuppressive tumor-associated microenvironments

Kawakami Yutaka, Yaguchi Tomonori, Sumimoto Hidetoshi, Kudo-Saito Chie, Tsukamoto Nobuo, Iwata-Kajihara Tomoko, Nakamura Shoko, Nishio Hiroshi, Satomi Ryosuke, Kobayashi Asuka, Tanaka Mayuri, Park Jeong Hoon, Kamijuku Hajime, Tsujikawa Takahiro, Kawamura Naoshi

307 - 323 2014.01

Cancer cells trigger multiple immunosuppressive cascades and generate immunosuppressive tumor-associated microenvironments including tumor and sentinel lymph nodes. Constitutive activation of various signaling pathways (e.g., MAPK, STAT3, NF-κB, β-catenin) in human cancer cells was found to trigger the multiple immunosuppressive cascades through the production of immunosuppressive cytokines, such as TGF-β, IL-10, IL-6, and VEGF, and induction of immunosuppressive immune cells, such as regulatory T cells, tolerogenic dendritic cells, and myeloid derived suppressor cells. Some of these cancer-derived cytokines impair various immune cells through activation of their signaling molecules such as STAT3 and NF-κB. Inhibitors for these activated signals could inhibit the multiple immunosuppressive cascades by acting on both cancer cells and immune cells. Since common signaling mechanisms are often utilized for some of the hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression), targeting these common signaling pathways may be an attractive strategy for cancer therapy, including immunotherapy.

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Towards the introduction of the 'Immunoscore' in the classification of malignant tumours

Galon Jérôme, Mlecnik Bernhard, Bindea Gabriela, Angell Helen K., Berger Anne, Lagorce Christine, Lugli Alessandro, Zlobec Inti, Hartmann Arndt, Bifulco Carlo, Nagtegaal Iris D., Palmqvist Richard, Masucci Giuseppe V., Botti Gerardo, Tatangelo Fabiana, Delrio Paolo, Maio Michele, Laghi Luigi, Grizzi Fabio, Asslaber Martin, D'Arrigo Corrado, Vidal-Vanaclocha Fernando, Zavadova Eva, Chouchane Lotfi, Ohashi Pamela S., Hafezi-Bakhtiari Sara, Wouters Bradly G., Roehrl Michael, Nguyen Linh, Kawakami Yutaka, Hazama Shoichi, Okuno Kiyotaka, Ogino Shuji, Gibbs Peter, Waring Paul, Sato Noriyuki, Torigoe Toshihiko, Itoh Kyogo, Patel Prabhu S., Shukla Shilin N., Wang Yili, Kopetz Scott, Sinicrope Frank A., Scripcariu Viorel, Ascierto Paolo A., Marincola Francesco M., Fox Bernard A., Pagès Franck

Journal of Pathology 232 ( 2 ) 199 - 209 2014.01

ISSN 0022-3417

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

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Targeting FSTL1 prevents tumor bone metastasis and consequent immune dysfunction

Kudo-Saito Chie, Fuwa Takafumi, Murakami Kouichi, Kawakami Yutaka

Cancer Research 73 ( 20 ) 6185 - 6193 2013.10

ISSN 0008-5472

Bone metastasis greatly deteriorates the quality of life in patients with cancer. Although mechanisms have been widely investigated, the relationship between cancer bone metastasis and antitumor immunity in the host has been much less studied. Here, we report a novel mechanism of bone metastasis mediated by FSTL1, a follistatinlike glycoprotein secreted by Snail⁺ tumor cells, which metastasize frequently to bone. We found that FSTL1 plays a dual role in bone metastasis-in one way by mediating tumor cell invasion and bone tropism but also in a second way by expanding a population of pluripotent mesenchymal stem-like CD45 ALCAM cells derived from bone marrow. CD45^- ALCAM ⁺ cells induced bone metastasis de novo, but they also generated CD8^low T cells with weak CTL activity in the periphery, which also promoted bone metastasis in an indirect manner. RNA interferencemediated attenuation of FSTL1 in tumor cells prevented bone metastasis along with the parallel increase in ALCAM⁺ cells and CD8^low T cells. These effects were accompanied by heightened antitumor immune responses in vitro and in vivo. In clinical specimens of advanced breast cancer, ALCAM⁺ cells increased with FSTL1 positivity in tumor tissues, but not in adjacent normal tissues, consistent with a causal connection between these molecules. Our findings define FSTL1 as an attractive candidate therapeutic target to prevent or treat bone metastasis, which remains a major challenge in patients with cancer.

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Sox6 Up-Regulation by Macrophage Migration Inhibitory Factor Promotes Survival and Maintenance of Mouse Neural Stem/Progenitor Cells

Ohta Shigeki, Misawa Aya, Lefebvre Véronique, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

PLoS One 8 ( 9 ) 2013.09

Macrophage migration inhibitory factor (MIF) has important roles in supporting the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs), but downstream effectors remain unknown. We show here that MIF robustly increases the expression of Sox6 in NSPCs in vitro. During neural development, Sox6 is expressed in the ventricular zone of the ganglionic eminence (GE) of mouse brains at embryonic day 14.5 (E14.5), cultured NSPCs from E14.5 GE, and NSPCs in the subventricular zone (SVZ) around the lateral ventricle (LV) of the adult mouse forebrain. Retroviral overexpression of Sox6 in NSPCs increases the number of primary and secondary neurospheres and inhibits cell differentiation. This effect is accompanied with increased expression of Hes1 and Bcl-2 and Akt phosphorylation, thus suggesting a role for Sox6 in promoting cell survival and/or self-renewal ability. Constitutive activation of the transcription factor Stat3 results in up-regulation of Sox6 expression and chromatin immunoprecipitation analysis showed that MIF increases Stat3 binding to the Sox6 promoter in NSPCs, indicating that Stat3 stimulates Sox6 expression downstream of MIF. Finally, the ability of MIF to increase the number of primary and secondary neurospheres is inhibited by Sox6 gene silencing. Collectively, our data identify Sox6 as an important downstream effector of MIF signaling in stemness maintenance of NSPCs.

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Immunochemoradiotherapy for patients with oral squamous cell carcinoma

Tano Tomoyuki, Okamoto Masato, Kan Shin, Bando Takashi, Goda Hiroyuki, Nakashiro Koh Ichi, Shimodaira Shigetaka, Koido Shigeo, Homma Sadamu, Fujita Tomonobu, Sato Mitsunobu, Yamashita Naomi, Hamakawa Hiroyuki, Kawakami Yutaka

Neoplasia (United States) 15 ( 7 ) 805 - 814 2013.07

ISSN 1522-8002

Eighty-one patients with oral squamous cell carcinoma (OSCC) received oral fluoropyrimidine UFT and radiotherapy (RT) with or without an immunotherapeutic agent OK-432. Both overall survival and progression-free survival of patients who received RT + UFT + OK-432 were significantly longer than those of patients who received RT + UFT (P =.0075 and P =.0175, respectively). Clinical response was also more favorable in RT + UFT + OK-432 group than in RT + UFT group (P =.0066). Next, in vitro experiments were conducted to examine the effect of 5-fluorouracil (5-FU) and X-ray irradiation in OK-432-induced immunity. Human peripheral blood mononuclear cells stimulated with OK-432 produced helper T cell 1 (Th1)-type cytokines as well as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), which are produced by Th2 and regulatory T cells (Tregs), respectively, and are inhibitory in antitumor immunity. OK-432-induced IL-10 and TGF-β but not Th1 cytokines were significantly inhibited by 5-FU and/or X-ray. 5-FU and X-ray also inhibited the expression of mRNAs for GATA-3 and Foxp3, which are transcription factors for Th2 and Tregs, respectively, but not for T-bet, a transcription factor for Th1. In addition, 5-FU and X-ray decreased the expression of mRNAs for suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Antisense oligonucleotides for SOCS1 and SOCS3 markedly reduced OK-432-induced IL-10 and TGF-β. This is the first report clearly demonstrating that OK-432-based immunotherapy significantly enhanced the therapeutic effects of chemoradiotherapy in patients with OSCC as well as elucidating the mechanism of the synergistic effect of immunochemoradiotherapy in which 5-FU and radiation enhanced OK-432-induced Th1 response mediated by the inhibition of SOCS1 and SOCS3 gene expression.

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Autocrine and paracrine loops between cancer cells and macrophages promote lymph node metastasis via CCR4/CCL22 in head and neck squamous cell carcinoma

Tsujikawa Takahiro, Yaguchi Tomonori, Ohmura Gaku, Ohta Shigeki, Kobayashi Asuka, Kawamura Naoshi, Fujita Tomonobu, Nakano Hiroshi, Shimada Taketoshi, Takahashi Takeshi, Nakao Ryuta, Yanagisawa Akio, Hisa Yasuo, Kawakami Yutaka

International Journal of Cancer 132 ( 12 ) 2755 - 2766 2013.06

ISSN 0020-7136

Lymph node metastasis is a poor prognostic factor for patients with head and neck squamous cell carcinoma (HNSCC). However, its molecular mechanism has not yet been fully understood. In our study, we investigated the expression of CCR4 and its ligand CCL22 in the HNSCC tumor microenvironment and found that the CCR4/CCL22 axis was involved in lymph node metastasis of HNSCC. CCR4 was expressed in 20 of 31 (64.5%) human tongue cancer tissues, and its expression was significantly correlated with lymph node metastasis (p < 0.01) and lymphatic invasion (p < 0.05). CCR4 was expressed in three of five human HNSCC cell lines tested. CCR4⁺ HNSCC cells, but not CCR4^- cells, showed enhanced migration toward CCL22, indicating that functional CCR4 was expressed in HNSCC cell lines. CCL22 was also expressed in cancer cells (48.4% of tongue cancer tissues) or CD206⁺ M2-like macrophages infiltrated in tumors and draining lymph nodes. CCL22 produced by cancer cells or CD206^high M2-like macrophages increased the cell motility of CCR4⁺ HNSCC cells in vitro in an autocrine or paracrine manner. In the mouse SCCVII in vivo model, CCR4⁺ cancer cells, but not CCR4 ^- cells, metastasized to lymph nodes which contained CCL22 producing M2-like macrophages. These results demonstrate that lymph node metastasis of CCR4⁺ HNSCC is promoted by CCL22 in an autocrine or M2-like macrophage-dependent paracrine manner. Therefore, the CCR4/CCL22 axis may be an attractive target for the development of diagnostic and therapeutic strategies for patients with HNSCC.

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Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland, Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Yaguchi Saori, Ogawa Yoko, Shimmura Shigeto, Kawakita Tetsuya, Hatou Shin, Satofuka Shingo, Nakamura Shigeru, Imada Toshihiro, Miyashita Hideyuki, Yoshida Satoru, Yaguchi Tomonori, Ozawa Yoko, Mori Takehiko, Okamoto Shinichiro, Kawakami Yutaka, Ishida Susumu, Tsubota Kazuo

PLoS One 8 ( 6 ) 2013.06

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2^d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.

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Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy

Annals of the New York Academy of Sciences 1284 ( 1 ) 80 - 86 2013.05

ISSN 0077-8923

Immunological status in tumor tissues varies among patients. Infiltration of memory-type CD8⁺ T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8⁺ T cell infiltration has not been well investigated. In general, tumor-associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune-activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

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