Tomonori Yaguchi

The mechanisms of cancer immunoescape and development of overcoming strategies

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 immunocompetence in cancer patients and improve current immunotherapy.

Immune Suppression and Resistance Mediated by Constitutive Activation of Wnt/β-Catenin Signaling in Human Melanoma Cells

Cancer-induced immunosuppression is a major problem reducing antitumor effects of immunotherapies, but its molecular mechanism has not been well understood. We evaluated immunosuppressive roles of activated Wnt/β-catenin pathways in human melanoma for dendritic cells (DCs) and CTLs. IL-10 expression was associated with β-catenin accumulation in human melanoma cell lines and tissues and was induced by direct β-catenin/TCF binding to the IL-10 promoter. Culture supernatants from β-catenin–accumulated melanoma have activities to impair DC maturation and to induce possible regulatory DCs. Those immunosuppressive culture supernatant activities were reduced by knocking down β-catenin in melanoma cells, partly owing to downregulation of IL-10. Murine splenic and tumor-infiltrating DCs obtained from nude mice implanted with human mutant β-catenin–overexpressed melanoma cells had less ability to activate T cells than did DCs from mice with control melanoma cells, showing in vivo suppression of DCs by activated Wnt/β-catenin signaling in human melanoma. This in vivo DC suppression was restored by the administration of a β-catenin inhibitor, PKF115-584. β-catenin–overexpressed melanoma inhibited IFN-γ production by melanoma-specific CTLs in an IL-10–independent manner and is more resistant to CTL lysis in vitro and in vivo. These results indicate that Wnt/β-catenin pathways in human melanoma may be involved in immunosuppression and immunoresistance in both induction and effector phases of antitumor immunoresponses partly through IL-10 production, and they may be attractive targets for restoring immunocompetence in patients with Wnt/β-catenin–activated melanoma.

Melanoma Cells Control Antimelanoma CTL Responses via Interaction between TIGIT and CD155 in the Effector Phase

Recently, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) was reported as a candidate for novel immune checkpoints. However, the impact of TIGIT on melanoma-specific cytotoxic T lymphocytes in the effector phase remains unclear. In this study, we demonstrated that melanoma cells control antimelanoma cytotoxic T lymphocyte responses via the TIGIT-CD155 interaction in the effector phase. TIGIT is an inhibitory receptor expressed on T cells, and CD155 is one of the cognate ligands expressed on the tumor cells or antigen-presenting cells. First, we confirmed that CD155 was constitutively expressed on melanoma cells. We then demonstrated that CD155 on melanoma cells suppressed cytokine release from melanoma-specific cytotoxic T lymphocytes via interaction with TIGIT. Overexpression of CD155 enhanced and its downregulation attenuated the suppressive effect. This suggested that antimelanoma cytotoxic T lymphocyte responses are controlled not only by an imbalance in CD226 (an activating molecule that binds to CD155) and TIGIT expression on T cells but also by the expression levels of CD155 on melanoma cells. In addition, the co-blockade of TIGIT and PD-1 signals synergistically elicited a response of tumor-infiltrating lymphocytes on autologous melanoma cells. These results suggest that the CD155-TIGIT interaction should be blocked for enhancement of antimelanoma immune responses.

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

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 CD206high 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.

Simultaneous suppression of MITF and BRAFV600E enhanced inhibition of melanoma cell proliferation

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 BRAFV600E. Thus to enhance the anti‐proliferative effect of MITF down‐regulation, we combined shRNA‐mediated MITF depletion with BRAFV600E 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)

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

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.

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

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-2d) 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.

Fibroblast growth factor-2 is an important factor that maintains cellular immaturity and contributes to aggressiveness of osteosarcoma

Osteosarcoma is the most frequent, nonhematopoietic, primary malignant tumor of bone. Histopathologically, osteosarcoma is characterized by complex mixtures of different cell types with bone formation. The role of environmental factors in the formation of such a complicated tissue structure as osteosarcoma remains to be elucidated. Here, a newly established murine osteosarcoma model was used to clarify the roles of environmental factors such as fibroblast growth factor-2 (Fgf2) or leukemia-inhibitory factor (Lif) in the maintenance of osteosarcoma cells in an immature state. These factors were highly expressed in tumor environmental stromal cells, rather than in osteosarcoma cells, and they potently suppressed osteogenic differentiation of osteosarcoma cells in vitro and in vivo. Further investigation revealed that the hyperactivation of extracellular signal–regulated kinase (Erk)1/2 induced by these factors affected in the process of osteosarcoma differentiation. In addition, Fgf2 enhanced both proliferation and migratory activity of osteosarcoma cells and modulated the sensitivity of cells to an anticancer drug. The results of the present study suggest that the histology of osteosarcoma tumors which consist of immature tumor cells and pathologic bone formations could be generated dependent on the distribution of such environmental factors. The combined blockade of the signaling pathways of several growth factors, including Fgf2, might be useful in controlling the aggressiveness of osteosarcoma. Mol Cancer Res; 10(3); 454–68. ©2012 AACR.

Suppression of Alkali Burn-Induced Corneal Neovascularization by Dendritic Cell Vaccination Targeting VEGF Receptor 2

PURPOSE To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization caused by alkali injury. METHODS H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGF receptor 2 (VEGFR2(400-408)) was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of VEGFR2(400-408)- or gp70-pulsed mature DCs three times at 6-day intervals. After the third immunization, corneal neovascularization was induced by alkali injury. Two weeks after the injury, the corneal vascularized area was evaluated by lectin angiography. To confirm the peptide-specific CTL activities in C57BL/6 mice, CD8(+) T cells from immunized mice were subjected to ELISA for interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and (51)Cr-release cytotoxicity assay. To determine the in vivo effector T cells, the immunized mice were intraperitoneally injected with an anti-CD4 or -CD8 depletion antibody. RESULTS Corneal neovascularization was significantly attenuated in mice immunized with VEGFR2(400-408) compared with those not immunized or immunized with gp70. VEGFR2(400-408) or gp70, but not beta-gal(96-103), application led to dose-dependent induction of IFN-gamma and TNF-alpha in the CD8(+) T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of major histocompatibility complex-matched cells expressing VEGFR2, but not beta-gal(96-103). In vivo depletion of CD8(+), but not CD4(+), T cells significantly reversed the suppressive effect of VEGFR2(400-408) immunization on corneal neovascularization to the level observed in nonimmunized or gp70-immunized animals. CONCLUSIONS These results indicate the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for corneal chemical injury.

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

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-&bgr;1 in the generation of immunosuppressive microenvironments of tumors and SLNs was investigated. Murine colorectal carcinoma CT26 transduced with TGF-&bgr;1 cDNA (CT26-TGF-&bgr;1) showed enhanced tumor growth compared with mock-transduced CT26 (CT26-Mock) when implanted in syngeneic Balb/c mice, even though CT26-TGF-&bgr;1 shows slower growth in vitro. This enhanced growth was not observed when implanted in immunodeficient mice, suggesting that TGF-&bgr;1 enhanced tumor growth by suppressing antitumor T-cell responses. Analysis of immune cells in CT26-TGF-&bgr;1-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-&agr; production. In addition, induction of tumor antigen–specific T cells from SLNs of the CT26-TGF-&bgr;1–implanted mice was significantly reduced. These results demonstrate that overproduction of TGF-&bgr;1 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-&bgr;1 is an attractive target for restoration of immunosuppressive condition in cancer patients.