Naoshi Kawamura

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.

Enhanced Cancer Immunotherapy Using STAT3-Depleted Dendritic Cells with High Th1-Inducing Ability and Resistance to Cancer Cell-Derived Inhibitory Factors

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/STAT3flox/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 RNA were 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.

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.

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.

Improvement of cancer immunotherapy by combining molecular targeted therapy.

In human cancer cells, a constitutive activation of MAPK, STAT3, β-catenin, and various other signaling pathways triggers multiple immunosuppressive cascades. These cascades result in the production of immunosuppressive molecules (e.g., TGF-β, IL-10, IL-6, VEGF, and CCL2) and induction of immunosuppressive immune cells (e.g., regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells). Consequently, immunosuppressive conditions are formed in tumor-associated microenvironments, including the tumor and sentinel lymph nodes. Some of these cancer-derived cytokines and chemokines impair immune cells and render them immunosuppressive via the activation of signaling molecules, such as STAT3, in the immune cells. Thus, administration of signal inhibitors may inhibit the multiple immunosuppressive cascades by acting simultaneously on both cancer and immune cells at the key regulatory points in the cancer-immune network. Since common signaling pathways are involved in manifestation of several hallmarks of cancer, including cancer cell proliferation/survival, invasion/metastasis, and immunosuppression, targeting these shared signaling pathways in combination with immunotherapy may be a promising strategy for cancer treatment.

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

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.

Aberrant Myosin 1b Expression Promotes Cell Migration and Lymph Node Metastasis of HNSCC.

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 node metastases 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. ©2014 AACR.

Expression and localization of aging markers in lacrimal gland of chronic graft-versus-host disease

Aging is commonly defined as the accumulation of diverse deleterious changes in cells and tissues with advancing age. To investigate whether aging changes are involved in the lacrimal glands of chronic graft-versus-host disease (cGVHD) model mice, we obtained the specimens from cGVHD model mice, untreated aged and young mice, and examined by histopathology, and immunoblotting. Oxidative stress markers, 8-OHdG, 4-HNE, and hexonoyl lesion (HEL), and other aging markers, p16 and p38, were used to assess the samples. The infiltrating mononuclear cells and endothelia of capillaries in the cGVHD and aged mice expressed the oxidative stress markers and other aging markers, but not in the young mice. Histological changes and the expression of aging markers in the samples from cGVHD mice exhibited similar features to those in aging mice. These results suggest that changes that typically appear with advanced age occur earlier in the lives of mice with lacrimal gland cGVHD.