Taketoshi Shimada

Interleukin-27 Activates Natural Killer Cells and Suppresses NK-Resistant Head and Neck Squamous Cell Carcinoma through Inducing Antibody-Dependent Cellular Cytotoxicity

Interleukin (IL)-27 is an IL-12 family cytokine playing a pivotal role in the induction of Th1 immune responses, although its action on natural killer (NK) cells has not been fully elucidated. Here, we show that IL-27 is capable of inducing phosphorylation of signal transducers and activators of transcription 1 and 3, as well as expression of T-bet and granzyme B in murine DX-5+ NK cells. IL-27 also enhances cytotoxic activity of NK cells both in vitro and in vivo, while the in vitro viability of NK cells is also improved by this cytokine. Therapeutic administration of the IL-27 gene drastically suppressed the growth of NK-unsusceptible SCCVII tumors that had been preestablished in syngenic mice, resulting in significant prolongation of the survival of the animals. This can likely be ascribed to the antibody-dependent cellular cytotoxicity machinery because IL-27 successfully induced tumor-specific IgG in the sera of the tumor-bearing mice, and supplementation of the sera enabled IL-27-activated NK cells to kill SCCVII cells in an Fcgamma receptor III-dependent manner. These findings strongly suggest that IL-27 may offer a powerful immunotherapeutic tool to eradicate head and neck squamous cell carcinoma and other poorly immunogenic neoplasms through activating NK cells and inducing tumor-specific immunoglobulin that may cooperatively elicit antibody-dependent cellular cytotoxicity activity.

Enhanced production and activation of progelatinase A mediated by membrane-type 1 matrix metalloproteinase in human oral squamous cell carcinomas: Implications for lymph node metastasis

We measured the production levels of seven different matrix metalloproteinases (MMP-1, 2, 3, 7, 8, 9 and 13) and two tissue inhibitors of metalloproteinases (TIMP-1 and 2) in the homogenates of human oral squamous cell carcinomas and control normal squamous epithelia by the corresponding sandwich enzyme immunoassay systems. The levels of MMP-1, 2, 3, 8, 9, 13 and TIMP-1 were significantly higher in the carcinoma samples than in the control. Among them, only the production level of MMP-2 was significantly higher in the carcinomas with cervical lymph node metastasis than in those without metastasis (P < 0.05). Gelatin zymography demonstrated that activation ratio of the zymogen of MMP-2 (proMMP-2) is significantly higher in the carcinomas with lymph node metastasis than in those without metastasis (P < 0.05) or normal control (P < 0.01). Quantitative RT-PCR for membrane-types 1, 2 and 3 MMPs (MT1, 2 and 3-MMPs), which activate proMMP-2 in vitro, demonstrated that MT1-MMP is predominantly expressed in the carcinoma tissues, and the expression level is significantly higher in the carcinomas with lymph node metastasis than in those without metastasis (P < 0.05) or the control samples (P < 0.05). Although MT2-MMP and MT3-MMP were detected in approximately 30% of the carcinoma cases, their expression levels were extremely lower compared with that of MT1-MMP. There was a direct correlation between the MT1-MMP expression level and proMMP-2 activation ratio (r = 0.62, P < 0.01). In situ hybridization and immunohistochemistry indicated that carcinoma cells and stromal cells adjacent to carcinoma cell nests express MT1-MMP transcripts and protein. MMP-2 and TIMP-2 were also immunolocalized to the carcinoma cells in the carcinoma samples. By in situ zymography, gelatinolytic activity was demonstrated in the carcinoma cell nests and abolished by the treatment with an MMP inhibitor, BB94. These results suggest that among seven different MMPs, the production of proMMP-2 and its activation mediated by MT1-MMP play an important role in the cervical lymph node metastasis of the human oral squamous cell carcinomas.

Interleukin‐21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

Interleukin‐21 (IL‐21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL‐21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC).

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.

RECQL1 and WRN Proteins Are Potential Therapeutic Targets in Head and Neck Squamous Cell Carcinoma

RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyper-recombination and repair. In this study, we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas, which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence, we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly, siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover, combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably, we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers.

Enhanced Production of Matrix Metalloproteinase-2 in Human Head and Neck Carcinomas is Correlated with Lymph Node Metastasis

Matrix metalloproteinase (MMP)-2 and -9 degrade type IV collagen, which is one of the major components of the basement membrane in normal tissue and expressed in the surroundings of the cancer nest in squamous cell carinoma. The degeneration of type IV collagen is an essential step in the metastasis to lymph nodes and distant organs. In this study, we examined MMP-2 and -9 levels of cancer tissue and serum obtained from patients with head and neck squamous cell carcinoma (HNSCC) in order to evaluate the relationship between the clinicopathologic features and MMPs. We examined the production of MMP-2 and -9 in cancer tissue homogenates of 73 patients who had HNSCC and the serum MMP levels of 16 patients with HNSCC and 8 healthy volunteers. We also studied the localization of MMP-2 in the carcinoma using an immunohistochemical approach. The concentrations of MMP-2 and -9 in the tissue homogenates and serum were measured by means of a sandwich enzyme immunoassay using a monoclonal antibody. Immunohistochemical analyses were performed with monoclonal antibody to MMP-2. The concentration of MMP-2 in the tumor tissue homogenates was unrelated to tumor size, but that in patients with lymph node metastases was significantly higher than in those without lymph node metastases. The concentration of MMP-9 was unrelated to lymph node metastasis and tumor size. The levels of both MMP-2 and -9 in serum were unrelated to lymph node metastasis. Immunohistochemistry indicated that MMP-2 was mainly expressed in cancer cells. Because MMP-2 degrades type IV collagen, the level of MMP-2 in carcinomas may be a useful indicator of the degree of invasion and metastasis.

Activation of pro-MMP-2 mediated by MT1-MMP in human salivary gland carcinomas: possible regulation of pro-MMP-2 activation by TIMP-2

Matrix metalloproteinases (MMPs), a family of extracellular matrix‐degrading enzymes, are considered to play important roles in cancer invasion and metastasis. The present study examined the production levels of eight different MMPs (MMP‐1, 2, 3, 7, 8, 9 and 13, and MT1‐MMP) and two tissue inhibitors of metalloproteinases (TIMP‐1 and 2) in homogenates of human salivary gland carcinomas [mucoepidermoid carcinomas (MECs), adenoid cystic carcinomas (ACCs), and adenocarcinomas (ADEs)] and non‐neoplastic control salivary glands using sandwich enzyme immunoassay systems. The levels of MMP‐1, MMP‐2, MMP‐13, MT1‐MMP, and TIMP‐1 were significantly higher in the carcinoma samples than in the controls (p < 0.05). Gelatin zymography demonstrated that the activation ratio of the MMP‐2 zymogen (pro‐MMP‐2) was significantly higher in the carcinomas than in the controls (p < 0.05). In addition, the activation ratio in MECs was significantly higher than that in ACCs or ADEs (p < 0.01) and also correlated with histological grade and lymph node metastasis in MECs (p < 0.05), whereas the ratio showed no such correlation in ACCs or ADEs. Although the production levels of pro‐MMP‐2 and MT1‐MMP were similar among these carcinoma groups, TIMP‐2 levels were significantly higher in ACCs and ADEs than in MECs (p < 0.01). In carcinoma samples, the pro‐MMP‐2 activation ratio correlated directly with the MT1‐MMP/TIMP‐2 ratio (r = 0.736, n = 23; p < 0.01). Immunohistochemistry and in situ zymography demonstrated localization of MMP‐2, MT1‐MMP, and TIMP‐2 to carcinoma cells, but only in MECs did carcinoma cell nests exhibit gelatinolytic activity, which was inhibited by 1,10‐phenanthroline. These results suggest that enhanced activation of pro‐MMP‐2 mediated by MT1‐MMP is implicated in the invasion and metastasis of MECs and that TIMP‐2 may regulate pro‐MMP‐2 activation in salivary gland carcinomas. Copyright

RB1CC1 activates the promoter and expression of RB1 in human cancer

RB1‐inducible coiled‐coil 1 (RB1CC1, also known as FIP200) is a tumor suppressor implicated in the regulation of RB1 (retinoblastoma 1) expression. However, the molecular mechanism of RB1 regulation by RB1CC1 has not been elucidated. Here, we demonstrate that nuclear RB1CC1 binds to the RB1 promoter using chromatin immunoprecipitation assays with anti‐RB1CC1 antibody. Luciferase assays with RB1 promoter reporter plasmids revealed that RB1CC1 activated the RB1 promoter through the 201 bp upstream GC‐rich region (from the initiation ATG). Electrophoretic mobility shift assay and Western blot analysis supported RB1CC1 binding to the GC‐rich region of the RB1 promoter. In addition, the C‐terminus of RB1CC1 was required for nuclear localization and subsequent RB1 promoter activation. Furthermore, the expression levels of RB1CC1 and RB1 significantly correlated with in vivo breast cancer tissues as determined by immunohistochemical analysis. These data indicate that nuclear RB1CC1 directly activates the RB1 promoter to enhance RB1 expression in cancer cells. Evaluation of RB1CC1 in various types of human cancer tissues is expected to provide useful information for clinical practice and future therapeutic strategies.

Expression of CC-chemokine receptor 7 (CCR7) and CXC-chemokine receptor 4 (CXCR4) in head and neck squamous cell carcinoma.

OBJECTIVES We tried to clarify the correlation of the expression of CCR7 and CXCR4 with lymph node and distant metastasis. MATERIALS AND METHODS We examined expression of CCR7 and CXCR4 in 9 HNSCC cell lines and 25 HNSCC tissues by semi-quantitative RT-PCR and immunohistochemistry study. We examined the expression levels of CCR7 and CXCR4 in undifferentiated and differentiated human normal keratinocyte. RESULTS All cell lines expressed CCR7 mRNA, and three expressed CXCR4 mRNA. CCR7 and CXCR4 mRNAs were significantly higher in HNSCC tissues than in non-neoplastic tissues (p<0.05, respectively) and correlated with lymph node metastasis (p<0.05, respectively). The level of CXCR4 mRNA also correlated with distant metastasis (p<0.05). Immunohistochemistry demonstrated localization of CCR7 and CXCR4 to carcinoma cells and lymphocytes and immunohistochemical staining scores of CCR7 and CXCR4 also showed similar correlation to lymph node and distant metastasis with CCR7 and CXCR4 mRNA levels. The level of CCR7 mRNA was significantly higher in poorly and moderately differentiated than in well-differentiated HNSCC (p<0.05). The level of CCR7 mRNA in undifferentiated keratinocyte was significantly higher than that in differentiated keratinocyte. CONCLUSION The expression of CCR7 in HNSCC increases by dedifferentiation and plays an important role in lymph node metastasis of HNSCC and CXCR4 plays an important role in lymph node metastasis as well as distant metastasis.

RB1CC1 activates RB1 pathway and inhibits proliferation and cologenic survival in human cancer.

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG) of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.