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Biomarkers are essential for improving the effectiveness of treatments for patients with cancer. The immunological status in a tumor microenvironment is closely associated with the prognosis of cancer patients. Here we investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in 50 patients with resectable oral squamous cell carcinoma (OSCC). We found that the relapse-free survival (RFS) 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, the infiltration of CD163-positive cells, and of CD66b-positive cells in the tumor tissues were correlated with the serum IL-8 level (p = 0.033, p = 0.038 and p = 0.044, respectively), and they were associated with poor clinical outcome (p = 0.007, p = 0.002 and p = 0.001 respectively, in DFS) in all patients. A multivariate analysis revealed that N status, tumor IL-8 expression, CD163 and CD66b infiltration significantly affected the DFS of the patients. A further analysis suggested that the combination of N status with serum IL-8, tumor IL-8, CD163 and CD66b infiltration in the tumor tissues may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Finally, the in vitro experiments clearly demonstrated that IL-8 enhanced the generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10 which suppress anti-tumor immunity. Furthermore, IL-8 increased the generation of arginase-producing, CD66b-overexpressing cells from peripheral blood polymorphonuclear cells. These findings indicate that IL-8 may be involved in poor clinical outcomes via the generation of CD163-positive M2 macrophages and CD66b-overexpressing, arginase-producing granulocytes, and that these factors may have prognostic value as well as may be new targets for treatment in patients with resectable OSCC. Citation Format: Masato Okamoto, Yohei Fujita, Hiroyuki Goda, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Interleukin-8 promotes generation of M2 macrophages and arginase-producing granulocytes: Prognostic significance of interleukin-8 and infiltration of immune cells positive for CD163 and CD66b in tumor tissues in patients with oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-219. doi:10.1158/1538-7445.AM2015-LB-219

Molecular target drugs are used in the treatment of various malignant tumors. However, the development of molecular targeted therapy for oral squamous cell carcinoma (OSCC) is lagging behind other cancers. In this study, we have attempted to identify an appropriate molecular target for treatment of patients with OSCC. We determined the gene expression profiles of human 9 OSCC cell lines and an immortalized non-neoplastic human keratinocyte cell line by microarray analysis, and then found cell division cycle associated 5 (CDCA5) as one of genes which were commonly overexpressed in OSCC cells. CDCA5 was initially identified as substrate of anaphase-promoting complex and as a regulator of sister chromatid cohesion in HeLa. In addition, it has been reported that CDCA5 plays a critical role in human lung carcinogenesis. However, function of CDCA5 in OSCC remains still unknown. First, we confirmed the overexpression of CDCA5 in 5 human OSCC cells by quantitative RT-PCR (qRT-PCR) and Western blotting. The expression levels of CDCA5 mRNA and protein were markedly elevated in all human OSCC cell lines compared to a non-neoplastic human epithelial cell line. Next, we examined the effect of synthetic small interfering RNAs specific for CDCA5 (siCDCA5) on the growth of human OSCC cells. RNAi effect of siCDCA5 was confirmed by Western blotting. Transfection of siCDCA5 suppressed the expression of its target protein in all types of cells. The growth inhibitory effect of siCDCA5 in OSCC cells was evaluated by WST-8 assay. Knockdown of CDCA5 significantly inhibited the growth of OSCC cells in vitro. Subsequently, we analyzed the influence of siCDCA5 on cell cycle by flow cytometry. Suppression of CDCA5 resulted in the decrease in percentage of cells in G0/G1 phase and increase in G2 phase. This indicated that the anti-proliferative effect of siCDCA5 was due to G2 arrest. Furthermore, we investigated the clinical significance of CDCA5 expression in OSCC. The expression of CDCA5 mRNA in tumor and adjacent normal tissues derived from the patient with OSCC was examined by qRT-PCR. Expression levels of CDCA5 mRNA in the OSCC tissues were significantly higher than in normal tissues. We also examined the expression of CDCA5 protein in 80 OSCC cases by immunohistochemistry with anti-CDCA5 antibodies. Tissue expression of CDCA5 was divided into high or low group at 50% stained tumor cells, resulting in 36 cases as high expression and 44 cases as low expression. We then examined the association of CDCA5 expression with various clinicopathological parameters of OSCC patients, and found a significant association between CDCA5 expression levels and pathological staging, metastasis, and recurrence. These results suggest that CDCA5 functions as a critical gene supporting the growth of human OSCC cells and targeting CDCA5 appears a useful therapeutic strategy for the patients with OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroshi Tanaka, Yohei Fujita, Kazuki Iwamoto, Hiroyuki Hamakawa. Overexpression and function of CDCA5 in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 476. doi:10.1158/1538-7445.AM2014-476

Squamous cell carcinoma is one of the most common malignancies in head and neck region. Primary tumors, especially derived from undifferentiated cells, tend to have aggressive phenotypes, which are mainly characterized by local recurrence and distant metastasis. In this study we found that Id-1, a negative regulator of tissue specific transcription factors, is highly expressed in head and neck squamous carcinoma cells (HNSCC), and that the expression is correlated with the malignant phenotype, i.e., cell proliferation and invasion. Id-1 knockdown dramatically reduces squamous cell invasion that is accompanied by profound morphologic changes and robust reduction in expression levels of mesenchymal markers.In addition, reduction in tumor growth and neoangiogenesis were observed in vivo, indicating that knock-down of Id-1 drastically suppresses VEGFA expression. Our results also suggested that VEGFA-autocrine loop activates tylosine kinase pathways including Srk and FAK, and therefore manifests invasive phenotype. With clinical materials obtained from HNSCC patients, significant correlation was observed between Id-1 and VEGFA. Furthermore, we demonstrated that a non toxic compound, cannabidiol, significantly downregulates Id-1 gene expression and associated squamous cell proliferation and invasiveness. Taken together, these results suggest that Id-1 controls cancer cell phenotype in culture and in vivo, and that drugs targeting Id-1 represent a novel and promising strategy for improving the therapy and outcome of patients with HNSCC. Citation Format: Ryuichi Murase, Sean McAllister, Yohei Fujita, Tomoki Sumida, Koichi Nakashiro, Pierre Desprez, Hiroyuki Hamakawa. Id-1 gene and protein as therapeutic target for head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2014-1017

Recent experiments have showed that it is possible to use pharmacological agents that inactivate oncogenes to treat at least some types of human malignancies. However, the development of molecular targeted therapy in oral squamous cell carcinoma (OSCC) is lagging behind compare to other cancers. In this study, we attempted to identify an appropriate target molecule and to determine whether targeting such a molecule is plausible therapeutic approach for the treatment of patients with OSCC. We determined the gene expression profiles of 9 human OSCC cell lines and non-neoplastic keratinocyte cell line by microarray analysis, and then identified Aurora kinase A (AURKA) as a cancer-related gene. AURKA has been shown to be related to the progression, survival, histological differentiation, and metastasis in various tumors. We revealed the overexpression of AURKA mRNA and protein in human OSCC cell lines and tissues. To clarify the function of AURKA in the cell proliferation of OSCC cells, we investigated the effect of small interfering RNA (siRNA) specific for AURKA (siAURKA) and MLN8237, an AURKA selective inhibitor, on the growth of human OSCC cells in vitro and in vivo. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of these cells compared to non-targeting siRNA. MLN8237 also markedly reduced the growth rate of human OSCC cells. Next, we assessed the growth inhibitory effect of siAURKA and MLN8237 using mouse model. Synthetic siAURKA/atelocollagen complexes (40 μM) were administrated into mouse tail vain every 3 days, and MLN8237 (20 mg/kg) was received orally for 14 consecutive days. We found both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors. To confirm the usefulness of targeting AURKA in OSCC, we attempted to culture the resected tumor tissues from 3 patients with OSCC and obtained primary cultured cells. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of OSCC primary cultured cells. Furthermore, the expression of AURKA mRNA in OSCC tissues resected from patients was examined. In 37 of 50 primary OSCC tissues, the expression levels of AURKA mRNA were more than 2-fold increase compared to normal oral mucosa tissues, and notably we found significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis. These results suggest that AURKA plays a critical role in the growth of human OSCC cells, and targeting AURKA appears to be a potentially useful therapeutic approach for patients with OSCC. Citation Format: Hiroshi Tanaka, Koh-ichi Nakashiro, Kazuki Iwamoto, Norihiko Tokuzen, Yohei Fujita, Rikimaru Shirakawa, Ryota Oka, Hiroyuki Goda, Hiroyuki Hamakawa. Aurora kinase A as a novel therapeutic target in oral squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2013-3003

Purpose: Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in initiating adaptive and innate immune responses. DC-based vaccination is widely tested as a promising therapeutic cancer vaccine in multiple clinical trials. Programed cell death 1(PD-1)/PD-1 Ligands (PD-Ls) pathway are involved in the negative regulation of T cell-functions. PD-1 is expressed on T cells, and PD-Ls are expressed on DCs as well as some cancer cells. More recent studies have indicated that certain chemotherapeutic agents might enhance the cancer vaccine-mediated immune responses. In the current study, we conducted the in vitro experiments to examine whether or not chemotherapeutic drugs which are used for patients with head and neck cancer, may enhance T cell-stimulating ability of DCs via regulation of the expression of PD-Ls. Methods: Healthy volunteer-derived human peripheral blood monocyte were cultured with IL-4 and GM-CSF for 5 days to generate immature DCs, and then the DCs were matured by the stimulation with OK-432 which is a streptococcal immune adjuvant, for 24 hours, followed by the treatment with each chemotherapeutic drug such as 5-FU, CDDP, docetaxel or gemcitabine for 24 hours. Viability of DCs was assessed by WST-8. IL-10 and IL-12 in the supernatants from the DC cultures were measured by ELISA. Expression of mRNAs and proteins for PD-Ls was analyzed by quantitative real-time PCR and flow cytometry, respectively. DCs were co-cultured with allogeneic T cells (E/T ratio=1/20, 1/200 or 1/2000), then IFN-γ in the supernatants were measured by ELISA to evaluate T cell-stimulating ability of the DCs. Results: IL-12 production by DCs was significantly enhanced by OK-432 stimulation. Although CDDP, docetaxel and gemcitabine inhibited the OK-432-induced IL-12 production on the DCs, 5-FU did not. 5-FU and gemcitabine significantly inhibited the IL-10 production by OK-432-stimulated DCs. Expression of PD-L1 and PD-L2 genes as well as of their proteins on the DCs which was increased by OK-432 stimulation, was significantly suppressed by the treated with 5-FU or docetaxel. OK-432-stimulated DCs-treated with 5-FU strongly enhanced both proliferation and IFN-γ production of allogeneic T cells as compared with DCs stimulated only with OK-432. Conclusions: The findings obtained from the current study suggested the possibility that 5-FU may enhance the T cell-stimulating ability of dendritic cells via regulation of the expression of PD-L1 and PD-L2, and that the combination therapy of DC-based cancer vaccine with 5-FU may elicit better anti-tumor efficacy than only vaccination. Citation Format: Tomoyuki Tano, Masato Okamoto, Hiroyuki Goda, Yohei Fujita, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Enhancement of the T cell-stimulating ability of dendritic cells by 5-FU via regulation of the expression of ligands for programed cell death 1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4722. doi:10.1158/1538-7445.AM2013-4722