Tomoko Ota

Humanized Anti-Interleukin-6 Receptor Antibody Suppresses Tumor Angiogenesis and In vivo Growth of Human Oral Squamous Cell Carcinoma

Purpose: The biological effect of interleukin-6 (IL-6) signaling in oral squamous cell carcinoma (OSCC) and whether IL-6 receptor (IL-6R)-mediated signaling can be a therapeutic target for OSCC are unclear. The aim of this study was to investigate the effects of inhibition of IL-6R–mediated signaling on OSCC progression and to evaluate the availability of tocilizumab, a humanized antihuman IL-6R antibody, as a therapeutic agent for OSCC. Experimental Design: We evaluated expression levels of IL-6 and IL-6R in 58 OSCC tissues and 4 OSCC cell lines by real-time quantitative reverse transcription-PCR and/or immunohistochemstry. We investigated the effects of tocilizumab on OSCC growth in vitro and in xenografts. Xenografts were analyzed by immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 (pSTAT3), Ki-67, and CD31, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay was done. Results: Expression levels of IL-6 at both mRNA and protein levels in OSCC tissues were significantly higher than those in normal mucosal tissues. In addition, OSCC cell lines expressed higher levels of both IL-6 and IL-6R mRNA than did HaCaT keratinocytes. Tocilizumab significantly reduced in vivo growth of SAS cells with a drastic reduction of STAT3 phosphorylation in tumor cells in mice. Inhibition of IL-6 signaling significantly decreased vascular endothelial growth factor mRNA expression in SAS, and microvessel density and vessel diameter in SAS tumors in tocilizumab-treated mice. Conclusions: Therapeutic approaches targeting IL-6R by tocilizumab may be effective for OSCC treatment by at least inhibiting angiogenesis. (Clin Cancer Res 2009;15(17):5426–34)

Interleukin‐6 signalling regulates vascular endothelial growth factor‐C synthesis and lymphangiogenesis in human oral squamous cell carcinoma

Lymph node metastasis is associated with resistance to conventional therapy and poor survival of patients with oral squamous cell carcinoma (OSCC). Although lymphangiogenesis is well known to be associated with the occurrence of lymph node metastasis in various cancers, the precise mechanisms of lymphangiogenesis in OSCC are largely unknown. IL‐6, a potent pro‐inflammatory cytokine, has been shown to play active roles in various cancers, including OSCC. This study aimed to investigate the involvement of IL‐6 signalling in lymphatic metastasis and to evaluate the efficacy of tocilizumab, a humanized anti‐human IL‐6 receptor antibody, as an anti‐lymphangiogenic agent for OSCC. This investigation confirmed that levels of expression of IL‐6 protein and VEGF‐C mRNA in OSCC tissues were significantly correlated with lymph node metastasis in patients with OSCC, as assessed by immunohistochemical analysis and real‐time quantitative RT–PCR. In vitro studies showed that IL‐6 regulated VEGF‐C mRNA expression in a human OSCC cell line, SAS cells, through the phosphoinositide 3‐kinase‐Akt pathway. In addition, treatment with tocilizumab led to markedly reduced VEGF‐C mRNA expression and OSCC‐related lymphangiogenesis in SAS xenografts. Together, these data suggest that tocilizumab acted as expected: it inhibited lymph node metastasis in OSCC by reducing tumour lymphangiogenesis. Copyright

Therapeutic approaches targeting midkine suppress tumor growth and lung metastasis in osteosarcoma.

Midkine (MK) plays important roles in tumorigenesis, however, the biological function of MK and whether MK can be a therapeutic target in osteosarcoma are unclear. Here, we found that osteosarcoma tissues showed high MK expression. MK knockdown by small interfering RNA significantly induced apoptosis in osteosarcoma cells, whereas recombinant MK increased cell proliferation. Inhibition of MK signaling by anti-MK monoclonal antibody (anti-MK mAb) suppressed growth of osteosarcoma cells both in vitro and in vivo. Moreover, inhibition of MK function significantly suppressed lung metastasis in xenograft transplantation model. Targeting MK by anti-MK mAb may have value in the treatment of osteosarcoma.

Midkine expression in malignant salivary gland tumors and its role in tumor angiogenesis

The aims of this study were to investigate midkine (MK) expression patterns in salivary gland tumors (SGTs) and to evaluate the correlation between MK expression and the degree of malignancy. We performed immunohistochemistry to examine MK expression in specimens of adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), and pleomorphic adenoma (PA). In addition, we performed immunohistochemistry for CD31 and measured microvessel density (MVD), which is an indicator of angiogenesis. Immunohistochemistry showed that MK protein expression was significantly higher in specimens of malignant SGTs (ACC [P<0.01] and MEC [P<0.001]) than in benign SGT (PA) samples. Furthermore, MVD values tended to be higher in cases that exhibited high expression of MK, which indicated a significant correlation between the degree of MK expression and MVD (P<0.001). These results suggest that MK may play important roles in malignant transformation and tumor angiogenesis in SGTs.

Stromal expression of neutrophil gelatinase-associated lipocalin correlates with poor differentiation and adverse prognosis in oral squamous cell carcinoma

Neutrophil gelatinase‐associated lipocalin (NGAL) is a member of the lipocalin superfamily. Although its overexpression in various cancers has been reported, little is known about its expression and clinical significance in oral squamous cell carcinoma (OSCC). This study aimed to elucidate the clinical significance of NGAL in OSCC.

Osteopontin‐integrin αvβ3 axis is crucial for 5‐fluorouracil resistance in oral squamous cell carcinoma

Clinical applications of a chemotherapeutic agent, 5‐fluorouracil (5‐FU) in oral squamous cell carcinoma (OSCC) have been limited because of drug resistance. This study aimed to identify novel mechanisms of 5‐FU resistance. Here we found increased osteopontin (OPN) gene expression in OSCC tissues with resistance to 5‐FU‐based chemoradiotherapy. OPN overexpression in OSCC cells led to 5‐FU resistance and abrogated the prosurvival effect of the drug in a mouse xenograft model. OPN‐induced 5‐FU resistance required integrin αvβ3. Targeting integrin αvβ3 reversed the resistance in a 5‐FU‐resistant clone highly expressing OPN. Our data suggest that the OPN‐integrin αvβ3 axis is crucial for 5‐FU resistance in OSCC.

Abstract 16: Downregulation of CYLD leads to acquisition of mesenchymal state and increased migration via ligand-independent TGFβR1 activation in human oral squamous cell carcinoma cells

The 5-year survival of patients with oral squamous cell carcinoma (OSCC) has not changed apparently for the past 30 years. Although cylindromatosis (CYLD) is thought as a potent tumor suppressor, its biological significances in malignancies are largely unknown. The aim of this study was to clarify the role of CYLD in OSCC progression. We investigated expression of CYLD in OSCC including intraepithelial neoplasia (IEN; n = 48) and invasive carcinomas (n = 133) tissues and normal oral mucosal tissues (n = 35) by immunohistochemistry. In addition, effects of CYLD knockdown by siRNA transfection on OSCC progression using 5 OSCC cell lines (SAS, Tu4, HSC3, Ca9-22, and SCC-NA) and HaCaT keratinocytes. Our immunohistochemical analyses revealed that CYLD expression was significantly reduced at invasive lesions in OSCC tissues whereas it was conserved in normal epithelium and IEN. In addition, lower CYLD expression was associated with the correlation with the increased tumor size, advanced clinical stage, and poor overall survival in invasive OSCC. Accordingly, CYLD knockdown led to acquisition of mesenchymal state and increased migratory activity in all the OSCC cell lines as well as HaCaT keratinocytes. Notably, such EMT-like changes were completely blocked by a TGFβR1 inhibitor in all the OSCC cell lines, but not in HaCaT keratinocytes. Furthermore, treatment with excess amount of anti-TGFβ antibody (1D11) did not inhibit the EMT-like changes induced by CYLD repression. These findings suggest that downregulation of CYLD promotes invasion through EMT-like changes via ligand-independent TGFβR1 activation in OSCC cells in mechanisms distinct from normal epithelium. Further studies for these mechanisms probably provide new insights into the biology including “TGFβ switch” and development of a novel therapy in OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 16. doi:1538-7445.AM2012-16

Abstract 5149: CYLD downregulation induces aerobic glycolysis via mTOR pathway in human oral squamous cell carcinoma cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The hypermetabolic nature of cancer cells and their increased reliance on “aerobic glycolysis,” as originally described by Otto Warburg et al, are considered metabolic hallmarks of cancer cells including oral squamous cell carcinoma (OSCC) cells. However, its precise mechanisms remain unknown. Cylindromatosis (CYLD) is recognized as a tumor suppressor gene whereas little is available about its impact on cancer progression. Our unpublished data showed that lower CYLD expression was associated with poor prognosis in OSCC patients. The objective of our study was to address specific contributions of CYLD to the signature metabolic features of cancer cells, including the so-called “Warburg effect”. We investigated effects of CYLD knockdown by siRNA on aerobic glycolysis in human OSCC cell lines. As the result of CYLD knockdown, both glucose consumption and lactate production significantly increased in several OSCC cell lines with increased expression of LDHA which is known to be important for glycolysis. Consistently, culture medium became more acidic after CYLD knockdown compared with the control. Repression of CYLD expression induced Akt phosphorylation. Importantly, the increase in aerobic glycolysis induced by CYLD repression was completely blocked by an mTOR inhibitor, rapamycin as well as a PIK inhibitor, LY294002. Our study indicats that dysfunction of CYLD contributs to the Warburg effect through Akt-mTOR pathway in OSCC. Further investigation on the advantage of such increased glycolysis and the change in metabolome profiling may lead to development of effective therapeutic strategies for OSCC and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5149. doi:1538-7445.AM2012-5149

Abstract 700: Reduced CYLD expression is an independent prognostic factor in breast cancer: A possible link with RANKL-RANK signaling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Deubiquitinase CYLD plays an important role in tumorigenesis and immune response. RANKL-RANK system is required for osteoclast development and is also involved in bone metastasis in various malignancies while it has been also implicated in tumorigenesis, metastasis to distant organs other than bone in breast cancer through unknown mechanisms. Importantly, a recent study has reported that CYLD negatively regulated RANKL-RANK signaling in osteoclastogenesis. However, not only the role of CYLD but also its involvement in RANKL-RANK signaling in breast cancer is largely unknown. The purpose of this study was to elucidate clinical and biological significance of CYLD as well as its association with RANKL-RANK signaling in breast cancer. Materials and Methods: We assessed CYLD mRNA expression in 305 primary invasive breast cancer tissues including 26 matched-pair of tumoral and adjacent non-tumoral tissues, and 9 breast cancer cell lines as well as a nonmalignant breast epithelial cell line, HMEC by quantitative real-time RT-PCR. In addition, effect of CYLD knockdown by using siRNA on cell proliferation, migratory activity and the biological activity of RANKL in MDA-MB-231 cells. Results: Expression levels of CYLD mRNA were lower in tumoral lesion than the matched non-tumoral lesion in all of 26 cases. Our statistical analysis revealed that lower CYLD mRNA expression was significantly associated with reduced disease free survival rate (rog-rank, p = 0.025), irrespectively of breast cancer subtype. Multivariate analyses for disease free survival confirmed that low CYLD mRNA expression (Hazard ratio: HR 2.23, 95% CI 1.09 - 4.36, p = 0.029) in addition to estrogen receptor status (HR 0.15, 95% CI 0.05 - 0.44, p = 0.0003) and lymph node metastasis (HR 2.21, 95% CI 1.10 - 4.62, p = 0.026) were significant factors. Consistent with our clinical data, CYLD gene expression was reduced in all the breast cancer cell lines compared with HMEC cells. Repression of CYLD gene expression led to increased cell number in MDA-MB-231 cells. Interestingly, combination of CYLD knockdown and RANKL stimulation promoted cell migration while no change was observed in the setting of each alone. Conclusion: Downregulation of CYLD was common and an independent poor prognostic factor in invasive breast cancer. CYLD repression may promote breast cancer development and/ or progression in coordination with RANKL-RANK signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 700. doi:1538-7445.AM2012-700