Akimitsu Hiraki

Expression of MMPs, MT-MMP, and TIMPs in squamous cell carcinoma of the oral cavity: Correlations with tumor invasion and metastasis

Matrix metalloproteinases (MMPs) that degrade the extracellular matrices (ECMs) have been thought to play an important role in both the invasion and metastasis of tumors. However, the detailed role of MMPs and TIMPs (tissue inhibitors of MMP) on the biological behavior of tumor cells has yet to be elucidated in vivo. The aim of the present study was thus to determine whether expression of MMPs on tumor cells is associated with such clinicopathological features as the invasive and metastatic potential.

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)

Immunohistochemical study of desmosomes in oral squamous cell carcinoma: Correlation with cytokeratin and E-cadherin staining, and with tumour behaviour

Reduction or loss of the intercellular junctions known as desmosomes may contribute to the invasive and metastatic behaviour of various carcinomas. Previous studies have shown that metastasis of oral squamous cell carcinomas of the head and neck correlates with a reduction in immunohistochemical staining for desmoplakin and desmoglein at the invasion front. The primary aim of the present study was to extend these observations to include a third component of desmosomes, the glycoprotein desmocollin. An additional aim was to determine whether the differentiation status of tumours is reflected in their staining for cytokeratins 1, 13, and 19, and, if so, whether these parameters correlate with desmosomal staining and/or metastasis. The study included 54 primary tumours of which 28 showed lymph node metastases. The results of this investigation show that tumours can be divided into three groups according to whether they have lost staining for no, one or more than one desmosomal component. A statistically significant correlation was found between the number of desmosomal components lost and metastasis. Tumours could also be divided into five groups according to their staining for different combinations of cytokeratins. Furthermore, differentiation status as indicated both histologically and by cytokeratin staining correlated with reduced desmosomal staining and metastasis. Tumours were also examined for intensity of staining for the adhesion molecule E‐cadherin. Reduction in E‐cadherin staining was correlated with mode of invasion and with reduction in desmosomal staining, but not with poor differentiation as indicated by cytokeratin staining. The results of this extensive study reinforce the view that adhesive junctions and adhesion molecules contribute to the suppression of tumour invasion and metastasis.

Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS

Purpose: The peptides derived from ideal cancer–testis antigens, including LY6K, CDCA1, and IMP3 (identified using genome-wide cDNA microarray analyses), were used in immunotherapy for head and neck squamous cell cancer (HNSCC). In this trial, we analyzed the immune response to and safety and efficacy of vaccine therapy. Experimental Design: A total of 37 patients with advanced HNSCC were enrolled in this trial of peptide vaccine therapy, and the OS, PFS, and immunologic response were evaluated using enzyme-linked ImmunoSpot (ELISPOT) and pentamer assays. The peptides were subcutaneously administered weekly with IFA. The primary endpoints were evaluated on the basis of differences between HLA-A*2402-positive [A24(+)] patients treated with peptide vaccine therapy and –negative [A24(−)] patients treated without peptide vaccine therapy among those with advanced HNSCC. Results: Our cancer vaccine therapy was well tolerated. The OS of the A24(+) vaccinated group (n = 37) was statistically significantly longer than that of the A24(−) group (n = 18) and median survival time (MST) was 4.9 versus 3.5 months, respectively; P < 0.05. One of the patients exhibited a complete response. In the A24(+) vaccinated group, the ELISPOT assay identified LY6K-, CDCA1-, and IMP3-specific CTL responses in 85.7%, 64.3%, and 42.9% of the patients, respectively. The patients showing LY6K- and CDCA1-specific CTL responses demonstrated a longer OS than those without CTL induction. Moreover, the patients exhibiting CTL induction for multiple peptides demonstrated better clinical responses. Conclusions: The immune response induced by this vaccine may improve the prognosis of patients with advanced HNSCC. Clin Cancer Res; 21(2); 312–21. ©2014 AACR.

Immunohistochemical staining of desmosomal components in oral squamous cell carcinomas and its association with tumour behaviour.

Desmosomes are intercellular junctions that have been shown to be down-regulated in certain types of carcinomas and that may play a role in suppression of invasion and metastasis. We have shown previously that immunohistochemical staining for the major desmosomal glycoprotein, desmoglein (Dsg), is reduced in some cases of squamous cell carcinoma (SCC) of the head and neck, and that reduced staining correlates with lymph node involvement. Desmosomes are multicomponent organelles. We therefore sought to determine whether another major desmosomal molecule, desmoplakin (Dp), showed similar reduced expression to that shown by desmoglein. We have stained 65 specimens of primary SCC of the oral cavity (37 non-metastatic and 28 metatastic) with monoclonal antibodies to both desmoglein and desmoplakin. We show that reduction of Dp staining correlates with loss of differentiation of the primary tumour, degree of invasion and presence of lymph node metastases. Similar correlations were found with Dsg staining. There was also correlation between reduction in Dp staining and reduction in Dsg staining. It is concluded that down-regulation of desmosomal expression occurs in some cases of SCC of the oral cavity and is associated with invasion and metastasis. Desmosomes may have an invasion and metastasis suppressor function.

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

Radiation-induced Parotid Gland Changes in Oral Cancer Patients: Correlation Between Parotid Volume and Saliva Production

OBJECTIVE To evaluate whether saliva production reflects the parotid volume during the course of radiation therapy (RT) in patients with head-and-neck cancer. METHODS Twenty patients with advanced oral squamous cell carcinomas, who were treated with preoperative chemo-RT, underwent morphological assessment with CT or MRI and functional assessment with the Saxon test. For the Saxon test, saliva production was measured by weighing a gauze pad before and 2 min after chewing without swallowing; the low-normal value is 2 g. Saliva production and parotid volumes before and 2 weeks after RT were compared with the paired t-test, the Spearman rank correlation test and the Fisher exact test. RESULTS After 30 Gy irradiation, mean saliva production was decreased from 4.2 to 1.0 g (P < 0.01); the reduction in saliva production ranged from 1.7 to 5.4 g (mean 3.2 g). The mean parotid volume was decreased from 68.2 to 47.9 cm(3) (P < 0.01); the post-RT:pre-RT parotid volume ratio ranged from 54% to 85% (mean 71%). Although the initial parotid ;volume was correlated with initial saliva production (r = 0.47, P = 0.04), no significant correlation was noted after RT (r = 0.08, P = 0.71), and there were considerable individual variations. The parotid volume ratio was inversely correlated with the saliva-reduction amount (r = - 0.79, P < 0.01). CONCLUSIONS There was a correlation between decreased parotid gland volume and decreased saliva production in patients with head-and-neck cancer undergoing RT. Parotid volume reduction may predict parotid gland function.

Involvement of hepatocyte growth factor in branching morphogenesis of murine salivary gland

We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5–12 (E11.5–E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real‐time reverse transcriptase‐polymerase chain reaction showed that the expression of the c‐met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14–E15.5. Although strong expression of the HGF and c‐met transcripts was observed in the tongue muscles, this expression was limited at E13.5–E14.5. Serum‐free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme‐free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme‐free epithelium failed to grow. When E13.5 or E14 mesenchyme‐free epithelium and a recombinant human HGF (rh‐HGF) ‐soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh‐HGF and anti‐HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh‐HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh‐HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c‐met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c‐Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme‐derived HGF and c‐met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage. Developmental Dynamics 228:173–184, 2003.

Overexpression of cIAP2 contributes to 5-FU resistance and a poor prognosis in oral squamous cell carcinoma

Background:Resistance to 5-fluorouracil (5-FU) is a major obstacle in treating oral squamous cell carcinoma (OSCC). However, little is known about apoptosis resistance, which contributes to 5-FU resistance in OSCC.Methods:We focussed on the cellular inhibitor of apoptosis protein 2 (cIAP2) on the basis of a DNA microarray data using parental and 5-FU-resistant OSCC cell lines. The effects of cIAP2 downregulation on 5-FU sensitivity and apoptosis were evaluated. An immunohistochemical analysis of cIAP2 and related proteins, cIAP1 and X-linked IAP, was performed in 54 OSCC patients who were treated with 5-FU-based chemoradiotherapy and surgery.Results:The downregulation of cIAP2 significantly enhanced the sensitivity of the 5-FU-resistant cells to 5-FU, with a significant increase in apoptosis. The immunohistochemical analysis demonstrated a high cIAP2 tumour expression to significantly correlate with the pathological response to chemoradiotherapy. Furthermore, a Cox regression analysis revealed the cIAP2 expression status (hazard ratio, 4.91; P=0.037) and the pathological response to chemoradiotherapy (hazard ratio, 0.418; P=0.016) to be significant prognostic factors for OSCC patients.Conclusion:These novel findings demonstrate that cIAP2 may represent a potentially useful therapeutic target for improving the treatment and survival of OSCC patients, particularly in the setting of 5-FU resistance.

Overexpression of fibronectin confers cell adhesion‑mediated drug resistance (CAM-DR) against 5-FU in oral squamous cell carcinoma cells

The tumor-associated microenvironment has been shown to protect tumor cells from treatment, and the extracellular matrix (ECM) is known to affect drug resistance as a key regulator of the tumor microenvironment. However, little is known about cell adhesion-mediated drug resistance (CAM-DR) due to cell-ECM contact in patients with oral squamous cell carcinoma (OSCC). In the present study, we evaluated the ECM molecule fibronectin (FN) using DNA microarray data obtained from parental and 5-FU-resistant OSCC cell lines. We investigated the effects of cell adhesion to FN on 5-FU resistance in OSCC cells and examined the activation of FN receptor β1 integrin-mediated survival regulators such as ILK, Akt and NF-κB. In addition, we investigated whether FNIII14, a 22-mer peptide derived from FN that potently prevents β1 integrin-mediated adhesion to FN, could overcome CAM-DR against 5-FU in OSCC cells and examined the activation of survival regulators and apoptosis-related molecules. Consequently, we obtained the following results. FN was extracellularly overexpressed in the 5-FU-resistant cells compared with that observed in the 5-FU-sensitive cells. Cell adhesion to FN enhanced 5-FU resistance and activated integrin-mediated ILK/Akt/NF-κB survival signaling in the 5-FU-resistant OSCC cells. Furthermore, the inhibition of cell adhesion to FN by FNIII14 enhanced chemosensitivity to 5-FU and apoptosis by suppressing ILK/Akt/NF-κB signaling in the 5-FU-resistant cells. These novel findings demonstrate that FN is a potentially useful biomarker and therapeutic target for improving the treatment of OSCC, particularly in the setting of 5-FU resistance.