Hideki Nakayama

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.

The pathological significance of Notch1 in oral squamous cell carcinoma

Notch signaling has been reported to be involved in several types of malignant tumors; however, the role and activation mechanism of Notch signaling in oral squamous cell carcinoma (OSCC) remains poorly characterized. The purpose of this study was to elucidate the pathological significance of Notch signaling and its activation mechanism in the development and progression of OSCC. In this study, we showed that the expression of Notch1 and intracellular Notch domain (NICD) are upregulated in OSCCs. In addition, Notch1 and NICD were found to be characteristically localized at the invasive tumor front. TNF-α, a major inflammatory cytokine, significantly activated Notch signaling in vitro. In a clinicopathological analysis, Notch1 expression correlated with both the T-stage and the clinical stage. Furthermore, loss of Notch1 expression correlated with the inhibition of cell proliferation and TNF-α-dependent invasiveness in an OSCC cell line. In addition, γ-secretase inhibitor (GSI) prevented cell proliferation and TNF-α-dependent invasion of OSCC cells in vitro. These results indicate that altered expression of Notch1 is associated with increased cancer progression and that Notch1 regulates the steps involved in cell metastasis in OSCC. Moreover, inactivating Notch signaling with GSI could therefore be a useful approach for treating patients with OSCC.

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.

Level of serum antibody against a periodontal pathogen is associated with atherosclerosis and hypertension

Inflammation has a role in the pathogenesis of atherosclerosis, which causes hypertension. Results from some studies have suggested links between periodontal disease and atherosclerosis, but links between periodontal disease and hypertension have been seldom studied. We investigated whether periodontal disease and serum antibody level were associated with hypertension. We studied 127 patients (93 men and 34 women, mean age 68±9 years) who were admitted with ischemic heart disease to our institution. A composite periodontal risk score was calculated from five periodontal vector scores. The levels of serum antibody against Porphyromonas gingivalis (Pg) were measured. Pulse pressure, mean blood pressure (BP) and pulse wave velocity were used as indices of atherosclerosis. We divided patients into two groups according to the levels of serum antibody against Pg: higher or equal to the median (high Pg antibody group) and lower than the median (low Pg antibody group).There was no difference in the use of antihypertensive agents between the two groups. The composite periodontal risk score (P=0.0003), systolic BP (P=0.030), diastolic BP (P=0.038), pulse pressure (P=0.050) and mean BP (P=0.055) were higher in the high Pg antibody group than in the low Pg antibody group. The composite periodontal risk score (r=0.320, P=0.0003), systolic BP (r=0.212, P=0.017), diastolic BP (r=0.188, P=0.035) and mean BP (r=0.225, P=0.011) correlated with the level of serum antibody against Pg, even after adjustment for age. An elevated antibody level against Pg indicates advanced periodontal disease and suggests advancement of atherosclerosis and hypertension.

Selective inhibition of nuclear factor-κB by nuclear factor-κB essential modulator-binding domain peptide suppresses the metastasis of highly metastatic oral squamous cell carcinoma

Nuclear factor‐κB (NF‐κB) activation contributes to the development of metastasis, thus leading to a poor prognosis in many cancers, including OSCC. However, little in vivo experimental data are available about the effects of NF‐κB inhibition on OSCC metastasis. OSCC sublines were established from a GFP‐expressing parental cell line, GSAS, and designated GSAS/N3 and N5 according to the in vivo passage number after cervical lymph node metastasis by a serial orthotopic transplantation model. In vitro migration and invasion were assessed in these cells, and the NF‐κB activities and expression of NF‐κB‐regulated metastasis‐related molecules were also examined. In in vivo experiments, the metastasis and survival of tumor‐engrafted mice were monitored. Furthermore, the effects of a selective NF‐κB inhibitor, NEMO‐binding domain (NBD) peptide, on metastasis in GSAS/N5‐engrafted mice were assessed, and engrafted tongue tumors were immunohistochemically examined. Highly metastatic GSAS/N3 and N5 cells showed an enhanced NF‐κB activity, thus contributing to increased migration, invasion, and a poor prognosis compared with the parent cells. Furthermore, the expression levels of NF‐κB‐regulated metastasis‐related molecules, such as fibronectin, β1 integrin, MMP‐1, ‐2, ‐9, and ‐14, and VEGF‐C, were upregulated in the highly metastatic cells. The NBD peptide suppressed metastasis and tongue tumor growth in GSAS/N5‐inoculated mice, and was accompanied by the downregulation of the NF‐κB‐regulated metastasis‐related molecules in engrafted tongue tumors. Our results suggest that the selective inhibition of NF‐κB activation by NBD peptide may provide an effective approach for the treatment of highly metastatic OSCC. (Cancer Sci 2012; 103: 455–463)

A low dicer expression is associated with resistance to 5-FU-based chemoradiotherapy and a shorter overall survival in patients with oral squamous cell carcinoma

BACKGROUNDnThe deregulation of microRNA (miRNA) is associated with multiple processes involved in cancer progression. RNase III endonucleases, Dicer and Drosha, are key enzymes for miRNA biogenesis, and it has been reported that altered expressions of these molecules affect the clinical outcomes of patients with various cancers. However, the clinical value of measuring the levels of Dicer and Drosha in oral squamous cell carcinoma (OSCC) patients is unclear. The purpose of this study was to determine the clinical significance of the expressions of Dicer and Drosha in patients with OSCC.nnnMETHODSnOral squamous cell carcinoma specimens were obtained from 61 patients who underwent surgery following 5-fluorouracil-based chemoradiotherapy at Kumamoto University Hospital between October 2003 and January 2009. Paraffin-embedded sections obtained from biopsy specimens were immunohistochemically analyzed. The associations between Dicer, Drosha, and various clinicopathological features were examined, and the effects of Dicer and Drosha on the prognosis were evaluated.nnnRESULTSnA low Dicer tumor expression was significantly correlated with the pathological response to chemoradiotherapy. Furthermore, a Cox regression analysis based on the overall survival revealed the Dicer expression status (hazard ratio, 0.34; Pxa0=xa00.048) and pathological response to chemoradiotherapy (hazard ratio, 0.21; Pxa0=xa00.014) to be significant prognostic factors in OSCC patients. On the other hand, the Drosha expression was not associated with any clinicopathological features or the prognosis.nnnCONCLUSIONnThese results suggest that Dicer is a potential biomarker for predicting the clinical response to 5-FU-based chemoradiotherapy and the overall survival in patients with OSCC.

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.

The tumour stromal features are associated with resistance to 5‐FU‐based chemoradiotherapy and a poor prognosis in patients with oral squamous cell carcinoma

It has been increasingly recognized that the tumour microenvironment is a critical factor involved in cancer progression. However, little is known about the clinical value of the stromal features in oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the clinical significance of cancer‐associated fibroblasts (CAFs) and tumour‐associated macrophages (TAMs) in OSCC. OSCC specimens were obtained from 60 patients who underwent surgery following 5‐fluorouracil‐based chemoradiotherapy. Paraffin‐embedded sections obtained from biopsy specimens were immunohistochemically analysed. The associations among CAFs, TAMs and various clinicopathological features were examined, and the effects of CAFs and TAMs on the prognosis were evaluated. In the group with a high level of CAFs, the incidence of advanced pT‐ and pN‐stage cases was significantly higher than that in the group with the low level. A high TAMs tumour expression was significantly correlated with a poor response to preoperative chemoradiotherapy. A Kaplan–Meier analysis revealed that higher numbers of CAFs and TAMs were significantly correlated with a poor prognosis. These findings suggest that TAMs are a potential biomarker for predicting the clinical response to 5‐FU‐based chemoradiotherapy, and the expression status of the CAFs and TAMs may be useful for making treatment decisions to improve the survival of OSCC patients.

Overexpression of nucleostemin contributes to an advanced malignant phenotype and a poor prognosis in oral squamous cell carcinoma.

Background:Nucleostemin (NS) is essential for the maintenance of stem cell properties, the functions of which remain poorly understood in cancer cells. The purpose of this study was to explore the impact of NS on malignancy and its clinical significance in oral squamous cell carcinoma (OSCC) patients.Methods:We investigated the effects of NS on the proliferation and invasion of OSCC using NS-overexpressing or -knockdown OSCC cells. We assessed the activation of the STAT3 (signal transducer and activator of transcription 3) signalling pathway and the downstream targets in the cells with different expression levels of NS. An immunohistochemical analysis of NS was also performed in 54 OSCC patients who were treated with preoperative chemoradiotherapy and surgery.Results:The overexpression of NS significantly enhanced the proliferation and invasive potential of OSCC cells. On the other hand, downregulation of NS suppressed the invasiveness of the cells. The alterations of these malignant phenotypes were associated with the activation of STAT3 signalling and its downstream targets. An immunohistochemical analysis demonstrated that a high NS tumour expression level significantly correlated with an advanced T-stage and N-stage. Furthermore, a Cox regression analysis revealed that the NS status (hazard ratio, 9.09; P=0.002) was a significant progression factor for OSCC patients.Conclusions:Our results suggest that targeting NS may provide a promising treatment for highly malignant OSCC.