IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma.

BACKGROUND: In promoting tumour malignancy IL-6 signalling is considered to have an important role. However, the biological roles of IL-6 on radiosensitivity in oral squamous cell carcinoma (OSCC) remain largely unclear. The objective of this study is to determine the effects and molecular mechanisms of IL-6 on radiosensitivity in OSCC. METHODS: Two OSCC cell lines, and OSCC tissue samples with radioresistant cells were used. We examined the effects of IL-6, or tocilizumab, a humanised anti-human IL-6 receptor antibody, or both on radiosensitivity and DNA damage after X-ray irradiation in vitro. In addition, we investigated the involvement of the Nrf2-antioxidant pathway in IL-6-mediated radioresistant mechanisms using OSCC cell lines and tissues. RESULTS: Increased levels of IL-6 suppressed radiation-induced cell death, and the blockade of IL-6 signalling by tocilizumab sensitised tumour cells to radiation. The radioresistant effect of IL-6 was associated with decreased DNA damage after radiation. We also found that IL-6 promotes the activation of not only the downstream molecule STAT3 but also the Nrf2-antioxidant pathway, leading to a significant decrease in oxidative stress by upregulating Mn-SOD. CONCLUSIONS: These results indicate that the blockade of IL-6 signalling combined with conventional radiotherapy could augment the treatment response and survival rate in patients with radioresistant OSCC.

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, ß1 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.

Association of high cellular expression and plasma concentration of angiopoietin-like 4 with tongue cancer lung metastasis and poor prognosis.

Angiopoietin-like 4 (ANGPTL4) promotes cancer cell migration through vessels and has been implicated in cancer metastasis. Our previous study identified a robust increase in ANGPTL4 mRNA expression in lung-metastasized tongue cancer (TC) cells. Therefore, the present study investigated the association of ANGPTL4 with lung metastasis and outcomes of patient with TC. ANGPTL4 expression in TC cells was investigated by immunohistochemical staining. Patients were classified into 'low (0-30%)' and 'high (>30%)' ANGPTL4-expression groups based on the proportion of ANGPTL4-positive TC cells. The high ANGPTL4-expression group included 15 of 48 patients with TC. Notably, a significantly greater proportion of patients with lung metastasis exhibited a high rate of ANGPTL4-expressing cancer cells compared with patients without lung metastasis (P=0.029). The overall 5-year survival rate was lower in the high (27%) ANGPTL4-expression group compared with the low (68%) ANGPTL4-expression group. Univariate and multivariate analyses revealed that patients with high ANGPTL4 expression in TC cells exhibited significantly lower overall survival (OS) rates [hazard ratio (HR), 2.99; 95% confidence interval (95% CI), 1.34-6.69; P=0.008 and HR, 2.72; 95% CI, 1.14-6.51; P=0.024, respectively]. High plasma ANGPTL4 concentrations as measured by ELISA were associated with lung metastasis (P<0.001). The optimal cut-point for prediction of TC lung metastasis was 9.1 ng/ml (P<0.001; 95% CI, 7.2-10.9). The OS of patients with plasma ANPTL4 above the cut-point was significantly lower than that of patients with plasma ANGPTL4 ≤9.1 ng/ml (P<0.001). These results suggest that a high level of ANGPTL4 in cancer cells and plasma may predict lung metastasis and/or a poor prognosis of patients with TC.

Enhancement of cancer invasion and growth via the C5a-C5a receptor system: Implications for cancer promotion by autoimmune diseases and association with cervical cancer invasion.

Autoimmune diseases are caused by immune complex-induced activation of the complement system and subsequent inflammation. Recent studies have revealed an association between autoimmune diseases and worse survival in patients with cancer; however, the underlying mechanism is still unknown. The C5a-C5a receptor (C5aR) system has been shown to enhance cancer activity and recruit myeloid-derived suppressor cells (MDSCs) that suppress the anti-tumor immune response. The Arthus reaction is inflammation caused by complement system activation by the immune complex and thus is a model of autoimmune diseases. To explore the effect of the Arthus reaction on cancer progression, mouse cancer cells were inoculated in syngeneic mouse skin, where the Arthus reaction was induced simultaneously. The Arthus reaction enhanced invasion and tumor growth of C5aR-positive cancer cells, but not control cells, and induced MDSC recruitment. Intravenous injection of C5a-stimulated C5aR-positive cancer cells into nude mice resulted in more lung nodules than injection of nontreated C5aR-positive cells and C5a-stimulated C5aR-negative cells, supporting C5a-C5aR-mediated enhancement of cancer growth. C5aR expression in uterine cervical carcinoma stage I cells, which invade into the deeper tissues, was significantly higher than that in CIN3 cells, which remain in the epithelium. These results indicate that cancer promotion by the C5a-C5aR system may underlie poor prognosis in cancer patients with autoimmune diseases, particularly in patients with C5aR-positive cancer, and may be associated with cervical cancer invasion. The enhancement of cancer cell invasion and growth by the C5a-C5aR system suggests that this system is a possible target of cancer therapy.

Predictive value of the combination of SMAD4 expression and lymphocyte infiltration in malignant transformation of oral leukoplakia.

Oral leukoplakia (OL) is a common, potentially malignant disorder of the oral cavity. SMAD4 was initially identified as a tumor suppressor and central mediator of transforming growth factor (TGF)-ß signaling. In this study, we aimed to determine the expression patterns of SMAD4 in OL, its relationship with the degree of inflammation, and its clinical implications as a biomarker for OL malignant transformation. A total of 150 patients with OL were enrolled in this study. Paraffin-embedded sections obtained from biopsy or resection specimens were subjected to immunohistochemical analysis. Associations among the status of epithelial SMAD4 expression, stromal lymphocyte infiltration, and malignant transformation of OL were examined. Malignant transformation was significantly associated with the status of SMAD4 expression (P = 0.0017) and lymphocyte infiltration status (P = 0.0054). Cox regression analysis, based on the event-free survival (EFS), revealed that a low SMAD4 expression was a significant prognostic factor in OL patients (hazard ratio, 2.632; P = 0.043). In addition, a low SMAD4 expression was closely correlated with high lymphocyte infiltration (P = 0.00035), resulting in a significant correlation between the combination of low SMAD4 expression and high lymphocyte infiltration with malignant transformation of OL (P = 0.00027). The combination of the status of epithelial SMAD4 expression and stromal lymphocyte infiltration may be a useful biomarker for predicting malignant transformation in OL patients. These results suggest that not only epithelial SMAD4 loss, but also stromal features, may regulate the risk of malignant transformation of OL.

Enhancement of active MMP release and invasive activity of lymph node metastatic tongue cancer cells by elevated signaling via the TNF-α-TNFR1-NF-κB pathway and a possible involvement of angiopoietin-like 4 in lung metastasis.

To study the role of TNF-α in tongue cancer metastasis, we made highly metastatic cells from a human oral squamous cell carcinoma cell line (SAS) by repeating the passage in which the cells were injected into a nude mouse tongue and harvested from metastasized cervical lymph nodes. Cancer cells after 5 passages (GSAS/N5) increased invasive activity 7-fold in a TNF-α receptor 1 (TNFR1)-dependent manner and enhanced mRNA expression of TNF-α and TNFR1. In the highly metastatic cells, NF-κB activation was upregulated via elevated phosphorylation of Akt and Ikkα/ß in the signaling pathway and secretion of TNF-α, active MMP-2 and MMP-9 increased. Suppression of increase of TNF-α mRNA expression and MMP secretion by NF-κB inhibitor NBD peptide suggested a positive feedback loop in GSAS/N5 cells; TNF-α activates NF-κB and activated NF-κB induces further TNF-α secretion, leading to increase of active MMP release and promotion of invasion and metastasis of the cells. GSAS/N5 cells that had been injected into the nude mouse tongue and harvested from metastasized lungs multiplied angiopoietin-like 4 (angptl4) expression with enhanced migration activity, which indicated a possible involvement of angptl4 in lung metastasis of the cells. These results suggest that TNF-α and angptl4 promote metastasis of the oral cancer cells, thus, these molecules may be therapeutic targets for patients with tongue cancer.

Ventral abdominal wall dysmorphogenesis of Msx1/Msx2 double-mutant mice.

Msx1 and Msx2 genes encode the homeodomain transcription factors. Several gene knockout mice and expression studies suggest that they possess functionally redundant roles in embryogenesis. In this study, we revealed that Msx1 and Msx2 were expressed during ventral body wall formation in an overlapping manner. Msx1/Msx2 double-mutant mice displayed embryonic abdominal wall defects with disorganized muscle layers and connective tissues. These findings indicate that Msx1 and Msx2 play roles in concert during embryonic ventral abdominal wall formation.

The morphological and histological characters of the male external genitalia of the house musk shrew, Suncus murinus.

External genitalia are the reproductive organs necessary for efficient copulation and internal fertilization in various mammalian species. Their morphogeneses display significant morphological and developmental differences among species. The house musk shrew, Suncus murinus (hereafter described as suncus) is a species of the order Insectivora, which has been considered as primitive and one of the earliest eutheria phylogenetically. Comparative anatomical analyses of phylogenetically different mammals will contribute to the better understanding of morphological diversity of external genitalia. This study performed various anatomical and histological analyses concerning the organization of the external genitalia of male suncus. It was shown that the external genitalia of suncus possessed a muscular structure, which we proposed as musculus ischiocavernosus dorsalis of suncus. The musculus ischiocavernosus dorsalis is originated from the inner surface of the tuber ischiadicum and was allocated adjacent to the corpus cavernosum penis. In addition, a pair of alpha-smooth muscle actin positive muscles was located bilaterally to the urethra. This unique morphology of the external genitalia of suncus males may provide a unique model system to investigate genital morphogenesis.

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.

Sonic hedgehog expression during early tooth development in Suncus murinus.

Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.

Comparison of expression patterns of fibroblast growth factor 8, bone morphogenetic protein 4 and sonic hedgehog in jaw development of the house shrew, Suncus murinus.

To elucidate the mechanism underlying jaw development in mammals, we used a new laboratory animal, Suncus murinus (house shrew, an insectivore) as the subject for the investigation, because Suncus has all types of teeth (incisor, canine, premolar and molar) in its upper and lower jaws and is thought to be a good model animal having a general mammalian tooth pattern. At the start, by use of degenerate primers we cloned Suncus homologues of fibroblast growth factor 8 (sFgf8), bone morphogenetic protein 4 (sBmp4) and sonic hedgehog (sShh) genes from cDNA library derived from whole Suncus embryos at day 12 (E12). Thereafter, we examined the expression patterns of these genes in the jaw development of Suncus E11-16 embryos (for mouse E9.5-12 embryos). sFgf8 and sBmp4 were expressed in E11 but not in E15 and onward during orofacial development. sShh was expressed from E11 onward, and its expression was increased in the orofacial area. The expression pattern of sFgf8 in the maxillary and mandibular arches of E14 coincided with the area of the presumptive tooth arch. However, sShh and sBmp4 were expressed only in the outer area (= buccal/labial side) of presumptive tooth arch. Thus, these 3 genes showed specific expression pattern in jaw development of Suncus, and their distributions did not overlap each other except in a few regions. These findings suggest that sFgf8, sBmp4 and sShh have a specific function respectively during jaw development in Suncus murinus.

Molecular cloning and characterization of the mouse reticulon 3 cDNA.

cDNA of mouse reticulon 3 (mRTN3) was cloned. The cloned cDNA is 1745 bp long and contains an open reading frame of 237 amino acids for a 30 kDa protein. The gene was mapped at band B of chromosome 19 by FISH. Two altematively spliced transcripts, 3.4 and 2.3 kb, of mRTN3 were found by Northem blot analysis. Both transcripts were expressed in many tissues and embryos and the highest expression of the 3.4 kb-transcript was observed in the brain, especially in neurons. The expression of 30 kDa-mRTN3 protein was also greatest in the brain. Both N and C-termini of mRTN3 faced the cytosol, indicating that they may recruit other proteins to the endoplasmic reticulum.

Regulation of outgrowth and apoptosis for the terminal appendage: external genitalia development by concerted actions of BMP signaling [corrected].

Extra-corporal fertilization depends on the formation of copulatory organs: the external genitalia. Coordinated growth and differentiation of the genital tubercle (GT), an embryonic anlage of external genitalia, generates a proximodistally elongated structure suitable for copulation, erection, uresis and ejaculation. Despite recent progress in molecular embryology, few attempts have been made to elucidate the molecular developmental processes of external genitalia formation. Bone morphogenetic protein genes (Bmp genes) and their antagonists were spatiotemporally expressed during GT development. Exogenously applied BMP increased apoptosis of GT and inhibited its outgrowth. It has been shown that the distal urethral epithelium (DUE), distal epithelia marked by the Fgf8 expression, may control the initial GT outgrowth. Exogenously applied BMP4 downregulated the expression of Fgf8 and Wnt5a, concomitant with increased apoptosis and decreased cell proliferation of the GT mesenchyme. Furthermore, noggin mutants and Bmpr1a conditional mutant mice displayed hypoplasia and hyperplasia of the external genitalia respectively. noggin mutant mice exhibited downregulation of Wnt5a and Fgf8 expression with decreased cell proliferation. Consistent with such findings, Wnt5a mutant mice displayed GT agenesis with decreased cell proliferation. By contrast, Bmpr1a mutant mice displayed decreased apoptosis and augmented Fgf8 expression in the DUE associated with GT hyperplasia. These results suggest that some of the Bmp genes could negatively affect proximodistally oriented outgrowth of GT with regulatory functions on cell proliferation and apoptosis. The DUE region can be marked only until 14.0 dpc (days post coitum) in mouse development, while GT outgrowth continues thereafter. Possible signaling crosstalk among the whole distal GT regions were also investigated.