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Oral squamous cell carcinomas unusually show distant metastasis to the lung after primary treatment, which can be difficult to differentiate from primary squamous cell carcinoma of the lung. While the location and number of tumor nodules is helpful in diagnosing cases, differential diagnosis may be difficult even with histopathological examination. Therefore, we attempted to identify molecules that can facilitate accurate differential diagnosis. First, we performed a comprehensive gene expression analysis using microarray data for OSCC-LM and LSCC, and searched for genes showing significantly different expression levels. We then identified KRT13, UPK1B, and nuclear receptor subfamily 0, group B, member 1 (NR0B1) as genes that were significantly upregulated in LSCC and quantified the expression levels of these genes by real-time quantitative RT-PCR. The expression of KRT13 and UPK1B proteins were then examined by immunohistochemical staining. While OSCC-LM showed no KRT13 and UPK1B expression, some tumor cells of LSCC showed KRT13 and UPK1B expression in 10 of 12 cases (83.3%). All LSCC cases were positive for at least one of these markers. Thus, KRT13 and UPK1B might contribute in differentiating OSCC-LM from LSCC.

Mutations in p53 are common in human oral squamous cell carcinoma (OSCC). However, in previous analyses, only detection of mutant p53 protein using immunohistochemistry or mutations in some exons have been examined. Full length mutant p53 protein in many cases shows a loss of tumor suppressor function, but in some cases possibly shows a gain of oncogenic function. In this study, we investigate relationships of outcomes with the mutational spectrum of p53 (missense and truncation mutations) in whole exon in OSCC. Specimens from biopsy or surgery (67 cases) were evaluated using next-generation sequencing for p53, and other oncogenic driver genes. The data were compared with overall survival (OS) and disease-free survival (DFS) using univariate and multivariate analyses. p53 mutations were detected in 54 patients (80.6%), 33 missense mutations and 24 truncation mutations. p53 mutations were common in the DNA-binding domain (43/52) and many were missense mutations (31/43). Mutations in other regions were mostly p53 truncation mutations. A comparison of cases with p53 mutations (missense or truncation) with wild-type p53 cases showed a significant difference in lymph node metastasis. DFS was significantly poorer in cases with p53 truncation mutations. Cases with p53 truncation mutations increased malignancy. In contrast, significant differences were not found between cases with p53 missense mutations and other mutations. The p53 missense mutation cases might include cases with mostly similar function to that of the wild-type, cases with loss of function, and cases with various degrees of gain of oncogenic function.

MicroRNAs (miRNAs) can act not only as tumor suppressor genes but also as oncogenes. Oncogenic miRNAs (oncomiRs) could therefore provide opportunities for the treatment of human malignancies. Here, we aimed to identify oncomiRs present in oral squamous cell carcinoma (OSCC) and addressed whether targeting these miRNAs might be useful in treatment for cancer. Functional screening for oncomiRs in a human OSCC cell line (GFP‐SAS) was carried out using the miRCURY LNA microRNA Knockdown Library – Human version 12.0. We identified a locked nucleic acid (LNA)/DNA antisense oligonucleotide against miR‐361‐3p (LNA‐miR‐361‐3p) which showed the largest degree of growth inhibition of GFP‐SAS cells. Transfection with a synthetic mimic of mature miR‐361‐3p resulted in an approximately 20% increase in the growth of GFP‐SAS cells. We identified odd‐skipped related 2 (OSR2) as a miR‐361‐3p target gene. Transfection of GFP‐SAS cells with LNA‐miR‐361‐3p caused a significant increase in the expression levels of OSR2. Cotransfection of a OSR2 3′‐UTR luciferase reporter plasmid and LNA‐miR‐361‐3p into GFP‐SAS cells produced higher levels of luciferase activity than in cells cotransfected with the LNA‐nontarget. We assessed the effect of LNA‐miR‐361‐3p on the in vivo growth of GFP‐SAS cells. We found that LNA‐miR‐361‐3p significantly reduced the size of s.c. xenografted GFP‐SAS tumors, compared to the control group treated with LNA‐NT. Finally, we observed that miR‐361‐3p is overexpressed in OSCC tissues. These results suggest that miR‐361‐3p supports the growth of human OSCC cells both in vitro and in vivo and that targeting miR‐361‐3p could be a useful therapeutic approach for patients with OSCC., This study shows that microRNA (miR)‐361‐3p supports the growth of human oral squamous cell carcinoma (OSCC) cells both in vitro and in vivo and that targeting miR‐361‐3p could be a useful therapeutic approach for patients with OSCC.

Background/aim Odontogenic diseases are diagnosed based on clinical course, imaging, and histopathology. However, a definitive diagnosis is not always possible. Patients and methods We analyzed whole exons of SMO, BRAF, PTCH1 and GNAS using next-generation sequencing (NGS) in 18 patients. Results Of the 6 patients with ameloblastoma, 2 patients had the same missense mutation in BRAF, and 1 patient with peripheral ameloblastoma had a missense mutation in PTCH1. Of the 7 patients with odontogenic keratocyst, 4 patients had a missense mutation in PTCH1, 2 patients had missense mutations in BRAF, and 1 patient had a missense mutation in SMO. The patient with odontoma had missense mutations in SMO, BRAF and PTCH1. One patient with cement-osseous dysplasia had missense mutations in SMO and PTCH1. The patient with adenomatoid odontogenic tumor had missense mutations in SMO. Conclusion Whole exome sequencing of the above genes by NGS would be useful for the differential diagnosis of odontogenic diseases.

Mammary analogue secretory carcinoma (MASC) has recently been recognized as a salivary gland tumour that is characterized by the ETV6–NTRK3 fusion gene. A case of locally advanced MASC of the parotid gland in a 67-year-old man is presented here. The patient visited the hospital due to a large right infra-auricular mass, which had been enlarging gradually over a period of 2 years. Contrast-enhanced computed tomography (CT) demonstrated a multilocular mass, 75 × 63 mm in size, containing a fluid component with non-uniform contrast effects in the interior portion. The mass had invaded the orbit, skull base, and parapharyngeal space. The patient had neither lymph node nor distant metastasis. The tumour showed tubular and ductal proliferation lined by a single layer of neoplastic cuboidal cells with clear foamy cytoplasm. Characteristic hobnail cells were observed. Expression of ETV6–NTRK3 fusion transcript in the tumour tissues was confirmed by RT-PCR. The final diagnosis was MASC (T4bN0M0, stage IVB). The patient received cetuximab together with radiotherapy at a total dose of 66 Gy. After treatment, CT showed a slightly reduced tumour volume, indicating stable disease. More than 56 months after treatment, the patient remains alive with no remarkable change in the tumour.

Human papillomavirus (HPV) is a possible carcinogenetic factor in oral squamous cell carcinoma (OSCC). Previous studies have reported the prevalence of HPV in patients with OSCC. However, the association between HPV and OSCC remains controversial. The present study aimed to clarify the association between HPV infection, p16 protein expression and the clinicopathological characteristics of OSCC. The expression level of HPV-16E6 mRNA and p16 protein, a known surrogate marker of HPV infection, was investigated in 100 OSCC cases using TaqMan reverse transcription-quantitative PCR and immunohistochemistry staining, respectively. HPV-16E6 mRNA expression level was only detected in one case (1%), and positive expression of p16 was found in 10 cases (10%), including an HPV-positive case. Subsequently, the association between p16 expression level and clinicopathological characteristic factors were analyzed; however, no significant association was found. These results suggested that HPV-16 infection was less likely to cause OSCC in Japan and p16 expression was not a suitable marker for HPV infection in OSCC.

Molecules that demonstrate a clear association with the aggressiveness of oral squamous cell carcinoma (OSCC) have not yet been identified. The current study hypothesized that tumor cells in OSCC have three different origins: Epithelial stem cells, oral tissue stem cells from the salivary gland and bone marrow (BM) stem cells. It was also hypothesized that carcinomas derived from less-differentiated stem cells have a greater malignancy. In the present study, sex chromosome analysis by fluorescence in situ hybridization and/or microdissection PCR was performed in patients with OSCC that developed after hematopoietic stem cell transplantation (HSCT) from the opposite sex. OSCC from 3 male patients among the 6 total transplanted patients were considered to originate from donor-derived BM cells. A total of 2/3 patients had distant metastasis, resulting in a poor prognosis. In a female patient with oral potentially malignant disorder who underwent HSCT, there were 10.7% Y-containing cells in epithelial cells, suggesting that some epithelial cells were from the donor. Subsequently, gene expression patterns in patients with possible BM stem cell-derived OSCC were compared with those in patients with normally developed OSCC by microarray analysis. A total of 3 patients with BM stem cell-derived OSCC exhibited a specific pattern of gene expression. Following cluster analysis by the probes identified on BM stem cell-derived OSCC, 2 patients with normally developed OSCC were included in the cluster of BM stem cell-derived OSCC. If the genes that could discriminate the origin of OSCC were identified, OSCCs were classified into the three aforementioned categories. If diagnosis can be performed based on the origin of the cancer cells, a more specific therapeutic strategy may be implemented to improve prognosis. This would be a paradigm shift in diagnostic and therapeutic strategies for OSCC.

Background/Aim: Odontogenic diseases are diagnosed based on clinical course, imaging, and histopathology. However, a definitive diagnosis is not always possible. Patients and Methods: We analyzed whole exons of SMO, BRAF, PTCH1 and GNAS using next-generation sequencing (NGS) in 18 patients. Results: Of the 6 patients with ameloblastoma, 2 patients had the same missense mutation in BRAF, and 1 patient with peripheral ameloblastoma had a missense mutation in PTCH1. Of the 7 patients with odontogenic keratocyst, 4 patients had a missense mutation in PTCH1, 2 patients had missense mutations in BRAF, and 1 patient had a missense mutation in SMO. The patient with odontoma had missense mutations in SMO, BRAF and PTCH1. One patient with cement-osseous dysplasia had missense mutations in SMO and PTCH1. The patient with adenomatoid odontogenic tumor had missense mutations in SMO. Conclusion: Whole exome sequencing of the above genes by NGS would be useful for the differential diagnosis of odontogenic diseases.

Administration of cetuximab (C-mab) in combination with paclitaxel (PTX) has been used for patients with head and neck squamous cell carcinoma (SCC) clinically. In this study, we attempted to clarify the molecular mechanisms of the enhancing anticancer effect of C-mab combined with PTX on oral SCC cells in vitro. We used two oral SCC cells (HSC4, OSC19) and A431 cells. PTX alone inhibited cell growth in all cells in a concentration-dependent manner. C-mab alone inhibited the growth of A431 and OSC19 cells at low concentrations, but inhibited the growth of HSC4 cells very weakly, even at high concentrations. A combined effect of the two drugs was moderate on A431 cells, but slight on HSC4 and OSC19 cells. A low concentration of PTX enhanced the antibody-dependent cellular cytotoxicity (ADCC) induced by C-mab in all of the cells tested. PTX slightly enhanced the anticancer effect of C-mab in this ADCC model on A431 and HSC4 cells, and markedly enhanced the anticancer effect of C-mab on OSC19 cells. These results indicated that PTX potentiated the anticancer effect of C-mab through enhancing the ADCC in oral SCC cells.

More than 90% of oral cancers are histopathologically squamous cell carcinomas (SCCs). According to clinical behavior and histopathological features, we hypothesize that oral SCC can originate from either oral squamous epithelium or minor salivary glands. Here, we examined whether some oral SCCs originate from minor salivary glands, and investigated whether these tumors show particularly aggressive biological behavior. The mRNA expression profiles of samples obtained from six patients with oral floor SCC (five men, one woman; mean age, 62.7 years) were analyzed using a microarray containing 32,878 probes. The six samples were divided into two groups by clustering of expression levels of 845 probes differentially expressed in normal oral squamous epithelium and normal salivary glands. The expression profile in four cases was similar to that of normal oral squamous epithelium, and in two cases was similar to that of normal salivary glands. Furthermore, we identified nine genes that reveal the origin of the oral SCC. Subsequently, we examined the expression levels of these nine marker genes by reverse transcriptase‐polymerase chain reaction to determine the origin of 66 oral SCCs. Twelve of the 66 oral SCCs were considered to originate from minor salivary glands, and these tumors showed high metastatic potential (p = 0.044, Chi‐square test). Furthermore, SCC derived from minor salivary glands showed a poor event‐free survival rate (p = 0.017, Kaplan–Meier analysis). In conclusion, determination of the origin of oral SCC is helpful in planning treatment for patients with oral SCC., What's new? In spite of several past attempts, no distinct factors controlling the biological aggressiveness of oral squamous cell carcinoma (SCC) have been identified. Determining the origin of the cancer cells could help predict biological aggressiveness, however. Here, the authors provide evidence that oral SCC has two different origins, as mucosal or salivary SCC. Furthermore, they identify nine genes able to reveal the origin of oral SCC. Based on their expression levels, the authors determined that twelve of the 66 oral SCCs in their sample originated from minor salivary glands. Salivary SCC tumors showed high metastatic potential and poor event‐free survival rate.

Background: Mechanisms underlying immune cells' recruitment and activation into the inflammatory lesions of lip salivary glands (LSGs) from primary Sjogren's syndrome (pSS) patients are incompletely understood. Chemokines play pivotal roles in these processes, so we investigated the clinical significance of chemokine receptor CXCR3 and its ligands in the autoimmune lesions of pSS. Methods: We histologically determined the grade of LSG samples from 22 pSS patients and subjected the samples to immunofluorescence analysis to determine the expressions of CXCR3 and its ligands: CXCL9, CXCL10, and CXCL11. To identify the immune cells expressing CXCR3 in the LSGs, we performed double immunofluorescence analysis using antibodies against CD3 (pan-T cells), CD80 (M1 macrophages), CD163 (M2 macrophage), and CD123 (plasmacytoid dendritic cells: pDCs). The relationship between the grade of lymphocytic infiltration and the number of positively stained cells was analyzed by Spearman's rank correlation test. Results: The expressions of CXCL9 and CXCL10 showed particularly intense staining in the LSG samples' ductal cells. The CXCR3 expression was detected mainly in CD80+ and CD163+ macrophages. The number of CXCR3+CD163+ macrophages inversely correlated with the LSG inflammatory lesions' severity (rs= −0.777, p

CXCL10, a CXC chemokine induced by interferon-gamma [IFN-γ], has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions. Although CXCL10 is known to be overexpressed in the salivary glands of individuals with primary Sjögren's syndrome (pSS), it is unclear which cells produce CXCL10 under what types of stimulations. Here, we investigated the precise molecular mechanisms by which CXCL10 was produced in human salivary gland ductal (NS-SV-DC) and acinar (NS-SV-AC) cell lines. Our results demonstrated that NS-SV-DC cells produced higher levels of CXCL10 compared to NS-SV-AC cells. In addition, our findings demonstrated that the regulator of the enhancement of CXCL10 was different between NS-SV-DC and NS-SV-AC cells, i.e., interferon-gamma (IFN-γ) had more potential than interferon-alpha (IFN-α), tumor necrosis factor (TNF)-α, and interleukin (IL)1-β in the induction of CXCL10 production in NS-SV-DC cells, whereas TNF-α had potential to induce CXCL10 production in NS-SV-AC cells. A Western blot analysis demonstrated that IFN-γ enhanced the production of CXCL10 via both the JAK/STAT1 pathway and the NF-κB pathway in NS-SV-DC cells, whereas TNF-α enhanced the production of CXCL10 via the NF-κB pathway in NS-SV-AC cells. The results of study suggest that the CXCL10 overexpression in the salivary glands is caused mainly by IFN-γ-stimulated salivary gland ductal cells. The enhanced production of CXCL10 by IFN-γ from ductal cells may result in the inflammation of pSS lesions.

Juvenile ossifying fibromas (JOFs) are rare fibro-osseous lesions. Conservative surgery, such as curettage and enucleation of the tumor, is the widely accepted therapeutic method; however, JOFs frequently recur after surgery. Here we report a case of a recurrent JOF, which had a hypervascular and aggressive phenotype. A 21-year-old woman presented with a painless, enlarged swelling at the left mandible. Clinical and biopsy findings indicated an ossifying fibroma. The initial tumor was treated via conservative curettage and recurred a year thereafter. The recurrent tumor was treated via segmental bone resection and recurred 2.5 years thereafter. With each recurrence, tumor cell proliferation and tumor hypervascularity increased. We describe the clinical and histological features of a twice-recurrent JOF. Our findings provide new insight into the mechanisms underlying recurrence and may facilitate the establishment of novel diagnostic methods for JOF.

Background The molecular mechanisms underlying aortic valve calcification are poorly understood. Here, we aimed to identify the master regulators of calcification by comparison of genes in valve interstitial cells (VICs) with calcified and noncalcified aortic valves. Methods Calcified aortic valves were surgically excised from patients with aortic valve stenosis who required aortic valve replacements. Noncalcified and calcified sections were obtained from aortic valve leaflets. Collagenase-digested tissues were seeded into dishes, and VICs adhering to the dishes were cultured for 3 weeks, followed by comprehensive gene expression analysis. Functional analyses of identified proteins were performed by in vitro calcification assays. Tissue localization was determined by immunohistochemical staining for normal (n = 11) and stenotic valves (n = 30). Results We found 87 genes showing greater than a twofold change in calcified tissues. Among these genes, 68 were downregulated and 19 were upregulated. Cyclooxygenase-1 (COX1) messenger RNA and protein levels were upregulated in VICs from calcified tissues. The COX1 messenger RNA and protein levels in VICs were also strongly increased by stimulation with osteoblast differentiation medium. These were VIC-specific phenotypes and were not observed in other cell types. Immunohistochemical staining revealed that COX1-positive VICs were specifically localized in the calcified area of aortic valve tissues. Conclusions The VIC-specific COX1 overexpression played a crucial role in calcification by promoting osteoblast differentiation in aortic valve tissues.

Orocutaneous fistulas are one of the most problematic postoperative complications after oral cancer surgery. Notably, in patients with mandibular plate exposure it is necessary to remove the plate. However, it takes longer for these patients to achieve complete fistula closure. The present report described an 84-year-old man with a postoperative orocutaneous fistula and exposed mandibular plate who was treated with the vacuum-assisted closure system. This system protects the wound from contamination while the negative pressure prevents tissue fluid retention, promotes blood flow, facilitates granulation tissue formation and decreases the bacterial cell count. Vacuum-assisted closure was successful in the present case, and complete fistula closure took 20 days. Additionally, there was no evidence of recurrence over the 11-month follow-up.

Several studies have suggested that an elevated neutrophil-lymphocyte ratio (NLR) is associated with a poorer prognosis in patients with pancreatic cancer (PC). The correlations between the NLR and immunohistochemical (IHC) analysis with regard to the prognosis of patients with PC remain to be elucidated. By using IHC findings, we determined the value of the NLR as a prognostic factor in patients with PC.We collected the clinico-pathological data of 28 consecutive patients who underwent surgical resection for PC between January 2008 and December 2012 at The Jikei University Kashiwa Hospital. We investigated whether the NLR and IHC results were related and ensured the consistency of the prognosis of patients with PC.The Kaplan-Meier curves for the disease-free survival (DFS) and the overall survival (OS) revealed that an NLR ≥ 5 is an implicit factor for decreased DFS and OS in patients with PC (p = 0.003, p 0.001, log-rank test). The density of CD163(+) macrophages and CD66b(+) neutrophils was significantly higher in the high NLR group; on the contrary, the density of CD20(+) lymphocytes was significantly higher in the low NLR group. Moreover, a Mann-Whitney U test showed that the NLR was significantly correlated with a high density of CD20(+) lymphocytes (p = 0.031) and CD163(+) macrophages (p = 0.023), while the NLR was not significantly correlated with CD66b(+) neutrophils (p = 0.397).Our results demonstrated the validity of the NLR by IHC analyses and we determined that a higher value of NLR is a trustworthy prognostic factor for patients with PC.

Cervical lymph node metastasis is an important prognostic factor in oral squamous cell carcinoma (OSCC), but its accurate assessment after sentinel node biopsy or neck dissection is often limited to the histopathological examination of only one or two sections. Previous our study showed the usefulness of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting keratin 19 (KRT19) mRNA for the genetic detection of lymph node metastasis, but the sensitivity was insufficient. Here, we have attempted to identify novel molecular markers for OSCC cells in lymph nodes. We performed microarray analysis to identify genes overexpressed in 7 metastatic lymph nodes from OSCC patients, compared to 1 normal lymph node and 5 salivary glands from non-cancer patients. We then used real-time quantitative RT-PCR (qRT-PCR) and RT-LAMP to compare the expression of these genes in newly resected metastatic and normal lymph nodes. Of 4 genes identified by microarray analysis, annexin A8 (ANXA8) and desmoglein 3 mRNA were detected by qRT-PCR in metastatic lymph nodes but not in normal lymph nodes. Furthermore, ANXA8 mRNA expression was detected in all KRT19-negative metastatic lymph nodes. Both KRT19 and ANXA8 mRNA may be useful markers for detecting lymph node metastases in OSCC patients.

Oral squamous cell carcinoma (OSCC) frequently metastasizes to cervical lymph nodes, which is the most known prognostic factor. Screening methods to identify sentinel lymph nodes (SLNs) are therefore of great interest for the management of potential neck metastasis. The purpose of this study was to evaluate the clinical benefit of double SLN mapping with indocyanine green (ICG) and 99m-technetium-tin colloid ((99m)Tc-tin colloid) for sentinel node navigation surgery (SNNS). Between 2007 and 2010, 16 patients diagnosed with OSCC were investigated by SLN biopsy using the double mapping method. (99m)Tc-tin colloid was injected into the peri-tumoural region on the preoperative day, and ICG was administered intraoperatively in the same position to assist in detecting nodes during surgery. Based on the gamma-ray signal and near-infrared (NIR) fluorescence of ICG, SLNs were identified and thereafter assessed pathologically and genetically for cancer involvement. Radio-guided detection was successful for all patients. ICG mapping identified a relatively larger number of nodes, suggesting that several non-SLNs were potentially involved. The double mapping method assisted surgeons to explore SLNs. Since the ICG fluorescence was shielded by the subcutaneous fatty tissue and the muscle layer including platysma and sternocleidomastoid, it was necessary to retract the tissue away from nodes.

Oncogene addiction can provide therapeutic opportunities in human malignancies. In this study, we aimed to identify critical oncogenes for oral squamous cell carcinoma (OSCC) development and progression. We determined gene expression profiles in 10 primary OSCCs and 10 human OSCC cell lines using Applied Biosystems Human Genome Survey Arrays. Akt1 was the only gene identified that was expressed in all OSCC tissues and cultured cells, but not in non-neoplastic tissues and cells. Subsequently, western blot analysis showed that Akt1 protein was overexpressed in OSCC tissues and cell lines. Immunohistochemistry also showed Akt1 protein expression in 59 of 63 (94%) primary OSCCs. To clarify the oncogenic function of Akt1 in human OSCC cells, we used RNA interference. We designed and synthesized 5 small interfering RNAs specific for Akt1 (siAkt1). Transfecting human OSCC cells with siAkt1 in vitro markedly suppressed their expression of Akt1 protein and significantly reduced their growth rate. Furthermore, the growth of human OSCC tumors which had been subcutaneously xenografted in athymic nude mice lacking interferon responses was markedly inhibited by atelocollagen-mediated systemic siAkt1 administration. We also found that synthetic siAkt1 had an inhibitory effect on the growth of primary cultured OSCC cells. Finally, we investigated the molecular mechanisms involved in the growth inhibitory effect of Akt1 suppression using microarray analysis of human OSCC cells transfected with siAkt1. Knockdown of Akt1 induced the expression of CDKN2B, a tumor suppressor gene, and reduced the expression of TGFBR1, which supports malignant phenotypes. These results suggest that Akt1 functions as a critical oncogene in human OSCC cells and may therefore be an appropriate target for novel OSCC therapies.

In our previous study, ribonucleotide reductase M2 (RRM2) was identified as a cancer-related gene commonly overexpressed in human oral squamous cell carcinoma (OSCC) cell lines. Herein, we attempted to determine whether targeting RRM2 may be a plausible therapeutic approach for the treatment of patients with OSCC. First, we examined the expression levels of RRM2 in human OSCC cell lines and tissues. Overexpression of RRM2 in OSCC was confirmed by western blot analysis. Subsequently, we investigated the effects of a synthetic small interfering RNA specific for RRM2 and gemcitabine (GEM), an inhibitor of RRM2 enzymatic activity, on the growth of human OSCC cell lines and primary cultured cells. Targeting RRM2 by RNA interference almost completely suppressed the expression of RRM2 and markedly suppressed the growth of both types of cells by >54.8%. GEM also reduced the growth rate of these cells by >83.0%. Finally, we evaluated the antitumor effects of GEM, cisplatin (CDDP), 5-fluorouracil (5-FU), and docetaxel (DOC) against OSCC cells using the collagen gel droplet embedded culture drug sensitivity test. OSCC cells were more sensitive to GEM and DOC than to CDDP and 5-FU, regardless of the expression level of RRM2 mRNA. These results suggested that RRM2 supported the growth of human OSCC cells and that targeting of RRM2, e.g., via GEM treatment, may be a promising therapeutic strategy for OSCC.

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

Mechanisms underlying immune cells' recruitment and activation into the inflammatory lesions of lip salivary glands (LSGs) from primary Sjögren's syndrome (pSS) patients are incompletely understood. Chemokines play pivotal roles in these processes, so we investigated the clinical significance of chemokine receptor CXCR3 and its ligands in the autoimmune lesions of pSS.We histologically determined the grade of LSG samples from 22 patients with pSS and subjected the samples to immunofluorescence analysis to determine the expressions of CXCR3 and its ligands: CXCL9, CXCL10, and CXCL11. To identify the immune cells expressing CXCR3 in the LSGs, we performed double immunofluorescence analysis using antibodies against CD3 (pan-T cells), CD80 (M1 macrophages), CD163 (M2 macrophages), and CD123 (plasmacytoid dendritic cells: pDCs). The relationship between the grade of lymphocytic infiltration and the number of positively stained cells was analyzed by Spearman's rank correlation test.The expressions of CXCL9 and CXCL10 showed particularly intense staining in the LSG samples' ductal cells. The CXCR3 expression was detected mainly in CD80Our results suggest that the enhanced production of CXCL9 and CXCL10 from ductal cells results in the CXCR3

Failure to detect recurrence and lymph node metastasis early represents a fundamental barrier to the improvement of survival rate in early stage oral squamous cell carcinoma (OSCC). The present study evaluated the association between serum interleukin-6 (IL-6) level and clinical outcomes in patients with early stage OSCC patients defined by sentinel node biopsy (SNB). A total of 53 patients with clinical stage I/II OSCC who underwent SNB were enrolled. SNB was determined by a radioisotope method, and was evaluated by histopathological examination and genetic analysis. Preoperative sera were measured for IL-6 by ELISA. In the clinical stage I/II patients, disease-free survival (DFS) was demonstrated to be higher in patients with negative SNB compared with patients with positive SNB. In total, 13 patients were demonstrated to exhibit lymph node metastasis by SNB or were reclassified to pathological stage T4 subsequent to analysis of the surgically resected specimens. Thus, 40 patients were diagnosed with early stage OSCC. Of these 40 patients, DFS of the patients with low serum IL-6 was significantly higher compared with the patients with high serum IL-6 (P=0.012). In 19 patients with negative SNB and low serum IL-6, the disease-free rate was 100%. These findings suggested that SNB staging and serum IL-6 level have a high prognostic value in patients with early stage OSCC. Additional investigation and longer follow-up times are warranted to improve understanding of the group of patients that may benefit from this procedure.

MicroRNAs (miRNAs) are small non-coding RNAs with size of 20-25 nucleotides, and inhibit protein translation by binding the 3’-untranslated region (3’-UTR) of target mRNA. Each miRNA can regulate multiple mRNAs and each mRNA can be targeted by a number of miRNAs. In cancer, miRNAs can act as not only tumor suppressor genes but also oncogenes. Addiction to oncogenic miRNAs (OncomiRs) may provide therapeutic opportunities in human cancers such as oncogene addiction. In this study, we have attempted to identify OncomiRs in human oral squamous cell carcinoma (OSCC) cells and considered whether targeting miRNAs can be possible for cancer therapy. Functional screening for OncomiRs in human OSCC cells was performed with the use of miRCURY LNATM microRNA Knockdown Library-Human v12.0 (EXIQON). We transfected 918 locked nucleic acid (LNA)/DNA antisense oligonucleotides for specific human mature miRNAs into human OSCC cells (GFP-SAS). After 80 hours, each cell growth was evaluated by WST-8 assay. We identified LNA/DNA antisense oligonucleotide against miR-361-3p (LNA-miR-361-3p) which showed remarkable growth inhibition in GFP-SAS cells. Subsequently, we confirmed the target specificity of LNA-miR-361-3p by quantitative RT-PCR (qRT-PCR). LNA-miR-361-3p significantly reduced the expression of miR-361-3p. Co-transfection of synthetic mature miR-361-3p abrogated the growth inhibitory effect of LNA-miR-361-3p in GFP-SAS cells. Furthermore, transfection of synthetic mature miR-361-3p resulted in 20% increase of cell growth in GFP-SAS cells. Next, we identified odd-skipped related 2 (OSR2) as a target gene of miR-361-3p by the use of microarray, GeneSpring GX, and Ingenuity Pathway Analysis (IPA). The expression of OSR2 after transfection with LNA-miR-361-3p was significantly induced, and co-transfection of the OSR2 3’-UTR luciferase reporter plasmid and LNA-miR-361-3p into GFP-SAS cells produced higher luciferase activity than cells in co-transfected with LNA-non target (LNA-NT). Finally, we assessed the effect of LNA-miR-361-3p on the in vivo growth of GFP-SAS cells. We administered LNA-miR-361-3p into the xenograft tumors every 3 days. We found that LNA-miR-361-3p significantly reduced the size of subcutaneously xenografted GFP-SAS tumors, compared to the control group treated with LNA-NT. The expression of miR-361-3p in excised tumors was examined by qRT-PCR. LNA-miR-361-3p suppressed the expression levels of miR-361-3p compared with the control tumors. These results suggest that miR-361-3p support the growth of human OSCC cells and that targeting miR-361-3p may be a useful therapeutic approach for patients with OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Himiko Ogawa, Hiroyuki Goda. Targeting miR-361-3p suppresses the growth of human oral squamous cell carcinoma cells in vitro & in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3553.

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

Antitumor functions of the host immune system are frequently compromised in patients with malignancies. In the current study, we evaluated the relationship between expression ratio of mRNAs for the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax (the Bcl-2/Bax ratio) in peripheral blood mononuclear cells and clinical outcomes in patients with head and neck carcinomas. The overall survival (OS) time of patients with Bcl-2/Bax ratios ≥ 1.2 tended to be longer than that of patients with Bcl-2/Bax ratios < 1.2 but not significantly so (P = .084, n = 61). Disease-free survival (DFS) of patients with Bcl-2/Bax ratios ≥ 1.2 was statistically significantly longer than that of patients with Bcl-2/Bax ratios < 1.2 (P = .001, n = 76). All of the patients whose Bcl-2/Bax ratio is ≥ 2.0 were alive after 36 months and survived without any evidence of disease for 24 months (Bcl-2/Bax ≥ 2.0 versus Bcl-2/Bax < 2.0; P = .035, n = 61 in OS, P < .001, n = 76 in DFS, respectively). In 56 patients who received immunochemoradiotherapy using UFT and OK-432 in combination with radiotherapy, a statistically significant relationship between the Bcl-2/Bax ratio and the therapeutic effect estimated using Response Evaluation Criteria in Solid Tumors was observed, as well as a relation with interferon-γ (IFN-γ) induction in response to the therapy [P = .002 in complete response versus partial response + stable disease; P = .046 in IFN-γ(+) versus IFN-γ(-)]. In addition, there were significant correlations of the Bcl-2/Bax ratio with both the absolute number of CD4+ T cells and the rate of CD4+ T cell and natural killer cell activity. These findings strongly suggest that the balance of expression of Bcl-2 and Bax genes in circulating immune cells has a high prognostic value in head and neck cancer patients.

Summary Objectives Lymph node stage is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC). We previously reported the clinical usefulness of sentinel lymph node biopsy diagnosed by genetic analysis using quantitative RT-PCR. However, this method takes about 3 h. In this study, we attempted to develop a more efficient method for the intraoperative genetic detection of lymph node metastasis in HNSCC. Materials and methods A total of 312 lymph nodes (65 patients) were diagnosed by the one-step nucleic acid amplification (OSNA) method using GD-100. OSNA consists of a short homogenization step followed by amplification of cytokeratin 19 (CK19) mRNA directly from the lysate. Each lymph node was divided into two to diagnose metastasis. One half was used for the OSNA assay, and the other was subjected to semi-serial sectioning, sliced at 200-μm intervals and examined by H&E and cytokeratin AE1/AE3 immunohistochemical staining. The accuracy of OSNA assay was evaluated based on histopathological diagnosis. Results Sixty-one of 312 lymph nodes were pathologically metastasis-positive. The overall concordance rate between the OSNA assay using breast cancer criteria and histopathology was 94.2%. The optimal cut-off for the copy number of CK19 mRNA in assessing lymph node metastasis of HNSCC was 300 copies/μl, which had the highest diagnostic accuracy (95.2%). The OSNA assay can be completed within 30 min. Conclusion The OSNA assay, which shows high sensitivity and specificity, suggests the possibility to be used as a novel tool for the genetic detection of lymph node metastasis in HNSCC patients.

Radiotherapy is commonly used to treat oral squamous cell carcinoma (OSCC), but its therapeutic effects are unpredictable. To determine which genes correlate with radiation resistance in oral cancer, the authors evaluated radiation sensitivity using a standard colony formation assay with a gene microarray system for seven OSCC cell lines. They found significant associations between dozens of gene-expression levels and radiation resistance of OSCC cell lines. Following analysis of the different radiosensitive cancer cell lines, the friend leukaemia insertion (Fli)-1 gene was selected as a prediction marker gene for OSCC radiotherapy resistance. Fli-1 expression was associated with radiation resistance in OSCC patients. These data help to predict the effects radiation therapy has on OSCC, in turn contributing to the development of alternative radiation therapies.

Spindle cell carcinoma is considered a subtype of squamous cell carcinoma. There is no effective chemotherapy for cases of recurrence after first-line treatment, and the prognosis is poor. The patient was an 85-year-old man with spindle cell carcinoma after irradiated squamous cell carcinoma of the left lower gingiva. Extended surgery was performed, but 1 month postoperatively recurrence in the contralateral lower lip, cervical lymph node metastasis, and liver metastasis were detected, and treatment with S-1 was started. After 1 month of treatment, the cervical lymph node metastasis had resolved, and the liver metastasis had regressed, but after 4 months of treatment new cervical lymph node metastasis, lung metastasis, and rapid enlargement of the liver metastasis were observed, and led to the patient's death. Treatment with S-1 alone had an adequate antitumor effect temporarily. Further chemotherapeutic trials including S-1 are recommended to treat spindle cell carcinoma.

Molecular target drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. The PI3K/AKT/mTOR and RAS/MAPK pathways are important pathways for the growth and survival of cancer cells. These pathways also have been activated such as AKT and ERK overexpression in OSCC. In this study, we investigated the antitumor effect of PI3K/mTOR inhibitor, NVP-BEZ235 and MEK1/2 inhibitor, Trametinib, in human OSCC cells. First, we confirmed the inhibitory effect of NPV-BEZ235 and Trametinib in PI3K/AKT/mTOR and RAS/MAPK pathways by Western blotting. NPV-BEZ235 and Trametinib were reduced phosphorylation of AKT, p70S6K, and MEK1/2 in OSCC cells. Next, we examined the anti-proliferative effect of NVP-BEZ235 and Trametinib using 4 human OSCC cell lines by WST-8 assay. NVP-BEZ235 and Trametinib significantly inhibited the growth of all OSCC cells in a dose-dependent manner in vitro. Further, we investigated potential synergistic effects of inhibiting both the PI3K/AKT/mTOR and RAS/MAPK pathways, by treating with NVP-BEZ235 and Trametinib. The anti-proliferative effect of this combination was measured in OSCC cells by calculating the Combination Index. Combination treatment with NVP-BEZ235 and Trametinib showed a synergistic effect on the cell growth of all OSCC cells in vitro. Subsequently, we analyzed the effect of NBP-BEZ235 and Trametinib on the cell cycle of green fluorescent protein (GFP)-SAS cells by flow cytometry. Treated with NVP-BEZ235 showed no enhanced effect compared to control. Treatment of Trametinib resulted in the decrease in percentage of cells in G2 phase and increase in G1 phase. This indicated that the anti-proliferative effect of Trametinib was due to G1 arrest. Furthermore, treatment of Trametinib increased apoptosis cells in GFP-SAS cells. Next, we assessed the effect of NVP-BEZ235 and Trametinib on the in vivo growth of GFP-SAS cells. GFP-SAS tumor-bearing mice were treated with single, combination, or vehicle by oral daily. Treatment with NVP-BEZ235 and Trametinib significantly reduced the size of tumors compared with vehicle control. However, combination treatment showed no synergistic effect compared to single treatment. Finally, we investigated the gene expression changes with combination treatment using microarray analysis. The suppression of PI3K/AKT/mTOR and RAS/MAPK pathways caused overexpression of genes related to cell cycle and DNA-repair. These results suggest that activation of PI3K/AKT/mTOR and RAS/MAPK pathways support the growth of OSCC cells and NVP-BEZ235 and Trametinib may be a useful therapeutic strategy for OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro. Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3468.

One of the most critical issues of cancer chemotherapy is to establish a method for predicting efficacy or toxicity of anti-cancer drugs for individual patients. To identify genes that correlate with chemosensitivity of oral cancer treatment, we performed cDNA microarray analysis of nine oral squamous cell carcinoma (OSCC) cell lines. We compared gene-expression profiling with the sensitivity of OSCC cell lines to five anti-cancer drugs (5-fluorouracil, cisplatin, docetaxel, mitomycin C and adriamycin), which were measured using the collagen gel droplet embedded culture-drug sensitivity test method. We found significant associations between dozens of gene-expression levels and chemosensitivity of OSCC cell lines. These data should prove useful in the identification of predictive drug sensitivity markers, which may eventually provide “personalized chemotherapy” for individual patients, as well as for the development of novel drugs against chemotherapeutic resistance.

Background The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes: SCCA1 and SCCA2. SCCA1 gene is up-regulated in squamous cell carcinoma cells. We analyzed the proximal region of the SCCA1 promoter and the antitumor effect of oncolytic adenovirus driven by the SCCA1 promoter in squamous cell carcinoma cells. Methods The SCCA1 promoter was analyzed by dual luciferase assay and substituted with the E1A promoter to construct the oncolytic adenovirus to determine the squamous cell carcinoma-specific cell lysis. Results Deletion analysis of SCCA1 promoter identified a 175-bp core promoter region and an enhancer region at −525 to −475 bp upstream of the transcription start site. The transcriptional activity of the SCCA1 promoter was up-regulated in squamous cell carcinoma cells. Five tandem repeats of enhancer increased SCCA1 promoter activity by four-fold. Oncolytic adenovirus driven by this SCCA1 enhancer-promoter complex specifically killed squamous cell carcinoma cells in vitro and in vivo. A549 carrier cells infected with the oncolytic adenovirus induced complete regression of syngeneic squamous cell carcinoma cell tumor by overcoming immunogenicity and adenovirus-mGM-CSF augmented the antitumor effect of carrier cells. Conclusions SCCA1 was up-regulated in squamous cell carcinoma cells and oncolytic adenovirus driven by SCCA1 promoter specifically killed these cells. These findings suggest that SCCA1 promoter is a potential target of gene therapy for squamous cell carcinoma. Copyright © 2010 John Wiley & Sons, Ltd.

Metastasis of malignant tumors to the oral cavity is rare. The authors report a case of thyroid carcinoma with mandibular osteoblastic metastasis. An 83-year-old female presented with lower jaw swelling and pain. An elastic hard subcutaneous mass was observed in the median mandible. X-ray images confirmed a tumor lesion with periosteal reaction spreading radially from the mandible. A biopsy revealed nests of large, polygonal tumor cells growing in a supporting fibrovascular framework. The patient's anamnesis included thyroid carcinoma with lung metastasis, 2 years ago, treated by total enucleation of the thyroid and excision of the superior lobe of the left lung. Biopsy, primary and metastatic tumor samples all tested positive for thyroglobulin, suggesting a thyroid follicular epithelial origin. Mandibular metastasis of poorly differentiated carcinoma of the thyroid gland was diagnosed. Consent for further treatment was not obtained. The patient died 1 year and 7 months later.

Early phase prostate cancer is usually androgen-dependent, with the androgen/androgen receptor (AR) signaling pathway playing a central role. At this stage, the cancer responds well to androgen ablation therapy, but prostate cancers eventually acquire androgen independence and more aggressive phenotypes. Several studies, however, have shown that the majority of tumors still express functional AR, which is often amplified and mutated. To determine if the AR is a plausible therapeutic target, we investigated the anti-tumor effect of small interfering RNAs targeting the AR (siAR) in the human prostate cancer cells, LNCaP and 22Rv1, which express mutated AR. In both types of cells, transfection of siAR suppressed mutated AR expression and significantly reduced cell growth. Furthermore, atelocollagen-mediated systemic siAR administration markedly inhibited the growth of 22Rv1 cells subcutaneously xenografted in castrated nude mice. These results suggest that the AR is still a key therapeutic target even in androgen-independent prostate cancer (AIPC). Silencing of AR expression in AIPC opens promising therapeutic perspectives.

Objective: 18F-Fluorodeoxyglucose positron emission tomography—computed tomography is increasingly being used for the diagnosis of primary malignant tumours, cervical lymph node metastases, and distant metastases. As cervical lymph node metastasis is a particularly important prognostic factor for patients with cancers of the head and neck, this study was performed to ascertain the efficacy of this technique for the diagnosis of such metastases in patients with oral squamous cell carcinoma. Patients and Methods: 123 cervical lymph nodes were analysed from 21 patients, who underwent preoperative diagnosis by 18F-fluorodeoxyglucose positron emission tomography—computed tomography in addition to other conventional diagnostic procedures, such as computed tomography and magnetic resonance imaging. Lymph nodes were removed by sentinel lymph node biopsy or neck dissection and were examined histopathologicallyfor metastasis. Results: Seven patients were metastasis-positive. The optimal cut-off for the maximum standardised uptake value for 18F-fluorodeoxyglucose positron emission tomography—computed tomography in assessing lymph node metastases was 4.1, which had the highest diagnostic accuracy (sensitivity, 100%; specificity, 100%; accuracy, 100%). Of the 13 lymph nodes identified as metastasis-positive by histological examinations, 12 (92.3%) also tested positive using 18F-fluorodeoxyglucose positron emission tomography—computed tomography. Conclusion: These results suggest that 18F-fluorodeoxyglucose positron emission tomographycomputed tomography is useful for detecting cervical lymph node metastasis.

We previously demonstrated that CD151 forms a functional complex with c-Met and integrin alpha3/alpha6 in human salivary gland cancer cells. In the current study, we investigated the involvement of CD151, c-Met, and integrin alpha3/alpha6 in the cellular morphogenesis of human breast cancer cells. Knockdown of CD151, integrin alpha3, or integrin alpha6 expression abolished branching morphogenesis. Decreased c-Met expression in these cells led to the formation of rudimentary networks and prevented their conversion. Furthermore, hepatocyte growth factor (HGF) promoted cellular morphogenesis by accelerating network reorganization. Immunoprecipitation revealed a specific association between CD151 and c-Met. The involvement of CD151 and integrin alpha3/alpha6 in HGF-dependent signaling was confirmed by the decreased Akt phosphorylation in cells lacking CD151, integrin alpha3, or integrin alpha6. Hence, the regulation of CD151 expression might contribute to changes in HGF/c-Met signaling and thereby modulate the phenotypic characteristics of cancer cells.