Possible Role of miR-375-3p in Cervical Lymph Node Metastasis of Oral Squamous Cell Carcinoma.

No clinically useful predictors of latent cervical lymph node metastasis (LNM) in early oral squamous cell carcinoma (OSCC) are available. In this study, we focused on the microRNAs (miRNAs) involved in the expression of numerous genes and explored those associated with latent cervical LNM in early OSCC (eOSCC). First, microarray and RT-PCR analyses revealed a significant downregulation of miR-375-3p expression in primary eOSCC tissues with latent cervical LNM. Next, we examined the effects of miR-375-3p mimics on the growth and migration of four human OSCC cell lines that do not express miR-375-3p. The overexpression of miR-375-3p significantly suppressed the cell proliferation and migration of human OSCC cells in vitro. Furthermore, miR-375-3p mimics markedly inhibited the subcutaneously xenografted human OSCC tumors. Finally, we found the genes involved in the PI3K-AKT pathway and cell migration as target gene candidates of miR-375-3p in human OSCC cells. These findings suggest that miR-375-3p functions as a tumor suppressive-miRNA in OSCC and may serve as a potential biomarker for the prediction of latent cervical LNM in eOSCC and a useful therapeutic target to suppress OSCC progression.

A Case of Nonocclusive Mesenteric Ischemia During Bioradiotherapy With Cetuximab.

Nonocclusive mesenteric ischemia (NOMI) causes mesenteric ischemia and intestinal necrosis despite the absence of organic obstruction, such as thrombi and emboli in mesenteric blood vessels, and it has an extremely poor prognosis. We report a case of NOMI developed during bioradiotherapy (BRT) with cetuximab for cervical lymph node metastasis of tongue cancer. The patient was a 73-year-old man who underwent right radical neck dissection for neck lymph node metastasis after tongue cancer surgery. Postoperatively, the patient received BRT with cetuximab. On the 34th day after BRT, the patient had abdominal distension and a decreased level of consciousness. Contrast-enhanced computed tomography revealed mesenteric ischemia without thrombi and extensive intestinal emphysema. The patient was diagnosed with NOMI. Furthermore, he had septic shock and was treated with vasopressors and antibacterial agents; however, the condition of the patient did not improve, and he died on the same day.

Prognostic Significance of Serum Interleukin-6 Levels in Oral Squamous Cell Carcinoma.

Introduction The prognosis of oral squamous cell carcinoma (OSCC) is often poor despite standard treatments. Additionally, no useful prognostic markers are available. Therefore, we aimed to investigate the relationship between serum Interleukin-6 (IL-6) levels and prognosis and explore its local and systemic effects in patients with OSCC. Methods Ninety-five new cases of OSCC were included, and the prognosis was compared between high and low serum IL-6 groups. The localization of IL-6 in OSCC tissues was examined. Furthermore, a comprehensive gene expression analysis was performed in OSCC tissues and compared between the two groups. Results A significant difference in overall survival and disease-free survival was observed. Furthermore, a substantial expression of IL-6 was localized in the stroma. Comprehensive gene expression analysis of tumor localization showed increased expression of genes related to oxidoreductase and lipid metabolism in the primary tissues of the group with high serum IL-6 levels. Regarding the correlation between blood tests and serum IL-6 levels, a strong positive correlation was observed between inflammatory responses and nutritional factors. Conclusion These results suggest that serum IL-6 may be a prognostic factor for metabolic abnormalities in patients with OSCC and that aggressive nutritional interventions may contribute to prognosis.

KRT13 and UPK1B for differential diagnosis between metastatic lung carcinoma from oral squamous cell carcinoma and lung squamous cell carcinoma.

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.

MicroRNA-1289 Functions as a Novel Tumor Suppressor in Oral Squamous Cell Carcinoma.

Recently, numerous tumor-suppressive microRNAs (TS-miRs) have been identified in human malignancies. Here, we attempted to identify novel TS-miRs in oral squamous cell carcinoma (OSCC). First, we transfected human OSCC cells individually with 968 synthetic miRs mimicking human mature miRs individually, and the growth of these cells was evaluated using the WST-8 assay. Five miR mimics significantly reduced the cell growth rate by less than 30%, and the miR-1289 mimic had the most potent growth inhibitory effect among these miRs. Subsequently, we assessed the in vivo growth-inhibitory effects of miR-1289 using a mouse model. The administration of the miR-1289 mimic-atelocollagen complex significantly reduced the size of subcutaneously xenografted human OSCC tumors. Next, we investigated the expression of miR-1289 in OSCC tissues using reverse transcription-quantitative PCR. The expression level of miR-1289 was significantly lower in OSCC tissues than in the adjacent normal oral mucosa. Furthermore, 15 genes were identified as target genes of miR-1289 via microarray and Ingenuity Pathway Analysis (IPA) microRNA target filtering. Among these genes, the knockdown of magnesium transporter 1 (MAGT1) resulted in the most remarkable cell growth inhibition in human OSCC cells. These results suggested that miR-1289 functions as a novel TS-miR in OSCC and may be a useful therapeutic tool for patients with OSCC.

Human papillomavirus-16 infection and p16 expression in oral squamous cell carcinoma.

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.

MicroRNA-361-3p is a potent therapeutic target for oral squamous cell carcinoma.

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.

Annexin A8 is a novel molecular marker for detecting lymph node metastasis in oral squamous cell carcinoma.

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.

Therapeutic potential of targeting cell division cycle associated 5 for oral squamous cell carcinoma.

Molecularly targeted 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. We have attempted to find appropriate molecular targets for OSCC and identified cell division cycle associated 5 (CDCA5) as a cancer-related gene which was overexpressed in all the human OSCC cells tested by microarray analysis. In this study, we investigated the expression and function of CDCA5 in OSCC. First, we confirmed that CDCA5 was overexpressed in 4 human OSCC cell lines by quantitative RT-PCR and Western blotting. We then tested the effect of synthetic small interfering RNAs specific for CDCA5 on the growth and invasion of human OSCC cells. Knockdown of CDCA5 markedly inhibited the growth of OSCC cells in vitro and in vivo. We also examined the expression of CDCA5 protein in 80 cases of OSCC immunohistochemically and found a significant association between CDCA5 expression levels and overall survival. These results suggest that CDCA5 functions as a critical gene supporting OSCC progression and that targeting CDCA5 may be a useful therapeutic strategy for OSCC.

Identification of Akt1 as a potent therapeutic target for oral squamous cell carcinoma.

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.

Ribonucleotide reductase M2 is a promising molecular target for the treatment of oral squamous cell carcinoma.

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.

Targeting Aurora kinase A suppresses the growth of human oral squamous cell carcinoma cells in vitro and in vivo.

OBJECTIVES: Oncogene addiction has provided therapeutic opportunities in many human malignancies, but molecular targeted therapy for oral squamous cell carcinoma (OSCC) is not yet available. In this study, we attempted to identify an appropriate target molecule for treatment of patients with OSCC. MATERIALS AND METHODS: Microarray analysis was performed to determine the gene expression profiles in nine human OSCC cell lines and a non-neoplastic keratinocyte cell line. The expression levels of Aurora kinase A (AURKA) mRNA and protein in human OSCC cells and tissues were examined. We investigated the effect of small interfering RNAs specific for AURKA (siAURKAs) and MLN8237, an AURKA selective inhibitor on the growth of OSCC cells in vitro and in vivo. We also analyzed clinical significance in AURKA mRNA expression levels in OSCC. RESULTS: AURKA was overexpressed in human OSCC cell lines and tissues. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of OSCC cells by 31-89%. MLN8237 also reduced the cellular growth rate by 38-74%. Both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors by 66% and 40%. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of primary cultured cells established from patients' OSCC tumors. Furthermore, we found a significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis. CONCLUSIONS: AURKA plays a critical role in the growth of human OSCC cells and targeting AURKA may be a useful therapeutic strategy for OSCC.