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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.

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.

2003年から2012年までの9年間に当科で診断・治療を行った歯原性腫瘍75症例について，2005年WHO分類に基づいて臨床的検討を行った．疾患別症例数は角化嚢胞性歯原性腫瘍が22例，エナメル上皮腫16例，歯牙腫16例，骨性異形性症13例，線維性異形性症3例，石灰化嚢胞性歯原性腫瘍2例，歯原性粘液腫，歯原性線維腫，腺様歯原性腫瘍が各1例であった．男女比は1.5対1であった．手術時年齢は，平均39.8歳であった．発生部位別症例数は，角化嚢胞性歯原性腫瘍，エナメル上皮腫は下顎大臼歯部に多く，歯牙腫は，上顎前歯部に多かった．治療方法は，角化嚢胞性歯原性腫瘍に対しては摘出後閉鎖7例，摘出後開放7例，生検後開窓5例，開窓後摘出3例であった．エナメル上皮腫は摘出後開放8例，区域切除3例，辺縁切除3例，開窓1例，開窓後摘出1例であった．歯牙腫16例に対しては摘出術を施行した．治療成績は75例中71例（94.7％）は再発を認めなかったが，一次症例で，エナメル上皮腫1例，角化嚢胞性歯原性腫瘍1例，歯原性粘液腫1例の3症例，他院で治療後のエナメル上皮腫1例に再発が認められた．

Aim We attempted to clarify the role of Peroxisome proliferator-activated receptor γ (PPARγ) and its ligand, troglitazone (TRO) on oral squamous cell carcinoma (SCC). Materials and methods The expression of PPARγ gene was examined in 47 human oral SCC tissues and two human oral SCC cell lines, CA9-22 and HSC-4. The effects of TRO on the growth and cell-cycle progression of human oral SCC cells were examined. Results PPARγ mRNA was detected in 20 of 47 oral SCC tissues and two human oral SCC cells. TRO significantly suppressed the growth of the cells, but did not induce apoptosis. CA9-22 cells treated with TRO showed an increased fraction in the G1 phase and decreased fractions in the S and G2-M phases. Conclusion TRO did not induce apoptosis in oral SCC cells, but did inhibit the growth of the cells by arresting the cell cycle at G1 phase.

Werner syndrome (WS) is an autosomal recessive disorder characterized by physical signs and symptoms, including premature aging and scleroderma-like skin changes. The gene responsible for WS is the WRN gene. A significant proportion of WS-related malignant tumours are non-epithelial types, and the incidence of oral squamous cell carcinoma (SCC) is rare. A case of oral SCC of the lower alveolus and gingiva arising in a 63-year-old woman with WS is reported here. Biopsy confirmed moderately differentiated SCC. Surgical resection was performed and there was no recurrence or metastasis at the 3-year follow-up. Mutation analysis using next-generation sequencing, detected no mutations in the genes encoding the molecules strongly involved in the development of oral SCC, such as TP53 or PIK3CA. No obvious mutations were detected. Based on the results of the study, the results of mutation analysis suggest that this case might be genetically different from the common mechanisms of SCC in the oral cavity.

We have previously demonstrated that the stromal cell‑derived factor (SDF‑1)/CXCR4 system is involved in the metastasis of head and neck cancer. Additionally, it has been revealed that the blockade of CXCR4 by subcutaneous daily injection with AMD3100, a CXCR4 antagonist, may be effective in preventing metastasis in CXCR4‑related head and neck cancer. Recent investigations have suggested that AMD070, a novel orally bioavailable inhibitor of CXCR4, may be minimally invasive compared with AMD3100. In the present study, we examined the effect of AMD070 on metastasis induced by the SDF‑1/CXCR4 axis in B88‑SDF‑1 oral cancer cells, which express high levels of SDF‑1 and CXCR4. Although treatment with AMD070 did not affect the anchorage‑dependent growth of B88‑SDF‑1 cells, it significantly suppressed the anchorage‑independent growth. Moreover, the SDF‑1/CXCR4‑dependent migration and invasion of B88‑SDF‑1 cells was significantly inhibited following treatment with AMD070. Subsequently, we performed an experimental therapy using AMD070 to prevent the distant metastasis of B88‑SDF‑1 cells in vivo. Daily oral administration of AMD070 significantly inhibited the lung metastasis of B88‑SDF‑1 cells in nude mice. These results indicated that AMD070 could be useful as a novel orally bioavailable inhibitor of oral cancer metastasis.

Oral cancer cells have a significantly augmented nuclear factor-κB (NF-κB) activity and the inhibition of this activity suppresses tumor growth. Bortezomib is a proteasome inhibitor and a drug used for molecular-targeted therapy (targets NF-κB). In this study, we investigated whether bortezomib would be effective as an inhibitor of proliferation and a radiosensitizer for the treatment of oral cancer. We demonstrate that bortezomib inhibits NF-κB activity and cell proliferation. The combined treatment with bortezomib and radiation (RT) suppressed NF-κB activity and cell growth in vitro and in vivo compared with RT treatment alone. To investigate the mechanisms by which bortezomib suppresses tumor growth, the expression of signaling molecules downstream of NF-κB were examined by ELISA. The combined treatment significantly inhibited the radiation-induced production of angiogenic factors and decreased the number of blood vessels in the tumor tissues. Although the expression of anti-apoptotic proteins was upregulated by RT, bortezomib downregulated the RT-induced expression of these proteins. Moreover, the expression of cleaved poly(ADP-ribose) polymerase in vitro and in vivo was enhanced by bortezomib, indicating that bortezomib inhibits tumor growth by inducing apoptosis. This study clearly demonstrates that bortezomib significantly inhibits tumor growth and that the combined treatment with bortezomib and RT results in a significant inhibition of tumor growth. The mechanisms underlying the inhibition of tumor growth by bortezomib include the suppression of angiogenesis and the induction of apoptosis. A novel molecular targeting therapy including bortezomib may be effective in the treatment of oral cancer by suppressing NF-κB activity.

Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs.

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.

We have previously demonstrated that the stromal cell-derived factor (SDF)-1/CXCR4 system is involved in the metastasis of head and neck cancer. Additionally, it has been shown that the blockade of CXCR4 by subcutaneous daily injection with AMD3100, a CXCR4 antagonist, may be effective in preventing metastasis in CXCR4-related head and neck cancer. Recent investigations have suggested that AMD070, a novel orally bioavailable inhibitor of CXCR4, may be minimally invasive compared with AMD3100. In the present study, we examined the effect of AMD070 on metastasis induced by the SDF-1/CXCR4 axis in B88-SDF-1 oral cancer cells, which express high levels of SDF-1 and CXCR4. Although treatment with AMD070 did not affect the anchorage-dependent growth of B88-SDF-1 cells, it significantly suppressed the anchorage-independent growth. Moreover, the SDF-1/CXCR4-dependent migration and invasion of B88-SDF-1 cells was significantly inhibited following treatment with AMD070. Subsequently, we performed an experimental therapy using AMD070 to prevent the distant metastasis of B88-SDF-1 cells in vivo. Daily oral administration of AMD070 significantly inhibited the lung metastasis of B88-SDF-1 cells in nude mice. Furthermore, AMD070 treatment did not induce body weight loss despite the tumor bearing-condition of the mice. In addition, we could not detect the apparent macroscopic organ abnormalities associated with the daily oral administration of AMD070. These results indicated that AMD070 could be useful as a novel orally bioavailable inhibitor of oral cancer metastasis. Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Kyoko Kuribayashi, Tomonori Hasegawa, Shuma Yagisawa, Hitoshi Kawamata. Effect of a novel orally bioavailable CXCR4 inhibitor, AMD070, on the metastasis of oral cancer 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 4191.

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system enhances the metastases of oral cancer cells via induction of mGluR5. In this study, we examined the transcriptional regulation of mGluR5 induced by the SDF-1/CXCR4 system, using oral cancer cells, B88, which express functional CXCR4 and have highly metastatic potentials. mGluR5 was markedly induced both by the exogenous SDF-1 addition and overexpression of SDF-1 in B88 cells. These induction was completely inhibited by a MEK inhibitor, U0126, and partially inhibited by a PI3K inhibitor, wortmannin. Furthermore, these inhibitors significantly inhibited the SDF-1/CXCR4 dependent cell-migration in the presence or absence of mGluR5 agonist, DHPG. Recently, several investigators demonstrated that activation of the ERK1/2 pathway is responsible for the transcription of several cancer-associated microRNAs (miRNAs). Thus, we performed miRNA microarray in SDF-1 stimulated B88 cells. we isolated miR-30 family which has predictive binding sites in 3'-UTR of mGluR5 mRNA in silico analysis. We now analyze the regulation of these miRNA by MEK/ERK1/2 and PI3K/Akt pathway. Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Sayaka Izumi, Kyoko Kuribayashi, Hitoshi Kawamata. Mechanism of transcriptional regulation of metabotropic glutamate recepter-5 induced by the CXCR4 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1328. doi:10.1158/1538-7445.AM2017-1328

We have demonstrated that CXCR4 system is involved in the metastatic process of oral cancer. Recently, we identified the miR-518c-5p as a downstream target of the CXCR4 system. In this study, we examined the function of miR-518c-5p on the metastasis of oral cancer. We transfected miR-518c expression vector into oral cancer cells, B88 and CAL27, and isolated stable transfectants, B88-518c and CAL27-518c, respectively. The growth and migration of both cells were significantly enhanced in compared with those of mock cells. LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of these cells, indicating that these phenomena were dependent on the expression of miR-518c-5p, not on that of miR-518c-3p. Next, we examined the function of miR-518c-5p in vivo. miR-518c or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with miR-518c transfectants. Furthermore, we examined the mRNA expression induced by miR-518c-5p using B88 cells and B88-518c cells by cDNA microarray analysis. Consequently, some of the genes involved in the cell growth and metastasis were upregulated in the B88-518c cells. These results indicate that CXCR4 system regulates the metastases of oral cancer via induction of miR-518c-5p. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Yuske Komiyama, Shuji Tsuchida, Hitoshi Kawamata. MicroRNA-518c-5p promotes the metastasis of oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2535. doi:10.1158/1538-7445.AM2017-2535

We have previously demonstrated that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) have been shown to be involved in the metastatic process of several types of cancers. However, the miRNAs that contribute to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. In this study, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system using B88-SDF-1 oral cancer cells, which exhibit functional CXCR4 and distant metastatic potential in vivo. Through miRNA microarray analysis, we identified the upregulation of miR-518c-5p in B88-SDF-1 cells, and confirmed the induction by real-time PCR analysis. Although an LNA-based miR-518c-5p inhibitor did not affect cell growth of B88-SDF-1 cells, it did significantly inhibit the migration of the cells. Next, we transfected a miR-518c expression vector into parental B88 cells and CAL27 oral cancer cells and isolated stable transfectants, B88-518c and CAL27-518c cells, respectively. The anchorage-dependent and -independent growth of miR-518c transfectants was significantly enhanced compared with the growth of mock cells. Moreover, we detected the enhanced migration of these cells. The LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of miR-518c transfectants, indicating that these phenomena were mainly dependent on the expression of miR-518c-5p. Next, we examined the function of miR-518c-5p in vivo. miR-518c transfectants or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with the miR-518c transfectants. These results indicated that miR-518c-5p regulates the growth and metastasis of oral cancer as a downstream target of the SDF-1/CXCR4 system.

We have demonstrated that the stromal cell-derived factor (SDF)-1/CXCR4 system is involved in metastatic processes in oral cancer. Moreover, we have also reported that blocking CXCR4 with AMD3100, a CXCR4 antagonist, may be a potent anti-metastatic therapy for CXCR4-related head and neck cancer. Recently, AMD070 has been described as a novel orally bioavailable inhibitor of CXCR4, being minimally invasive than AMD3100. In this study, we examined the effect of AMD070 on the SDF-1/CXCR4 dependent-metastases in oral cancer cells, B88-SDF-1, which express high levels of SDF-1 and CXCR4. We examined the effect of CXCR4-specific antagonists, AMD3100 and AMD070 on the cell growth, cell migration and cell invasion of B88-SDF-1 cells. Both antagonists did not affect the anchorage-dependent growth of B88-SDF-1 cells, but did inhibit the SDF-1/CXCR4 dependent-migration of the cells. Moreover, AMD070 significantly suppressed anchorage-independent growth and invasion of B88-SDF-1 cells. Next we examined the effect of AMD070 to distant metastasis in vivo. Daily administration of AMD070 per os significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results indicated that AMD070 might be a novel orally bioavailable inhibitor on the metastases of oral cancer by the SDF-1/CXCR4 system. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Takahiro Wakui, Kyoko Kuribayashi, Maki Okubo, Masahiro Saito, Eri Masuyama, Hitoshi Kawamata. AMD070, a novel orally bioavailable CXCR4 inhibitor, inhibits the metastases of oral cancer via SDF-1/CXCR4 system. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4117.

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system enhances the metastases of oral cancer cells via induction of metabotropic glutamate receptor (mGluR) 5. However, the mechanism(s) of mGluR5 expression induced by the SDF-1/CXCR4 system are largely unknown. Recently, small non-coding RNAs, microRNAs (miRNAs) are involved in the metastatic process of several types of cancers. In this study, we examined the miRNA association involved in the mGluR5 expression using oral cancer cells, B88, which express functional CXCR4 and exhibit highly metastatic potentials. We examined the metastasis-related miRNAs in SDF-1 stimulated B88 cells by use of a miRNA microarray analysis. Consequently, we isolated miR-30 family which has predictive binding sites in 3’-UTR of mGluR5 mRNA in silico analysis. Among these miRNAs, we confirmed the downregulation of all miR-30 family in SDF-1 stimulated B88 cells by the real-time PCR analysis. Next, we transfected these miR-30 families in SDF-1 stimulated B88 cells. We confirmed the downregulation of mGluR5 by the miR-30a-5p and miR-30c-5p overexpression. These results indicated that SDF-1/CXCR4 system might regulate the metastases of oral cancer by the upregulation of mGluR5 via downregulation of miR-30 family. Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Chikako Koshiji, Sayaka Izumi, Kembun Hakata, Yuske Komiyama, Shuji Tsuchida, Tomonori Hasegawa, Hitoshi Kawamata. Regulation of metabotropic glutamate receptor 5 expression by the miR-30 downregulation induced by the SDF-1/CXCR4 system in oral cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1925.

We have previously demonstrated that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (OSCC). In this study, we investigated whether the blockade of CXCR4 inhibits lymph node metastasis in B88 OSCC cells. These cells harbour a functional CXCR4 and have the potential to metastasise to the lymph node in vivo. Following introduction of a vector that expresses short hairpin small interfering RNA (shRNA) against CXCR4, we isolated three clones (shCXCR4-16, -17 and -21) that showed decreased expression of CXCR4 mRNA. These clones also had reduced CXCR4 protein levels and showed impairments in calcium flux and cell migration in response to SDF-1. These cells were orthotopically inoculated into the masseter muscle of nude mice. Lymph node metastases, loss in body weight and tumour volumes were significantly inhibited in mice inoculated with shCXCR4-17 cells compared to mice inoculated with control cells. SDF-1-induced migration of B88 cells was significantly inhibited in vitro by the treatment with 1,1′-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist. Subcutaneous administration of AMD3100 significantly inhibited the lymph node metastases of B88 cells when they were orthotopically inoculated into the masseter muscle of nude mice. Moreover, the enhanced production of interleukin (IL)-6 and IL-8 in response to SDF-1 was inhibited by shRNA against CXCR4 or by treatment with AMD3100. These results suggest that blockade of CXCR4 may be a potent anti-metastatic therapy against lymph node metastases in cases of CXCR4-related OSCC.

We have demonstrated that CXCR4 system is involved in the metastatic process of oral cancer. Recently, we identified the miR-518c-5p as a downstream target of the CXCR4 system, which was involved in the metastasis of B88, a CXCR4-expressing oral cancer cell line (2014 AACR annual meeting). In this study, we examined the function of miR-518c-5p in an oral cancer cell line, CAL27, which did not express CXCR4 and did not exhibit metastatic potential in vivo. We transfected a miR-518c expression vector into parental CAL27 cells and isolated a stable transfectant, CAL27-518c. We could detect the upregulation of miR-518c-5p in the miR-518c transfectants compared with mock cells, which was completely inhibited by treatment with the miR-518c-5p LNA inhibitor. The growth and migration of CAL27-518c cells were significantly enhanced compared with those of mock cells. The miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of CAL27-518c cells, indicating that these phenomena were mainly dependent on the expression of miR-518c-5p. Next, we examined the function of miR-518c-5p in vivo. miR-518c or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with miR-518c transfectants. These results indicate miR-518c-5p promotes the metastasis of oral cancer in a CXCR4-dependent and -independent manner. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Tetsuya Tamatani, Hirokazu Nagai, Hitoshi Kawamata, Youji Miyamoto. miR-518c-5p promotes the metastasis of oral cancer cells in a CXCR4-dependent and -independent manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3059. doi:10.1158/1538-7445.AM2015-3059

We identified a metastasis-related gene, metabotropic glutamate receptor (mGluR)-5, as a target gene of stromal cell-derived factor (SDF)-1/CXCR4 system in oral cancer. In this study, we examined the role of mGluR5 on the lymph node metastasis of oral cancer. mGluR5-specific antagonists, 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) did not affect the growth of oral cancer cells, but did inhibit the SDF-1/CXCR4 dependent-migration of the cells. When the CXCR4 expressing-oral cancer cells were orthotopically transplanted into masseter muscle of nude mice, these antagonists significantly inhibited lymph node metastases of the cells. Immunohistochemical analysis revealed that there was a significant association between CXCR4 and mGluR5 protein expression. Furthermore, the mGluR5 protein expression in clinical specimen was significantly associated with the lymph node metastases and the modes of invasion. Moreover conditioned media derived from the oral cancer cells acquiring SDF-1/CXCR4 system enhanced the migration of lymphatic endothelial cells, which was significantly suppressed by the treatment with MPEP or MTEP. Although SDF-1/CXCR4 dependent-production of angiogenic factors, including interleukin-6, interleukin-8, and vascular endothelial growth factor (VEGF)-A were observed, mGluR5 antagonists did not affect the production of these factors. However, induction of a lymphangiogenic factor, VEGF-C was specifically impaired by the treatment with these mGluR5 antagonists. These results suggested that mGluR5 promotes the lymph node metastases of oral cancer cells by the SDF-1/CXCR4 system via enhancement of cancer cell migration and induction of lymphangiogenesis by VEGF-C. Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Makoto Kinouchi, Tetsuya Tamatani, Hirokazu Nagai, Youji Miyamoto. The role of metabotropic glutamate receptor 5 on the lymph node metastases in oral cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1993. doi:10.1158/1538-7445.AM2014-1993

We have demonstrated that stromal-cell derived factor (SDF)-1/CXCR4 system is involved in the metastatic process of oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) are involved in the metastatic process of several types of cancers. However, the miRNAs contributed to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. Thus, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system, using oral cancer cells, B88-SDF-1 which exhibits functional CXCR4 and distant metastatic potential, in vivo. We examined metastasis-related miRNAs induced by the SDF-1/CXCR4 system using miRNA microarray analysis. Among these miRNAs, we confirmed the upregulation of miR-518c-5p in B88-SDF-1 cells by the real-time PCR analysis. Although LNA-based miR518c-5p inhibitor did not affect the cell growth, but did significantly inhibit the migration of B88-SDF-1 cells. Next, we transfected miR-518c expression vector into parental B88 cells, and isolated stable transfectant, B88-518c. The anchorage dependent and independent growth of B88-518c cells were significantly enhanced in compared with those of mock cells. Moreover, we could detect the enhanced migration of B88-518c cells. LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of B88-518c cells, indicating that these phenomenon were dependent on the expression of miR-518c-5p, not on that of miR-518c-3p. Next, we examined the function of miR-518c-5p in vivo. B88-518c cells or mock cells were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with B88-518c cells. These results indicate that SDF-1/CXCR4 system regulates the metastases of oral cancer via induction of miR-518c-5p. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Tetsuya Tamatani, Hirokazu Nagai, Youji Miyamoto. Isolation of a novel metastasis-related microRNA, miR-518c-5p, induced by the stromal cell-derived factor (SDF)-1/CXCR4 system in oral cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1446. doi:10.1158/1538-7445.AM2014-1446

[Background] A cancer stem cell (cancer initiating cell, CSC) is considered capable of self -replication, self-differentiation, drug resistance, and immune evasion. Recently, CSC has become increasingly important in the treatment of malignant tumors. Cancer stem cells express specific molecules termed CSC marker, including ATP-binding cassette, G2 subfamily (ABCG2), aldehyde dehydrogenase (ALDH) 1A1, CD44, and CD44 variant 9 (CD44v9), and their expression has been reported to be the potential prognostic values. However, the prognostic values of ABCG2, ALDH1A1, CD44, and CD44v9 expression in patients with oral cancer are less understood. [Purpose] The aims of present study were to evaluate the expression of ABCG2, ALDH1A1, CD44, and CD44v9 in oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the CSC marker expression, clinical stages, histological differentiation, classification of invasion mode, lymph node metastasis and disease-free survival rate. [Materials and Methods] Tissue specimens were obtains from 99 patients with OSCC after surgery or biopsy. Immunohistochemistry was used to assess ABCG2, ALDH1A1, CD44, and CD44v9 protein using at least 10% staining-positive cells as the definition of positive staining. [Results] Immunohistochemical analysis of 99 cases of OSCC showed that CD44 and CD44v9 expression were strongly detected in all OSCC compared with normal epithelial cells. Twenty eight (28.2%) cases of 99 OSCC patients expressed ABCG2. There was no relationship between ABCG2 expression and clinical stages, histological differentiation, metastasis, or disease-free survival rate. There was significant association between ABCG2 expression and classification of invasion mode. Otherwise, thirty one (31.3%) cases of 99 OSCC patients expressed ALDH1A1. There was no relationship between ALDH1A1 expression and clinical stagesor metastasis. However, there was significant association between ALDH1 expression and histological differentiation, or classification of invasion mode or disease-free survivalrate. [Conclusions] These findings suggested that the expression of ALDH1A1 as CSC markers in OSCC may be good marker indicating survival in patients with OSCC. Citation Format: Tetsuya Tamatani, Natsumi Takamaru, Makoto Kinouchi, Nobuyuki Kuribayashi, Daisuke Uchida, Hirokazu Nagai, Youji Miyamoto. Expression of ABCG2, ALDH1A1, CD44, and CD44 variant 9 in human oral squamous cell carcinoma and its relationship with clinical factors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3888. doi:10.1158/1538-7445.AM2014-3888

We have previously demonstrated that stromal cell-derived factor (SDF)-1/CXCR4 system is involved in the metastatic process of oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) are involved in the metastatic process of several types of cancers. However, the miRNAs contributed to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. Thus, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system, using oral cancer cells, B88-SDF-1, which exhibits functional CXCR4 and distant metastatic potential, in vivo. We examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system by use of a miRNA microarray representing approximately 1700 miRNA. Consequnetly, we identified 4 kinds of upregulated-miRNAs in B88-SDF-1 cells whose functions are unknown. Among these miRNAs, we confirmed the upregulation of miR-5XX in B88-SDF-1 cells by the real-time PCR analysis. Although LNA-based miR-5XX inhibitor did not affect the growth of the cells, it did significantly inhibit the migration of B88-SDF-1 cells. Thus, we examined the target gene(s) of miR-5XX in the SDF-1/CXCR4 system. cDNA microarray analysis revealed that 6 kinds of mRNA containing the predictive binding site of miR-5XX were upregulated by the treatment with miR-5XX inhibitor. Furthermore, gene A mRNA and protein was downregulated in B88-SDF-1 cells, and upregulated by the treatment with miR-5XX inhibitor. These results indicating that SDF-1/CXCR4 system might regulate the metastases of oral cancer by the downregulation of gene A via upregulation of miR-5XX. Citation Format: Makoto Kinouchi, Daisuke Uchida, Nobuyuki Kuribayashi, Tetsuya Tamatani, Hirokazu Nagai, Youji Miyamoto. Functional analysis of metastasis-related microRNA induced by the stromal-cell derived factor (SDF)-1/CXCR4 system in oral cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2697. doi:10.1158/1538-7445.AM2013-2697

Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. On the contrary to the CXCR4 expression, CXCR7, a recently identified second receptor for SDF-1, was detected in all SGC cell lines, expression levels were considerably lower on average compared with that of CXCR4 expression. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs. Citation Format: Nobuyuki Kuribayashi, Daisuke Uchida, Yasusei Kudo, Makoto Kinouchi, Tetsuya Tamatani, Hirokazu Nagai, Youji Miyamoto. Expression and function of CXCR4 in human salivary gland cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2701. doi:10.1158/1538-7445.AM2013-2701

Previously, we identified a metastasis-candidate gene, metabotropic glutamate receptor (mGluR)-5, which is a target gene of stromal cell-derived factor (SDF)-1/CXCR4 system in oral cancer. In this study, we examined the role of mGluR5 on the lymph node metastasis of oral cancer. We used 2 kinds of mGluR5-specific antagonists, 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP). These antagonists did not affect the growth of oral cancer cells, but did inhibit the SDF-1/CXCR4 dependent-migration of the cells. In order to confirm the drug toxicities, these antagonists were intraperitoneally injected into wild-type mice for 4 weeks. All the mice injected with MPEP (5 mg/kg) and MTEP (5 mg/kg) did not show side effects, such as hematotoxicity, allergic reaction and body weight loss. When the CXCR4 expressing-oral cancer cells were transplanted into masseter muscle of nude mice, these antagonists significantly inhibited lymph node metastases of the cells. Although SDF-1/CXCR4 dependent production of angiogenic factors, including interleukin-6, interleukin-8, and vascular endothelial growth factor (VEGF)-A were observed, mGluR5 antagonists did not influence the production of these factors. However, induction of a lymphangiogenic factor, VEGF-C was specifically inhibited by the treatment with these mGluR5 antagonists. These results suggested that SDF-1/CXCR4 system induce lymph node metastases by the production of VEGF-C via upregulation of mGluR5. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5329. doi:1538-7445.AM2012-5329

Previously, we identified a metastasis-candidate gene, metabotropic glutamate receptor (mGluR)-5 by using an oral cancer cell line, B88-SDF-1, which have an autocrine stromal cell-derived factor (SDF)-1/CXCR4 system and distant metastatic potentials in vivo. In this study, we examined the effect of mGluR5 antagonists on the SDF-1/CXCR4 dependent-metastases in oral cancer. We used 2 kinds of mGluR5-specific antagonists, 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP). These antagonists did not affect the growth of B88-SDF-1 cells, but did inhibit the enhanced-migration of the cells. Under these conditions, actin polymerization and pseudopodia formation were also inhibited by the treatment with MPEP and MTEP. In order to confirm the drug toxicities, these antagonists were intraperitoneally injected into wild-type mice for 4 weeks. All the mice injected with MPEP (5 mg/kg) and MTEP (5 mg/kg) did not show side effects, such as hematotoxicity, allergic reaction and body weight loss. When the B88-SDF-1 cells were transplanted into the vein of nude mice, these antagonists significantly inhibited the lung metastases of the cells. These results suggested that blockade of mGluR5, using antagonists such as MPEP and MTEP, may be effective at preventing metastasis in patients with CXCR4-related oral cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2440. doi:10.1158/1538-7445.AM2011-2440

We have previously demonstrated that distant metastasis might require the autocrine stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system in cases of oral squamous cell carcinoma (SCC). In this study, we examined the expression of metastasis-related genes in B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and distant metastatic potentials in vivo. Microarray analysis revealed that 418 genes were upregulated in B88-SDF-1 cells. To confirm the specificity of the microarray analysis, 3 highly upregulated genes, metabotropic glutamate receptor 5 (mGluR5), plasticity related gene 1 (PRG1), and unknown gene were reanalyzed by RT-PCR. These 3 genes were upregulated in B88-SDF-1 cells in comparison with those in mock cells, but AMD3100, a specific antagonist of CXCR4, abrogated the upregulation of 2 (mGluR5 and PRG1) out of 3 genes in B88-SDF-1 cells. Thus, we analyzed the expression of metabotropic glutamate receptor 5 (mGluR5), one of the genes that were upregulated in B88-SDF-1 cells. A specific mGluR5 antagonist, MPEP did not affect the growth of B88-SDF-1 cells, but did inhibit the enhanced-migration of B88-SDF-1 cells. When the B88-SDF-1 cells were transplanted into the vein, numerous metastatic foci were detected. Under the present conditions, MPEP significantly inhibited the lung metastasis of the B88-SDF-1 cells, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that mGluR5 might be one of a possible metastasis-related gene induced by an autocrine SDF-1/CXCR4 system in oral SCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 412.

Background We have demonstrated that the stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (SCC). Chemotherapy is a powerful tool for the treatment of oral cancer, including oral SCC; however, the effects of chemotherapeutic agents on the expression of CXCR4 are unknown. In this study, we examined the expression of CXCR4 associated with the chemotherapeutic agents in oral cancer cells. Results The expression of CXCR4 was examined using 3 different chemotherapeutic agents; 5-fluorouracil, cisplatin, and vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2-(1H)-quinolinone) in B88, a line of oral cancer cells that exhibits high levels of CXCR4 and lymph node metastatic potential. Of the 3 chemotherapeutic agents that we examined, only vesnarinone downregulated the expression of CXCR4 at the mRNA as well as the protein level. Vesnarinone significantly inhibited lymph node metastasis in tumor-bearing nude mice. Moreover, vesnarinone markedly inhibited 2.7-kb human CXCR4 promoter activity, and we identified the transcription factor, Krüppel-like factor 2 (KLF2), as a novel vesnarinone-responsive molecule, which was bound to the CXCR4 promoter at positions -300 to -167 relative to the transcription start site. The forced-expression of KLF2 led to the downregulation of CXCR4 mRNA and impaired CXCR4 promoter activity. The use of siRNA against KLF2 led to an upregulation of CXCR4 mRNA. Conclusion These Results indicate that vesnarinone downregulates CXCR4 via the upregulation of KLF2 in oral cancer.