Norihiko Tokuzen

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.

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.

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.

Abstract 4461: Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells

Exosomes are small vesicles, ranging from 30 to 100 nm in size, secreted from most types of cells. They contain proteins, lipids, and nucleic acids (e.g. DNA, mRNA, and microRNA) and are thought to play an important role in cell proliferation, invasion, and metastasis in human malignancies. Recently, circulating exosomes have also been of interest as a source for liquid biopsies. In this study, we examined the usefulness of exosomal RNA as a biomarker and attempted to detect the tumor-specific gene mutations from exosomal RNA derived from human oral squamous cell carcinoma (OSCC) cells. We used 3 human OSCC cells (HSC2, KT-T, KT-N). KT-T and KT-N cells were established from the lower gingival tumor and lymph node metastasis, respectively. First, we detected the somatic mutations in genomic DNA extracted from these cells by next-generation sequencing (NGS) with the use of HaloPlex Cancer Research Panel (Agilent Technologies). PIK3CA H1047R mutation was identified in all OSCC cells, and KT-N cells had the additional HRAS Q61R mutation. We confirmed these mutations of PIK3CA and HRAS in OSCC cells by TaqMan® SNP Genotyping Assays (Thermo Fisher Scientific). Subsequently, we evaluated the gene mutations in exosomal RNA from these OSCC cells. Exosomal RNA was isolated from the conditioned medium using exoRNeasy Serum/Plasma Maxi Kit (Qiagen) according to the manufacture’s protocol. In exosomal RNA, we found the PIK3CA H1047R mutation from all OSCC cells and HRAS Q61R mutation from KT-N cells by genotyping assays. Furthermore, we assessed the detection of these mutations in tumor xenograft mouse models. KT-T and KT-N cells (5 x 106) complexed with Matrigel® (BD) in 100-µl aliquots were injected subcutaneously space in the flank of male athymic nude mice. Exosomal RNA was isolated from the serum of tumor-bearing nude mice using exoRNeasy Serum/Plasma Midi Kit (Qiagen). By genotyping assays using serum exosomal RNA, PIK3CA H1047R mutation was observed in both types of xenograft mice and HRAS Q61R mutation was positive in KT-N xenograft mice. Cancer cell-derived exosomal RNA had the same gene mutations in those cells, and could be detected in the serum of tumor-bearing nude mice. These results suggest that exosomal RNA is useful for detecting somatic mutations in OSCC cells. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroyuki Hamakawa. Detection of somatic mutations in exosomal RNA from human oral squamous cell carcinoma cells [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 4461. doi:10.1158/1538-7445.AM2017-4461

Abstract 1137: Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells

Whole-exome sequencing studies showed the mutational landscape of head and neck squamous cell carcinoma including oral cavity. Recent several reports also demonstrated the somatic mutations of oncogenes and tumor suppressor genes in oral squamous cell carcinoma (OSCC), and identified the mutations of HRAS, BRAF, FGFR3, SMAD4, KIT, PTEN, NOTCH1, AKT1, CTNNB1, and PTPN11 to predict disease-free survival of OSCC patients. In this study, we have attempted to elucidate somatic mutations of these genes and their functions in human OSCC cells. We isolated and cultured tumor cells from the resected primary tumor (KT-T), metastatic lymph node (KT-N), and cancerous pleural effusion (KT-M) in the same patient with OSCC. KT-T cells showed epithelial-like shape, whereas both KT-N and KT-M cells indicated the morphological changes to fibroblast-like spindle shape. Genomic DNA was extracted from these cells, and then we performed the mutational analysis by ultra-deep targeted sequencing using Haloplex Cancer Research Panel. All types of KT cells had the mutation of TP53 (R141P, C135S, and E182*) and PIK3CA (H1047R), and an additional active mutation of HRAS (Q61R) was detected in only metastatic KT-N and KT-M cells. Subsequently, we transfected all KT cells with synthetic small interfering RNA (siRNA) specific for PIK3CA (siPIK3CA) and/or HRAS (siHRAS) at the concentration of 10 nM complexed with Lipofectamine RNAiMAX. We confirmed the knockdown effects of these siRNAs in KT cells by quantitative RT-PCR. After transfection of these siRNAs for 72 hours, the growth of KT cells was evaluated by WST-8 assay. Knockdown of PIK3CA or HRAS expression significantly suppressed the growth of KT-T cells only, whereas double knockdown of PIK3CA and HRAS inhibited the growth of KT-N and KT-M cells as well. Next, we investigated the function of mutant HRAS (Q61R) in KT-T cells. Overexpression of mutant HRAS did not change the growth rate and morphology of KT-T cells. Finally, we examined the effects of BEZ-235 (dual PI3K/mTOR inhibitor) and trametinib (MEK1/2 inhibitor) on the growth of KT-T, KT-N, and KT-M cells. BEZ-235 (100 nM) almost completely suppressed the growth of these cells. Trametinib (2 μM) also reduced the growth rate by 16.9% in KT-T, 13.5% in KT-N, and 45.7% in KT-M cells. Furthermore, BEZ-235 combined with trametinib inhibited the cell growth more effectively even at low concentrations. These results suggest that constitutive active mutations of HRAS and PIK3CA support the growth of human OSCC cells and these signaling pathways may be useful therapeutic targets for the patients with OSCC. Citation Format: Hitoshi Akiyama, Koichi Nakashiro, Norihiko Tokuzen, Hiroshi Tanaka, Satoshi Hino, Hiroyuki Hamakawa. Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma 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 1137.

Abstract 1147: CDCA5 is a novel therapeutic target molecule in 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 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 9 human OSCC cells tested by microarray analysis. CDCA5 was initially identified as a substrate of anaphase-promoting complex and as a regulator of sister chromatid cohesion in HeLa cells. In addition, CDCA5 has been reported to be overexpressed in the majority of human lung cancers and urothelial cancers and to play a critical role in carcinogenesis. However, function of CDCA5 in OSCC remains still unknown. In this study, we investigated the expression and function of CDCA5 in OSCC. First, we confirmed the expression level of CDCA5 in 4 human OSCC cell lines and an immortalized non-neoplastic human keratinocyte cell line, HaCaT, by quantitative RT-PCR (qRT-PCR) and Western blotting. The expression levels of CDCA5 mRNA and protein were markedly elevated in all human OSCC cell lines compared to HaCaT cells. Next, we tested the effect of synthetic small interfering RNAs specific for CDCA5 (siCDCA5) on the growth of human OSCC cells. The RNA interference effect of siCDCA5 was confirmed by Western blotting. Transfection of siCDCA5 suppressed the expression of its target protein in all types of cells. The growth inhibitory effect of siCDCA5 in OSCC cells was evaluated by WST-8 assay. The Knockdown of CDCA5 significantly inhibited the growth of OSCC cells in vitro. Subsequently, we assessed the effect of siCDCA5 on the in vivo growth of human OSCC cells, green fluorescent protein (GFP)-SAS. We administered siCDCA5/atelocollagen complexes into the subcutaneous spaces around the xenograft tumors every 3 days. We found that these complexes significantly reduced the size of subcutaneously xenografted GFP-SAS tumors, compared to the control group treated with synthetic siRNA specific for GFP (siGFP)/atelocollagen complexes. Next, we analyzed the influence of siCDCA5 on the cell cycle of OSCC cells by flow cytometry. Suppression of CDCA5 resulted in the decrease in percentage of cells in G0/G1 phase and the increase in G2 phase. This indicated that the anti-proliferative effect of siCDCA5 was due to G2 arrest. Finally, we investigated the clinical significance of CDCA5 expression in OSCC. We examined the expression of CDCA5 in OSCC tissues by immunohistochemistry, and then 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. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroshi Tanaka, Kazuki Iwamoto, Hitoshi Akiyama, Hiroyuki Hamakawa. CDCA5 is a novel therapeutic target molecule in oral squamous cell carcinoma. [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 1147.

Abstract 3107: MicroRNA-1289 is a novel tumor suppressive microRNA in human oral squamous cell carcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA MicroRNAs (miRNAs) are small non-coding double-stranded RNA with sizes of approximately 22 nucleotides, and those inhibit protein translation by binding the 3′-untranslated region of target mRNAs. Each miRNA generally can regulate multiple mRNAs and each mRNA can be inhibited by a number of miRNAs. MiRNAs can behave as not only oncogenes but also tumor suppressive (TS) genes in human malignancies. In fact, a number of TS miRNAs (TS-miRs) were found in human many types of cancer, such as let-7, miR-1, miR-34, miR-145, miR-200, miR-206 and more. In this study, we have attempted to identify a novel TS-miR in human oral squamous cell carcinoma (OSCC) using function-based screening. First, we transfected human OSCC cells (GFP-SAS) with approximately 1,000 synthetic mimicking human mature miRNAs at the concentration of 20 nM complexed with Lipofectamine RNAiMAX. After transfection for 72 hours, the growth of these cells was evaluated by WST-8 assay. As a result, miR-1289 had the most potent growth inhibitory effect against GFP-SAS cells. The growth rate of these cells transfected with miR-1289 was reduced by 86.4%. MiR-1289 also remarkably inhibited the growth of other human OSCC cells (Ca9-22, HSC2, HSC3 and primary cultured cells). Subsequently, we assessed the growth inhibitory effect of miR-1289 in vivo using a mouse model. GFP-SAS cells (2 × 106) complexed with matrigel in 100 μl aliquots were injected subcutaneously in nude mice. After tumor formation, we administered 100 ng of miRNA with 0.5% atelocollagen complexes (100 μl) around tumor every 3 days for a total of 5 injections. MiR-1289/atelocollagen complex administration group significantly reduced the size of subcutaneously xenografted GFP-SAS tumors compared with the control group. Next, we investigated the expression of miR-1289 in OSCC tissues by real-time quantitative RT-PCR. The expression levels of miR-1289 were significant decreased in OSCC tissues compared to adjacent normal oral mucosa tissues. Finally, target genes of miR-1289 were explored by microarray and Ingenuity Pathway Analysis (IPA). Microarray analysis revealed that the expression levels of 51 genes were commonly down-regulated in OSCC cells (GFP-SAS, Ca9-22 and HSC2) by transfection with miR-1289. Among these genes, 15 genes were identified as target genes of miR-1289 by IPA. These results suggest that miR-1289 functions as a novel TS-miR in OSCC, and may be a useful therapeutic tool for the patients with OSCC. Citation Format: Kazuki Iwamoto, Koh-ichi Nakashiro, Hiroshi Tnaka, Norihiko Tokuzen, Hiroyuki Hamakawa. MicroRNA-1289 is a novel tumor suppressive microRNA in human oral squamous cell carcinoma. [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 3107. doi:10.1158/1538-7445.AM2015-3107

Abstract 476: Overexpression and function of CDCA5 in oral squamous cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Molecular target drugs are used in the treatment of various malignant tumors. However, the development of molecular targeted therapy for oral squamous cell carcinoma (OSCC) is lagging behind other cancers. In this study, we have attempted to identify an appropriate molecular target for treatment of patients with OSCC. We determined the gene expression profiles of human 9 OSCC cell lines and an immortalized non-neoplastic human keratinocyte cell line by microarray analysis, and then found cell division cycle associated 5 (CDCA5) as one of genes which were commonly overexpressed in OSCC cells. CDCA5 was initially identified as substrate of anaphase-promoting complex and as a regulator of sister chromatid cohesion in HeLa. In addition, it has been reported that CDCA5 plays a critical role in human lung carcinogenesis. However, function of CDCA5 in OSCC remains still unknown. First, we confirmed the overexpression of CDCA5 in 5 human OSCC cells by quantitative RT-PCR (qRT-PCR) and Western blotting. The expression levels of CDCA5 mRNA and protein were markedly elevated in all human OSCC cell lines compared to a non-neoplastic human epithelial cell line. Next, we examined the effect of synthetic small interfering RNAs specific for CDCA5 (siCDCA5) on the growth of human OSCC cells. RNAi effect of siCDCA5 was confirmed by Western blotting. Transfection of siCDCA5 suppressed the expression of its target protein in all types of cells. The growth inhibitory effect of siCDCA5 in OSCC cells was evaluated by WST-8 assay. Knockdown of CDCA5 significantly inhibited the growth of OSCC cells in vitro. Subsequently, we analyzed the influence of siCDCA5 on cell cycle by flow cytometry. Suppression of CDCA5 resulted in the decrease in percentage of cells in G0/G1 phase and increase in G2 phase. This indicated that the anti-proliferative effect of siCDCA5 was due to G2 arrest. Furthermore, we investigated the clinical significance of CDCA5 expression in OSCC. The expression of CDCA5 mRNA in tumor and adjacent normal tissues derived from the patient with OSCC was examined by qRT-PCR. Expression levels of CDCA5 mRNA in the OSCC tissues were significantly higher than in normal tissues. We also examined the expression of CDCA5 protein in 80 OSCC cases by immunohistochemistry with anti-CDCA5 antibodies. Tissue expression of CDCA5 was divided into high or low group at 50% stained tumor cells, resulting in 36 cases as high expression and 44 cases as low expression. We then examined the association of CDCA5 expression with various clinicopathological parameters of OSCC patients, and found a significant association between CDCA5 expression levels and pathological staging, metastasis, and recurrence. These results suggest that CDCA5 functions as a critical gene supporting the growth of human OSCC cells and targeting CDCA5 appears a useful therapeutic strategy for the patients with OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro, Hiroshi Tanaka, Yohei Fujita, Kazuki Iwamoto, Hiroyuki Hamakawa. Overexpression and function of CDCA5 in oral squamous cell carcinoma. [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 476. doi:10.1158/1538-7445.AM2014-476

Abstract 3003: Aurora kinase A as a novel therapeutic target in oral squamous cell carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Recent experiments have showed that it is possible to use pharmacological agents that inactivate oncogenes to treat at least some types of human malignancies. However, the development of molecular targeted therapy in oral squamous cell carcinoma (OSCC) is lagging behind compare to other cancers. In this study, we attempted to identify an appropriate target molecule and to determine whether targeting such a molecule is plausible therapeutic approach for the treatment of patients with OSCC. We determined the gene expression profiles of 9 human OSCC cell lines and non-neoplastic keratinocyte cell line by microarray analysis, and then identified Aurora kinase A (AURKA) as a cancer-related gene. AURKA has been shown to be related to the progression, survival, histological differentiation, and metastasis in various tumors. We revealed the overexpression of AURKA mRNA and protein in human OSCC cell lines and tissues. To clarify the function of AURKA in the cell proliferation of OSCC cells, we investigated the effect of small interfering RNA (siRNA) specific for AURKA (siAURKA) and MLN8237, an AURKA selective inhibitor, on the growth of human OSCC cells in vitro and in vivo. All siAURKAs almost completely suppressed the expression of AURKA protein, and significantly inhibited the growth of these cells compared to non-targeting siRNA. MLN8237 also markedly reduced the growth rate of human OSCC cells. Next, we assessed the growth inhibitory effect of siAURKA and MLN8237 using mouse model. Synthetic siAURKA/atelocollagen complexes (40 μM) were administrated into mouse tail vain every 3 days, and MLN8237 (20 mg/kg) was received orally for 14 consecutive days. We found both siAURKA and MLN8237 significantly reduced the size of subcutaneously xenografted OSCC tumors. To confirm the usefulness of targeting AURKA in OSCC, we attempted to culture the resected tumor tissues from 3 patients with OSCC and obtained primary cultured cells. Knockdown of AURKA expression and MLN8237 induced the growth inhibition of OSCC primary cultured cells. Furthermore, the expression of AURKA mRNA in OSCC tissues resected from patients was examined. In 37 of 50 primary OSCC tissues, the expression levels of AURKA mRNA were more than 2-fold increase compared to normal oral mucosa tissues, and notably we found significant association between AURKA mRNA expression levels and histological differentiation and lymph node metastasis. These results suggest that AURKA plays a critical role in the growth of human OSCC cells, and targeting AURKA appears to be a potentially useful therapeutic approach for patients with OSCC. Citation Format: Hiroshi Tanaka, Koh-ichi Nakashiro, Kazuki Iwamoto, Norihiko Tokuzen, Yohei Fujita, Rikimaru Shirakawa, Ryota Oka, Hiroyuki Goda, Hiroyuki Hamakawa. Aurora kinase A as a novel therapeutic target in oral squamous cell carcinoma. [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 3003. doi:10.1158/1538-7445.AM2013-3003