Hiroki Kasama

ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

Angiopoietin‐like 3 (ANGPTL3), which is involved in new blood vessel growth and stimulation of mitogen‐activated protein kinase (MAPK), is expressed aberrantly in several types of human cancers. However, little is known about the relevance of ANGPTL3 in the behavior of oral squamous cell carcinoma (OSCC). In this study, we evaluated ANGPTL3 mRNA and protein in OSCC‐derived cell lines (n = 8) and primary OSCCs (n = 109) and assessed the effect of ANGPTL3 on the biology and function of OSCCs in vitro and in vivo. Significant (P < 0.05) ANGPTL3 upregulation was detected in the cell lines and most primary OSCCs (60%) compared with the normal counterparts. The ANGPTL3 expression level was correlated closely (P < 0.05) with tumoral size. In patients with T3/T4 tumors, the overall survival rate with an ANGPTL3‐positive tumor was significantly (P < 0.05) lower than that of ANGPTL3‐negative cases. In vitro, cellular growth in ANGPTL3 knockdown cells significantly (P < 0.05) decreased with inactivated extracellular regulated kinase (ERK) and cell‐cycle arrest at the G1 phase resulting from upregulation of the cyclin‐dependent kinase inhibitors, including p21Cip1 and p27Kip1. We also observed a marked (P < 0.05) reduction in the growth in ANGPTL3 knockdown‐cell xenografts with decreased levels of phosphorylated ERK relative to control‐cell xenografts. The current data indicated that ANGPTL3 may play a role in OSCCs via MAPK signaling cascades, making it a potentially useful diagnostic/therapeutic target for use in patients with OSCC.

Down-Regulation of Nucleolar and Spindle-Associated Protein 1 (NUSAP1) Expression Suppresses Tumor and Cell Proliferation and Enhances Anti-Tumor Effect of Paclitaxel in Oral Squamous Cell Carcinoma

Background Nucleolar and spindle-associated protein 1 (NUSAP1) is an important mitotic regulator. In addition to its crucial function in mitosis, NUSAP1 has recently received attention due to the interesting roles in carcinogenesis. The aim of this study was to reveal functional mechanisms of NUSAP1 in oral squamous cell carcinoma (OSCC). Methods mRNA and protein expression levels of NUSAP1 in 9 OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. The correlation between the NUSAP1 expression profile and the clinicopathological factors was evaluated by immunohistochemistry (IHC) in clinical OSCC samples (n = 70). The NUSAP1 knockdown cells were established with short hairpin RNA (shRNA) in OSCC cells, and functional assays were performed using these cells. In addition to the evaluation of cellular proliferation and cell cycle, we also investigated the potential role of NUSAP1 in paclitaxel (PTX)-induced cellular responses. Results mRNA and protein expression of NUSAP1 were significantly up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (P < 0.05). IHC revealed that NUSAP-1 expression is closely associated with primary advanced T stage (P<0.05). Suppression of NUSAP1 expression levels led to significant (P < 0.05) inhibition of cellular proliferation. Furthermore, apoptosis induced by PTX was enhanced in NUSAP1 knockdown OSCC cells. Conclusions NUSAP1 may be a crucial biomarker for OSCC. Moreover, down-regulated NUSAP1 expression suppresses tumor proliferation and also enhances anti-tumor effect of PTX by activating apoptotic pathways. Thus, the present study strongly suggests that regulating NUSAP1 expression should contribute to the therapy for OSCC.

Decorin in human oral cancer: a promising predictive biomarker of S-1 neoadjuvant chemosensitivity.

We reported previously that decorin (DCN) is significantly up-regulated in chemoresistant cancer cell lines. DCN is a small leucine-rich proteoglycan that exists and functions in stromal and epithelial cells. Accumulating evidence suggests that DCN affects the biology of several types of cancer by directly/indirectly targeting the signaling molecules involved in cell growth, survival, metastasis, and angiogenesis, however, the molecular mechanisms of DCN in chemoresistance and its clinical relevance are still unknown. Here we assumed that DCN silencing cells increase chemosusceptibility to S-1, consisted of tegafur, prodrug of 5-fluorouracil. We first established DCN knockdown transfectants derived from oral cancer cells for following experiments including chemosusceptibility assay to S-1. In addition to the in vitro data, DCN knockdown zenografting tumors in nude mice demonstrate decreasing cell proliferation and increasing apoptosis with dephosphorylation of AKT after S-1 chemotherapy. We also investigated whether DCN expression predicts the clinical responses of neoadjuvant chemotherapy (NAC) using S-1 (S-1 NAC) for oral cancer patients. Immunohistochemistry data in the preoperative biopsy samples was analyzed to determine the cut-off point for status of DCN expression by receiver operating curve analysis. Interestingly, low DCN expression was observed in five (83%) of six cases with complete responses to S-1 NAC, and in one (10%) case of 10 cases with stable/progressive disease, indicating that S-1 chemosensitivity is dramatically effective in oral cancer patients with low DCN expression compared with high DCN expression. Our findings suggest that DCN is a key regulator for chemoresistant mechanisms, and is a predictive immunomarker of the response to S-1 NAC and patient prognosis.

SIPA1 promotes invasion and migration in human oral squamous cell carcinoma by ITGB1 and MMP7

ABSTRACT Signal‐induced proliferation‐associated protein 1 (SIPA1) is known to be a GTPase activating protein. Overexpressed SIPA1 is related to metastatic progression in breast and prostate cancers; however, the relevance of SIPA1 in oral squamous cell carcinoma (OSCC) is still unknown. The aim of this study was to examine SIPA1 expression and its functional mechanisms in OSCC. SIPA1 mRNA and protein expressions were analyzed by quantitative reverse transcriptase‐polymerase chain reaction, Western blot analysis, and immunohistochemistry. The expressions of SIPA1 were up‐regulated significantly in vitro and in vivo. Moreover, SIPA1 expression was correlated with regional lymph node metastasis. We next assessed the cellular functions associated with tumoral metastasis using SIPA1 knockdown (shSIPA1) cells and analyzed the downstream molecules of SIPA1, i.e., bromodomain containing protein 4(BRD4), integrin beta1 (ITGB1), and matrix metalloproteinase 7 (MMP7). The shSIPA1 cells showed decreased invasiveness and migratory activities, however cellular adhesion ability was maintained at a high level. In addition, ITGB1 expression was greater in shSIPA1 cells, whereas MMP7 expression was lower than in control cells. This research is the first to establish that SIPA1 promotes cancer metastasis by regulating the ITGB1 and MMP7. Therefore, SIPA1 might be a novel therapeutic target for patients with lymph node metastasis of OSCC. HighlightsSIPA1 expression was up‐regulated in oral squamous cell carcinoma (OSCC).SIPA1‐positive OSCCs were correlated with regional lymph node metastasis.SIPA1 controlled BRD4 and influenced transcription of ITGB1and MMP7.SIPA1 induced cellular invasion and migration and decreased cellular adhesion.SIPA1 might be a potential biomarker of cancer metastasis for OSCC.

FBLIM1 enhances oral cancer malignancy via modulation of the epidermal growth factor receptor pathway

Filamin‐binding LIM protein 1 (FBLIM1) is related to regulation of inflammatory responses, such as chronic recurrent multifocal osteomyelitis; however, the relevance of FBLIM1 in oral squamous cell carcinoma (OSCC) is unknown. The aim of the current study was to elucidate the possible role of FBLIM1 in the carcinogenesis of OSCC. We analyzed FBLIM1 expression using quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR), immunoblot analysis, and immunohistochemistry. The expression levels of FBLIM1 were up‐regulated significantly (P < 0.05) in OSCC‐derived cell lines and primary OSCCs specimens compared with normal counterparts. FBLIM1 expression also was correlated with the primary tumoral size (P < 0.05) and vascular invasion (P < 0.05). We then assessed tumoral progression after treatment with FBLIM1 siRNA and clopidogrel, an antiplatelet agent. Similar to the FBLIM1 knockdown effect, clopidogrel‐treated cells had attenuated functions of proliferation, migration, and invasiveness. Interestingly, clopidogrel treatment led to down‐regulation of epidermal growth factor receptor (EGFR) and FBLIM1. These findings identify FBLIM1 as a putative therapeutic target by using clopidogrel for inhibiting over activation of EGFR signaling to prevent OSCC malignancy.

Critical role of deoxynucleotidyl transferase terminal interacting protein 1 in oral cancer

Deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1) forms a complex with histone deacetylase (HDAC); however, the relevance of DNTTIP1 in cancer remains unknown. The aim of this study was to examine DNTTIP1 expression and its functional mechanisms in oral squamous cell carcinomas (OSCCs). DNTTIP1 expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting analysis, and immunohistochemistry. The expression of DNTTIP1 was upregulated significantly in vitro and in vivo, and in patients with OSCC in whom DNTTIP1 was overexpressed and the expression level was correlated significantly (P < 0.05) with tumoral growth. DNTTIP1 knockdown (siDNTTIP1) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with high acetylation of p53 and upregulation of p21Cip1. Moreover, resveratrol, a HDAC inhibitor, controlled not only acetylated p53 status but also DNTTIP1 expression, leading to a similar phenotype of siDNTTIP1 cells. A marked (P < 0.05) reduction of tumoral growth in mouse xenograft models was observed with lower DNTTIP1 expression under the presence of this chemical reagent. Taken together, our results suggested that DNTTIP1–HDAC interaction promotes tumoral growth through deacetylation of p53 and that DNTTIP1 might be a critical therapeutic target in OSCCs.This study demonstrates that deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1), a binding partner of histone deacetylase (HDAC), plays a crucial role in progression of oral squamous cell carcinoma (OSCC) by controlling p53 acetylation. Resveratrol, a HDAC inhibitor, controls both acetylated p53 status and DNTTIP1 expression. Thus, DNTTIP1 might be a novel therapeutic target in OSCC.

Overexpression of Translocation Associated Membrane Protein 2 Leading to Cancer-Associated Matrix Metalloproteinase Activation as a Putative Metastatic Factor for Human Oral Cancer

Translocation associated membrane protein 2 (TRAM2) has been characterized as a component of the translocon that is a gated channel at the endoplasmic reticulum (ER) membrane. TRAM2 is expressed in a wide variety of human organs. To date, no information is available regarding TRAM2 function in the genesis of human cancer. The purpose of this study was to investigate the status of the TRAM2 gene in oral squamous cell carcinoma (OSCC) cells and clinical OSCC samples. Using real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting analysis, and immunohistochemistry, we detected accelerated TRAM2 mRNA and protein expression levels both in OSCC-derived cell lines and primary tumors. Moreover, TRAM2-positive OSCC tissues were correlated closely (P<0.05) with metastasis to regional lymph nodes and vascular invasiveness. Of note, knockdown of TRAM2 inhibited metastatic phenotypes, including siTRAM2 cellular migration, invasiveness, and transendothelial migration activities with a significant (P<0.05) decrease in protein kinase RNA(PKR) - like ER kinase (PERK) and matrix metalloproteinases (MMPs) (MT1-MMP, MMP2, and MMP9). Taken together, our results suggested that TRAM2 might play a pivotal role in OSCC cellular metastasis by controlling major MMPs. This molecule might be a putative therapeutic target for OSCC.

Multiple coagulation factor deficiency protein 2 as a crucial component in metastasis of human oral cancer

ABSTRACT Multiple coagulation factor deficiency protein 2 (MCFD2), a binding partner of lectin mannose binding 1 (LMAN1), causes combined deficiencies of coagulation factors V and VIII. MCFD2 function in inherited hematologic disorders is well elucidated; however, little is known about its role in human tumorigenesis. The aim of the current study was to investigate the states of MCFD2 in oral squamous cell carcinoma (OSCC). The expression of MCFD2 was up‐regulated significantly in all cell lines examined. Evaluation of the cellular functions associated with tumoral metastasis showed that MCFD2 knockdown (shMCFD2) cells exhibited significantly lower cellular invasiveness and migration and higher cellular adhesion compared with shControl cells. Of note, shMCFD2 cells also showed weak immunoreactivity of LMAN1 and a lower secretion level of galactoside‐binding soluble 3 binding protein (LGALS3BP). In addition to in vitro validation, clinical data on 70 patients with OSCC indicated that state of MCFD2 expression level is associated with regional lymph node metastasis. Altogether, we have demonstrated that MCFD2 promotes cancer metastasis by regulating LMAN1 and LGALS3BP expression levels. Hence, MCFD2 may represent a promising candidate for a novel therapeutic target for patients with metastatic OSCCs. HighlightsExpression of MCFD2 was up‐regulated in human oral squamous cell carcinoma (OSCC).MCFD2 expression is associated with regional lymph node metastasis.MCFD2 induced cellular invasion and migration and decreased cellular adhesion.MCFD2 regulated secretion of galectin 3 binding protein via MCFD2‐LMAN1 interaction.MCFD2 might be a novel therapeutic target for patients with metastatic OSCCs.

Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression

ABSTRACT Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2‐arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real‐time quantitative reverse transcription‐polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up‐regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC‐derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell‐cycle arrest at G1 phase. Furthermore, we found that DAGLA‐positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs. HIGHLIGHTSDAGLA was up‐regulated in human oral squamous cell carcinoma (OSCC) cells compared with human normal oral keratinocytes (HNOKs).Overexpression of DAGLA in OSCCs controlled cell proliferation through cell‐cycle progression.The tumoral volume of the orlistat‐treated group was clearly smaller than that of the control group in vivo.DAGLA expression level might be a useful diagnostic indicator and a new therapeutic target for OSCCs.