UNC13C Suppress Tumor Progression via Inhibiting EMT Pathway and Improves Survival in Oral Squamous Cell Carcinoma

Potential function of UNC13C in variety of cancers including, oral squamous cell carcinoma (OSCC) remains obscure. In the present study, immunohistochemical staining in tissue microarrays containing 268 OSCC samples showed that UNC13C protein levels were inversely correlated with AJCC Stage III and IV (P = 0.002) and death (P = 0.0134). Patients with lower UNC13C expression had a significantly shorter survival (P = 0.0231) than those with higher UNC13C expression. We also identified decreased overall UNC13C expression in oral cancer cell lines. In addition, our functional analysis of UNC13C shows that overexpression of UNC13C inhibited migration and invasion capacities of SCC-9 and SAS cells compared with the empty plasmid transfected cells. Further experiments suggested that transcription factors (Slug, Snail, Twist, and ZEB1) and mesenchymal marker (Vimentin) were down regulated and Tight Junction Protein (Claudin1) was up regulated after UNC13C overexpression in SCC9 and SAS cells. The novel role of UNC13C is revealed for the first time in OSCC. In summary, these results suggest that UNC13C as a novel tumor suppressor and an essential regulator of EMT signaling pathway during OSCC progression, and thus it could be used as a target for preventing oral cancer metastasis.

Potential function of UNC13C in variety of cancers including, oral squamous cell carcinoma (OSCC) remains obscure. In the present study, immunohistochemical staining in tissue microarrays containing 268 OSCC samples showed that UNC13C protein levels were inversely correlated with AJCC Stage III and IV (P = 0.002) and death (P = 0.0134). Patients with lower UNC13C expression had a significantly shorter survival (P = 0.0231) than those with higher UNC13C expression. We also identified decreased overall UNC13C expression in oral cancer cell lines. In addition, our functional analysis of UNC13C shows that overexpression of UNC13C inhibited migration and invasion capacities of SCC-9 and SAS cells compared with the empty plasmid transfected cells. Further experiments suggested that transcription factors (Slug, Snail, Twist, and ZEB1) and mesenchymal marker (Vimentin) were down regulated and Tight Junction Protein (Claudin1) was up regulated after UNC13C overexpression in SCC9 and SAS cells. The novel role of UNC13C is revealed for the first time in OSCC. In summary, these results suggest that UNC13C as a novel tumor suppressor and an essential regulator of EMT signaling pathway during OSCC progression, and thus it could be used as a target for preventing oral cancer metastasis.

INTRODUCTION
Oral squamous cell carcinoma (OSCC) is one of the most common cause of cancer death in the worldwide (1), particularly in Taiwan (2). OSCC is a prevalent malignancy that represents the fourth most common cancer affecting males (M:F = 10.8:1; 6,308:582 new cases in 2012, per the Taiwan Cancer Registry; http://tcr.cph.ntu.edu.tw/main.php?Page=A1). Despite of the improved diagnostic techniques and treatment methodology, survival rate of oral cancer patients has not improved (3) because most of OSCC cases were detected at advanced stage, the main reasons is due to lack of early molecular marker. Early detection is one of the effective ways to improve patient survival. Therefore, it is important to screen better biomarkers to improve the detection efficiency in oral cancer patients.
UNC13C or Munc13-3 plays a role in vesicle maturation during exocytosis and in neurotransmitter release. Previous study has found that, UNC13C was frequently altered (10-22% of patients) in Gingivo-buccal oral squamous cell carcinoma (OSCC-GB) patients (4). UNC13C is predicted in regulating neurotransmitter release (5); alterations in UNC13C could be associated to predisposition to tobacco addiction, which might enhance the risk of oral cancer. However, the function of UNC13C in tumor initiation and progression is unclear. Moreover, there are currently no reports describing the role of UNC13C in OSCC.
In this study, we showed that UNC13C expression in OSCC tissues was significantly down-regulated compared with surrounding normal marginal tissues and reduced UNC13C expression predicted poor survival rate in OSCC patients. In vitro experiments confirmed the inhibitory effects of UNC13C on cell migration and invasion. Additionally, we investigated the possible roles and molecular mechanisms of UNC13C in oral cancer cells using in vitro models. Our study provides novel insights into the tumor suppressive role of UNC13C in OSCC, restoring UNC13C expression may be a novel treatment strategy for OSCC.

UNC13C Down-Regulated in Human OSCC Tissues
To determine the role of UNC13C in oral cancer, the expression level of UNC13C was first examined in oral cancer tissues (n = 268) using Immunohistochemistry staining. The patients' characteristics, including their sex, age, tumor stage, lymph node status, AJCC cancer stage, and histological grade are listed in Table 1. UNC13C expression was evaluated and scored by two pathologists, tumor tissues showed positive and negative staining of UNC13C ( Figure 1A). Of the OSCC specimens examined, low UNC13C expression was detected in 190 of the 257 (74.0%) patients and high UNC13C expression was detected in 67 of the 257 (26.0%) patients.

Association of UNC13C Expression With Clinico-Pathological Parameters
We then analyzed the relationship between UNC13C expression and clinicopathological variables in OSCC ( Table 2). UNC13C expression was significantly correlated with AJCC stage (P = 0.002) and death (P = 0.0134) ( Table 2). However, UNC13C expression was not associated with the other clinicopathological features, such as TNM stage and histologic grade, was observed (P > 0.05) in our study. Survival analysis showed that patients with lower UNC13C expression had a shorter survival time than those with higher UNC13C expression ( Figure 1B, P < 0.05).

UNC13C Influenced Migration and Invasion of Oral Cancer Cells
Firstly, we measured the expression of UNC13C in different OSCC cell lines, and the results showed that UNC13C was not expressed in OSCC cell lines (Data not shown). To elucidate the functional relevance of UNC13C in OSCC progression, we performed functional analysis in SAS and SCC-9 cell lines after UNC13C expression. As shown in Figure 2A, transfection of  the plasmid pUNC13C-Myc into SAS and SCC-9 cells led to a significant increase in endogenous UNC13C compared with empty plasmid transfected cells (Figure 2A).
In order to determine the role of UNC13C in metastasis of OSCC, we performed transwell assay to assess the impact of UNC13C expression on cell migration and invasion abilities of SCC9, SAS, and OSC-20 cells. Transwell assays revealed that the migration and invasion abilities of the OSCC cells were significantly impaired following upon UNC13C overexpression. As shown in Figure 2B, SCC-9 and SAS cell lines showed high metastasis and invasive ability.
It is well-known that epithelial-mesenchymal transition (EMT) is an important event involved in the initiation of tumor invasion; the impact of UNC13C on the expression of EMT markers was analyzed in SCC-9 and SAS cells. UNC13C overexpression resulted in repression of E-cadherin, Vimentin, Twist, TCF8/ZEB1, Snail, and Slug, as well as induction of Claudin-1 expression in SCC-9 and SAS cells (Figure 3), while β-catenin exhibited no significant difference after UNC13C overexpression in SCC-9 and SAS cells. Taken together, these results demonstrate that the loss of UNC13C induces EMT and promotes migration, invasion of OSCC cells.

DISCUSSION
Here, we showed for the first time that UNC13C down regulation in OSCC tissues, consistently with these results, UNC13C was reduced in OSCC cell lines (Data not shown). In addition, UNC13C expression was significantly correlated with clinical stage, death, and overall survival of OSCC patients. Furthermore, multivariate analysis identified that UNC13C protein level may serve as an independent prognostic factor in patients with OSCC. For validating the role of UNC13C in EMT pathway in vitro, oral cancer cells analyzed for migration and invasion ability. As expected, impaired migration, and invasion were observed following UNC13C overexpression. All of these data indicate that UNC13C acts as a tumor suppressor in OSCC and that the downregulation of UNC13C may promote progression and metastasis of OSCC.
Metastasis is a multifactorial process (6), during the metastatic event epithelial cells lose their polarity, and acquire migratory and invasive abilities (7,8). We observed that UNC13C overexpression in oral cancer cells down regulated cellular migration and invasion and undergo a morphologic change like EMT. Recently, several factors that regulate EMT have been identified, SNAIL (SNAI1), SLUG (SNAI2), TWIST1, ZEB1, and ZEB2 (9, 10). In correlation with these altered migration and invasive capacities, we found that UNC13C stimulation induced tight Junction Protein -Claudin-1 expression and inhibited mesenchymal marker-Vimentin and transcription factors -Slug, Snail, Twist, and ZEB1 in both SCC-9 and SAS cell lines. Whereas, in SAS cell line, UNC13C overexpression decreased Vimentin, ZEB1, Snail and Slug expression and increased tight junction proteins Claudin-1. Decreased Ecadherin expression plays an important factor in cancer metastasis (11), however in our study UNC13C expression was negatively associated with E-cadherin expression in both SCC9 and SAS cell lines. The importance of the EMT in UNC13C-related metastatic functions of oral cancer needs further research. These results prove that UNC13C could inhibit cell migration, and invasion of OSCC cells through Caludin-1/Snail/Slug/Twist/ZEB1/Vimentin-signaling pathway.
In conclusion, this is the first study to examine the expression of UNC13C in OSCC. UNC13C expression was down regulated in OSCC samples, which predicts poor survival in patients with OSCC. Detection of UNC13C down-regulation in OSCC might be used to identify patients with poor prognosis in the future. In addition, our results suggest that restoration of UNC13C function represents a potential therapeutic strategy for treatment of metastatic OSCC.

Participants and Clinical Tissues
To evaluate the association of UNC13C expression with clinical/pathological factors and patient survival, a total of 268 oral cancer tissues samples (from January 2000 to December 2008) were obtained from 253 male and 15 female patients The migration and invasion capacities were determined by Transwell assay. Bar graphs represent quantitative data from three independent experiments. *P < 0.05 vs. non-transfected cells.  Cancer). The main treatment was tumor removal and radical neck dissection, including post-operation irradiation as well as selective patients treated with 5-FU and cisplatin chemotherapy.

Tissue Microarray and Immunohistochemical Staining
Tissue microarray and Immunohistochemical staining was performed as described previously (12). Then slides were incubated with UNC13C antibody (ab122725, Abcam, Cambridge, UK) in room temperature for 20 min. After washing three times with PBS, the sections were incubated with appropriate peroxidase-labeled secondary antibodies for 30 min at room temperature. The sections were washed three times with PBS and then labeled by diaminobenzidine and counterstained with Mayer's haematoxylin, dehydrated and mounted. Two experienced pathologists (Kun-Tu Yeh and Chang Wei-Hsiang) independently assessed the results of immunohistochemical staining and a final agreement was obtained for each score at a discussion microscope. The UNC13C expression levels were divided to three groups, negative (−), low expression (1+) and high expression (2+).

Cell Transfection
Prior transfection, cells were plated on 6-cm tissue culture dishes at a density of 4 × 10 5 cells/dish for overnight. A mixture of 4 µg DNA of pUNC13C-Myc, a constructed UNC13C expression plasmid (OriGene), and 12 µl TurboFect reagent (Thermo Fisher Scientific) in 400 µl DMEM/F12 (serum free) was added to cells and incubated for 24 h.

Cell Migration and Invasion Assays
Cell migration and invasion was assayed according to the methods described previously (13,14). After transfection for 24 h, the SCC-9 and SAS cells were harvested and seeded to upper Transwell (Greiner Bio-One, North Carolina, USA), and DMEM-F12 medium supplemented with 10% FBS (600 µl) was added in advance into Transwell lower chambers in a 24-well plate, then incubated for 24 h at 37 • C. The invasion assay was carried out as described above used matrigel (25 mg/50 mL, 60 µl; BD Biosciences, MA) coating on the upper Transwell. Finally, the migrating or invading cells were fixed with methanol and stained with Giemsa for 2 h, and used a light microscope to count the cells.

Western Blot Analysis
The cell lysates preparation using the methods as previously described (15,16). The cell extracts were separated by 8 or 10% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in TBS-T buffer (20 mM Tris, 137 mM NaCl, pH 7.4) with 5% nonfat milk for 1 h, and the blots were probed with the indicated primary antibodies for 24 h at 4 • C. After washing steps with TBS-T (10 min each) for three times, the blots were incubated with horseradish peroxidase anti-mouse IgG or goat anti-rabbit for 1 h at room temperature. Last, the blots were visualized by ECL reagent (Millipore), and the protein expression was detected by chemiluminescence.