Ⅰ. INTRODUCTION
According to the Globocan report in 2018, lip and oral cavity cancer is the 17th most common cancer worldwide, accounting for about 2.2% of all cancers.1 More than 90% of oral cancers are diagnosed as oral squamous cell carcinoma (OSCC). Although there has been much understanding of the molecular pathogenesis of oral cancer over the past few decades, the majority of patients with oral cancer still die from local recurrence and metastasis of tumors. It is wellknown that cervical lymph node (LN) metastasis is a major prognostic marker affecting patient survival in OSCC.2 Therefore, it is very important to develop reliable molecular markers that can predict LN metastasis and block the formation of metastatic lesions to improve patient’s prognosis in OSCC.
The KiSS-1 metastasis suppressor gene was first identified in human melanoma cell lines.3 The KiSS-1 gene encodes 145-amino acid protein, which is subsequently cleaved into several types of Kisspeptins, including Kisspeptin-54.4 Kisspeptins bind to the KiSS-1 receptor (KiSS-1R) and have diverse physiologic functions, such as gonadotropin-releasing hormone release, puberty initiation, placentation, and regulation of reproduction.5 Kisspeptin-54 (also called metastin) is known to play an anti-metastatic role by suppressing colonization at secondary sites.6 Downregulated expression of KiSS-1 has been reported in various human malignancies including melanoma, breast, stomach, pancreatic, and kidney cancers.6 Our previous study demonstrated that 36.4% of OSCC cases showed decreased expression of KiSS-1 and its downregulation significantly correlated with cervical LN metastasis, worse clinical outcome, and lower survival rate of patients.7 It suggested that KiSS-1 seems to play a role in the tumor progression of OSCC and may be a new molecular target to inhibit the LN metastases in patients with OSCC. Therefore, further investigation was needed to identify the function of KiSS-1 using OSCC cell line and to evaluate the possibility of KiSS-1 as a new therapeutic target. In the present study, we examined the expression of KiSS-1 and KiSS-1R in OSCC cell lines and investigated whether recovery of KiSS-1 can suppress proliferation and/or migration of OSCC cells or not.
Ⅱ. MATERIALS and METHODS
1. Cell lines
Nine OSCC cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, HO-1-N-1, HO-1-U-1, KOSCC-11, KOSCC-25B, and KOSCC-33A) were cultured in Dulbecco’s modified Eagle’s medium (DMEM), a mixture of DMEM and F12, or RPMI 1640. Each media was supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic antimycotics. All media and FBS were purchased from WELGENE Inc. (Seoul, Korea). All cell lines were cultured in a 37ﾟC incubator with 5% CO2.
2. Western blot analysis
Cells were lysed on ice in RIPA buffer with 1.0% mammalian protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). The mixture was centrifuged for 30 min at 12,000g at 4ﾟC. The BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA) was used to determine the concentration of protein. Samples (30 μg) were separated by 12% SDS/ polyacrylamide gels and transferred to PVD membranes (Pall Corporation, Port Washington, NY, USA). After blocking for 1h with 3% skim milk at room temperature, the membranes were incubated overnight at 4ﾟC with anti-human KiSS-1 rabbit polyclonal antibody (1:500, ab19028; Abcam, Cambridge, UK) or incubated with anti-human KiSS-1R rabbit polyclonal antibody (1:1000, ab137483; Abcam) for 1h at room temperature right after overnight at 4ﾟC. After washing, membranes were then probed with 1:5000 diluted secondary antibody for 2 h 30 min at room temperature. The membrane was developed with a West-Q Chemiluminescent Substrate Plus Kit (GenDEPOT, Barker, TX, USA).
3. In vitro cell proliferation assay
To assess the function of KiSS-1 on the cell growth of OSCC cells, KiSS-1 peptide (metastin) was purchased from Phoenix Pharmaceuticals Inc (Burlingame, CA, USA). Among the 9 OSCC cell lines, KOSCC-25B cells and Ca9-22 cells were selected and treated with metastin. Cells were seeded into 96-well plates at 1x104 cells/well and cultured using complete media. After 24h, media containing metastin (1μ mol/L or 10μmol/L) was replaced to each well. For a negative control, PBS was added instead of metastin. After treatment for 24, 48, or 72h, cells were counted using WST-1 assay kit (Roche Applied Science, Mannheim, Germany).
4. In vitro cell migration assay
500μL DMEM (1% FBS) containing 7x104 cells were added to each upper chamber of 24 well-plate and supplemented with metastin (10μmol/L) or PBS. The lower chamber was filled with 500μL of media containing 10% FBS. After a 48h incubation, cells on the upper surface of filter were removed. Migrated cells on the lower surface were fixed and stained with Diff-Quick and counted in five randomly selected fields at a magnification of x100.
5. Statistical analysis
Student t-test was used to analyze the data from the In vitro assay. P<0.05 was considered statistically significant.
Ⅲ. RESULTS
1. Expression analyses of KiSS-1 and KiSS-1R in OSCC cell lines by western blotting
Expression of KiSS-1 and KiSS-1R protein was evaluated in 9 OSCC cell lines by western blot analysis. Most of OSCC cell lines showed absence or low expression of KiSS-1 protein (Fig. 1A). In addition, almost all kinds of OSCC cell lines showed more than moderate expression of KiSS-1R (Fig.1B). Ca9-22, KOSCC-25B, and KOSCC-33A showed relatively strong expression of KiSS-1R. Among these 3 cell lines showing high expression, KOSCC-25B and Ca9-22 cell lines were selected for the treatment of metastin.
2. Effect of metastin on the growth of KOSCC-25B and Ca9-22 cells
The effect of KiSS-1 on cell proliferation was observed at two different concentrations of metastin (1 μmol/L and 10 μmol/L). Control group was treated with PBS instead of metastin. In the cell proliferation assay, there was no effect of metastin on cell growth of both two cell lines (Fig.2). These data indicate that KiSS-1 doesn’t affect the tumor cell growth as it has been known.
3. Effect of metastin on the migration of KOSCC- 25B and Ca9-22 cells
To determine the effect of metastin on the cell migration of KOSCC-25B and Ca9-22 cells, the transwell migration assay was performed. After a 48h incubation with the treatment of metastin (10 μmol/L), there was a statistically significant decrease in cell migration of both KOSCC-25B and Ca9-22 cells compared to the control group (Fig.3A and 3B; P<0.001, respectively). These data indicate that KiSS-1 can suppress the cell migration of OSCC In vitro.
Ⅳ. DISCUSSION
Cervical LN metastasis has been well known to be an important prognostic factor closely related to the survival of patients with OSCC. Nodal disease can decrease survival rates of patients by 50% or more, being estimated to be 35% to 45%.2 Therefore, prevention and early detection of metastasis are very important to increase the survival rate of patients. Metastasis is one of the hallmarks of cancer and a very complex multistep process. The metastatic process can be divided into six sequential steps: local invasion, intravasation, survival in the circulation, arrest at distant organ site and extravasation, micrometastasis formation, and metastatic colonization.8 Each of steps is controlled through the complex interactions between the metastasis promoters and the metastasis suppressors. Therefore, therapeutic intervention may be either to develop drugs that inhibit metastasis promoters or to develop drugs capable of restoring the expression of a metastatic suppressor or mimicking its functions.9 Metastasis suppressor genes are defined as molecules that inhibit the formation of metastatic lesions without affecting the growth of primary tumor.10 Since 1986, more than thirty metastasis suppressor genes including KiSS-1 have been identified. Metastasis suppressors have diverse mechanisms of action and each of them regulates different steps of the metastatic cascades.11
The role of KiSS-1 as a metastasis suppressor has been investigated using human cancer tissues since the first study of KiSS-1 in cutaneous melanoma.12 In various malignancies such as bladder and nasopharyngeal cancers, loss of KiSS-1 expression has been shown to significantly correlate with metastasis, advanced clinical stage, and worse survival of patients.13,14 In addition, several studies demonstrated that KiSS-1 expression could be an independent prognostic factor for overall survival, disease-specific survival or metastasis- free survival of patients with bladder, nasopharygeal, or ovarian cancers.13,14,15KiSS-1 expression in OSCC was also found to be an independent prognostic marker for overall and disease-free survivals.7
Despite the significant correlation between loss of KiSS-1 expression and adverse clinical outcome of cancer patients, the underlying molecular mechanism of this phenomenon is still unclear. Therefore, In vitro and/or in vivo studies have been conducted to elucidate molecular mechanisms of downregulation of metastasis suppressor. According to In vitro studies of several malignancies, re-expression of KiSS-1 inhibited cell migration and invasion in kidney, prostate, and head and neck cancer.13,16,17 In addition, several in vivo experiments demonstrated re-expression of KiSS-1 could reduce lung metastasis by 50-80% as compared to controls.17-19 Our study also showed that recovery of KiSS-1 expression using treatment of metastin could suppress the migration of OSCC cells.
The mechanism by which KiSS-1 regulates the cell migration of OSCC is still unclear. According to the study in urothelial carcinoma, expression of KiSS-1 inhibited the transcriptional activity of NF-kB and consequently downregulated the expression of MMP-9, thereby reducing the migration and invasion of bladder cancer cells.19 Therefore, it is necessary to further investigate whether KiSS-1 can inhibit the cell migration through regulation of MMP-9 expression in OSCC.
In conclusion, our study showed that KiSS-1 re-expression could significantly inhibit cell migration of OSCC cells. This result supports our previous report demonstrating down- regulation of KiSS-1 may play an important role in the progression and metastasis of OSCC. Therefore, recovery of KiSS-1 expression may be one of the new therapeutic strategies to suppress metastasis of OSCC by regulating cancer cell migration.
