Tie2 Regulates Tumor Metastasis of Oral Squamous Cell Carcinomas

Abstract

The endothelial-specific receptor, tyrosine kinase with immunoglobulin-like loops and epidermal growth factor homology domains-2 (Tie2) is a member of the tyrosine kinase family and is ubiquitous in normal tissues; however, little is known about the mechanisms and roles of Tie2 in oral squamous cell carcinomas (OSCCs). In the current study, we investigated the expression status of Tie2 in OSCCs by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemistry and the functional mechanisms of Tie2 using its overexpressed OSCC (oeTie2) cells and Tie2 blocking by its antibody. We found that Tie2 expression was down-regulated significantly (p < 0.05) in OSCCs compared with normal counterparts in vitro and in vivo. Interestingly, oeTie2 cells showed higher cellular adhesion (p < 0.05) and lower cellular invasion (p < 0.05) compared with control cells; whereas there was similar cellular proliferation in both transfectants. Furthermore, cellular adhesion was inhibited and invasion was activated by Tie2 function-blocking antibody (p < 0.05), indicating that Tie2 directly regulates cellular adhesion and invasion. As expected, among the clinical variables analyzed, Tie2-positivity in patients with OSCC was correlated closely with negative lymph node metastasis. These results suggested for the first time that Tie2 plays an important role in tumor metastasis and may be a potential biomarker for OSCC metastasis.

Evidence for critical role of Tie2/Ang1 interaction in metastatic oral cancer

“…oeTie2 cells, which were established in our previous study (14), were grown in Dulbecco's modified Eagle medium (DMEM) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Sigma-Aldrich; Merck KGaA) and 50 units/ml of penicillin and streptomycin (Sigma-Aldrich; Merck KGaA). mRNA expression analysis.…”

“…The protein concentration was measured using a commercial Bradford reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Immunoblot analysis was performed as described previously (14,(34)(35)(36)(37). Briefly, protein extracts (20 µg) were electrophoresed on 4-12% Bis-Tris gel (Invitrogen; Thermo Fisher Scientific, Inc.), transferred to polyvinylidene fluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc.), and blocked for 1 h at room temperature in Blocking One (Nacalai Tesque Inc., Kyoto, Japan).…”

“…cDNA was generated from 5 µg of total RNA using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare Life Sciences, Little Chalfont, UK) and oligo (dT) primers (Hokkaido System Science Co., Ltd., Sapporo, Japan). As described previously (14), real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed using the LightCycler 480 apparatus (Roche Diagnostics GmbH, Mannheim, Germany). Primers were designed using the Universal Probe Library Assay Design Center (http://lifescience.roche.com/), which specifies the most suitable set.…”

“…An adhesion assay was performed as described previously (14,40). Briefly, the cells were seeded in collagen I-coated 96-well plates, incubated for 1 h at 37˚C at a density of 2x10 4 cells/well, and incubated for 1 h in DMEM, washed once with PBS, fixed in methanol, stained with crystal violet, and photographed.…”

“…Therefore, elucidation of the molecular mechanisms involved in cancer metastasis clearly is needed to improve the prognosis (6,13). We reported previously that endothelial cell-specific tyrosine-protein kinase receptor (Tie2) is related closely to OSCC metastasis using overexpressed Tie2 (oeTie2) cells and its neutralization technique (14).…”