Ephrin-B2 reverse signaling regulates progression and lymph node metastasis of oral squamous cell carcinoma

<div><p>Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck and frequently metastasizes to cervical lymph nodes. Aggressive local invasion and metastasis of OSCC are significant factors for poor prognosis. In this study, we investigated whether ephrin-B2 expressed in OSCC contributed to tumor progression and lymph node metastasis. Clinical specimens from patients with OSCC had robust ephrin-B2-positive tumor cells and ephrin-B2 protein level was associated with clinical stage, lymph node metastasis, and poor survival outcomes. We also determined that ephrin-B2 protein level was increased in OSCC cell lines compared to normal human oral keratinocytes and that its levels were associated with the migratory and invasive potential of OSCC cell lines. Transfection of an <i>EFNB2</i>–specific small interfering RNA (siRNA) into SAS-L1 cells significantly reduced proliferation, attachment, migration, and invasion through phosphorylation of the epidermal growth factor receptor, FAK, ERK1/2, p38, AKT, and JNK1/2 pathways. Furthermore, knockdown of <i>EFNB2</i> significantly suppressed adhesion and transmigration of SAS-L1 cells toward human lymphatic endothelial cells. In addition, the growth rate of tumor xenografts and cervical lymph node metastases of OSCC were suppressed by local injection of <i>EFNB2</i> siRNA. These results suggest that ephrin-B2 overexpression and activation of the ephrin-B2 reverse signaling pathway in tumor microenvironment in OSCC facilitates progression and lymph node metastasis via enhancement of malignant potential and interaction with surrounding cells.</p></div

Associations between ephrin-B2 protein level and clinicopathological factors.

<p>Associations between ephrin-B2 protein level and clinicopathological factors.</p

Correlation between ephrin-B2 protein level and malignant potential of OSCC cell lines.

<p>(<b>A</b>) Total cell lysates were extracted, and ephrin-B2 protein level was determined by western blot analysis. The numbers of (<b>B</b>) viable, (<b>C</b>) migrating, and (<b>D</b>) invading cells were determined using the WST-8, wound healing, and invasion assays, respectively. Values are presented as means ± standard deviation (SD); n = 8 per group.</p

Effect of <i>EFNB2</i> knockdown on tumor growth and lymph node metastasis through interactions with HLECs.

<p>(<b>A</b>) Control and <i>EFNB2</i> siRNA-transfected SAS-L1 cells were seeded on HLEC cultures as a monolayer in the presence of 2 μg/mL Fc, ephrin-B2-Fc, or Eph-B4-Fc. GFP-positive cells attached to HLECs were counted. <b>(B</b>) Control and <i>EFNB2</i> siRNA-transfected SAS-L1 cells were labeled with CytoTracker™ and placed over the HLEC monolayer. After 24 h, SAS-L1 cells that transmigrated toward the HLEC monolayer were lysed, and fluorescence intensity was measured. Values are presented as means ± standard deviation (SD); n = 8 per group. *<i>p</i> < 0.05 compared to Fc-treated cells and ^†<i>p</i> < 0.05 compared to mock-transfected cells. <b>(C)</b> BALB/c nu/nu mice were treated as described in the Materials and Methods. Each group contained 8 mice. Volume of xenograft tumors elicited by injection of SAS-L1 cells to the dorsal flank region of mice was assessed for 40 days. Tumor size was determined every 4 days. Values are presented as means ± standard deviation (SD). ^†<i>p</i> < 0.05 compared to control mice. (<b>D</b>) SCID mice were treated as described in the Materials and Methods. Each group contained 8 mice. Number of metastatic cervical lymph nodes of control and <i>EFNB2</i> siRNA-treated mice was determined. Values are presented as means ± standard deviation (SD). ^†<i>p</i> < 0.05 compared to control mice. (<b>E</b>) Ephrin-B2 protein levels in tongues of eight control and <i>EFNB2</i> siRNA-treated SCID mice on day 19 was examined by IHC.</p

Effect of <i>EFNB2</i> knockdown on phosphorylation of protein kinases in SAS-L1 cells.

<p>Phosphorylation status of kinases in whole cell lysates from control and <i>EFNB2</i> siRNA-transfected SAS-L1 cells was analyzed. Changes in ten representative proteins, which were approximately 40% less hyper-phosphorylated in <i>EFNB2</i> knockdown cells compared to control cells, are shown.</p