Tumor cell-derived vascular endothelial growth factor (VEGF)-C has been primarily implicated in promoting lymphangiogenesis by activating Flt-4 (VEGFR-3) expressed on lymphatic endothelial cells via a paracrine mechanism. Flt4 has also been shown to be expressed selectively in subsets of cancer cells. However, little is known about the functional role of VEGF-C/Flt4 signaling via an autocrine mechanism, as well as the clinicopathological implication of the VEGF-C/Flt4 axis and its downstream effector molecules, in head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC). In the present study, we detected Flt-4 expression selectively in several HNSCC cell lines by quantitative PCR, and its internalization reflecting receptor activation was confirmed by immunocytochemistry in SAS and HO1U1 cells. Flt-4 stimulation upregulated the expression of contactin-1 (CNTN-1, a neural cell adhesion molecule) and VEGF-C itself in SAS cells, while Flt-4 inhibition downregulated the expression of CNTN-1 in both SAS and HO1U1 cells and that of VEGF-C itself in SAS cells. In vitro cell proliferation and migration assays using SAS cells demonstrated that both cell proliferation and migration were promoted by Flt-4 stimulation, while those were suppressed by Flt-4 inhibition. Clinicopathological factors and immunohistochemical expression of Flt-4, VEGF-C, and CNTN-1 in tumor cells were evaluated using surgical specimens from patients with tongue squamous cell carcinoma. We found a significant correlation of CNTN-1 expression with both VEGF-C and Flt-4 expression, but not between VEGF-C and Flt-4. Multivariate logistic regression analysis revealed that T classification (P = 0.003), lymphatic invasion (P = 0.024), and Flt-4 expression in tumor cells (P = 0.046) were independently predictive of neck lymph node metastasis. These results suggest that the VEGF-C/Flt-4 axis in tumor cells enhances tumor cell proliferation and migration via upregulating the expression of VEGF-C itself and CNTN-1 in an autocrine manner, thereby contributing to cancer progression of OSCC, including neck metastasis. Hence, targeting the VEGF-C/Flt-4 axis in tumor cells can be an attractive therapeutic strategy for the treatment of cancer.
Keywords: Flt-4; VEGF-C; autocrine manner; contactin-1; metastasis; migration; oral squamous cell carcinoma; proliferation; tumor cell.