Autocrine loop between vascular endothelial growth factor (VEGF)-C and VEGF receptor-3 positively regulates tumor-associated lymphangiogenesis in oral squamoid cancer cells

Numerous past studies have suggested a critical role of the paracrine effect between tumor vascular endothelial growth factor (VEGF)-C and lymphatic VEGFR3/Flt-4 in solid tumor-associated lymphangiogenesis. In contrast, the pathophysiological role of tumor cell-associated VEGFR3/Flt-4 in tumor progression remains to be elucidated. Here, we investigated this role using a tumor implantation model. SAS cells, an oral squamous carcinoma cell line expressing both VEGF-C and VEGFR3/Flt-4 but neither VEGFR2/KDR/Flk-1/VEGFR2/KDR/Flk-1 nor VEGF-D were adopted for experiments. Stable transformants of dominant-negative (dn) SAS cells were established in which the cytoplasmic domain-deleted VEGFR3/Flt-4 was exogenously overexpressed, which can lead to inactivation of endogenous VEGFR3/Flt-4 through competitive antagonism and is associated with down-activation of endogenous FLT-4-related intracellular signals. In vitro and in vivo proliferation assays showed lower proliferative activity of dn-SAS cells. An immunohistochemical study revealed that the tumor lymphangiogenesis was significantly suppressed, and the level of human VEGF-C mRNA was significantly lower in dn-SAS cell-derived tumor tissues. Moreover, in vitro studies demonstrated that the significant suppression of VEGF-C and VEGF-A expression was evident in dn-SAS cells or wild-type SAS cells treated with either the VEGFR3/Flt-4 kinase inhibitor MAZ51 or the inhibitor of FLT-4-related signals. These findings together suggested that the VEGF-C/VEGFR3/Flt-4 autocrine loop in tumor cells was a potential enhancer system to promote Cancer progression, and VEGFR3/Flt-4 in tumor tissue might become an effective target for Cancer therapy.