FoxO3a represses lymphangiogenesis in gastric cancer

Abstract

Lymph node metastasis is the most important prognostic factor and is associated with about 60% of gastric cancer. For cancer cells to metastasize to lymph nodes, lymphangiogenesis is required. FoxO3a, a tumor suppressive transcription factor, is known to be a negative regulator of angiogenesis. Despite that lymphangiogenesis and angiogenesis in cancer are interrelated processes, the role of Foxo3a in lymphangiogenesis remains unknown. Thus, the goal of the study is to investigate the clinical significance and the role of FoxO3a in lymph node metastasis and lymphangiogenesis in gastric cancer. Foxo3a expression was analyzed across a large number of gastric cancer patient tissue samples with reverse phase protein microarray (RPPA). To study the biological function, FoxO3a was knocked down with RNA interference or overexpressed with an expression vector in gastric cancer cells. Tube formation and migration assays were carried out in human lymphatic endothelial cells (HLECs) with conditioned media of FoxO3a-silenced or overexpressed gastric cancer cells, respectively. To assess the DNA binding activity of FoxO3a, electrophoretic mobility shift assay (EMSA) was performed. RPPA analysis revealed that FoxO3a expression was inversely correlated with lymph node metastasis and high expression of phosphorylated FoxO3a was a poor prognostic factor in gastric cancer patients. Silencing of FoxO3a in gastric cancer cells profoundly induced VEGF-C expression and secretion, increased tube formation, and migration of HLECs treated with the conditioned media while overexpression of FoxO3a in gastric cancer cells showed the opposite effects. EMSA with supershift assay demonstrated that FoxO3a binds to the FHRE in the promoter region of VEGF-C. Treatment of LY294002, a PI3K pathway inhibitor, reversed the phosphorylation of FoxO3a and increased the nuclear localization of FoxO3a in gastric cancer cells. Collectively, our data clearly illustrate that FoxO3a binds to VEGF-C promoter and transcriptionally represses the expression of VEGF-C thereby inhibiting tumor lymphangiogenesis.