Inhibition of proliferation, sprouting, tube formation and Tie2 signaling of lymphatic endothelial cells by the histone deacetylase inhibitor SAHA.

Histone deacetylase (HDAC) inhibitors exert potent inhibitory effects on various types of human cancer. The pioneer drug suberoylanilide hydroxamic acid (SAHA) is currently used in the clinic for cancer treatment. However, the effect of SAHA on tumor lymphangiogenesis is unclear. We recently showed that SAHA suppresses the expression and production of pro-lymphagenic factor vascular endothelial growth factor‑C (VEGF-C) in breast cancer cells. In the present study, the effect of SAHA on lymphatic endothelial cells (LECs) was examined. We generated a lymphatic-like endothelial cell line (named FP01) by overexpressing the master LEC transcription factor PROX1 in EA.hy926 endothelial cells. This cell line exhibited a gene expression pattern and phenotype similar to primarily cultured LECs. SAHA inhibited cell cycle progression and proliferation of FP01 cells. In addition, SAHA suppressed sprouting and tube formation in these cells. Moreover, SAHA attenuated the angiopoietin (Ang)/Tie signaling pathway which plays important roles in the regulation of LEC function. FP01 cells expressed Ang1, Ang2, Tie1 and Tie2, and SAHA dose-dependently reduced the expression of Tie2 in these cells. Tie2 promoter activity was attenuated by SAHA indicating a transcriptional repression. Importantly, Tie2 protein was significantly reduced by SAHA at the concentration in which no alteration of Tie2 mRNA was detected. We found that SAHA enhanced Tie2 protein degradation via the ubiquitin-proteasome pathway, and the expression of c-Cbl, the E3 ligase for Tie2 ubiquitination, rapidly increased after SAHA treatment. Knockdown of c-Cbl reversed SAHA‑induced Tie2 protein degradation. Taken together, our results demonstrate that SAHA impairs the proliferation, sprouting and tube formation of LECs and attenuates Ang/Tie signaling in LECs by downregulating Tie-2 via transcriptional and post-transcriptional mechanisms.