Transcriptional factor snail controls tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma

BACKGROUND Snail, a transcriptional factor and repressor of E-cadherin is well known for its role in cellular invasion. It can regulate epithelial to mesenchymal transition (EMT) during embryonic development and in epithelial cells. Snail also mediates tumor progression and metastases. Silencing of Snail and its associate member Slug in human A2780 ovarian epithelial carcinoma cell line was investigated to identify its role in tumor neovascularization. METHODS Live cell sialidase, WST-1 cell viability and immunohistochemistry assays were used to evaluate sialidase activity, cell survival and the expression levels of tumor E-cadherin, N-cadherin, VE-cadherin, and host endothelial CD31+(PECAM-1) cells in archived paraffin-embedded ovarian A2780, A2780 Snail shRNA GIPZ lentiviral knockdown (KD) and A2780 Slug shRNA GIPZ lentiviral KD tumors grown in RAGxCγ double mutant mice. RESULTS Oseltamivir phosphate (OP), anti-Neu1 antibodies and MMP-9 specific inhibitor blocked Neu1 activity associated with epidermal growth factor (EGF) stimulated A2780 ovarian epithelial carcinoma cells. Silencing Snail in A2780 cells abrogated the Neu1 activity following EGF stimulation of the cells compared to A2780 and A2780 Slug KD cells. OP treatment of A2780 and cisplatin-resistant A2780cis cells reproducibly and dose-dependently abated the cell viability with a LD50 of 7 and 4 μm, respectively, after 48 h of incubation. Heterotopic xenografts of A2780 and A2780 Slug KD tumors developed robust and bloody tumor vascularization in RAG2xCγ double mutant mice. OP treatment at 50 mg/kg daily intraperitoneally did not significantly impede A2780 tumor growth rate but did cause a significant reduction of lung metastases compared with the untreated and OP 30mg/kg cohorts. Silencing Snail in A2780 tumor cells completely abrogated tumor vascularization, tumor growth and spread to the lungs in RAGxCγ double mutant mice. A2780 and A2780 Slug KD tumors expressed high levels of human N- and VE-cadherins, and host CD31+ endothelial cells, while A2780 Snail KD tumors expressed E-cadherin and reduced host CD31+ cells. OP 50mg/kg cohort tumors had reduced numbers of host CD31+ cells compared to a higher expression levels of CD31+ cells in tumors from the untreated control and OP 30mg/kg cohorts. CONCLUSION Snail transcriptional factor is an important intermediate player in human ovarian tumor neovascularization.