Slug protects from apoptosis and reduces invasion in in vitro and in vivo neuroblastoma models. Free

B185

Neuroblastoma (NB) derives from embryonic neural crest precursors that undergo malignant transformation at some stage of differentiation into neural crest derivatives. Despite intensive multimodal treatment, metastatic NB is still characterized by poor prognosis. Slug is a transcriptional regulator that belongs to a small family of development-regulatory genes. During embryonic development, Slug is involved in the delamination of neural crest cells and in their migration to distant sites. In this study, we assessed the relevance of Slug expression for apoptosis resistance and for invasion potential of NB cells in vitro and in vivo. Slug expression was detectable in 8 out of 10 human NB cell lines analyzed. Two Slug-expressing cell lines (LAN-5 and HTLA-230) were infected with the lentiviral vector pLKO-Slug that encodes a miRNA directed against Slug mRNA. After selection, both pLKO-Slug infected cell lines expressed strongly reduced levels of Slug. The expression of some pro-apoptotic and anti-apoptotic markers (p53, Bax, Bcl-2), previously described as Slug targets, was analyzed by immunoblotting. Bcl-2 expression was decreased in pLKO-Slug infected cells. Slug down-regulation also caused an increased sensitivity to apoptosis induced by etoposide (1 μM) treatment. Invasion capability of pLKO-Slug infected cells was strongly reduced in Matrigel-coated invasion chambers. Interestingly, the selective tyrosine kinase inhibitor Imatinib Mesylate (IM) also reduced invasion of NB cells in similar assays. In vivo, we tested the ability of Slug-down-regulated NB cells to invade using a pseudo-metastatic model. pLKO-Slug HTLA-230 and control (pLKO-GFP infected HTLA-230) cells were injected in the tail vein of SCID mice. Injected animals were divided in 4 groups: pLKO-Slug untreated; pLKO-Slug treated with IM (200 mg/Kg/day); control (pLKO-GFP) untreated; control (pLKO-GFP) treated with IM (200 mg/Kg/day). After 35 days mice were sacrificed and tumor burden was evaluated. Animals injected with pLKO-Slug had fewer tumors than both untreated and IM-treated pLKO-GFP controls. Tumor inhibition was significantly more evident in pLKO-Slug injected animals treated with IM. Our data demonstrate that Slug protects NB cells from apoptosis induced by cytotoxic drugs and increases their invasion capability in vitro and in vivo. Inhibition of Slug expression, possibly combined with exposure to IM, could represent a suitable strategy to decrease NB metastatic potential.