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Slug (SNAI2) Down-Regulation by RNA Interference Facilitates Apoptosis and Inhibits Invasive Growth in Neuroblastoma Preclinical Models

Authors' Affiliations: ¹ Section of Toxicology and Biomedical Sciences, ENEA Research Center Casaccia; ² Department of Experimental Oncology, Regina Elena Cancer Institute; ³ Department of Pediatrics, La Sapienza University; ⁴ Laboratory of Oncology, Bambino Gesù Children's Hospital, Rome, Italy; ⁵ Department of Biological Technologies, Wyeth Research, Cambridge, Massachusetts; ⁶ Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania; and ⁷ Division of Child Health, School of Reproductive and Developmental Medicine, Liverpool University, Liverpool, United Kingdom

Requests for reprints: Giuseppe Raschellà, Section of Toxicology and Biomedical Sciences, ENEA Research Center Casaccia, Via Anguillarese, 301-00123 Rome, Italy. Phone: 39-0630483172; Fax: 39-0630486559; E-mail: raschella{at}casaccia.enea.it.

Purpose: We assessed the relevance of Slug (SNAI2) for apoptosis resistance and invasion potential of neuroblastoma cells in vitro and in vivo.

Experimental Design: We evaluated the effect of imatinib mesylate on invasion and analyzed the genes modulated by imatinib mesylate treatment in neuroblastoma cells. Slug expression, inhibited by imatinib mesylate treatment, was knocked down in neuroblastoma cells by RNA interference, and the effects on invasion and apoptosis were evaluated in vitro. A pseudometastatic model of neuroblastoma in severe combined immunodeficient mice was used to assess the effects of Slug silencing alone or in combination with imatinib mesylate treatment on metastasis development.

Results: Microarray analysis revealed that several genes, including Slug, were down-regulated by imatinib mesylate. Slug expression was detectable in 8 of 10 human neuroblastoma cell lines. Two Slug-expressing cell lines were infected with a vector encoding a microRNA to Slug mRNA. Infected cells with reduced levels of Slug were tested for the expression of apoptosis-related genes (p53, Bax, and Bcl-2) identified previously as Slug targets. Bcl-2 was down-regulated in Slug-interfered cells. Slug down-regulation increased sensitivity to apoptosis induced by imatinib mesylate, etoposide, or doxorubicin. Invasion of Slug-silenced cells was reduced in vitro. Animals injected with Slug-silenced cells had fewer tumors than controls and the inhibition of tumor growth was even higher in animals treated with imatinib mesylate.

Conclusions: Slug down-regulation facilitates apoptosis induced by proapoptotic drugs in neuroblastoma cells and decreases their invasion capability in vitro and in vivo. Slug inhibition, possibly combined with imatinib mesylate, may represent a novel strategy for treatment of metastatic neuroblastoma.