Fenretinide (4-HPR) Targets Caspase-9, ERK 1/2 and the Wnt3a/β-Catenin Pathway in Medulloblastoma Cells and Medulloblastoma Cell Spheroids.

Barbara Bassani,Elisa Principi,Arianna Pagani,Massimo Zollo,Desirèe Bartolini,Adriana Albini,Antonino Bruno,Douglas M Noonan,Javier S Castresana

doi:10.1371/journal.pone.0154111

Abstract

Medulloblastoma (MB), a neuroectodermal tumor arising in the cerebellum, represents the most frequent childhood brain malignancy. Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl) retinamide (4-HPR, fenretinide), a synthetic analogue of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopreventive and chemotherapeutic agent for various neoplasms, from breast cancer to neuroblastoma. Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB. Flow cytometry analysis was performed to evaluate 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects. Functional analysis to determine 4-HPR ability to interfere with MB cell migration and invasion were performed. Western Blot analysis were used to investigate the crucial molecules involved in selected signaling pathways associated with apoptosis (caspase-9 and PARP-1), cell survival (ERK 1/2) and tumor progression (Wnt3a and β-catenin). We show that 4-HPR induces caspase 9-dependent cell death in DAOY and ONS-76 cells, associated with increased ROS generation, suggesting that free radical intermediates might be directly involved. We observed 4-HPR induction of cell cycle arrest in G1/S phase, inactivated β-catenin, and inhibition of MB cell migration and invasion. We also evaluated the ability of 4-HPR to target MB cancer-stem/cancer-initiating cells, using an MB spheroids model, followed by flow cytometry and quantitative real-time PCR. 4-HPR treatment reduced DAOY and ONS-76 spheroid formation, in term of number and size. Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities. Finally, we analyzed 4-HPR ability to inhibit MB tumor cell growth in vivo in nude mice. Taken together, our data suggest that 4-HPR targets both parental and MB tumor stem/initiating cell-like populations. Since 4-HPR exerts low toxicity, it could represent a valid compound in the treatment of human MB.

Highlights

Medulloblastoma (MB) is a highly aggressive pediatric tumor of the cerebellum, usually located in the posterior fossa and represents the most common malignancy of the cerebellum in childhood, accounting for 13–20% of all pediatric central nervous system tumors [1, 2].Current treatments include the combination of surgical resection, whole brain and spinal cord radiation and aggressive systemic multidrug-chemotherapy [3, 4]
Mittal et al demonstrated that fenretinide exhibited a relevant anti-tumor activity on endometrial cancer cells, both in vitro and in vivo [69]. 4-HPR has been reported to decrease cell viability of endometrial cells inducing apoptosis mediated by poly ADP-ribose polymerase (PARP) and caspase-9 activation [69]
Antiangiogenic and angiopreventive properties by 4-HPR have been reported [31, 60, 70]. 4-HPR shows potential synergy with cytotoxic drugs: the combinations 4HPR/cisplatin, 4HPR/paclitaxel and 4HPR/etoposide were more than additive compared with monotherapy in human lung cancer cells [19, 71]and the combination of fenretinide/indole-3carbinol has increased the cytotoxic activity compared with agents administered as monotherapy by enhancing apoptosis in human breast cancer cell lines [72]

Summary

Introduction

Medulloblastoma (MB) is a highly aggressive pediatric tumor of the cerebellum, usually located in the posterior fossa and represents the most common malignancy of the cerebellum in childhood, accounting for 13–20% of all pediatric central nervous system tumors [1, 2]. The knowledge of the MB molecular profiling has led to several attempts at targeted therapies [14, 15] in preclinical studies and still open clinical trials that focused their attention mainly on SHH pathway antagonists, and among all the inhibitors of Smoothened (SMO) [11, 13] Mostly of these molecules might be ineffective in a clinical context due to secondary resistence onset in treated patients, suggesting that further studies are needed [12, 13]. Our data demonstrated that 4-HPR is able to target both parental and MB tumor stem/initiating cell-like populations, suggesting that fenretinide might represent a valid compound in the treatment of human MB

Materials and Methods

Findings

Discussion