Abstract 2477: Autophagy is correlated with chemoresistance in neuroblastoma

Abstract

Abstract Neuroblastoma (NB) is a frequent pediatric tumor. After combined treatments of chemotherapy, bone marrow transplantation, surgery and/or radiotherapy, metastatic NBs still have a poor prognosis. Therefore, finding new therapeutic strategies to increase the survival rate of patients with NB is essential. Autophagy is a self-degradative process induced primarily by starvation and, with the intermediate of lysosomes, damaged macromolecules and cell organelles are degraded. This degradation insures cell survival by adapting to stress conditions. In addition, recent studies proposed that autophagy may contribute to cancer resistance to chemotherapy and radiotherapy. However, in some circumstances, autophagy can induce non-apoptotic programmed cell death. The aim of this study is to determine how autophagy is regulated and whether it is associated with chemoresistance in NB. Firstly, tissue Microarray blocks containing 184 patients NB were used for an immunohistochemistry study in order to identify the expression of LC3, a cytosolic protein required for autophagic vacuole (autophagosome) formation, and beclin 1, a positive regulator of autophagy. Secondly, in vitro and in vivo (NOD/SCID/IL2Rαc-null mice) studies were performed to determine the level of autophagy in NB cells following chemotherapy. Finally, autophagy was inhibited in NB cells with shRNA targeting Atg5 (an essential protein for autophagy) or with hydroxychloroquine (HCQ), a pharmacological inhibitor of autophagy. NB cells were further treated with conventional drugs used in NB treatments to evaluate if they retain their ability to resist to chemotherapy. Cell survival was measured using MTT cell proliferation assay. Autophagy was detected by labelling the autophagic vacuoles with monodansylcadaverine (MDC) and by Western blot analysis of LC3 cleavage and Atg5 expression. Our study demonstrated that autophagy is present at low levels in a majority of NB. LC3 expression was not correlated with any clinical pathological data. On the other hand, Beclin1 expression in NB was higher in children older than one year of age who have a poor prognosis. Also, it had a higher level in primitive tumors than in metastases. In our in vitro and in vivo studies, autophagy, which was detected by cleavage of LC3 and by MDC test, was correlated with increasing concentrations of therapeutic agents. Interestingly, inhibition of autophagy with either Atg5 shRNA or HCQ strongly increased the sensitivity of NB cells to chemotherapy.Overall, these results suggest that autophagy contributes to NB cells resistance to chemotherapy. Therefore, inhibition of autophagy in combination with current treatments may be of great interest in order to improve therapeutic strategies of NB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2477. doi:1538-7445.AM2012-2477