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Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma

Oncogene volume 35, pages 3839–3853 (2016)Cite this article

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Abstract

A limiting factor in the therapeutic outcome of children with high-risk neuroblastoma is the intrinsic and acquired resistance to common chemotherapeutic treatments. Here we investigated the molecular mechanisms by which the hemisynthetic cardiac glycoside UNBS1450 overcomes this limitation and induces differential cell death modalities in both neuroblastic and stromal neuroblastoma through stimulation of a cell-type-specific autophagic response eventually leading to apoptosis or necroptosis. In neuroblastic SH-SY5Y cells, we observed a time-dependent production of reactive oxygen species that affects lysosomal integrity inducing lysosome-associated membrane protein 2 degradation and cathepsin B and L activation. Subsequent mitochondrial membrane depolarization and accumulation of mitochondria in phagophores occurred after 8h of UNBS1450 treatment. Results were confirmed by mitochondrial mass analysis, electron microscopy and co-localization of mitochondria with GFP-LC3, suggesting the impaired clearance of damaged mitochondria. Thus, a stress-induced defective autophagic flux and the subsequent lack of clearance of damaged mitochondria sensitized SH-SY5Y cells to UNBS1450-induced apoptosis. Inhibition of autophagy with small inhibitory RNAs against ATG5, ATG7 and Beclin-1 protected SH-SY5Y cells against the cytotoxic effect of UNBS1450 by inhibiting apoptosis. In contrast, autophagy progression towards the catabolic state was observed in stromal SK-N-AS cells: here reactive oxygen species (ROS) generation remained undetectable preserving intact lysosomes and engulfing damaged mitochondria after UNBS1450 treatment. Moreover, autophagy inhibition determined sensitization of SK-N-AS to apoptosis. We identified efficient mitophagy as the key mechanism leading to failure of activation of the apoptotic pathway that increased resistance of SK-N-AS to UNBS1450, triggering rather necroptosis at higher doses. Altogether we characterize here the differential modulation of ROS and mitophagy as a main determinant of neuroblastoma resistance with potential relevance for personalized anticancer therapeutic approaches.

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Abbreviations

ATG:

autophagy-related

CG:

cardiac glycoside

FOXO:

Forkhead box O

GFP:

green fluorescent protein

LAMP-2:

lysosome-associated membrane protein 2

LC3:

microtubule-associated protein 1A/1B light-chain 3

PKB/AKT:

protein kinase B

ROS:

reactive oxygen species

SQSTM1:

sequestosome-1

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Acknowledgements

FR thanks ‘Een Haerz fir kriibskrank Kanner’ association for initial support. This manuscript is dedicated to the memory of Marian Aldred. The authors are very thankful to ThinkPinkLux and Europadonna Luxembourg for their generous support. FR and AG are supported by a Télévie grant (Fonds National de la Recherche Scientifique, Belgium). CC is supported by a ‘Waxweiler grant for cancer prevention research’ from the Action LIONS ‘Vaincre le Cancer’. Research at LBMCC is financially supported by the Fondation de Recherche Cancer et Sang, the Recherches Scientifiques Luxembourg association, the ‘Een Haerz fir kriibskrank Kanner’ association, the Action LIONS ‘Vaincre le Cancer’ association and the Télévie Luxembourg. Research at SNU is supported by the National Research Foundation (NRF) by the MEST of Korea for Tumor Microenvironment Global Core Research Center (GCRC) grant (grant number 2012–0001184) and by Brain Korea (BK21) PLUS program. The authors thank Justine Paoli for technical assistance with electron microscopy.

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Authors and Affiliations

  1. Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, Luxembourg, Luxembourg

    F Radogna, C Cerella, A Gaigneaux & M Dicato

  2. de Médecine de Nancy, Université de Lorraine, Vandoeuvre-les-Nancy, France

    C Christov

  3. College of Pharmacy, Seoul National University, Gwanak-gu, Seoul, Korea,

    M Diederich

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  1. F Radogna
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Correspondence to M Diederich.

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Radogna, F., Cerella, C., Gaigneaux, A. et al. Cell type-dependent ROS and mitophagy response leads to apoptosis or necroptosis in neuroblastoma. Oncogene 35, 3839–3853 (2016). https://doi.org/10.1038/onc.2015.455

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