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LAPTM4B facilitates late endosomal ceramide export to control cell death pathways

Nature Chemical Biology volume 11, pages 799–806 (2015)Cite this article

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Abstract

Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase–dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.

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Figure 1: LAPTM4B deficiency causes ceramide accumulation.
Figure 2: LAPTM4B-depleted cells accumulate ceramide in LEs.
Figure 3: LAPTM4B overexpression suppresses ceramide accumulation.
Figure 4: LAPTM4B interacts with ceramide.
Figure 5: LE ceramide sequestration confers protection against chemotherapy-induced caspase-3 cleavage.
Figure 6: LAPTM4B affects lysosome-associated cell death pathways by regulating LE sphingolipid content.

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Acknowledgements

The authors thank K. Sandhoff for helpful comments on the manuscript; A. Uro and D.J. Baek for technical support; S. Hautaniemi, R. Louhimo and S. Karinen for help with bioinformatics and the Biomedicum imaging unit for help with microscopy. This study was supported by Academy of Finland grants 273533 and 266092 (T.B.), 131489, 263841 and 272130 (E.I.), the Liv och Hälsa Foundation (T.B. and N.B.), the University of Helsinki Research Fund Grant (T.B.), the Ruth and Nils-Erik Stenbäck Foundation (T.B.), the Finnish Medical Foundation (E.I. and R.B.), the Sigrid Juselius Foundation (E.I.), the Magnus Ehrnrooth Foundation (N.B.), the Perklén Foundation (N.B.), the Swiss National Science Foundation (H.R.), the NCCR Chemical Biology (H.R.) and SystemsX.ch (H.R.).

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Authors and Affiliations

  1. Department of Anatomy, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Tomas Blom, Shiqian Li, Andrea Dichlberger, Nils Bäck & Elina Ikonen

  2. Minerva Foundation Institute for Medical Research, Helsinki, Finland

    Tomas Blom, Shiqian Li, Andrea Dichlberger & Elina Ikonen

  3. Department of Chemistry and Biochemistry, Queens College, City University of New York, Flushing, New York, USA

    Young Ah Kim & Robert Bittman

  4. Department of Biochemistry, University of Geneva, CH-1211 Geneva 4, Switzerland.,

    Ursula Loizides-Mangold & Howard Riezman

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Contributions

E.I. and T.B. conceived the project, designed and analysed experiments. T.B., S.L. and A.D. designed, carried out and analyzed experiments. N.B. carried out and analysed the electron microscopy experiments. R.B. conceived and designed the synthesis of the fluorescent ceramide probes, and Y.A.K. carried out the synthesis. H.R. planned and U.L.-M. carried out the lipid mass spectrometry and analysed the data. E.I. and T.B. wrote the manuscript; all authors read and commented on it.

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Correspondence to Tomas Blom or Elina Ikonen.

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Supplementary Text and Figures

Supplementary Figures 1–23, Supplementary Tables 1 and 2 and Supplementary Note (PDF 2772 kb)

Movement of LAPTM4B-Cherry and ceramide-BODIPY positive organelles along the ER

Stable A431 cells overexpressing LAPTM4B-Cherry were transfected with BFP-KDEL cDNA to label the ER. Starting at 24 h post-transfection, the cells were serum starved for 16 h. Cells were then pulsed with ceramide-BODIPY/LDL for 1 h, chased for 30 min in serum free medium and imaged by confocal microscopy. The image capture rate was ~0.4fps. See also Supplementary Figure 18. (MOV 10127 kb)

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Blom, T., Li, S., Dichlberger, A. et al. LAPTM4B facilitates late endosomal ceramide export to control cell death pathways. Nat Chem Biol 11, 799–806 (2015). https://doi.org/10.1038/nchembio.1889

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