Letter | Published:

PTEN counteracts FBXL2 to promote IP3R3- and Ca2+-mediated apoptosis limiting tumour growth

Nature volume 546, pages 554558 (22 June 2017) | Download Citation

Abstract

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the ‘quasisynaptical’ feeding of calcium to the mitochondria to promote oxidative phosphorylation1. However, persistent Ca2+ release results in mitochondrial Ca2+ overload and consequent apoptosis2. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca2+-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes3) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca2+ influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca2+ release from the endoplasmic reticulum and sensitization to Ca2+-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer4. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten/ mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca2+ mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca2+-dependent cytotoxicity after irradiation with visible light5,6. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor7, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca2+ overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.

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Acknowledgements

The authors thank W. Dai, R. Parsons, M. Phillips and K. Tanaka for reagents; E. DeMarchi, C. Espiritu, G. Rona and E. Schenkein for their contribution to this study, and L. Cantley and B. Neel for advice. M.P. and P.P. are grateful to T. M. Thor and C. degli. Scrovegni, respectively, for continuous support. This work was funded by grants from the NIH to MP, and grants from AIRC (IG-18624 and MFAG-13521), funds from Ferrara’s University (5x1000), the Italian Ministry of Health, and Cariplo to S.M., C.G. and P.P. A.S., L.F. and M.P.W. are supported by the Stowers Institute for Medical Research. M.P. is an Investigator with the Howard Hughes Medical Institute.

Author information

Author notes

    • Shafi Kuchay
    •  & Carlotta Giorgi

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA

    • Shafi Kuchay
    • , Carlotta Giorgi
    • , Daniele Simoneschi
    • , Julia Pagan
    •  & Michele Pagano
  2. NYU Perlmutter Cancer Center, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA

    • Shafi Kuchay
    • , Carlotta Giorgi
    • , Daniele Simoneschi
    • , Julia Pagan
    •  & Michele Pagano
  3. Howard Hughes Medical Institute, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA

    • Shafi Kuchay
    • , Julia Pagan
    •  & Michele Pagano
  4. Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy

    • Carlotta Giorgi
    • , Sonia Missiroli
    •  & Paolo Pinton
  5. The Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA

    • Anita Saraf
    • , Laurence Florens
    •  & Michael P. Washburn
  6. Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA

    • Michael P. Washburn
  7. Department of Pathology at Icahn School of Medicine at Mount Sinai, New York, New York 10029 USA

    • Ana Collazo-Lorduy
    • , Mireia Castillo-Martin
    •  & Carlos Cordon-Cardo
  8. Spanish Society of Medical Oncology, Madrid, Spain

    • Ana Collazo-Lorduy
  9. Department of Pathology at Champalimaud Centre for the Unknown, Lisbon, Portugal

    • Mireia Castillo-Martin
  10. Drug Discovery Department, Moffitt Cancer Center, and Department of Oncologic Sciences, University of South Florida, Tampa, Florida 33612, USA

    • Said M. Sebti

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Contributions

S.K. and C.G. planned and performed most experiments and helped to write the manuscript. D.S. generated the CRISPR knock-in and heterozygous clones. M.P. directed and coordinated the study, oversaw all results, and wrote the manuscript. P.P. supervised the study and oversaw the results. A.L., M.M. and C.C. performed immunohistochemical analysis. S.S. provided advice for the in vivo experiments. A.S., L.F. and M.W. performed the mass spectrometry analysis. J.P. and S.M. helped with some experiments. All authors discussed the results and commented on the manuscript.

Competing interests

S.S. is co-inventor of GGTI-2418 and Chief Scientific Officer of Prescient Therapeutics that has licensed GGTI-2418. All other authors declare no competing financial interests.

Corresponding authors

Correspondence to Paolo Pinton or Michele Pagano.

Reviewer Information Nature thanks R. Parsons, C. Taylor, W. Wei and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    This file contains the uncut scans of the Western blots presented in main figures and Extended Data figures.

  2. 2.

    Supplementary Table 1

    This file contains the ANOVA statistical analysis. Source data analyzed by ANOVA for the indicated figures is summarized in a tabular format.

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https://doi.org/10.1038/nature22965

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