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Development of a yeast-based system to identify new hBRAFV600E functional interactors

Oncogene volume 38, pages 1355–1366 (2019)Cite this article

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Abstract

BRAFV600E is a mutant Ser–Thr protein kinase that plays a crucial role in many types of cancer, including melanoma. Despite several aspects of BRAFV600E biology have been already elucidated, the proteins that regulate its expression and activity remain largely unknown, hampering our capacity to control its unrestrained effects. Here, we propose yeast Saccharomyces cerevisiae as a model system that can be used to achieve a better understanding of the regulation of human BRAFV600E.

By showing that in osmotic stress conditions hBRAFV600E can rescue the growth of strains carrying a double or triple deletion in MAPKKK belonging to the HOG pathway, we demonstrate that this oncogenic kinase is active in yeast even if it does not have an ortholog. Moreover, we report that, in the yeast ptp3∆ptc1∆ strain that is deleted in the genes encoding for two phosphatases responsible for Hog1 de-phoshorylation, hBRAFV600E mimics the toxicity observed in the presence of constitutive Hog1 activation. Finally, we exploit such a toxicity to perform a functional screening of a human cDNA library, looking for cDNAs able to rescue yeast growth. In this way, we identify SMIM10, a mitochondrial protein that in melanoma cells selectively downregulates BRAFV600E RNA and protein levels, by acting indirectly at the post-transcriptional level. Upon SMIM10 overexpression, BRAFV600E melanoma cells show disrupted mitochondrial structure/function and undergo senescence. They also show decreased ability to proliferate and form colonies, as well as increased sensitivity to the BRAF inhibitor vemurafenib. Interestingly, the analysis of TCGA melanoma samples indicates that patients with higher SMIM10 levels have a better prognosis. Therefore, these data suggest that SMIM10 exerts an oncosuppressive role in melanoma cells.

Taken together, our results unveil the potential of S. cerevisiae to study hBRAFV600E, to populate the network of its functional interactors and, in doing so, to uncover new cancer-associated genes with therapeutic potential.

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Acknowledgements

The authors would like to thank all the Poliseno and Galli lab members, as well as A. Tuccoli, for helpful discussions. They also thank E. O’Shea (Department of Molecular and Cellular Biology, Harvard University, Cambridge, United States, USA) for providing the ECY46 yeast strain. This work was supported by: Progetto Giovani Ricercatori (University of Padova) to GC; Start up funding ITT and Progetto Giovani Ricercatori #GR-2011-02348535 (Ministero della Salute) to LP. It was also partially supported by AIRC grant #MFAG 17095 to LP; Fondazione Pisa grant #127/16 as well as AIRC grant #IG 14477 to AG.

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Authors and Affiliations

  1. Oncogenomics Unit, Core Research Laboratory, ISPRO, Pisa, Italy

    Simone Lubrano, Andrea Marranci & Laura Poliseno

  2. Institute of Clinical Physiology, IFC-CNR, Pisa, Italy

    Simone Lubrano, Laura Comelli, Chiara Piccirilli, Andrea Marranci, Mario Chiariello, Alvaro Galli, Laura Poliseno & Tiziana Cervelli

  3. Department of Biology, University of Pisa, Pisa, Italy

    Simone Lubrano & Federica Gemignani

  4. Signal Transduction Unit, Core Research Laboratory, ISPRO, Siena, Italy

    Francesca Dapporto & Mario Chiariello

  5. Scuola Normale Superiore, Pisa, Italy

    Elena Tantillo

  6. FPS-Pisa Science Foundation, Pisa, Italy

    Elena Tantillo

  7. Department of Pharmacology, UC San Diego Moores Cancer Center, La Jolla, CA, USA

    J. Silvio Gutkind

  8. Unit of Experimental Biology and Genetics, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy

    Alessandra Salvetti

  9. Lab of Cancer Genomics, Fondazione Edo and Elvo Tempia, Biella, Italy

    Giovanna Chiorino

  10. Department of Molecular Medicine, University of Padova, Padova, Italy

    Giorgio Cozza

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  1. Simone Lubrano
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Correspondence to Alvaro Galli, Laura Poliseno or Tiziana Cervelli.

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Lubrano, S., Comelli, L., Piccirilli, C. et al. Development of a yeast-based system to identify new hBRAFV600E functional interactors. Oncogene 38, 1355–1366 (2019). https://doi.org/10.1038/s41388-018-0496-5

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  • DOI: https://doi.org/10.1038/s41388-018-0496-5

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