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RBL2/p130 is a direct AKT target and is required to induce apoptosis upon AKT inhibition in lung cancer and mesothelioma cell lines

Oncogene volume 37, pages 3657–3671 (2018)Cite this article

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Abstract

The retinoblastoma (RB) protein family includes RB1/p105, RBL1/p107, and RBL2/p130, which are key factors in cell-cycle regulation and stand at the crossroads of multiple pathways dictating cell fate decisions. The role of RB proteins in apoptosis is controversial because they can inhibit or promote apoptosis depending on the context, on the apoptotic stimuli and on their intrinsic status, impacting on the response to antitumoral treatments. Here we identified RBL2/p130 as a direct substrate of the AKT kinase, a key antiapoptotic factor hyperactive in multiple cancer types. We showed that RBL2/p130 and AKT1 physically interact and AKT phosphorylates RBL2/p130 Ser941, located in the pocket domain, but not when this residue is mutated into Ala. We found that pharmacological inhibition of AKT, through the highly selective AKT inhibitor VIII (AKTiVIII), impairs RBL2/p130 Ser941 phosphorylation and increases RBL2/p130 stability, mRNA expression and nuclear levels in both lung cancer and mesothelioma cell lines, mirroring the more extensively studied effects on the p27 cell-cycle inhibitor. Consistently, AKT inhibition reduced cell viability, induced cell accumulation in G0/G1, and triggered apoptosis, which proved to be largely dependent on RBL2/p130 itself, as shown upon RBL2/p130 silencing. AKT inhibition induced RBL2/p130-dependent apoptosis also in HEK-293 cells, in which re-expression of a short hairpin-resistant RBL2/p130 was able to rescue AKTiVIII-induced apoptosis upon RBL2/p130 silencing. Our data also showed that the combination of AKT and cyclin-dependent kinases (CDK) inhibitors, which converge on the re-activation of RBL2/p130 antitumoral potential, could be a promising anticancer strategy.

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Acknowledgements

This work was supported by the AIRC- Associazione Italiana per la Ricerca sul Cancro, IG 2014-15690 to AG and by the Italian Ministry of Health. We are grateful to the Sbarro Health Research Organization (www.shro.org) for its support and for providing the custom phosphoRBL2/p130S941 antibody, and to the Commonwealth of Pennsylvania. We are grateful to our intern students Flora Magnotti, Michela Napolitano, Caterina Miro and Sonia Sodano for technical help. We are grateful to Flavio Rizzolio for sharing RBL2/p130 silencing vectors and to Enrico Bucci for helpful discussion. AG is also Director of the Cell Cycle and Cancer Research Line at CROM, Istituto Nazionale Tumori; Naples. FP is also Adjunct Associate professor at Temple University, Department of Biology, Philadelphia, PA, USA.

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Authors and Affiliations

  1. Oncology Research Center of Mercogliano (CROM), Istituto Nazionale Tumori – IRCCS, “Fondazione G. Pascale”, 80131, Napoli, Italy

    Francesca Pentimalli, Iris M. Forte, Luca Esposito, Carmelina A. Iannuzzi, Luigi Alfano, Caterina Costa & Daniela Barone

  2. Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Scienceand Technology, Temple University, Philadelphia, PA, 19122, USA

    Paola Indovina & Antonio Giordano

  3. Department of Medicine, Surgery and Neuroscience, University of Siena, 53100, Siena, Italy

    Carmelina A. Iannuzzi, Daniela Barone & Antonio Giordano

  4. Division of Thoracic Surgery, Department of Thoracic Surgery and Oncology, Istituto Nazionale Tumori “Fondazione G. Pascale”; IRCCS, 80131, Napoli, Italy

    Gaetano Rocco

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  1. Francesca Pentimalli
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Pentimalli, F., Forte, I.M., Esposito, L. et al. RBL2/p130 is a direct AKT target and is required to induce apoptosis upon AKT inhibition in lung cancer and mesothelioma cell lines. Oncogene 37, 3657–3671 (2018). https://doi.org/10.1038/s41388-018-0214-3

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