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AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation

Nature Cell Biology volume 17, pages 20–30 (2015)Cite this article

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A Corrigendum to this article was published on 30 April 2015

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Abstract

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

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Figure 1: Ambra1 hemizygosity affects cell proliferation.
Figure 2: AMBRA1 is an interactor of PP2A
Figure 3: AMBRA1 affects c-Myc dephosphorylation at Ser 62.
Figure 4: Inhibition of mTOR affects c-Myc phosphorylation at Ser 62 in an AMBRA1-dependent manner.
Figure 5: AMBRA1 affects c-Myc phosphorylation and cell proliferation through its direct binding to PP2A-C.
Figure 6: Ambra1 affects tumorigenesis.

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Change history

  • 01 April 2015

    In the version of this Article originally published, incorrect western blot scans were provided for the actin panels in Figure 4h,i. These panels have been corrected online. All samples in 4i were collected and processed simultaneously, on the same or on parallel gels/blots.

References

  1. Parzych, K. R. & Klionsky, D. J. An overview of autophagy: morphology, mechanism, and regulation. Antioxid. Redox Signal. 20, 460–473 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Neufeld, T. P. Autophagy and cell growth–the yin and yang of nutrient responses. J. Cell Sci. 125, 2359–2368 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease. Cell 149, 274–293 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hosokawa, N. et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol. Biol. Cell 20, 1981–1991 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jung, C. H. et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol. Biol. Cell 20, 1992–2003 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hara, T. et al. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells. J. Cell Biol. 181, 497–510 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hosokawa, N. et al. Atg101, a novel mammalian autophagy protein interacting with Atg13. Autophagy 5, 973–979 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Nazio, F. et al. mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nat. Cell Biol. 15, 406–416 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Banreti, A., Lukacsovich, T., Csikos, G., Erdelyi, M. & Sass, M. PP2A regulates autophagy in two alternative ways in Drosophila. Autophagy 8, 623–636 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Sutter, B. M., Wu, X., Laxman, S. & Tu, B. P. Methionine inhibits autophagy and promotes growth by inducing the SAM-responsive methylation of PP2A. Cell 154, 403–415 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Puustinen, P. et al. CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation. J. Cell Biol. 204, 713–727 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Eichhorn, P. J., Creyghton, M. P. & Bernards, R. Protein phosphatase 2A regulatory subunits and cancer. Biochim. Biophys. Acta 1795, 1–15 (2009).

    CAS  PubMed  Google Scholar 

  13. Virshup, D. M. & Shenolikar, S. From promiscuity to precision: protein phosphatases get a makeover. Mol. Cell 33, 537–545 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Arroyo, J. D. & Hahn, W. C. Involvement of PP2A in viral and cellular transformation. Oncogene 24, 7746–7755 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Janssens, V. & Goris, J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417–439 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Janssens, V., Goris, J. & Van Hoof, C. PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 15, 34–41 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Sears, R. et al. Multiple Ras-dependent phosphorylation pathways regulate Myc protein stability. Genes Dev. 14, 2501–2514 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yeh, E. et al. A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells. Nat. Cell Biol. 6, 308–318 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Junttila, M. R. & Westermarck, J. Mechanisms of MYC stabilization in human malignancies. Cell Cycle 7, 592–596 (2008).

    Article  CAS  PubMed  Google Scholar 

  20. Fimia, G. M. et al. Ambra1 regulates autophagy and development of the nervous system. Nature 447, 1121–1125 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Di Bartolomeo, S. et al. The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy. J. Cell Biol. 191, 155–168 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cecconi, F. & Levine, B. The role of autophagy in mammalian development: cell makeover rather than cell death. Dev. Cell 15, 344–357 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Benato, F. et al. Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis. Autophagy 9, 476–495 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Malumbres, M. & Barbacid, M. Cell cycle, CDKs and cancer: a changing paradigm. Nat. Rev. Cancer 9, 153–166 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Hara, K. et al. Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action. Cell 110, 177–189 (2002).

    Article  CAS  PubMed  Google Scholar 

  26. Jayadeva, G. et al. B55alpha PP2A holoenzymes modulate the phosphorylation status of the retinoblastoma-related protein p107 and its activation. J. Biol. Chem. 285, 29863–29873 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Zhang, H. et al. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J. Clin. Invest. 112, 1223–1233 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dyson, H. J. & Wright, P. E. Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6, 197–208 (2005).

    Article  CAS  PubMed  Google Scholar 

  29. Lim, W. A., Richards, F. M. & Fox, R. O. Structural determinants of peptide-binding orientation and of sequence specificity in SH3 domains. Nature 372, 375–379 (1994).

    Article  CAS  PubMed  Google Scholar 

  30. Evans, B. J. et al. Physical association of GPR54 C-terminal with protein phosphatase 2A. Biochem. Biophys. Res. Commun. 377, 1067–1071 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Holzinger, A. et al. Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumor diagnosis. Hybridoma 15, 49–53 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Szabo, E. Lung epithelial proliferation: a biomarker for chemoprevention trials? J. Natl Cancer Inst. 93, 1042–1043 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Kauffman, S. L. Cell proliferation in the mammalian lung. Int. Rev. Exp. Pathol. 22, 131–191 (1980).

    CAS  PubMed  Google Scholar 

  34. Ayers, M. M. & Jeffery, P. K. Proliferation and differentiation in mammalian airway epithelium. Eur. Respir. J. 1, 58–80 (1988).

    CAS  PubMed  Google Scholar 

  35. White, E. Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 12, 401–410 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mathew, R. et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Marino, G. et al. Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3. J. Biol. Chem. 282, 18573–18583 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Takamura, A. et al. Autophagy-deficient mice develop multiple liver tumors. Genes Dev. 25, 795–800 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Inami, Y. et al. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J. Cell Biol. 193, 275–284 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Sontag, E. Protein phosphatase 2A: the Trojan Horse of cellular signaling. Cell Signal. 13, 7–16 (2001).

    Article  CAS  PubMed  Google Scholar 

  41. Kolupaeva, V. & Janssens, V. PP1 and PP2A phosphatases–cooperating partners in modulating retinoblastoma protein activation. FEBS J. 280, 627–643 (2013).

    Article  CAS  PubMed  Google Scholar 

  42. Millward, T. A., Zolnierowicz, S. & Hemmings, B. A. Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. 24, 186–191 (1999).

    Article  CAS  PubMed  Google Scholar 

  43. Cho, U. S. & Xu, W. Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445, 53–57 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Xu, Y. et al. Structure of the protein phosphatase 2A holoenzyme. Cell 127, 1239–1251 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Mathew, R. & White, E. Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night. Curr. Opin. Genet. Dev. 21, 113–119 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Guo, J. Y. et al. Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis. Genes Dev. 27, 1447–1461 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Guo, J. Y. et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 25, 460–470 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Qu, X. et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J. Clin. Invest 112, 1809–1820 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Liang, X. H. et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672–676 (1999).

    Article  CAS  PubMed  Google Scholar 

  50. Thoresen, S. B., Pedersen, N. M., Liestol, K. & Stenmark, H. A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic. Exp. Cell Res. 316, 3368–3378 (2010).

    Article  CAS  PubMed  Google Scholar 

  51. Helgason, G. V., Holyoake, T. L. & Ryan, K. M. Role of autophagy in cancer prevention, development and therapy. Essays Biochem. 55, 133–151 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Smith, K. & Dalton, S. Myc transcription factors: key regulators behind establishment and maintenance of pluripotency. Regen. Med. 5, 947–959 (2010).

    Article  CAS  PubMed  Google Scholar 

  53. Burgess, A. et al. Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc. Natl Acad. Sci. USA 107, 12564–12569 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Junttila, M. R. et al. CIP2A inhibits PP2A in human malignancies. Cell 130, 51–62 (2007).

    Article  CAS  PubMed  Google Scholar 

  55. Nusslein-Volhard, C. & Dahm, R. Zebrafish. A Practical Approach (Oxford Univ. Press, 2002).

    Google Scholar 

Download references

Acknowledgements

We wish to thank M. Canney, V. Unterkircher, R. Laricchia and M. Salomé for excellent technical assistance, and M. Acuña Villa and M. W. Bennett for editorial and secretarial work. We also thank S. Campello for critical reading of the manuscript. We are indebted to R. Sears (Portland, Oregon, USA), A. C. Gingras (Toronto, Canada) and A. Teleman and K. Dimitriadis (Heidelberg, Germany) for providing us with V5–Flag–c-Myc and Flag–PR65A constructs and Tsc2 MEFs, respectively, and to S. Cannata (Rome) for his advice on histopathology. This work was supported by grants from KBVU (R72-A4408), Lundbeck Foundation (R167-2013-16100), Novo Nordisk Foundation (7559), The Bjarne Saxhof Foundation, AIRC (IG2010 and IG2012 to both F.C. and M.P.), and in part from FISM (2009), the Telethon Foundation (GGP10225), the Italian Ministry of University and Research (PRIN 2009 and FIRB Accordi di Programma 2011) and the Italian Ministry of Health (RF 2009). V.C. is supported by the Lundbeck Foundation (R165-2013-15982). Also, we are grateful to the Spanish Ministry of Economy and Competitiveness (MINECO) (PS09/01401; PI12/02248, FR2009-0052 and IT2009-0053) and to Fundación Mutua Madrileña (AP101042012) for funding the laboratory of G.V.

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Author notes

  1. Maria Salazar & Pier Federico Gherardini

    Present address: Present addresses: Cell Division and Cancer Group, Spanish National Cancer Research Centre, Madrid E-28029, Spain (M.S.); Department of Microbiology and Immunology, Centre for Clinical Science Research, Stanford University School of Medicine, 94305 Stanford, California, USA (P.F.G.).,

Authors and Affiliations

  1. Unit of Cell Stress and Survival, Danish Cancer Society Research Center, 2100 Copenhagen, Denmark

    Valentina Cianfanelli, Daniela De Zio & Francesco Cecconi

  2. Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, 00143 Rome, Italy

    Valentina Cianfanelli, Francesca Nazio, Matteo Bordi & Francesco Cecconi

  3. Department of Biology, University of Rome ‘Tor Vergata’, 00133 Rome, Italy

    Claudia Fuoco, Pier Federico Gherardini, Manuela Antonioli, Melania D’Orazio, Manuela Helmer-Citterich, Mauro Piacentini, Sabrina Di Bartolomeo & Francesco Cecconi

  4. Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain

    Mar Lorente, Maria Salazar & Guillermo Velasco

  5. Instituto de Investigaciones Sanitarias San Carlos (IdISSC), 28040 Madrid, Spain

    Mar Lorente, Maria Salazar & Guillermo Velasco

  6. Skin and Extracellular Matrix Research Group, Anatomy NUI Galway, Ireland

    Fabio Quondamatteo

  7. National Institute for Infectious Diseases IRCCS ‘L. Spallanzani’, 00149 Rome, Italy

    Manuela Antonioli, Gian Maria Fimia & Mauro Piacentini

  8. Department of Biology, University of Padua, 35131 Padua, Italy

    Tatjana Skobo & Luisa Dalla Valle

  9. Unit of Cell Death and Metabolism, Danish Cancer Society Research Center, 2100 Copenhagen, Denmark

    Mikkel Rohde

  10. Department of Dermatology, University Freiburg Medical Center, 79104 Freiburg, Germany

    Christine Gretzmeier & Joern Dengjel

  11. ZBSA Center for Biological Systems Analysis, University of Freiburg, 79104 Freiburg, Germany

    Christine Gretzmeier & Joern Dengjel

  12. Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce 73100, Italy

    Gian Maria Fimia

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Contributions

V.C. performed most experiments with crucial help from: C.F., M.B. and F.Q. (immunohistochemistry analysis); M.L. and M.S. (xenograft assay); P.F.G., M.R. and M.H-C. (bioinformatic analysis); D.D.Z. (real-time PCR); F.N. and M.A. (mutagenesis and cloning); M.D’O. (gel-filtration assay); T.S. and L.D.V. (experiment in zebrafish); C.G. and J.D. (mass-spectrometry analysis). G.M.F. and G.V. provided critical reagents. S.D.B. discussed the results and commented on the manuscript; V.C. and F.C. wrote the manuscript, with suggestions from M.P., G.M.F. and G.V.; F.C. and V.C. conceived and designed the research.

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Correspondence to Francesco Cecconi.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the interaction region between AMBRA1 and PP2A.

(a) HEK293 cells were transfected with vectors encoding for Flag-AMBRA1 and Flag-WD40 AMBRA1. Protein extracts were immunoprecipitated using an anti-PP2A-C antibody. Purified complexes and corresponding total extracts were analysed by western blot using an anti-Flag and anti-PP2A-C antibodies. The arrow and the bracket indicate bands corresponding to Flag-AMBRA1 and Flag-WD40 AMBRA1, respectively. (b) HEK293 cells were transfected with vectors encoding for Myc-AMBRA1 and AMBRA1 deletion constructs, called Myc-F1, Myc-F2, Myc-F3. Protein extracts were immunoprecipitated using an anti-PP2A-C antibody. Purified complexes and corresponding total extracts were analysed by western blot using an anti-Myc and anti-PP2A-C antibodies. (c) HEK293 cells were transfected with vectors encoding for AMBRA1 deletion constructs called Myc-F1, Myc-F1ΔWD40, Myc-F3, Myc-F3a and Myc-F3b. Protein extracts were treated as in a. Asterisks (∗) indicate the specific bands.

Supplementary Figure 2 Analysis of the banding patterns of overexpressed AMBRA1 and endogenous C-MYC.

(a) HeLa cells were transfected with increasing concentrations of AMBRA1 cDNAs. Protein extracts were analysed by Western Blot analysis using antibodies against AMBRA1 and TUBULIN. Different bands can be appreciated on AMBRA1 over-expression, with the band indicated by the arrowhead corresponding to the expected size-range for full-length AMBRA1. The other bands, with lower molecular weights, correspond to AMBRA1 full-length cleavage products. (b) HeLa cells were transfected with control oligos and plasmid (first lane), AMBRA1 cDNA alone (second lane) or AMBRA1 cDNA in combination with siRNA against AMBRA1 (third lane). Protein extracts were analysed by Western Blot analysis with the same antibodies as in b. (c) Primary MEFs have been knocked-down for c-Myc by specific siRNAs (c-Myc siRNA); unspecific oligos have been used as a control (CTRL siRNA). Protein extracts were analysed by Western Blot using antibodies against c-Myc (anti-c-Myc N262) and Tubulin. Unspecific bands [indicated by asterisks (∗)] are not affected by c-Myc siRNA, while central bands, corresponding to c-Myc protein, are down-regulated by the c-Myc siRNA treatment. We did not report the unspecific bands in the c-Myc Western Blots throughout the manuscript for a major clarity. (d) HeLa cells have been knocked-down for C-MYC by specific siRNAs (C-MYC siRNA); unspecific oligos have been used as a control (CTRL siRNA). Protein extracts were analysed by Western Blot using antibodies against C-MYC (anti C-MYC N262) and Tubulin. Unspecific bands [indicated by asterisks (∗)] are not affected by C-MYC siRNA, while central bands, corresponding to C-MYC protein, are down-regulated by the C-MYC siRNA treatment. We did not report the unspecific bands in the C-MYC Western Blots throughout the manuscript for a major clarity. Two exposure of the same Western Blot are shown: in the longer exposure it is possible to appreciate the residual signal corresponding to overexpressed AMBRA1 in silenced cells (third lane), probably due to a small percentage of cells not transfected with the siRNA. (e) NCI-H1299 and A549 cells were knocked-down for AMBRA1 and protein extracts were analysed by Western Blot using antibodies against AMBRA1 and HSP90, as a loading control.

Supplementary Figure 3 Analysis of c-Myc regulators and PP2A-C substrates in AMBRA1-defective cells.

(a) Protein extracts from AMBRA1 knocked-down cells were analysed by western blot using antibodies against AKT, phospho-AKT, GSK3B and phospho-GSK3B. (b) Western blot analysis of phospho-Erk1/2 and total Erk1/2 in Ambra1 +/+ and Ambra1gt/gt MEFs.

Supplementary Figure 4 Characterization of AMBRA1NT-PX and AMBRA1CT-PXP mutants.

(a) HEK293 cells were transfected with vectors encoding for AMBRA1WT and AMBRA1NT-PXP. Protein extracts were immunoprecipitated using an anti-PP2A-C antibody and the immunocomplexes were analysed by western blot, using an anti-AMBRA1 and anti-PP2A-C antibodies. (b) HEK293 cells were transfected with vectors encoding for AMBRA1WT and AMBRA1CT-PXP. Protein extracts were treated as in a. (c) HeLa cells, transfected with βGal, as a control, AMBRA1WT or AMBRA1PXP, were lysed and proteins extracts were analysed for p62 levels and LC3I to LC3II conversion by western blot analysis. (d) HEK293 cells were transfected with vectors encoding for AMBRA1WT and AMBRA1PXP. Endogenous BECLIN 1 was immunoprecipitated from protein extracts and the immunocomplexes were analysed by western blot, using antibodies against BECLIN 1 and AMBRA1. (e) HEK293 cells were transfected as in d. Endogenous ULK1 was immunoprecipitated from protein extracts and the immunocomplexes were analysed by western blot, using antibodies against ULK1 and AMBRA1. The vertical line represents a splice mark of samples on the same gel. The brackets indicate bands corresponding to over-expressed AMBRA1.

Supplementary Figure 5 Analysis of Ambra1 levels, cell proliferation and autophagy in Ambra1 +/gt mice.

(a) Analysis of Ambra1-mRNA levels in tissues of, wild-type (+/+) and heterozygous (+/gt) 3-months old Ambra1 mice. Data are presented as means ± s.e.m. and significance is ∗∗P < 0.005 (n = 3 mice per each genotype). (b) We extracted proteins from lung, liver and kidney organs of wild-type or heterozygous for Ambra1 3-months old mice. Ambra1 levels were analysed by western blot using an anti-Ambra1 and anti-Actin antibody. (c) Analysis of Ambra1 levels in lung and liver organs from wild-type (Ambra1 +/+) or heterozygous (Ambra1 +/gt) mice. We selected both tumour tissue and the surrounding healthy tissue from the same animal. Ambra1 protein levels were monitored by western blot, using an anti Ambra1 antibody. (d) p62 immunostaining on liver of Ambra1 heterozygous (Ambra1 +/gt) mice. T: tumour. Scale bar, 100 μm. (e) Western blot analysis of p62 in liver of Ambra1 heterozygous (Ambra1 +/gt) mice. Both tumour tissue and the surrounding healthy tissue were analysed from the same animal.

Supplementary Figure 6 Study of the functional correlation between AMBRA1 and P C-MYCS62 levels in MCF-7.

(a) Western blot analysis of AMBRA1 and P C-MYCS62 in human breast cell lines (MCF-10A cells, as a control, and the breast cancer derived cells, MCF-7). We observe an inverse correlation between AMBRA1 and P C-MYCS62 levels. On the right panel, MCF-7 cells were reconstituted for AMBRA1 and the levels of P C-MYCS62 were analysed by western blot. Brea. E.: Breast epithelium; Brea. C.: Breast cancer. (b) Graph showing the correlation between AMBRA1 (white columns) and P C-MYCS62 (black columns) protein levels in lung cell lines analysed in a. Data are presented as means ± s.d. and significance is ∗P < 0.05; ∗∗P < 0.005 (n = 6 independent experiments). (c) MCF-7 cells, overexpressing AMBRA1 or βGal as control, by retroviral infection, were analysed by MTS assay at different time points. Data are presented as means ± s.d. and significance is ∗∗P < 0.005 (n = 3 independent experiments). (d) The tumorigenicity of the cells used in c was assessed by a colony formation assay in soft agar. Data are presented as means ± s.d. and significance is ∗∗P < 0.005 (n = 4 independent experiments).

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Cianfanelli, V., Fuoco, C., Lorente, M. et al. AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation. Nat Cell Biol 17, 20–30 (2015). https://doi.org/10.1038/ncb3072

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