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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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.
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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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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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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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DOI: https://doi.org/10.1038/ncb3072
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