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AKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions

Nature Cell Biology volume 21, pages 226–237 (2019)Cite this article

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Abstract

Aberrant activation of AKT disturbs the proliferation, survival and metabolic homeostasis of various human cancers. Thus, it is critical to understand the upstream signalling pathways governing AKT activation. Here, we report that AKT undergoes SETDB1-mediated lysine methylation to promote its activation, which is antagonized by the Jumonji-family demethylase KDM4B. Notably, compared with wild-type mice, mice harbouring non-methylated mutant Akt1 not only exhibited reduced body size but were also less prone to carcinogen-induced skin tumours, in part due to reduced AKT activation. Mechanistically, the interaction of phosphatidylinositol (3,4,5)-trisphosphate with AKT facilitates its interaction with SETDB1 for subsequent AKT methylation, which in turn sustains AKT phosphorylation. Pathologically, genetic alterations, including SETDB1 amplification, aberrantly promote AKT methylation to facilitate its activation and oncogenic functions. Thus, AKT methylation is an important step, synergizing with PI3K signalling to control AKT activation. This suggests that targeting SETDB1 signalling could be a potential therapeutic strategy for combatting hyperactive AKT-driven cancers.

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Fig. 1: AKT methylation promotes its activity and oncogenic functions.
Fig. 2: Methylation-deficient Akt1 knock-in mice display reduced body size and weight and are resistant to chemical carcinogen-induced skin tumorigenesis in vivo.
Fig. 3: SETDB1 methylates AKT on K140 and K142 to promote its kinase activity.
Fig. 4: Oncogenic function of SETDB1 depends on the activation of AKT.
Fig. 5: SETDB1-mediated methylation of AKT synergizes with PI3K to activate AKT.
Fig. 6: KMD4B demethylates AKT to inhibit AKT kinase activity.
Fig. 7: Deficiency of SETDB1 inhibits AKT kinase activity and oncogenic function.

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Data availability

The MS-based screening data generated in this study have been deposited in ProteomeXchange under the accession code PXD011657. The SETDB1, EZH2, PTEN, EGFR, PIK3CA and AKT1 genetic alterations in TCGA datasets were integrated from the cBioPortal database (www.cbioportal.org), with the query of genes “ESET”, “EZH2”, “PTEN”, “EGFR”, “PIK3CA” and “AKT1” for both mutation and copy number alterations (CNAs) in different cancer types, such as BRCA and melanoma. Information regarding each cancer study is as follows: one dataset for BRCA (TCGA, provisional); one dataset for melanoma (TCGA, provisional). The source data for Figs. 1e,f,h,j–m, 2b,c,g,h, 3n,p,q, 4c,f,h,j,l,n, 6m–o and 7b,d,j,l,n, and Supplementary Figs. 1l,m, 2e,f, 3o, 4c–f,h and 7e,g,i have been provided as Supplementary Table 2. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors thank B. North, J. Zhang, F. Dang and other Wei Lab members for critical reading of the manuscript, as well as members of the Pandofi and Toker Laboratory for helpful discussions. The authors thank H. Okada (Kindai University of Medicine) for the generation of Kdm4bflox/flox MEFs. W.G. is supported by K99CA207867 from the National Cancer Institute. W.W. is a LLS research scholar. This work was supported in part by the NIH grant CA177910 (to W.W. and A.T.). The MS work was partially supported by NIH grants P01CA120964 (to J.A.) and P30CA006516 (to J.A.)

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Authors and Affiliations

  1. The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

    Jianping Guo

  2. Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Jianping Guo, Xiangpeng Dai, Nana Zheng, Wenjian Gan, Alex Toker & Wenyi Wei

  3. Institute of Translational Medicine, The First Hospital, Jilin University, Changchun, China

    Xiangpeng Dai

  4. Division of Newborn Medicine and Epigenetics Program, Department of Medicine, Boston Children’s Hospital, Boston, MA, USA

    Benoit Laurent & Yang Shi

  5. Key Laboratory of Systems Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai, China

    Jian Zhang

  6. Cell Signaling Technology Inc., Danvers, MA, USA

    Ailan Guo

  7. Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Min Yuan & John M. Asara

  8. Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Pengda Liu

  9. Division of Genetics, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

    Pier Paolo Pandolfi

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Contributions

J.G. designed and performed most of the experiments with assistance from X.D., B.L., W.G., P.L. W.W., P.P.P. and Y.S. A.T. supervised the study. J.G., N.Z. and J.Z. performed the revision. A.G. performed the IAP-LC-MS/MS screen. M.Y. and J.M.A. performed the MS work. J.G. and W.W. wrote the manuscript. All authors commented on the manuscript.

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Correspondence to Wenyi Wei.

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Integrated supplementary information

Supplementary Figure 1 Mass spectrometry-based non-biased approach to identify Akt methylation.

a, Tri-methylation sites and proteins were identified from OVCAR5 cells with the IAP-LC-MS approach (also see Table S1). This screen was repeated twice. b, Immunoblot (IB) analysis of Akt immunoprecipitates (IP) products and whole cell lysates (WCL) derived from OVCAR5 and A375 cells treated with/without methyltransferase inhibitors DZneP (5 μM) 12 hrs before harvesting. c, A schematic graph represents the methylation occurring in Akt1 identified by MS. Where indicated, mono-, di- and tri-methylation of Akt1 were respectively. d, IB analysis of HA-IP products and WCL derived from HEK293 cells transfected with indicated constructs. e,f, IB analysis of WCL derived from cells generated from pancreas or lung tissues of Smyd3-knockout mice (Kras;p53 and Kras;p53;Smyd3-/-). g, HEK293 cells were transfected with indicated constructs, and stimulated with IGF (100 ng/ml) after serum-starvation, for GST pull-down assays and IB analysis. h-j, AKT1/2-/--DLD1 cells were lentivirally infected with indicated constructs. Cells were serum-starved and stimulated with insulin (0.1 μM) (h-i) or EGF (10 ng/ml) (j) before harvesting for IB analysis. k, Akt in vitro kinase assays were performed with bacterially purified recombinant His-GSK3β as the substrate, and the recombinant Akt1 immunopurified from HEK293 cells as the source of kinase. l,m, Cells generated in (i) were subjected to glucose uptake (l) and lactate production (m) assays. The experiments were performed twice independently with three repeats, exhibited similar results. The relative glucose or lactate levels derived from two independent experiments were plotted in (l,m). n,o, Cells generated in (i) were subcutaneously injected into nude mice, and the tumors were monitored (n) and dissected. The tumor lysates were subjected for IB analysis (o). p, A sequence alignment of nuclear acids between WT and K140/142R mutant form of AKT1 indicates the strategy for generating the AKT1-K140/142R knock-in mutation. (q) The gel image illustrates the Sac II-digesting genomic DNAs derived from HEK293 knock-in cells. Corresponding DNAs were subjected for sequencing (r). s-u, Cells generated in (s) were serum-starved for IB analysis in different time periods after EGF (10 ng/ml) (t) or IGF (100 ng/ml) (u) stimulation. Statistical source data for l and m are shown in Supplementary Table 2. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 2 Depletion of SETDB1 decreases Akt kinase activity.

a, A sequence alignment of nuclear acids between WT and K140/142R mutant form of Akt1 indicates the strategy for generating the Akt1-K140/142R knock-in (KI) mice. As indicated, TGG sequence in WT-Akt1 was selected as the sgRNA PAM sequence. b,c, The gel image illustrates the Sac II-digesting genomic DNAs derived from litters selected from knock-in mice, and the corresponding DNAs were subjected for sequencing (c). d-f, Frequency of genotypes produced from Akt1 KI/WT mouse intercrosses (d). Numbers in parentheses indicate the expected number by Mendelian ratio. Growth of Akt1WT versus Akt1KI/KI female and male mice. Data were shown as mean+/-s.e.m. (male, n = 26 mice in Akt1WT group and n = 18 mice in Akt1KI/KI group; female, n = 19 mice in Akt1WT group and n = 14 mice in Akt1KI/KI group). g,h, The mice derived from the same litter were imaged at age of 4 weeks old, and were euthanized and their organs were dissected (g) and weighed, and further subjected to H&E and IHC staining (h). Scale bar, 50 μm. The experiment in h was performed twice, independently, with similar results. i, IB analysis of IP and WCL derived from HEK293 cells transfected with indicated constructs. j, Genomic alterations of SETDB1 are mutually exclusive with the alterations of the PI3K/Akt pathway-related genes (PTEN, EGFR, PIK3CA and AKT1) from the TCGA database including melanoma and breast cancer. k, A schematic illustration of the potential regulation of Akt1 with SETDB1 or EZH2, respectively. Where indicated, SETDB1 is the upstream regulator of Akt capable of methylating Akt, whereas EZH2 has been reported to be the downstream substrate of Akt, which can be phosphorylated by Akt. l,m, IB analysis of GST pulldown and WCL derived from HEK293 cells transfected with indicated constructs. Statistical source data for e and f are shown in Supplementary Table 2. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 3 Depletion of SETDB1 decreases Akt kinase activity.

a-c, IB analysis of GST pull-down (a,b) or IP (c) products and WCL derived from HEK293T cells transfected with indicated constructs. d, IB analysis of cell fractionations separated from HEK293, DLD1 and A375 cells, respectively. e-g, IB analysis of dot blot (e) and IP products derived from HEK293 cells transfected with the indicated constructs (f,g). h, In vitro methylation assays were performed with IP Flag-SETDB1 derived from HEK293T cells as the source of methyltransferase, and the commercially purified His-Akt1 as the substrate. i-k, IB analysis of IP products, GST pull-down and WCL derived from HBL cells lentivirally infected with shRNAs against SETDB1 (i), or derived from HEK293 cells transfected with the indicated constructs (j,k). l-o, IB analysis of Setdb1 conditional knockout MEFs treated with or without 4-OHT (500 nM) in different time points (l,m). Resulting cells were subjected to colony formation assays (n). The experiment was performed twice independently with three repeats, and exhibit similar results (n). Representative images were shown in n and relative colony numbers derived from two independent experiments were plotted in o. Source data for o are shown in Supplementary Table 2. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 4 Depletion of SETDB1 decreases Akt oncogenic functions.

a-e, IB assays of WCL derived from OVCAR5 (a) and DLD1 (b) cells infected with shRNAs against SETDB1. Resulting cells were subjected for colony formation and soft agar assays (c-e, top panel). The experiment was performed twice independently with three repeats, and exhibit similar results (c-e). Representative images were shown in (c-e, top panel) and relative colony numbers derived from two independent experiments were plotted in (c-e, bottom panel). f, The glucose uptake was measured with FACS as mentioned in Material section. The experiment was performed twice independently with three repeats, and exhibit similar results (f). Relative glucose levels derived from two independent experiments were plotted in f. g,h, IB analysis of WCL derived from A375 cells infected with lentivirus against SETDB1, and sequentially infected with retrovirus encoding myr-Akt1 (g). Resulting cells were subjected to colony formation assays (h, bottom panel). The experiment was performed twice independently with three repeats, and exhibit similar results (h). Representative images were shown in (h, top panel) and relative colony numbers derived from two independent experiments were plotted in (h, bottom panel). i-k, IB analysis of HA- or Akt1-IP as well as WCL derived from A375 or HEK293 cells transfected with the indicated constructs. Resulting cells were serum-starved for 18 hrs and stimulated with IGF (30 ng/ml) (i,k) or insulin (0.03 μM) (j) in different time points before harvest for IB analysis. l,m, IB analysis of IP products and WCL derived from HEK293 cells transfected with the indicated constructs treated with (l) or without (m) PI3K inhibitors. Source data for c, d, e, f and h are shown in Supplementary Table 2. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 5 PIP3 enhances the interaction of SETDB1 with Akt1 PH motif.

a-c, A cartoon illustration of the workflow describing the method for in vitro binding assays (a). Briefly, 293T cells transfected with different Akt1 encoding constructs were harvested for GST pull-down assay after treatment with the PI3K inhibitor, LY294002. The pull-down Akt1 was eluted from the beads (b) and incubated with flag-bead immunoprecipitated SETDB1 (c) in the presence/absence of PIP3 (20 μM) for 8 hrs in 4 degrees. Then the flag-beads were washed 3 times and subjected for IB analysis. d,e, His-Akt1 was purified from insect cells and eluted (d), then subjected to in vitro binding assay in the presence or absence of PIP3, and detected with IB analysis (e). f, HCT116-PTEN-/- and counterpart cells were starved and stimulated with IGF (100 ng/ml), then subjected to IB analysis. g,h, IB analysis of GST pull-down and WCL derived from HEK293 cells transfected with the indicated constructs. i-k, IB analysis of cell fractionations separated from SETDB1 knockdown A375 cells (i), AKT1K140/142R-edited and parental HEK293 cells (j) and Setdb1-deleted MEFs (k). Where indicated, MEFs were treated with 4-OHT (500 nM) or vehicle for 48 hrs, and resulting cells were serum-starved for 12 hrs and insulin-stimulated in different time points before harvest. l,m, IB analysis of WCL derived from HeLa cells transfected with C-terminal GFP-fusion Akt1. Cells were serum-starved 12 hrs and stimulated with or without insulin for 30 minutes, then subjected to DAPI staining and detected with fluorescence microscope. The nuclear DNA was stained with DAPI. Scale bar, 20 μm. The experiment in m was performed twice, independently, with similar results (m). n, A schematic illustration of the proposed model in which full Akt activation is achieved by a coordinated action of the SETDB1-mediated Akt methylation events and the canonical PI3K-PDK1 dependent kinase cascades leading to Akt phosphorylation. o-r, IB analysis of GST pull-down (o,q), his-tagged ubiquitination (p,r) and WCL derived from HEK293 cells transfected with indicated encoding constructs. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 6 KDM4B demethylates Akt1.

a, IB analyses of GST pull-down and WCL derived from HEK293 cells transfected with the indicated constructs. b-e, Coomassie staining image to illustrate mammalian purified GST-KDM4 (b) and bacterially purified GST-KDM4B catalytic domains (d), which were used for in vitro demethylation assays with Akt1 methylated peptides (c) or cell purified HA-Akt1 (insulin treated before harvesting) (e) as substrates. f-l, IB analyses of GST pull-down and WCL derived from HEK293 cells transfected with the indicated constructs. m-o, IB analysis of WCL derived from A375 cells (m), OVCAR5 cells (n) or HEK293 cells (o) lentivirally infected with shRNAs against KDM4B. p, IB analysis of WCL derived from AKT1K140/142R-edited HEK293 cells infected with lentivirus against KDM4B. q,r, IB analysis of cell fractionations separated from HEK293 cells infected with lentiviruses against KDM4B. s, IB analysis of cell fractionations separated from HEK293 cells infected with shRNA against KDM4B. t, HEK293 cells were transfected with indicated constructs and serum starved for 12 hrs. Resulting cells were stimulated with insulin (0.1 μM) in different time points before harvesting for GST pull-down and IB analysis. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 7 Inhibition of SETDB1 by Mithramycin A reduces colony formation in vitro and tumor growth in vivo.

a, A schematic illustration of the proposed model for the molecular mechanism underlying the tight regulation of Akt kinase activity and oncogenic functions by the synergy of SETDB1 and PI3K signaling axis. Where indicated, SETDB1 inhibitors were highlighted to combat tumors by reprogramming epigenome and repressing Akt oncogenic signaling. b, IB analysis of GST pull-down and WCL derived from DLD1 cells transfected with indicated constructs treated with different doses of Mithramycin A for 72 hrs before harvest. c-e, IB analysis of WCL derived from DLD1-AKT1/2-/- infected with WT or K140/142R-Akt1 lentivirus post treatment of different doses of Mithramycin A for 72 hrs (c). The resulting cells were subjected to colony formation assays (d). The experiment was performed twice independently with three repeats, and exhibit similar results (d). Representative images were shown in (d) and relative colony numbers derived from two independent experiments were plotted in (e). (e). f-i, Mithramycin A treatment retards in vivo tumorigenesis of xenografted DLD1 cells. When the tumors of xenografted DLD1 cells reached 100 mm3, the mice were treated with Mithramycin A (0.2 mg/kg) or PBS (as a negative control). Tumor sizes were monitored (f,g) and tumor mass were weighed and presented (h,i). Error bars are mean ± s.e.m, n = 8 mice. P value were calculated using two-way ANOVA analysis (g) or two-tailed unpaired Student’s t test (i). j, IB analysis of tumor lysates derived from the mice bearing A375 xenografted tumors treated with Mithramycin A or PBS. k, A schematic illustration of the proposed model for the molecular mechanisms underlying the tight regulation of Akt kinase activity and oncogenic functions by the SETDB1-KDM4B signaling axis. Where indicated, SETDB1 distributes both in nucleus and cytoplasm to repress gene expressions and promote oncogenic functions by methylating histone substrate H3K9 and non-histone substrate Akt, respectively. Conversely, KDM4B could de-methylate H3K9, Akt-K140/142 and other substrates to integratedly exert its physiological or pathological functions. Detailed statistical tests were described in Methods. Statistical source data for e,g and i are shown in Supplementary Table 2. All Western-blots above were performed twice independently with similar results. Scanned images of unprocessed blots are shown in Supplementary Fig. 8.

Supplementary Figure 8

Unprocessed scans of key blots.

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Supplementary Information

Supplementary Figures 1–8, and legends for Supplementary Tables 1 and 2

Reporting Summary

Supplementary Table 1

The list of lysine trimethylated peptides derived from IAP-LC-MS/MS based high throughput screening from OVCAR5 cells

Supplementary Table 2

Statistics source data

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Guo, J., Dai, X., Laurent, B. et al. AKT methylation by SETDB1 promotes AKT kinase activity and oncogenic functions. Nat Cell Biol 21, 226–237 (2019). https://doi.org/10.1038/s41556-018-0261-6

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