Abstract
The phosphoinositide 3-kinase (PI3K)–Akt axis is one of the most frequently activated pathways and is demonstrated as a therapeutic target in Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutated colorectal cancer (CRC). Targeting the PI3K–Akt pathway has been a challenging undertaking through the decades. Here we unveiled an essential role of E3 ligase SMAD ubiquitylation regulatory factor 1 (Smurf1)-mediated phosphoinositide-dependent protein kinase 1 (PDK1) neddylation in PI3K–Akt signaling and tumorigenesis. Upon growth factor stimulation, Smurf1 immediately triggers PDK1 neddylation and the poly-neural precursor cell expressed developmentally downregulated protein 8 (poly-Nedd8) chains recruit methyltransferase SET domain bifurcated histone lysine methyltransferase 1 (SETDB1). The cytoplasmic complex of PDK1 assembled with Smurf1 and SETDB1 (cCOMPASS) consisting of PDK1, Smurf1 and SETDB1 directs Akt membrane attachment and T308 phosphorylation. Smurf1 deficiency dramatically reduces CRC tumorigenesis in a genetic mouse model. Furthermore, we developed a highly selective Smurf1 degrader, Smurf1-antagonizing repressor of tumor 1, which exhibits efficient PDK1–Akt blockade and potent tumor suppression alone or combined with PDK1 inhibitor in KRAS-mutated CRC. The findings presented here unveil previously unrecognized roles of PDK1 neddylation and offer a potential strategy for targeting the PI3K–Akt pathway and KRAS mutant cancer therapy.
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Data availability
The data that support the findings of this study—including clinical information—are available within the paper and its source data in Supplementary Information. The raw files of proteome datasets can be obtained from the iProX database (www.iprox.org)—interactome of PDK1 (accession PXD041705), interactome of Smurf1 (accession PXD045430), identification of Neddylation site in PDK1 (accession PXD041708) and proteome (accessions PXD041740 and PXD051870). Source data are provided with this paper.
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Acknowledgements
We thank the members of the proteomics platform at the National Center for Protein Sciences (Beijing) for their help with MS analysis and SPR assay. We thank P. Wang (Tongji University Cancer Center, Tongji University, Shanghai, China) for providing the Smurf1−/− mice. We also thank P. Xie (Department of Cell Biology, Capital Medical University, Beijing, China) for the kind gift of Smurf1−/− and Smurf1C426A MEFs and X. Zheng (School of Life Sciences, Peking University, Beijing, China) for NEDP1+/+ and NEDP1−/− HEK293T cells. We thank C. H. Liu (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China) for the kind gift of WGA dyes. This study was jointly supported by the National Key R&D Program of China (2021YFA1300200 to L.Z. and 2022YFC3401500 to C.C.) and the National Science Foundation of China (32200574 to X.Z., 81974428 to L.Z. and 82273931 to C.C.).
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Contributions
The project was conceived by L.Z., C.P.C., Xueli Zhang and Y.R. The experiments were designed by L.Z., C.P.C., Y.R. and Z.P. Most of the modification experiments were performed by Z.P. and W.F. The cell biology experiments were contributed by W.F., Z.P., S.L. and J.W. The animal experiments were contributed by W.F., Z.P., S.L., Xin Zhang and L.J. The CRC sample collection and IHC analysis were contributed by S.L., Z.P., Xueli Z. and H.Q. The PROTAC experiments were contributed by B.W., M.H., Y.R., M.L., L.J. and Yange Wei. The MS experiments were contributed by Z.P., B.W., Y.G., Yinghua Wei, W.F. Y.Z. and J.J. The bioinformatics and statistical analysis were completed by Y.H., Z.P., S.L. and Z.C. Data were analyzed by L.Z., C.P.C. and Z.P. The manuscript was written by L.Z., C.P.C., Z.P., B.W., W.F. and S.L.
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Nature Chemical Biology thanks Baishan Jiang, Yi Sun and the other, anonymous, reviewers for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Smurf1 interacts with PDK1 and activates Akt signaling.
a, Immunoblot (IB) analysis of immunoprecipitate and whole-cell lysates (WCLs) derived from HEK293 cells transfected with indicated constructs. b, GST pull-down assay showed that PDK1 interacts with Smurf1 directly. c, IB analysis of PDK1 IP products and WCLs derived from HEK293 cells transfected with the indicated constructs treated with PI3K inhibitor AZD8166 for 12 h (1 μM) before being subjected to treatment with insulin. d, An in vitro kinase assay showed that PDK1 phosphorylated p70S6K but not Smurf1, as determined by a phosphoserine/threonine (p-S/T) antibody. e, IB analysis of WCLs derived from HEK293 cells transfected with Flag-Smurf1. f, IB analysis of WCLs derived from Smurf1 knockdown HCT116 cells. g, Immunoprecipitated Akt was incubated with PIP3 beads, and the binding was performed at 4 °C for 4 h, followed by IB analysis. h, Smurf1 WT and KO cells were starved for 24 h with glucose-free DMEM, and then glucose uptake probe-green was added in medium for 15 min. Fluorescence was recorded by microscope and quantified by ImageJ 1.8.0.345. (n = 3 independent experiments). i, Medium of WT and Smurf1 KO MEFs were quantified by the absorbance at 450 nm using microplate reader, and the standard curve of LDH activity was calculated from samples containing certain concentrations of pyruvate sodium (n = 3 independent experiments). j, Medium of WT and Smurf1 KO MEFs were quantified by the absorbance at 570 nm using microplate reader, and the standard curve of lactate concentration was calculated from samples containing certain concentrations of lactate (n = 3 independent experiments). k, IB analysis by pan lysine lactylation antibody in Smurf1+/+ and Smurf1−/− cells. l, Schematic diagram of AOM–DSS-induced colitis-associated CRC model in Smurf1−/− mice. m,n, Representative images (m) and statistical result of colon long (n) were displayed (n = 6 mice). o, The overall survival curves of mice from indicated groups (n = 6 mice). p, IB analysis of WCLs from Smurf1C426A/C426A and Smurf1+/+ MEFs treated with insulin. q, IB analysis of WCLs and PIP3 pull-down products from HCT116 cells expressing Smurf1 WT or C426A mutant. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. Statistical analyses were performed by unpaired two-tailed Student’s t test (h–j,n).
Extended Data Fig. 2 Smurf1 promotes PDK1 neddylation at K163.
a, Depletion of Smurf1 specifically inhibited PDK1 neddylation in cells after insulin treatment. IB and IP products and WCL derived from HEK293 cells transfected with indicated constructs. b, Deletion of Smurf1 inhibited PDK1 neddylation and Akt signaling pathway after insulin treatment in MEFs (30 min). IB analysis of Nedd8 IP products and WCLs derived from WT and Smurf1 Knockout MEFs. c, Smurf1 specifically promotes PDK1 neddylation in cell-free system. In vitro covalent neddylation assay was performed as described in the Methods section to determine the effects of Smurf1, Smurf1-C426A and Smurf1C699A on PDK1 neddylation in cell-free system. d, Nedd8 K0 (no lysine residue) mutant inhibited PDK1 neddylation in cells. IB and IP products and WCL derived from HEK293 cells transfected with indicated constructs. e, Quantitative mass spectrometry (MS) result showed that depletion of Smurf1 inhibited K48-Nedd8 linkage of PDK1 in Nedd8 R74K HEK293 cells under insulin treatment (n = 5 independent peptides). f, A schematic graph represents the identified potential neddylation sites of PDK1 upon insulin stimulation by MS. g, PDK1K163R mutant inhibited PDK1 neddylation in cells. IB and IP products and WCL derived from HEK293 cells transfected with indicated constructs. h, MS result showed the abundance of PDK1 neddylation at K163 site in Smurf1 knockdown HEK293 cells. i, A schematic diagram showing the evolutionarily conserved Lys163 (K163) residue. j, PDK1-silenced HCT116 cells stably expressing PDK1 WT or K163R were serum-starved and treated with insulin for 0 or 30 min. Immunofluorescence (IF) of Akt (green) and PDK1 (red) are displayed. The nuclei were stained with DAPI. Scale bars, 10 μm. k, PDK1-silenced HCT116 were re-expressed with PDK1 WT or K163R mutant, and cells were subjected to in vitro PIP3 binding assay. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. Statistical analyses were performed by unpaired two-tailed Student’s t test (e).
Extended Data Fig. 3 PDK1 neddylation recruited SETDB1 to activate Akt in the cytoplasm.
a, The ATP binding assay of PDK1 or PDK1K163R mutant was detected by MANT-ATP (n = 3 technical repeats from one of three independent experiments). In vitro ATP binding assays were performed with recombinant GST-PDK1 WT and K163R proteins purified from E. coli. The binding was performed at room temperature for 0.5 h, followed by fluorescence and absorbance analysis. Error bars denote the mean ± s.e.m. b, PDK1 specifically interacts with SETDB1 under insulin treatment. IB and IP products and WCL derived from HEK293 cells transfected with indicated constructs. c, The PDK1-silenced HCT116 cells stably expressing PDK1 WT or K163R were serum-starved and treated with insulin for 0 or 30 min, followed by IF with anti-Akt (red), anti-SETDB1 (green) and DAPI (blue). Scale bars, 10 μm. d, PDK1K163R mutant abolished Smurf1-mediated effect of interaction between PDK1 and SETDB1 in cells. IB and IP products and WCL derived from HEK293 cells or Smurf1 knockdown HEK293 cells transfected with indicated constructs. e, In vitro binding assays were performed with recombinant Flag-SETDB1 protein purified from mammalian cells, and GST beads bound with PDK1. The binding was performed at 4 °C for 4 h incubated and subjected to IB analysis. f, HEK293 cells transfected with Myc-SETDB1, HA-Nedd8 or HA-Ub were immunoprecipitated with Myc antibody and subjected to IB analyses. g, The identity and similarity of amino acid sequence of SETDB1 and CUBAN domain were analyzed by CLUSTAL OMEGA 2.1. h, The conservative amino acid residues of Nedd8-binding domain of SETDB1 in different species. i, HCT116 cells were starved and treated with insulin for indicated times and subjected to cytosolic and membrane fractions, WCLs were collected for IP PDK1 and IB analysis.
Extended Data Fig. 4 Smurf1 is positively correlated with activation of PDK1–Akt axis in KRAS-mutated CRC.
a, Smurf1 mRNA level in tumor tissues from CRC patients with WT or mutations (MUT) of KRAS from TCGA dataset. Error bars denote the mean ± s.d. b,c, Kaplan–Meier plots showing the overall survival of KRAS mutant CRC patients (b) and KRAS WT CRC patients (c) from the TCGA database. The 95% CIs were calculated using stratified Cox proportional hazards regression models, and P values were calculated using a stratified log-rank test. d, Representative images from immunohistochemical staining of Smurf1, p-PDK1 (S241), p-Akt (T308), PDK1 neddylation (K163) in tumors and matched adjacent tissue. Scale bars, 50 µm. e–j, Quantification of IHC staining of p-PDK1 (S241; e,g,i) and p-Akt (T308; f,h,j) in KRAS WT and mutant CRC patients from our local CRC cohort by Image-Pro Plus 6. k, Smurf1 is positively correlated with PDK1 neddylation level and p-Akt (T308) level in our local CRC cohort. Error bars denote the mean ± s.d. Statistical analyses were performed by unpaired two-tailed Student’s t test (a,e–j). The correlation analyses were performed by Pearson (k).
Extended Data Fig. 5 Smurf1 is upregulated via KRAS/AP-1 axis.
a, Representative images of HE and IHC staining against indicated proteins in colon tissues from KrasG12D/+ Villin-Cre and Kras+/+ Villin-Cre mice. Scale bars, 10 μm. b, Quantification of IHC of Smurf1, p-Akt (T308) and Ki-67 of colon tissues from KrasG12D/+ Villin-Cre and the control mice by Image-Pro Plus 6 (n = 3 mice). c, Protein extracts of intestinal tissues from KrasG12D/+ Villin-Cre and the control mice were immunoprecipitated with PDK1 antibody and analyzed by IB. d, Relative Smurf1 mRNA level in colon tissues from KrasG12D/+ Villin-Cre and the control mice (n = 5 mice). e,f, KRAS G12D inhibitor MRTX1133 reduced Smurf1 protein (e) and mRNA (f) levels in KRAS G12D mutant HCT116 cells (n = 4 technical repeats from one of three independent experiments). Cells treated with MRTX1133 for indicated times (10 mM). g–i, c-Fos or c-Jun was stably knocked down in HCT116 cells, followed by transfection with WT or G12D mutant KRAS plasmids. qPCR analysis was performed to measure the mRNA level of c-Fos (g), c-Jun (h) and Smurf1 (i) in indicated cells (n = 4 technical repeats from one of three independent experiments). Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. Statistical analyses were performed by unpaired two-tailed Student’s t test (b,d,f,g–i).
Extended Data Fig. 6 The chemical characterization of SMART1.
a, Structure–activity relationship of the tricyclic system. IB analysis of Smurf1 and Smurf2 in HCT116 cells treated with indicated compounds (1 μM, 12 h). b,c, Representative images of SPR binding assay of SMART1d (b) or SMART1e (c) to full-length Smurf1. d, Dose–response curves for HCT116 cells treated with SMART1, SMART1d or SMART1e for 72 h (data are presented as mean ± s.e.m. of 4 technical repeats from one of three independent experiments). Immunoblots are representative of three independent experiments.
Extended Data Fig. 7 SMART1-induced Smurf1 selective degradation is dependent on the ubiquitin-proteasome system.
a–d, Representative images of SPR binding assay of SMART1 to full-length Smurf1 (a), CRBN-TBD (thalidomide-binding domain; (b), Smurf1-HECT (c) or Smurf2 (d). e, IB analysis of Smurf1 and Smurf2 in HCT116 cells treated with SMART1 (0.5 μM), Smurf1-L (ligand binding to Smurf1; 0.5 μM) or pomalidomide (Poma; 0.5 μM) for 12 hours. f, IB analysis of Smurf1 and Smurf2 in HCT116 cells treated with SMART1 for indicated times (0.5 μM). g, IB analysis of indicated proteins in HCT116 cells treated with SMART1, in combination with increasing concentrations of Smurf1-L or Poma (0.5 μM, 5 μM, 50 μM) for 12 hours. h, The interaction between Smurf1 and CRBN in HCT116 cells treated with SMART1 (0.2 μM, 1 μM, 5 μM), Smurf1-L (1 μM) or pomalidomide (1 μM) as indicated. i, In vitro binding assay showed Smurf1 Y439A and R660A mutants inhibited the Smurf1-SMART1-CRBN complex formation (1 μM SMART1). In vitro binding assays were performed with recombinant GST-CRBN-TBD and His-Smurf1 HECT WT, Y439A, R660A and DM proteins were purified from E. coli. j, Smurf1 Y439A and R660A mutants inhibited SMART1-mediated Smurf1 degradation. IB analysis of indicated proteins in HCT116 cells treated with or without SMART1. k, SMART1-mediated Smurf1 degradation is dependent on CRBN. IB analysis of indicated proteins in HCT116 cells or CRBN KO HCT116 cells treated with SMART1 (0.5 μM), Smurf1-L (ligand binding to Smurf1; 0.5 μM) or Poma (0.5 μM). l, SMART1 promoted ubiquitination of Smurf1 (SMART1: 0.2 μM, 1 μM, 5 μM, SMART1-L: 1 μM or Poma: 1 μM). IB and IP products and WCL derived from HEK293 cells transfected with indicated constructs. m, The heat map illustrates relative abundance of the HECT domain-containing proteins identified in DIA-MS following SMART1 treatment. n, IB analysis of indicated protein in HCT116 cells treated with SMART1 at indicated concentration (12 h).
Extended Data Fig. 8 SMART1-induced selective degradation of Smurf1 in vivo.
a, Dose–response curves for control (shCtrl) and Smurf1 silenced (shSmurf1) HCT116 cells treated with SMART1 for 72 h (n = 4 independent experiments). b, IB analysis of Smurf1 expression in HCT116 cells with Smurf1 knockdown and protein levels were quantified by ImageJ 1.8.0.345. c, Compartment of IC50 between A01, A17, SMART1Et and SMART1 (72 h) in HCT116 cells (n = 4 independent experiments). d, Representative images of SPR binding assay of SMART1Et (left) or Smurf1-L (right) to full-length Smurf1. e, SMART1 inhibited Smurf1 protein level and PDK1 neddylation in cells (Smurf1-L: 0.5 μM, SMART1Et: 0.5 μM or SMART1: 0.5 μM). IB and Nedd8 IP products and WCL derived from HEK293 cells treated with different compounds. The indicated protein levels were quantified by ImageJ 1.8.0.345. f, Effect of SMART1Et (0.5 μM, 12 h) on the proteome of HCT116 cells. Data plotted log2 of the fold change versus DMSO control against −log10 of the P-value per protein (3 independent experiments). g, MS result showed SMART1Et inhibited Akt signaling pathway. GSEA enrichment plot was displayed. h, Dose–response curves for control (sgCtrl) and CRBN KO (sgCRBN) HCT116 cells treated with SMART1 or SMART1Et for 72 h (4 independent experiments). i, Dose–response curves for control (shCtrl) and Smurf1 knockdown (shSmurf1-1/shSmurf1-2) HCT116 cells treated with SMART1 or SMART1Et for 72 h (4 independent experiments). j, IB analysis of indicated proteins in HCT116 cells with varying degrees of Smurf1 knockdown and Smurf1 protein levels were quantified by ImageJ 1.8.0.345. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m.
Extended Data Fig. 9 SMART1 promoted the sensitivity of AR12.
a, The competitive effect of AR12 on ATP binding of PDK1 and PDK1K163R mutant. In vitro ATP binding assays were performed with recombinant Myc-PDK1-WT and K163R proteins purified from mammalian cells. The binding was performed at room temperature for 0.5 h incubated and subjected to add AR12 as indicated concentrations, followed by fluorescence and absorbance analysis. b, The influence of increasing concentrations of SMART1 on the competitive effect of AR12 on ATP binding of PDK1. In vitro ATP-binding assays were performed with recombinant Myc-PDK1 proteins purified from mammalian cells treated with SMART1 (0, 0.1 and 0.5 μM). The binding was performed at room temperature for 0.5 h incubated and subjected to add AR12 as indicated concentrations, followed by fluorescence and absorbance analysis. c, Dose–response curves of CRC cells treated with AR12 (0.3 μM) in combination with DMSO or SMART1 at indicated concentrations (left). Dose–response curves of CRC cells treated with SMART1 (20 nM) in combination with DMSO or AR12 at indicated concentrations (right). n = 3 biologically independent samples. d, Schematic outlining in vivo study of AR12 or SMART1 alone and the combinatorial regimens in the CDX model (n = 6 mice). e, Tumor growth curve in the HCT116 CRC xenograft mice model treated with SMART1 alone or in combination with AR12 (n = 6 mice). f, Individual tumor (top) and tumor weight (bottom) from CRC xenograft mice as indicated. g, Tumor lysates of CDX models were collected for PDK1 neddylation assay and followed by IB analysis. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. Statistical analyses were performed by unpaired two-tailed Student’s t test (a,b), two-way ANOVA with Bonferroni’s post hoc test (e) or Fisher’s LSD test (f).
Extended Data Fig. 10 The combinatorial regimen of SMART1 and AR12 induces potent tumor suppression in KRAS-mutated CRC.
a–c, Tumor tissues from CRC patients were injected subcutaneously into NOD-SCID mice, and two CRC PDX models (CO-04-0700 and CO-04-0114) were generated. Tumor volume (a) and weight (b) were monitored, and individual tumors (c) were shown (n = 4 mice). d, The heatmap of relative mRNA level of genes involved in PI3K/Akt/mTOR signaling in tumor tissue from these two patients. e, Tumor lysates of PDX models treated with SMART1 were collected for PDK1 neddylation assay and followed by IB analysis. f,g, Individual tumors (left) and tumor weight (right) of PDX models (CO-04-0070 (f) and CO-04-0108 (g)) treated with different combinatorial regimens. h, IB and IP product for PDK1 neddylation and indicated proteins in the tumor tissues from two PDX models. i,j, Body weight measurements showing MLN4924/AR12 or SMART1/AR12 does not affect weight of tumor-bearing mice. Immunoblots are representative of three independent experiments. Error bars denote the mean ± s.e.m. Statistical analyses were performed by unpaired two-tailed Student’s t test.
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Peng, Z., Fang, W., Wu, B. et al. Targeting Smurf1 to block PDK1–Akt signaling in KRAS-mutated colorectal cancer. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01683-5
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DOI: https://doi.org/10.1038/s41589-024-01683-5
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