An ID2-dependent mechanism for VHL inactivation in cancer

Subjects

Abstract

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors, most notably HIF2α (also known as EPAS1), are expressed in and required for maintenance of cancer stem cells (CSCs). However, the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex, displaces VHL-associated Cullin 2, and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2–VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma, ID2 positively modulates HIF2α activity. Conversely, elevated expression of DYRK1 phosphorylates Thr27 of ID2, leading to HIF2α destabilization, loss of glioma stemness, inhibition of tumour growth, and a more favourable outcome for patients with glioblastoma.

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Figure 1: Inhibition of DYRK1-mediated phosphorylation of ID2 at Thr27 promotes NSC properties.
Figure 2: DYRK1 kinases and ID2 Thr27 phosphorylation are inhibited by hypoxia and enhanced by PHD1.
Figure 3: The DYRK1–ID2–Thr27 pathway controls GSCs and HIF2α.
Figure 4: Unphosphorylated ID2 binds to and disrupts the VCB–Cul2 complex.
Figure 5: DYRK1 kinases inhibit human glioma growth by repressing an ID2–HIF2α network.

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Acknowledgements

We thank N. Sherman for phospho-ID2 and ID2-associated proteins analysis by mass spectrometry, C. Warnecke for the plasmid expressing HIF2α-TM, Z. Ronai for Flag–PHD1, Flag–PHD2, and Flag–PHD3, plasmids, A. Flores-Morales for the plasmid expressing Flag–SOCS2, M. Pagano for cDNAs for RBX1, Elongin B, Elongin C, and K. H. Kim for pcDNA-VHL. We thank D. D’Arca for preparation of VHL and HIFα constructs. This work was supported by National Institute of Health grants to A. L. (R01CA101644 and R01CA131126), and A. I. (R01CA178546 and R01NS061776 and a grant from The Chemotherapy Foundation). V. F. is supported by a fellowship from the American Brain Tumor Association (ABTA). S. B. L. was supported by NRF-2013R1A6A3A03063888 fellowship.

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Authors

Contributions

A.I. and A.L. conceived the project, coordinated the study, oversaw the results and wrote the manuscript. S.B.L. designed and performed most biochemical and cell biology experiments and helped with writing the manuscript; V.F. generated lentiviral vectors, performed in vitro GSC infections, and RT–PCR and binding studies; A.M.C. generated and analysed glioma xenografts and assisted in GSC experiments; G.L. conducted the in vitro screening assay that identified DYRK1 as the ID2 Thr27 kinase. M.B. and A.C. conducted gene expression and bioinformatics analyses. J.N.B. provided excess tissue from human GBM for GSC isolation. D.S., K.H. and T.C. generated the computational molecular docking model. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Antonio Iavarone or Anna Lasorella.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 ID2 is phosphorylated on Ser5, Ser14 and Thr27.

Chromatographic results of mass spectrometry analysis of ID2 protein immunoprecipitated from IMR32 human neuroblastoma cells. a, The peptide identified as ID2 A3–R8 shows phosphorylation of Ser5. b, The peptide identified as ID2 K12–R24 shows phosphorylation of Ser14. c, The peptide identified as ID2 S25–L36 shows phosphorylation of Thr27.

Extended Data Figure 2 T27A missense mutation in ID2 in human cancer cells and Thr27 phosphorylation of ID2 by DYRK1 kinases.

a, Sequence analysis of genomic DNA from the neuroblastoma cell line IMR32 shows the wild-type sequence (left). Sequencing of DNA from the colon cancer cell line HRT-18 shows a heterozygous mutation resulting in the change of codon-27 from ACC (Thr) to GCC (Ala) (right). b, Both wild-type and mutant ID2(T27A) are expressed in HRT-18 colon cancer cells. Sequence analysis of representative clones (out of 20 clones) derived from HRT-18 cDNA demonstrates expression of wild-type (left panel) and mutant (right panel) alleles. c, Amino acid sequence flanking Thr27 of ID2 (marked in red), including the DYRK1 consensus motif (bold), is evolutionarily conserved. d, In vitro kinase assay using bacterially expressed GST–ID proteins and recombinant DYRK1A. e, U87 cells transfected with Flag–ID2, Flag–ID2(T27A) or the empty vector were immunoprecipitated with Flag antibody. Co-precipitated proteins were analysed by western blot using DYRK1A, DYRK1B and Flag antibodies. β-actin was used as control for loading. WCL, whole cellular lysate. f, U87 stably transfected with Flag–ID2 were treated with harmine (10 μM) or vehicle for 24 h and analysed by western blot using the indicated antibodies.

Extended Data Figure 3 DYRK1 kinase activity and Thr27 phosphorylation of ID2 are inhibited by hypoxia.

a, U87 glioma cells were treated with 100 μM CoCl2 for the indicated times. Cellular lysates were analysed by western blot using the indicated antibodies. b, SK-N-SH cells were treated with 300 μM CoCl2 for the indicated times and assayed by western blot using the indicated antibodies. c, Stoichiometric evaluation of pThr-27-ID2 in SK-N-SH cells untreated or treated with CoCl2 for 24 h. Cellular lysates prepared in denaturing buffer were immunoprecipitated using pT27-ID2 antibody or normal rabbit IgG. Aliquots of whole cellular lysates (WCL, μg) and immunoprecipitates were assayed by western blot using pT27-ID2 and non-phosphorylated ID2 antibodies (upper panels). The efficiency of immunoprecipitation with anti-pT27-ID2 antibody from untreated cells was determined to calculate the percent of the pT27-ID2 in the absence and in the presence of CoCl2 (lower panel). d, 293T cells expressing GFP–DYRK1 proteins untreated or treated with 100 μM CoCl2 for 12 h were used as a source of active kinase. The kinase activity of the anti-GFP–DYRK1 immunoprecipitates was tested in vitro using bacterially expressed and purified Flag–ID2 as substrate. Kinase reactions were evaluated by western blot using p-T27-ID2 antibodies (top). Analysis of kinase reactions by Flag immunoblot shows similar amount of ID2 protein in each kinase reaction (middle). Immunocomplexes were analysed by western blot using GFP antibody (bottom). e, Lysates from U251 cells expressing GFP–DYRK1 proteins untreated or treated with 100 μM CoCl2 for 6 h were immunoprecipitated using GFP antibodies. Western blot was performed using anti-p-Tyrosine (p-Tyr) or GFP antibodies. Analysis of WCL shows similar expression levels of DYRK1 proteins. α-tubulin was used as control for loading. f, Lysates from 293T cells expressing GFP–DYRK1A untreated or treated with 100 μM CoCl2 for 12 h were immunoprecipitated with anti-p-Tyr antibodies and analysed by western blot using antibodies against GFP. α-tubulin was used as control for loading. g, Lysates from 293T cells expressing GFP–DYRK1B untreated or treated with 100 μM CoCl2 for 12 h were immunoprecipitated with anti-p-Tyr antibodies and analysed by western blot using antibodies against GFP. α-tubulin was used as control for loading. h, U87 transfected with GFP–DYRK1A, GFP–DYRK1B or GFP and Flag–PHD1, Flag–PHD2, or Flag–PHD3 were immunoprecipitated using anti-hydroxyproline antibody. Western blot was performed using GFP antibody (upper panels). HC, IgG heavy chain. Lower panels, WCL.

Extended Data Figure 4 The DYRK1–ID2 Thr27 pathway controls GSCs and HIF2α.

a, GSC line 48 cells were transduced with lentiviruses expressing ID2(WT), ID2(T27A), or the empty vector. b, Cells were analysed by in vitro LDA. Representative regression plot used to calculate gliomasphere frequency in panel c. c, The frequency of cells capable of forming gliomaspheres by in vitro LDA. Data in the histograms represent means of 3 biological replicates ± s.d.; **P = 0.00163. d, The microphotographs show representative gliomasphere cultures of cells treated as in a. e, HIF2α mRNAs from cells treated as in a were analysed by semi-quantitative RT–PCR. f, U87 cells stably expressing Flag–ID2 or Flag–ID2(T27A) were analysed by western blot using the indicated antibodies. Arrow points to specific band. Arrowhead indicates Flag–ID2. Asterisk indicates endogenous ID2. g, GSC line 34 cells were transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Cells were analysed by western blot using the indicated antibodies. Arrow points to specific band. Asterisk indicates a non-specific band. h, qRT–PCR from cells treated as in g. Data in the histograms represent means ± s.d. (n = 9, triplicate experiments each performed in triplicate; ***P = 8.44524 × 10−7 for TGFA). i, GSC line 31 was transduced with lentiviruses expressing DYRK1B-V5 or empty vector. Expression of HIF2α, DYRK1B-V5 and α-tubulin was analysed by western blot. j, mRNAs from experiment shown in Fig. 3a–c were analysed by semiquantitative RT–PCR for HIF2α. k, GSC line 31 cells were transduced with lentiviruses expressing DYRK1B and ID2, ID2(T27A), or the empty vector. Cells were analysed by LDA. Representative regression plot used to calculate gliomasphere frequency in Fig. 3b. l, GSC cell line 31 cells were transduced with lentiviruses expressing DYRK1B or the empty vector in the absence or in the presence of undegradable HIF2α (HIF2α-TM). Cells were analysed by in vitro LDA. Representative regression plot used to calculate the frequency of gliomaspheres in cultures from three independent infections (Vect plus Vect = 13.55%; DYRK1B-Vect = 4.36%; DYRK1B-HIF2α-TM = 9.73%).

Extended Data Figure 5 The DYRK1–ID2-Thr27 pathway modulates HIFα stability by regulating the interaction between ID2 and VHL.

a, In vivo ubiquitylation of HIF1α protein. U87 cells transfected with the expression plasmids HIF1α and MYC-ubiquitin were co-transfected with Flag–ID2, Flag–ID2(T27A), or the empty vector in the presence or in the absence of GFP–DYRK1B. After treatment with MG132 (20 μM) for 6 h, lysates were prepared in denaturing buffer and identical aliquots were immunoprecipitated with antibodies directed against MYC. An anti-HA antibody was used to detect HIF1α ubiquitin conjugates (left); Cellular lysates, WCL, were analysed by western blot using the indicated antibodies (right). b, U87 cells were co-transfected with plasmids expressing HA–HIF2α and GFP–DYRK1B or GFP-vector. Cells were treated with 50 μg ml−1 of CHX for the indicated times and analysed by western blot. c, Quantification of HIF2α protein from the experiment in panel b as the log2 of the percent of HIF2α relative to untreated cells. d, IMR32 cells were co-transfected with ID2 and Flag–VHL or Flag–HIF1α expression vectors. Immunoprecipitation was performed using Flag antibody and immunocomplexes and whole cellular lysates (WCL) were analysed by western blot using the indicated antibodies. e, IMR32 cells transfected with Flag–VHL expression vector were used for IgG or ID2 antibody immunoprecipitation. Immunocomplexes and WCL were analysed by western blot. Arrow points to the specific Flag–VHL band; asterisk indicates IgG light chain. f, Flag immunoprecipitation of binding reactions of in vitro translated Flag–ID and HA–Elongin C proteins. Immunocomplexes were analysed by western blot for HA and Flag. g, Flag-ID proteins and HA–VHL were translated and incubated in vitro. Flag immunocomplexes were analysed by western blot for HA and Flag. h, In vitro streptavidin pulldown assay of biotinylated ID2 peptides (amino acid 14–34 (WT), pT27, and T27W) and in vitro translated HA–VHL. Bound polypeptides were detected by western blot.

Extended Data Figure 6 Molecular docking of an ID2 (15–31) peptide on the VHL–Elongin C complex.

a, Ribbon representation of the backbone of the VHL-Elongin C complex and the predicted binding conformation of the ID2 peptide. VHL (red ribbon), Elongin C (blue ribbon) and the docked ID2 peptide (purple ribbon). Cul2 contact residues are colored yellow ribbon in both VHL and Elongin C. Arrow indicates the ID2 peptide. b, Docking result for the phospho-Thr-27-ID2 peptide shown from the same perspective as in panel a. c, The view and complex in a is rotated 90 degrees around an axis parallel to the page so that the perspective is from the arrow shown in panel a. d, Electrostatic molecular surface representation of the VHL–Elongin C complex with the docked ID2 peptide. The perspective is the same as in panel c. The T27 side chain is shown as space-filling spheres and is indicates by the red arrow. The N-terminus and C-terminus of the ID2 peptide are indicated by purple arrows.

Extended Data Figure 7 DYRK1-mediated phosphorylation of ID2 prevents dissociation of the VCB–Cul2 complex.

a, In vivo binding assay using lysates from U87 cells co-transfected with HA–VHL and Flag–ID2 or Flag–ID2(T27E) expression vectors. Flag immunocomplexes were analysed by western blot using HA and Flag antibodies. Whole cell lysates, WCL, were analysed by western blot using the indicated antibodies. Binding of Flag–ID2 and Flag–ID2(T27E) to the bHLH protein E47 is shown as control for ID2 binding. b, U87 cells were transfected with Flag–ID2, Flag–ID2(T27A) or Flag–ID2(T27E) plasmids. Cellular lysates were analysed by western blot using the indicated antibodies. c, In vitro binding between purified Flag-ID2 and His-VHL following in vitro kinase reaction using recombinant DYRK1B and Flag–ID2. d, Analysis of the HA–Elongin C immunocomplexes in U87 cells transfected with HA–Elongin C in the absence or presence of Flag–ID2(T27A). Anti-HA immunoprecipitation reactions and WCL were analysed by western blot using antibodies against Cul2, HA (Elongin C), and Flag (ID2). e, Analysis of the Flag–SOCS2 immunocomplexes in U87 cells transfected with ID2, ID2(T27A) or the empty vector. Flag immunoprecipitation reactions and WCL were analysed by western blot using antibodies against Cul5, ID2, and Flag (SOCS2). f, Stoichiometric analysis of ID2 and VHL in cellular lysates. Decreasing amount of WCL from 1 × 106 U87 cells and purified proteins were assayed by western blot (left). Regression plots of densitometry analysis were used to determine ID2 and VHL protein concentration and the ID2:VHL ratio (right). g, Immunoprecipitation of endogenous VHL in U87 cells in the presence and in the absence of CoCl2. Western blot for Cul2 and VHL are analysed by western blot. Vinculin is shown as loading control.

Extended Data Figure 8 DYRK1 kinase inhibits proliferation of human glioma.

a, Malignant glioma were induced in Id1Flox/Flox-Id2Flox/Flox-Id3-/- mice via injection of lentivirus expressing H-RAS-V12-IRES-CRE-ER linked to U6-shp53 cassette into the dentate gyrus as described9. Mice were treated for 5 days with tamoxifen or vehicle and euthanized 2 days later. Tumours were analysed by immunohistochemistry using HIF2α and OLIG2 antibodies. Nuclei were counterstained with haematoxylin. b, Western blot analysis of DYRK1B in U87 cells stably expressing a doxycycline inducible DYRK1B or the empty vector. Cells were treated with 0.75 μg ml−1 doxycycline or vehicle for 36 h. Lysates of adult mouse cortex (CX) and cerebellum (CB) were used to compare exogenous DYRK1B with endogenous levels of the protein. c, Tissue sections from experiment in Fig. 5e, f were analysed by immunostaining using BrdU antibodies. d, Quantification of BrdU positive cells from the experiment in c. Data in the histograms represent means ± s.d. (n = 5; ***P = 3.065 × 10−7, DYRK1B – Dox versus DYRK1B + Dox). Asterisks indicate statistical significance by two-tailed t-test. e, Western blot analysis of ectopically expressed V5-DYRK1B, V5-DYRK1B-K140R in U87 cells. f, Brain cross-sections of mice intracranially injected with U87 cells in e were analysed by immunofluorescence using V5 antibody (red, upper panels) to identify exogenous DYRK1B and human vimentin antibody (red, lower panels) to identify human glioma cells. Nuclei were counterstained with DAPI (blue). T, tumour; B, brain.

Extended Data Figure 9 Analysis of DYRK1A, DYRK1B and ID2 expression in human GBM.

a, Scatter plot showing the expression of DYRK1A and DYRK1B in GBM. Blue and red dots indicate GBM samples with high or low expression of both DYRK1A and DYRK1B, respectively. GBM samples were used for Kaplan–Meier survival analysis to evaluate the prognostic power of the expression of DYRK1A and DYRK1B shown in Fig. 5i. b, Distribution of ∆ESrand, representing the null model, for ID2 activity (left) and ID2 expression (right). This distribution is used to calculate the P value for enrichment of ∆ES. Red dot (or vertical black bar) represents the ∆ES using HIF2α targets. The P value is calculated as ratio of number of times ∆ESrand is greater than ∆ES (falls in green regions) over the total trials (= 1000).

Extended Data Figure 10 Model for the regulation of HIFα stability by the DYRK1 kinase and ID2 pathway.

In cellular contexts that favour HIFα protein instability (normal oxygen levels, but also low ID2 expression and high DYRK1 expression) prolyl hydroxylases (PHD1) is active and positively regulates DYRK1 kinases. Active, tyrosine phosphorylated DYRK1 kinases keep ID2 under functional constraint by phosphorylation of Thr27. The VCB–Cul2 ubiquitin ligase complex efficiently ubiquitylates HIFα (left). With decreasing oxygenation and PHD1 inactivation but also in the presence of downregulation of DYRK1, elevated expression of ID2, or ID2(T27A) mutation, the un-phosphorylated/un-phosphorylatable pool of ID2 exerts an inhibitory function towards the VCB–Cul2 complex by binding directly VHL and Elongin C proteins and displacing Cul2. This results in HIFα accumulation (right). The transcriptional activation of the ID2 gene, a HIFα target, by HIF2α generates a feed-forward ID2–HIF2α loop that amplifies the effects.

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Lee, S., Frattini, V., Bansal, M. et al. An ID2-dependent mechanism for VHL inactivation in cancer. Nature 529, 172–177 (2016). https://doi.org/10.1038/nature16475

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