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SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer

Nature volume 510, pages 283–287 (2014)Cite this article

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Abstract

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics1. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours2,3,4. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

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Figure 1: SMYD3 loss inhibits Ras-driven pancreatic tumorigenesis.
Figure 2: SMYD3 loss inhibits the development of Ras-driven lung adenocarcinoma.
Figure 3: SMYD3 methylates MAP3K2 in cancer cells.
Figure 4: SMYD3 methylation of MAP3K2 activates MAP kinase signalling pathways and repels PP2A.

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  • 11 June 2014

    Affiliation 4 has been updated.

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Acknowledgements

We thank members of the Gozani and Sage laboratories for critical reading of the manuscript, A. Smits for help with mass spectrometry data visualization and P.J. Utz and the Floren Family Trust for providing ProtoArrays. This work was supported in part by grants from the NIH to O.G. and J.S. (RO1 CA172560) and an NIH Innovator grant (DP2 OD007447) from the Office of the Director for B.A.G.; M.V. was supported by a grant from NWO-VIDI, P.K.M. was supported by the Tobacco-Related Disease Research Program, a Dean’s Fellowship from Stanford University, and the Child Health Research Institute and Lucile Packard Foundation for Children’s Health at Stanford. N.R. was supported by a grant from the Fondation pour la Recherche Médicale. J.S. is the Harriet and Mary Zelencik Scientist in Children's Cancer and Blood Diseases.

Author information

Author notes

  1. Dashyant Dhanak & Michiel Vermeulen

    Present address: Present addresses: Janssen Research and Development, 1400 McKean Road, Spring House, Pennsylvania 19477, USA (D.D.); Department of Molecular Biology, Faculty of Science, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University, 6525GA Nijmegen, The Netherlands (M.V.).,

  2. Pawel K. Mazur, Nicolas Reynoird, Julien Sage and Or Gozani: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatrics, Stanford University School of Medicine, 94305, California, USA

    Pawel K. Mazur, Atul J. Butte & Julien Sage

  2. Department of Genetics, Stanford University School of Medicine, 94305, California, USA

    Pawel K. Mazur, Atul J. Butte & Julien Sage

  3. Department of Biology, Stanford University, 94305, California, USA

    Nicolas Reynoird, Alex W. Wilkinson & Or Gozani

  4. and Department of Medicine, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, 94305, California, USA

    Purvesh Khatri

  5. Department of Molecular Cancer Research and Department of Medical Oncology, University Medical Center Utrecht, 3508 AB Utrecht, The Netherlands,

    Pascal W. T. C. Jansen & Michiel Vermeulen

  6. Epigenetics Program and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Shichong Liu & Benjamin A. Garcia

  7. Cancer Epigenetics DPU, Oncology R&D, GlaxoSmithKline, Collegeville, USA, 19426, Pennsylvania

    Olena Barbash, Glenn S. Van Aller, Michael Huddleston, Dashyant Dhanak, Peter J. Tummino & Ryan G. Kruger

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Contributions

N.R. and P.K.M. contributed equally to this work and are listed alphabetically. They were responsible for the experimental design, execution, data analysis and manuscript preparation. P.K. and A.J.B. performed the bioinformatics meta-analysis. P.W.T.C.J. and M.V. performed the SILAC experiments. S.L. and B.A.G. performed the methylated peptide mass spectrometry experiments. A.W.W. generated recombinant H3 and H3K4R protein. O.B., G.S.V., M.H., D.D., P.J.T. and R.G.K. generated SMYD3 and MAP3K2me antibodies, the MAP3K2 peptides, and determined the catalytic efficiency of SMYD3. O.G. and J.S. were equally responsible for supervision of research, data interpretation and manuscript preparation.

Corresponding authors

Correspondence to Julien Sage or Or Gozani.

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Competing interests

O.G. is a co-founder of EpiCypher, Inc. O.B., G.S.V., M.H., D.D., P.J.T. and R.G.K. are employees of GSK.

Extended data figures and tables

Extended Data Figure 1 SMYD3 is a highly overexpressed KMT in Ras-associated cancers.

a, Analysis of seven publically available human pancreatic ductal adenocarcinoma (PDAC) gene expression studies from the NCBI GEO and EBI ArrayExpress for SMYD3 levels. The red line indicates expression of SMYD3 in pancreatic cancer biopsies (n = 203); the blue line marks normal pancreas samples (n = 91). The scale shows relative expression levels (log2). b, A bioinformatics meta-analysis identified 5 lysine methyltransferase overexpressed in human pancreatic ductal adenocarcinoma (PDAC). Meta-effect size and statistical tools are described in the Methods. FDR, false discovery rate. c, d, Summary of SMYD3 expression levels in seven (n = 294 independent samples) publicly-available expression data sets of PDAC and six data sets (n = 319 tumours and n = 147 normal independent samples) of non-small cell lung cancer (NSCLC), respectively. Detailed statistical description in the Methods section.

Extended Data Figure 2 Analysis of SMYD3 expression in human and mouse PDAC and lung adenocarcinoma (LAC).

a, Immunohistochemical analysis of SMYD3 expression in mouse and human WT pancreas, PanIN lesions, and PDAC. The expression pattern was further analysed using a Smyd3LacZ reporter knock-in strain. Smyd3LacZ mice were crossed to p48;KrasG12D (Kras) mice and studied at progressing stages of disease. Analysis of LacZ activity by X-gal staining as a surrogate for Smyd3 expression is shown (lower panel) (see Extended Data Fig. 3 for a cartoon of the knock-in allele). b, Immunoblot analysis with the indicated antibodies on tumour biopsy lysates from wild-type pancreas and from the pancreas of Kras mutant mice at 4.5 and 9 months of age when mice develop PanIN and PDAC, respectively (each time point represents two biological replicates). c, IHC analysis of SMYD3 expression in normal lung, atypical adenomatous hyperplasia (AAH), and lung adenocarcinoma (LAC). X-gal analysis of LacZ activity in Kras-driven tumours with the Smyd3LacZ reporter strain (lower panel). All images shown are representative. Arrowheads indicate nuclear localization of SMYD3. Scale bars, 50 μm. d, Smyd3 knockout allele diagram. In this allele, insertion of a LacZ cassette with a strong splice acceptor in intron 2 of the Smyd3 gene creates a mutant allele serving additionally as a reporter (Smyd3LacZ). Expression of the Cre recombinase in cells removes the LacZ cassette and further deletes Smyd3 exon 2, resulting in a null allele Smyd3KO. SA, splice acceptor; pA, polyadenylation signal.

Extended Data Figure 3 Smyd3 deletion inhibits pancreatic tumorigenesis.

a, Analysis of pancreatic tumorigenesis at 6 months in Kras and Kras;Smyd3 mutant mice. Representative serial histology section (HE), IHC for pERK1/2 and the PanIN marker MUC5. b, Pancreatic cancer phenotypes in Kras;p53 and Kras;p53;Smyd3 mutant mice. Representative IHC for pERK1/2. Arrowheads indicate areas with intact acinar cells. c, Quantification of intact normal acinar area (amylase-positive area) in Kras;p53 and Kras;p53;Smyd3 mutant mice. Data are represented as mean ± s.e.m. ***P value < 0.001 (two-tailed unpaired Student’s t-test). d, Representative HE and pERK1/2 IHC images of lung sections from Kras and Kras;Smyd3 mutant mice 12, 16 and 20 weeks after Ad-Cre infection. pERK1/2 is a marker of Ras activity and advanced tumours. e, f, IHC analysis of SMYD3 expression in the PDAC (e) and LAC (h) mouse models. Arrowheads indicate cytoplasmic localization of SMYD3. Scale bars, 50 μm. g, h, Immunoblot analysis with the indicated antibodies probing pancreatic adenocarcinoma (g) or lung adenocarcinoma tumour lysates (h) dissected from Kras and Kras;Smyd3 mutant mice. Active Ras corresponds to Ras protein in the GTP-bound state pulled down with the RAF Ras-binding domain (RBD) (see Methods). *Tubulin loading control as in Fig. 1j and 2f, respectively.

Extended Data Figure 4 SMYD3 functions to maintain the tumorigenic characteristics of human and murine cancer cells.

a–c, Cell proliferation rates (top panels) and colony formation in soft agar assays (bottom panels) of murine LAC cell line LKR10 (a), human LAC cell line A549 (b), or human PDAC cell line CFPac1 (c) with or without SMYD3 depletion by stable shRNA (respective immunoblot in middle panels). d–f, SMYD3 depletion in CFPac1 attenuates tumour growth in mouse xenografts. d, Macroscopic picture of xenografts from control and SMYD3 knock-down tumours at the end of the experiment. Scale bar, 1 cm. e, Volume analysis shows that shSMYD3 significantly inhibits the expansion of pancreatic tumours (n = 6 for each group). f, HE of the tumours and IHC confirmation of SMYD3 expression and knock-down. All scale bars, 50 μm. *P value < 0.05; **P value < 0.01; ***P value < 0.001 (two-tailed unpaired Student’s t-test). Data are represented as mean ± s.e.m.

Extended Data Figure 5 Lentiviral reconstitution of SMYD3 in pancreatic acinar-to-ductal-metaplasia (ADM) assays and in lung cancer cells in vivo.

a, IHC analysis of SMYD3 reconstitution in the lung (from Fig. 3a). b, Immunofluorescent detection of SMYD3 expression in wild-type and transduced acinar clusters (left panel). Acini (asterisk) transduced with lenti-Cre carrying wild-type SMYD3 but not catalytically inactive SMYD3(F183A) undergo ADM and form ducts (arrowhead) ex vivo. c, Quantification of acinar and ductal clusters after lentiviral infection (each treatment represents four independent biological replicates). Data are represented as mean ± s.e.m. *P value < 0.05 (two-tailed unpaired Student’s t-test).

Extended Data Figure 6 SMYD3 specifically methylates MAP3K2 at lysine 260 in vitro.

a, SMYD3 methylates MAP3K2 on protein arrays. Representative image (n = 3 independent experiments) showing a SMYD3 methylation assay on a ProtoArray. The close-up shows the two independent MAP3K2 spots on the array being methylated. b, SMYD3 is detected in the cytoplasm and not the nucleus in LKR10 cells. Immunoblot analysis with the indicated antibodies of LKR10 cell lysates biochemically separated into cytoplasmic, nuclear and chromatin fractions (see Methods). c, SMYD3 catalytic efficiency is two orders of magnitude greater on MAP3K2 than on H4. kcat, KM, and kcat/KM values of SMYD3 activity on recombinant H4 and MAP3K2 as substrates are shown. d, Schematic of the H3K4* mutant form used in e. Note that the only lysine available to be methylated in H3 is present at K4. e, In vitro methylation assay on full-length recombinant MAP3K2, H3 or H3K4* with recombinant SMYD3 and PRDM9. Top panels, short and long exposure autoradiograms of the methylation assay. No signal was detected for SMYD3 on H3 and H3K4* after long exposures. The asterisk and line indicate breakdown products of MAP3K2 that contain K260 and can be detected in this methylation assay upon long exposure. Bottom panel, Coomassie stain of proteins in the reaction. f, Positive control of activity for enzymes used in Fig. 3f, g on their known respective substrates (MAP3K2, histone H3, nucleosome or RelA as indicated). g, In vitro methylation assays using MAP3K2-K260meo, me1, me2 or me3 peptides as SMYD3 substrates. Dot blot is shown as control of peptide’s comparable concentration used for the methylation assay. h, Mass spectrometry analysis of SMYD3 methylation activity on unmodified MAP3K2-K260 peptide. i, Specificity of the indicated MAP3K2-K260me antibodies in dot blot assays using MAP3K2-K260meo, me1, me2 or me3 peptides. j, MAP3K2 is methylated in cells upon SMYD3 overexpression. Immunoblot analysis with the indicated antibodies from 293T cells lysates after Flag immunoprecipitation in cells overexpressing Flag–MAP3K2 and/or HA–SMYD3.

Extended Data Figure 7 SMYD3 and MAP3K2 knockdown both impair MAP kinase signalling.

a–d, Immunoblot analysis with the indicated antibodies of LKR10 (a, b), A549 (c), and CFPac1 (d) lysates. Asterisk indicates a slower migrating ERK5 species that is phosphorylated. Stimulation, 10% serum-complemented media for 15 min (b) or EGF for 15 min at 25 ng μl−1 (a, c, d). Immunoblots are representative of 3 independent biological replicates. e, f, SMYD3 methylation of MAP3K2 does not alter the intrinsic kinase activity of MAP3K2. e, Immunoblot analysis with the indicated antibodies from lysates of 293T cells transfected with control vector, wild-type SMYD3, catalytically dead SMYD3(F183A), wild-type MAP3K2, MAP3K2(K260A), or kinase dead MAP3K2(K385M). f, Methylation of MAP3K2 does not alter its in vitro kinase activity. In vitro kinase assays were performed with the indicated recombinant versions of MAP3K2 (wild-type, SMYD3-resistant K260A mutant, or kinase dead K385 mutant) pre-methylated with wild-type SMYD3 or as a control, inactive SMYD3, using MEK1 as a substrate. MEK1 phosphorylation was detected by immunoblot analysis with the indicated antibody.

Extended Data Figure 8 SMYD3 knockout augments the effects of the MEK1/2 inhibitor trametinib (GSK1120212) in vivo.

a, Schematic of the caerulein pancreatitis-induced tumorigenesis protocol. Mice were treated with a normal dose of trametinib (1 mg per kg intraperitoneally daily) or a low dose (0.1 mg per kg intraperitoneally daily) or vehicle control. b, Immunoblot analysis with indicated antibodies of two independent pancreas biopsies per treatment group. c, Quantification of MUC5-positive lesions in caerulein-treated pancreata from Kras and Kras;Smyd3 mutant mice treated with trametinib or vehicle control (n = 5, each treatment). *P value < 0.05; ***P value < 0.001 (two-tailed unpaired Student’s t-test). Data are represented as mean ± s.e.m. d, Representative macroscopic pictures of pancreata from each treatment group. Scale bar, 1 cm. e, Representative serial HE staining and IHC for pERK1/2, a marker of Ras activity, and MUC5, a marker of PanIN lesions. All scale bars, 50 μm.

Extended Data Figure 9 SMYD3 depletion augments the effects of the MEK1/2 inhibitor trametinib (GSK1120212) in Ras-driven cancer cells.

a–c, Relative cell proliferation rates (bottom panel) of murine LAC cell line LKR10 (a), human LAC cell line A549 (b), or human PDAC cell line CFPac1 (c) with or without SMYD3 depletion by stable shRNA (SMYD3 proteins levels are shown in top panel) in response to the indicated doses of trametinib. Experiments shown represent an average of 3 independent experiments performed in triplicates for each cancer line. Values represent the number of cells relative to control shRNA cells without treatment at 48 h. b, Constitutively active MEK1 (MEK1-DD) increases EGF-mediated ERK1/2 activation in SMYD3 depleted-cells. Immunoblot analysis with the indicated antibodies using lysates from A549 cells stably expressing shControl or shSMYD3 and transfected with HA–MEK1-DD. Stimulation: EGF treatment for 15 min at 25 ng μl−1.

Extended Data Figure 10 Treatment with the PP2A inhibitor cantharidin phenocopies SMYD3 function in vivo.

a, Schematic of the caerulein pancreatitis-induced tumorigenesis protocol. Mice were treated with the PP2A inhibitor cantharidin (iPP2A, 0.15 mg kg−1 intraperitoneally twice a day) or vehicle control. b, Immunoblot analysis with indicated antibodies on two independent pancreas biopsies per treatment group. c, Macroscopic pictures of WT and Kras;Smyd3 mutant pancreata. Note that treatment with the PP2A inhibitor leads to the development of enlarged, ‘hard’ pancreata characteristic of tumorigenic development even in Kras;Smyd3 mutant mice. Scale bar, 1 cm. d, Representative serial haematoxylin and eosin (HE) staining and IHC for pERK1/2, a marker of Ras activity, and MUC5, a marker of PanIN lesions. All scale bars, 50 μm. e, Summary model for SMYD3 regulation of MAP kinase signalling after MAP3K2 methylation. Oncogenic Ras activates several kinase cascades that play important roles in pancreas and lung cancer development, including four major MAPK pathways (ERK1/2, ERK5, JNK, and p38) as well as AKT signalling. SMYD3 is frequently overexpressed in pancreatic and lung cancers, two cancer types that are commonly driven by oncogenic Ras signalling. Overexpression of SMYD3 and the resulting methylation of MAP3K2 at K260 potentiate activation of kinases like ERK1/2 and ERK5 in response to stimuli like oncogenic Ras. We postulate a mechanism in which the PP2A complex is unable to bind methylated MAP3K2, which decreases the ability of this enzyme to terminate activating phosphorylation events on MAP3K2 and/or MAP3K2 downstream targets. Under conditions with excessive SMYD3 protein, the physiological relationship between PP2A and MAP3K2 is disrupted and results in an increased pathological MAP3K2 signalling, which cooperates with Ras to promote tumorigenesis.

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Mazur, P., Reynoird, N., Khatri, P. et al. SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer. Nature 510, 283–287 (2014). https://doi.org/10.1038/nature13320

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