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Letterhttps://doi.org/10.1038/s41586-019-1173-8
Proteomics reveals NNMT as a master metabolic regulator of cancer-associated fibroblastsMark A. eckert1,9, Fabian Coscia2,3,9, Agnieszka Chryplewicz1, Jae Won Chang4, Kyle M. Hernandez5, Shawn Pan1, Samantha M. tienda1, Dominik A. Nahotko1, Gang Li4, Ivana Blaženović6, ricardo r. Lastra7, Marion Curtis1, S. Diane Yamada1, ruth Perets8, Stephanie M. McGregor7, Jorge Andrade5, Oliver Fiehn6, raymond e. Moellering4, Matthias Mann2,3 & ernst Lengyel1*
High-grade serous carcinoma has a poor prognosis, owing primarily to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the proteogenomics of ovarian cancer1,2, but a systematic examination of both the tumour and stromal compartments is critical in understanding ovarian cancer metastasis. Here we develop a label-free proteomic workflow to analyse as few as 5,000 formalin-fixed, paraffin-embedded cells microdissected from each compartment. The tumour proteome was stable during progression from in situ lesions to metastatic disease; however, the metastasis-associated stroma was characterized by a highly conserved proteomic signature, prominently including the methyltransferase nicotinamide N-methyltransferase (NNMT) and several of the proteins that it regulates. Stromal NNMT expression was necessary and sufficient for functional aspects of the cancer-associated fibroblast (CAF) phenotype, including the expression of CAF markers and the secretion of cytokines and oncogenic extracellular matrix. Stromal NNMT expression supported ovarian cancer migration, proliferation and in vivo growth and metastasis. Expression of NNMT in CAFs led to depletion of S-adenosyl methionine and reduction in histone methylation associated with widespread gene expression changes in the tumour stroma. This work supports the use of ultra-low-input proteomics to identify candidate drivers of disease phenotypes. NNMT is a central, metabolic regulator of CAF differentiation and cancer progression in the stroma that may be therapeutically targeted.
All high-grade serous ovarian cancers contain TP53 mutations3 but other recurrent mutations are rare and serous cancers are generally characterized by copy-number variants1,4. High-grade serous carci-noma (HGSC) has a high proportion of stroma, but little is known about how interactions between the cancer cells and the surrounding extracellular microenvironment affect tumour growth. Several reports describe the proteome of human ovarian cancer2,5,6 but none have dif-ferentiated between proteins in the stroma and the epithelial tumour compartment. Given that the stroma has a tumour-supporting role and co-evolves with the epithelial compartment during progression and metastasis7,8, we set out to evaluate the proteome of both compartments in a systematic method. We combined developments in ultra-high- sensitivity mass-spectrometry-based proteomics9,10 with microdissection technology to obtain an integrated picture of cancer progression.
To elucidate the proteomic changes that underlie ovarian cancer progression in both the tumour and stroma, we identified a cohort of 11 patients with HGSC with tissue from serous tubal in situ carci-noma (STIC), invasive fallopian tube lesions, invasive ovarian lesions and omental metastases (Fig. 1a, Supplementary Table 1). All tissues were collected prospectively during the initial debulking surgery and
all patients were chemotherapy-naive. For each patient and every anatomic site, both tumour and stromal compartments were micro-dissected and proteins extracted using an optimized high-sensitivity, label-free proteomic workflow for low-input samples (Methods). In total, we quantified 6,944 unique protein groups from 107 analysed samples, both tumour and benign, at a protein and peptide false- discovery rate (FDR) of less than 1%. A median of 4,942 and 4,428 proteins were quantified per tumour or stromal sample, respectively, at similar dynamic ranges and with excellent reproducibility (Pearson r = 0.98; Fig. 1b, Extended Data Fig. 1a, b, Supplementary Table 2). Unsupervised hierarchical clustering robustly segregated tumour and stromal proteomes (Extended Data Fig. 1c). Tumour compartments were enriched for known markers of HGSC (PAX8, MSLN, MUC16 (CA-125), EPCAM) and DNA replication and repair pathways, whereas stromal compartments were characterized by expression of extracel-lular matrix components and pathways that included activated fibro-blast markers (for example, collagens, vimentin, versican, tenascins and myosins) (Fig. 1c, Extended Data Fig. 1c–e, Supplementary Tables 3, 4).
Pairwise proteomic comparison of primary (invasive fallopian tube and ovarian lesions) and metastatic tumour compartments revealed only one protein, FABP4—previously reported to be upregulated in omental metastases and expressed at the tumour–stromal interface—as significantly higher in omental tumour metastases11 (FDR < 0.01, Fig. 1d, Extended Data Fig. 2a). The tumour compartment was instead characterized by patient-specific protein signatures (analysis of variance (ANOVA) FDR < 0.01, Extended Data Fig. 2b–e) that probably reflect the molecular heterogeneity of HGSC between patients1,2. By contrast, stromal proteomes clearly clustered by anatomic site, which reveals a conserved stromal response to metastasis that is shared between patients (Extended Data Fig. 2b–e). Differential expression analysis between the stromal compartments at the four anatomic sites (STIC, fallopian tube, ovary and omentum) identified 128 differentially expressed protein groups, with most contributed by the omental-metastasis- associated stroma (FDR < 0.05, Extended Data Fig. 2d). Among these, we identified a set of 62 protein groups that were universally up- or downregulated in all omental metastases compared to all pri-mary (invasive fallopian tube or ovarian lesion) stromal sites (FDR < 0.01, Fig. 1d, Extended Data Fig. 2f).
The stromal signature consisted of 21 up- and 41 downregulated proteins, which included proteins known to have tumour-supporting roles in the stroma such as FAP, LOX, TNC and VCAN and had con-siderable overlap with The Cancer Genome Atlas (TCGA) mesenchy-mal subtype (Fig. 1e, Extended Data Fig. 2g, Supplementary Table 5). Downregulated proteins included negative regulators of TGF-β signalling such as LTBP4 and SDPR. Other proteins, such as ENPP1 and COPZ2, had no previously described roles in the tumour stroma
1Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, IL, USA. 2Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany. 3Clinical Proteomics Group, Proteomics Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark. 4Department of Chemistry, University of Chicago, Chicago, IL, USA. 5Center for Research Informatics, University of Chicago, Chicago, IL, USA. 6West Coast Metabolomics Center, University of California Davis Genome Center, Davis, CA, USA. 7Department of Pathology, University of Chicago, Chicago, IL, USA. 8Division of Oncology, Clinical Research Institute at Rambam, Rambam Health Care Campus, Haifa, Israel. 9These authors contributed equally: Mark A. Eckert, Fabian Coscia. *e-mail: elengyel@uchicago.edu
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or the biology of CAFs. Owing to its biochemical activity and roles in epigenetic regulation12, upregulation of NNMT in the omental stroma was an interesting target. NNMT transfers a reactive methyl group from S-adenosyl methionine (SAM) to nicotinamide to generate S-adenosyl homocysteine (SAH) and the metabolically inert product 1-methyl nicotinamide (1-MNA). SAM is the universal methyl donor for histones, non-histone proteins, DNA, RNA, lipids and other metabolites. This activity generates a methyl sink in the form of 1-MNA, which leads to depletion of SAM and reduces the global methylation potential of the cell13,14. NNMT-mediated SAM depletion regulates gene expression by attenuating histone methylation in cancer cells, adipocytes and embry-onic stem cells14–17 (Fig. 2a).
The proteomic analysis revealed that NNMT expression was increased in the stroma of peritoneal and omental metastases com-pared to the benign omental, fallopian tube and ovarian stroma, including early micrometastases, which was also confirmed in a tissue microarray containing both primary and metastatic ovarian cancer samples (Fig. 2b, c, Extended Data Fig. 3a–d). Tumour expres-sion of NNMT did not vary significantly by anatomic site (Extended Data Fig. 3e). In both syngeneic and autochthonous18 mouse models of HGSC, NNMT was highly expressed in the stroma of metastases (Extended Data Fig. 3f). High stromal NNMT was also observed in breast and colon cancer stroma, which suggests that NNMT expres-sion is a feature of CAFs in multiple cancer types (Extended Data Fig. 3g).
CAFs are differentiated from normal fibroblasts by production of oncogenic extracellular matrix components, cytokine secretion, cytoskeletal rearrangements associated with smooth muscle actin
(SMA) expression, increased ability to contract collagenous matrices and expression of CAF markers8. Knockdown of NNMT in CAFs led to a reversion of cell morphology to one that more-closely resembled normal omental fibroblasts (Fig. 2d, Extended Data Fig. 4a–c). Knockdown or overexpression of NNMT led to a significant pertur-bation of its enzymatic activity, as assessed by 1-MNA production using mass spectrometry (Fig. 2e). CAF markers, including SMA and fibronectin, were decreased upon NNMT knockdown and increased with overexpression (Fig. 2f, Extended Data Fig. 4d, e).
NNMT was associated with expression of epithelial–mesenchymal transition markers, and transcriptional regulators of the epithelial–mesenchymal transition and inhibition of NNMT attenuated the acquisition of CAF markers by normal stromal cells in response to TGF-β (Extended Data Fig. 4f). NNMT was necessary and sufficient to induce collagen contractility (Fig. 2g) and globally regulated expression of thousands of genes, including pro-tumorigenic cytokines (Fig. 2h, Extended Data Fig. 4g, Supplementary Tables 6, 7). In further support of NNMT as a central regulator of CAF gene expression, genes differ-entially expressed by NNMT in fibroblasts or CAFs were significantly enriched for gene signatures associated with the epithelial– mesenchymal transition, TCGA mesenchymal subtype, and proteins that were highly expressed in the stroma of omental metastases com-pared to primary sites (Extended Data Fig. 4h–k). Functionally, CAFs support and accelerate tumour growth, progression and metastasis8,19,20. Overexpression of NNMT in normal fibroblasts promoted cancer cell proliferation; conversely, knockdown of NNMT in CAFs attenuated cancer cell proliferation and chemotaxis (Fig. 2i, j, Extended Data Fig. 4l, m).
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Fig. 1 | Compartment-resolved proteomics of ovarian cancer progression reveals a stromal signature of HGSC metastasis. a, Tumour and stromal compartments were microdissected from different anatomic sites (STIC, invasive fallopian tube (FT) lesions, ovarian (Ov) lesions and omental (Om) metastases) and label-free, quantitative shotgun proteomics performed to identify proteins differentially expressed across all anatomic sites. b, Number of unique proteins quantified by MaxLFQ in each anatomic compartment (n = 11 patients). c, Ranking of proteins by expression in tumour compartment (green; n = 43 samples) versus stromal compartment (purple; n = 42 samples) shows known markers.
d, Volcano plots comparing primary sites (fallopian tube and ovary) to omental metastases in tumour (left) and stromal (right) compartments. Significantly differentially expressed proteins are highlighted in green (tumour) or purple (stroma). Two-sided t-test FDR < 0.01, n = 11 patients. e, Heat map of proteins upregulated in omental stromal signature of metastasis across all patients (rows) and anatomic sites (STIC, fallopian tube, ovary and omentum). Undetected values are black; missing samples are white. The box plots in b define the range of the data (whiskers), 25th and 75th percentiles (box), and medians (solid line).
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We hypothesized that high stromal NNMT expression drives gene expression changes and acquisition of the CAF phenotype through hypomethylation of DNA, RNA or histones through attenuation of the SAM:SAH ratio (that is, methylation potential of the cell)14. Indeed, methylation potential was directly regulated by NNMT expression, as assessed by metabolite profiling with mass spectrometry. NNMT knockdown increased the SAM:SAH ratio, whereas NNMT overex-pression led to a decrease (Fig. 3a). Nicotinamide levels were negatively associated with NNMT expression (Fig. 3a). Knockdown of NNMT led to increased levels of NAD+(H) and was associated with increased expression of NAD-dependent sirtuin target genes such as catalase and CD36, and decreased acetylation of H3K9 and α-tubulin15,21 (Extended Data Fig. 5). Targeted metabolomics identified conserved metabolic changes upon both knockdown and overexpression of NNMT (Extended Data Fig. 6a, Supplementary Table 8). In particular, NNMT regulated the polyamine pathway and increased levels of 5-methylthioadenosine, an inhibitor of SAH hydrolase that contrib-utes to global hypomethylation22 (Extended Data Fig. 6b–d). NNMT expression induced genome-wide DNA-methylation changes that significantly altered methylation status in the promoter regions of genes, and was enriched for genes involved in collagen production and myosin-driven contractility (Fig. 3b, Extended Data Fig. 7a, b, Supplementary Tables 9, 10). Inhibition of DNA methylation with 5-azacytidine increased expression of CAF markers and collagen
contractility, which supports a direct role for DNA hypomethylation in regulating the CAF phenotype (Extended Data Fig. 7c, d).
To understand how NNMT affects histone methylation, we per-formed targeted histone proteomic analyses, using a multi-reaction monitoring approach to quantify relative levels of histone lysine and arginine methylation23. NNMT knockdown increased histone methylation at residues associated with transcriptional regulation, including an increase in H3K4 and H3K27 trimethylation (me3; Fig. 3c). Immunoblotting confirmed that H3K27 and H3K4 trimeth-ylation was perturbed upon knockdown or overexpression of NNMT (Fig. 3d). Histone methylation was metabolically sensitive to cellular methylation potential and extracellular methionine concentration (Extended Data Fig. 7e, f). Genes regulated by NNMT overexpres-sion were associated with gene sets regulated by H3K27 demethylation (Extended Data Fig. 8a). Chromatin immunoprecipitation sequenc-ing (ChIP-seq) of cells that overexpress NNMT found that NNMT activity reduced global H3K27 occupancy, including at the promot-ers of NNMT-regulated genes (Fig. 3e, f, Extended Data Fig. 8b, c, Supplementary Table 11). These data suggest that NNMT mediates genome-wide epigenetic and transcriptional changes through hypo-methylation of repressive chromatin marks (Extended Data Fig. 8d). Cartilage oligomeric matrix protein (COMP)—an extracellular matrix protein24, which was the most upregulated protein in all metastatic stroma samples (Fig. 1d)—is highly expressed in the stroma of omental
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Fig. 2 | NNMT is upregulated in the stroma of HGSC metastases and regulates the CAF phenotype. a, NNMT catalyses the transfer of a reactive methyl group from SAM to nicotinamide, generating SAH and the metabolically inert product 1-MNA, thus depleting SAM and reducing global cellular methylation potential. b, NNMT immunohistochemistry (IHC) finds increased expression in the stroma of omental metastases. NNMT is not expressed in the stroma of normal fallopian tube, ovary or omentum. Scale bars, 100 μm (25 μm for omental metastases 4×). c, Stromal NNMT expression is increased in omental and peritoneal metastases compared to ovarian sites (chi-squared test). d, Knockdown of NNMT in CAFs leads to a more elongated morphology resembling normal omental fibroblasts (GFP). Scale bar, 10 μm. e, Production of 1-MNA is attenuated upon knockdown and enhanced upon overexpression of NNMT. Two-sided t-test, n = 3 biological replicates. f, Immunoblot of
CAF markers (fibronectin, Fn1; SMA) upon knockdown or overexpression of NNMT. g, Effect of NNMT overexpression or knockdown on collagen contractility. Two-sided t-test, n = 3 biological replicates. h, Gene expression analysis identified thousands of genes that were significantly differentially expressed (red or blue) upon knockdown (left) or overexpression (right) of NNMT. FDR ANOVA < 0.01, n = 3 biological replicates. i, Proliferation (doubling time) of HeyA8 and TYK-nu ovarian cancer cells following treatment with the indicated conditioned medium. Proliferation rate increases (doubling time decreases) with NNMT overexpression and decreases (doubling time increases) upon knockdown. Two-sided t-test, n = 3 biological replicates. j, Quantification of chemotaxis in response to conditioned medium from CAFs expressing shCtrl or shNNMT constructs. Two-sided t-test, n = 3 biological replicates. All bar graphs represent mean of data and error bars are s.e.m.
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metastases, and its transcription is tightly regulated by NNMT expres-sion (Extended Data Fig. 8e–g). Histone methylation at the COMP promoter was increased upon knockdown of NNMT, which supports a central role for NNMT in functionally regulating histone methyla-tion of genes that are differentially expressed (Extended Data Fig. 8g). Treatment of CAFs that express short-hairpin RNA (shRNA) targeting NNMT (shNNMT) with the EZH2 histone methyltransferase inhibitor DZNep, the general histone methyltransferase inhibitor 3DZA or knockdown of EZH2 restored expression of CAF markers and promoted collagen contractility8 (Fig. 3g, h, Extended Data Fig. 9).
In a syngeneic model of HGSC metastasis, cancer cell metastasis to the omentum was significantly increased when ID8 cells were pre-treated with conditioned medium from fibroblasts overexpressing NNMT (Fig. 4a). The co-injection of HGSC cells with CAFs expressing control shRNA (shCtrl) or shNNMT constructs found that knockdown of stromal NNMT also reduced in vivo proliferation and overall tumour burden (Fig. 4b, Extended Data Fig. 10a). Recently, a small molecule inhibitor of NNMT was used to treat high-fat-diet-induced obesity in mouse models21. The inhibitor, 5-amino-1-methylquinolin-1-ium (NNMTi), was effective at inhibiting NNMT at micromolar concentra-tions and demonstrated target engagement as assessed by the cellular thermal shift assay (Extended Data Fig. 10b–d). Treatment of human CAFs with the NNMTi increased histone methylation, decreased tubu-lin acetylation and was specific to cells expressing NNMT (Fig. 4c, Extended Data Fig. 10e, f). NNMT inhibition did not affect the viability of CAFs or ovarian cancer cells (Extended Data Fig. 10g–i). In vivo treatment with the NNMTi decreased tumour burden in an orthotopic intraperitoneal model of ovarian cancer metastasis (Fig. 4d), reduced tumour cell proliferation and increased stromal H3K27 trimethylation (Extended Data Fig. 10j–m).
Although NNMT was primarily expressed in the stroma of omen-tal metastases, a subset of patients had high stromal expression of
NNMT in primary sites (Figs. 1e, 2c). The tissue microarray used to validate stromal NNMT expression was also used to evaluate the prognostic role of NNMT in chemotherapy-naive HGSC25. We found that high stromal NNMT protein expression in primary sites was associated with a significantly worse recurrence-free and overall survival and platinum resistance (median survival of 349 versus 598 days and 737 versus 1,489 days, respectively; Supplementary Table 12, Fig. 4e, Extended Data Fig. 10n, o). By contrast, expression of NNMT in the tumour compartment was not predictive of survival or recurrence (Extended Data Fig. 10p, q), which highlights the importance of compartment-resolved studies.
Using laser-capture microdissection combined with an optimized high-sensitivity proteomic pipeline, we quantified up to 5,000 unique protein groups per sample from as little as 5,000 cancer cells. This approach enabled compartment-resolved proteomic analysis of both tumour and stromal compartments across the HGSC progression series from STIC to metastatic tumours and revealed a metastatic stromal signature. Our results emphasize the molecular heterogeneity of ovarian cancer, and reveal that tumour proteomes within individual patients are relatively stable during progression—as was also recently observed in breast cancer26. The absence of a proteomic signature that differentiates STIC from advanced cancers suggests that STIC likewise already possesses the molecular aberrations at both the genomic and proteomic levels associated with a fully developed cancer, as sug-gested by recent genomic studies27,28. Despite the marked genetic and proteomic heterogeneity of epithelial ovarian cancer across patients, the metastatic stromal proteome was notably uniform and characterized by high NNMT expression and NNMT-regulated gene and protein expression. NNMT metabolically reprograms the epigenome of the stroma to co-opt NNMT-dependent processes that occur during stem cell priming17, metabolic syndrome15 and tumour aggressiveness14 (Fig. 4f). Whereas CAF gene expression can be regulated by chromatin
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Fig. 3 | NNMT regulates DNA and histone methylation to drive the CAF phenotype. a, Quantification of SAM, SAH and nicotinamide (NA) upon knockdown or overexpression of NNMT, n = 3 biological replicates. b, Distribution of significantly different β-values (Benjamini–Hochberg-adjusted P value < 0.01) corresponding to the degree of DNA methylation within 1,500 bp of transcriptional start sites in CAFs as assessed by global DNA methylation arrays. n = 2 biological replicates. c, Quantitative histone methylation proteomics in CAFs expressing shCtrl or shNNMT constructs following chromatin extraction. me1, mono-methylation; me2, dimethylation; me3, trimethylation. d, Immunoblotting of H3K4me3 and H3K27me3 in primary human CAFs when NNMT is inhibited (sh) or
in primary fibroblasts overexpressing NNMT. e, H3K27me3 ChIP-seq of normal fibroblasts overexpressing NNMT reveals that NNMT expression reduces H3K27me3 occupancy adjacent to transcriptional start sites (TSS) of genes significantly upregulated upon NNMT expression (Benjamini–Hochberg-adjusted P value < 0.05, n = 3 biological replicates). f, Enrichment of H3K27me3 at the Comp promoter as determined by qPCR in fibroblasts overexpressing NNMT. ANOVA, n = 3 biological replicates. g, h, Immunoblot of fibroblast markers (g) and collagen contractility of CAFs expressing shNNMT and treated with the EZH2 histone methyltransferase inhibitor DZNep (h). Two-sided t-test, n = 3 biological replicates. All bar graphs represent mean of data and error bars are s.e.m.
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modifiers and DNA methylation29,30, we find that metabolically defined histone methylation has a central role in defining the pro-tumorigenic role of the stroma. Inhibition of NNMT activity led to a reversion of the CAF phenotype, which suggests that stromal methyltransferase activ-ities can be targeted to normalize the metastatic stroma and should be further explored as treatment targets for cancer.
Online contentAny methods, additional references, Nature Research reporting summaries, source data, statements of data availability and associated accession codes are available at https://doi.org/10.1038/s41586-019-1173-8.
Received: 7 May 2017; Accepted: 27 March 2019; Published online 1 May 2019.
1. Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature 474, 609–615 (2011).
2. Zhang, H. et al. Integrated proteogenomic characterization of human high-grade serous ovarian cancer. Cell 166, 755–765 (2016).
3. Kuhn, E. et al. TP53 mutations in serous tubal intraepithelial carcinoma and concurrent pelvic high-grade serous carcinoma-evidence supporting the clonal relationship of the two lesions. J. Pathol. 226, 421–426 (2012).
4. Alexandrov, L. B. et al. Signatures of mutational processes in human cancer. Nature 500, 415–421 (2013).
5. Francavilla, C. et al. Phosphoproteomics of primary cells reveals druggable kinase signatures in ovarian cancer. Cell Reports 18, 3242–3256 (2017).
6. Coscia, F. et al. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status. Nat. Commun. 7, 12645 (2016).
7. Polyak, K., Haviv, I. & Campbell, I. G. Co-evolution of tumor cells and their microenvironment. Trends Genet. 25, 30–38 (2009).
8. Kalluri, R. The biology and function of fibroblasts in cancer. Nat. Rev. Cancer 16, 582–598 (2016).
9. Aebersold, R. & Mann, M. Mass-spectrometric exploration of proteome structure and function. Nature 537, 347–355 (2016).
10. Altelaar, A. F. & Heck, A. J. Trends in ultrasensitive proteomics. Curr. Opin. Chem. Biol. 16, 206–213 (2012).
11. Nieman, K. M. et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat. Med. 17, 1498–1503 (2011).
12. Pissios, P. Nicotinamide N-methyltransferase: more than a vitamin B3 clearance enzyme. Trends Endocrinol. Metab. 28, 340–353 (2017).
13. Aksoy, S., Szumlanski, C. L. & Weinshilboum, R. M. Human liver nicotinamide N-methyltransferase. cDNA cloning, expression, and biochemical characterization. J. Biol. Chem. 269, 14835–14840 (1994).
14. Ulanovskaya, O. A., Zuhl, A. M. & Cravatt, B. F. NNMT promotes epigenetic remodeling in cancer by creating a metabolic methylation sink. Nat. Chem. Biol. 9, 300–306 (2013).
15. Kraus, D. et al. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity. Nature 508, 258–262 (2014).
16. Hong, S. et al. Nicotinamide N-methyltransferase regulates hepatic nutrient metabolism through Sirt1 protein stabilization. Nat. Med. 21, 887–894 (2015).
17. Sperber, H. et al. The metabolome regulates the epigenetic landscape during naive-to-primed human embryonic stem cell transition. Nat. Cell Biol. 17, 1523–1535 (2015).
18. Perets, R. et al. Transformation of the fallopian tube secretory epithelium leads to high-grade serous ovarian cancer in Brca;Tp53;Pten models. Cancer Cell 24, 751–765 (2013).
19. Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121, 335–348 (2005).
20. Olumi, A. F. et al. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 59, 5002–5011 (1999).
21. Neelakantan, H. et al. Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice. Biochem. Pharmacol. 147, 141–152 (2018).
22. Ferro, A. J., Vandenbark, A. A. & MacDonald, M. R. Inactivation of S-adenosylhomocysteine hydrolase by 5′-deoxy-5′-methylthioadenosine. Biochem. Biophys. Res. Commun. 100, 523–531 (1981).
23. Zheng, Y. et al. Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3. Proc. Natl Acad. Sci. USA 109, 13549–13554 (2012).
24. Leung, C. S. et al. Calcium-dependent FAK/CREB/TNNC1 signalling mediates the effect of stromal MFAP5 on ovarian cancer metastatic potential. Nat. Commun. 5, 5092 (2014).
25. Kenny, H. A. et al. Targeting the urokinase plasminogen activator receptor inhibits ovarian cancer metastasis. Clin. Cancer Res. 17, 459–471 (2011).
26. Pozniak, Y. et al. System-wide clinical proteomics of breast cancer reveals global remodeling of tissue homeostasis. Cell Syst. 2, 172–184 (2016).
27. Eckert, M. A. et al. Genomics of ovarian cancer progression reveals diverse metastatic trajectories including intraepithelial metastasis to the fallopian tube. Cancer Discov. 6, 1342–1351 (2016).
28. Labidi-Galy, S. I. et al. High grade serous ovarian carcinomas originate in the fallopian tube. Nat. Commun. 8, 1093 (2017).
29. Hu, M. et al. Distinct epigenetic changes in the stromal cells of breast cancers. Nat. Genet. 37, 899–905 (2005).
30. Zong, Y. et al. Stromal epigenetic dysregulation is sufficient to initiate mouse prostate cancer via paracrine Wnt signaling. Proc. Natl Acad. Sci. USA 109, E3395–E3404 (2012).
Ctrl NNMTNormal mouse �broblasts
Mouse ID8 ovarian cancer cellsGFP/luciferase
Conditioned �broblast medium
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HR 2.015 (95% CI: 1.241–3.272)P = 0.0012 (log-rank)
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Fig. 4 | Stromal NNMT supports HGSC progression and is associated with a poor prognosis. a, Schematic of experimental design (top). Representative images and quantification of omental adhesion following intraperitoneal injection of luciferase- or GFP-labelled ID8 mouse ovarian cancer cells treated with conditioned medium from fibroblasts expressing the indicated constructs, n = 7 mice per group. Two-sided t-test. Scale bar, 500 μm. AU, arbitrary unit. b, In vivo proliferation and total tumour burden of luciferase-labelled ovarian cancer cells co-injected with CAFs expressing shCtrl or shNNMT constructs, n = 9 tumours per group. Scale bar, 1 cm. c, Treatment of CAFs with the NNMTi led to increased
histone methylation. d, Tumour burden of nude mice intraperitoneally injected with HeyA8 Ovarian cancer cells after 10 days of treatment with vehicle control (ctrl; PBS; n = 9) or NNMTi (n = 10). Two-sided t-test. e, Kaplan–Meier survival curves for patients with low (black) or high (red) stromal expression of NNMT in ovarian sites, as assessed by IHC. Two-tailed test. f, Stromal NNMT drives ovarian cancer progression by metabolic regulation of histone methylation which causes epigenetic and transcriptional changes in stromal cells promoting cancer cell proliferation, migration and metastasis. ECM, extracellular matrix. All bar graphs represent mean of data and error bars are s.e.m.
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Acknowledgements We thank H. A. Kenny, K. Watters and A. Mukherjee from the University of Chicago ovarian cancer laboratory for helpful discussions; and G. Isenberg, University of Chicago, for editing the manuscript. This work was supported by a Marsha Rivkin Foundation award (M.A.E.), National Cancer Institute (NCI) grants CA111882 and CA211916 (E.L.), the Ludwig Institute for Cancer Research (E.L.), the Arthur L. and Lee G. Herbst Professorship (E.L.), funding support from S. and J. Harris, M. Field, J. Kane and A. Gerry (M.A.E. and S.D.Y.), NIH grant CA175399 and DP2GM128199 (R.E.M.), V Foundation for Cancer Research (V2016-020 to R.E.M.), the Körber Foundation/Körber European Science Prize (M.M.), the Max-Planck Society for the Advancement of Science (M.M.), the Novo Nordisk Foundation (grant agreement NNF14CC0001 and NNF15CC0001; F.C. and M.M.), and University of Chicago Cancer Center Support Grant P30CA014599.
Reviewer information Nature thanks Amina Qutub and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Author contributions The study was conceived by M.A.E. and E.L. Proteomic sample preparation, analysis and interpretation were performed by F.C. under supervision of M.M. Experiments were designed by M.A.E., F.C., R.E.M., M.M. and E.L. Tissues were microdissected by M.A.E. and M.C. Experiments with primary and cancer cells were performed by M.A.E., S.P., A.C., D.A.N. and S.M.T. Animal experiments were performed by M.A.E., S.P., S.M.T. and A.C. Targeted metabolite analyses and inhibitor synthesis were performed by J.W.C and G.L. under the supervision of R.E.M. Data were analysed by M.A.E.,
F.C. and J.W.C. Human tissues for isolation of primary cells and proteomic analysis were identified and collected by S.D.Y. and E.L. Global metabolomics were performed, analysed and interpreted by I.B. under the supervision of O.F. Bioinformatics analyses of sequencing data were performed by K.M.H. under the supervision of J.A. Autochthonous mice were maintained and tissues collected and provided by R.P. Tissue microarrays were interpreted and scored by S.M.M. and R.R.L. Figures were prepared by M.A.E., F.C. and K.M.H. The paper was written by M.A.E. and E.L. The paper was edited by M.A.E., F.C., M.M., R.E.M. and E.L. All authors reviewed and provided feedback on the manuscript.
Competing interests The authors declare no competing interests.
Additional informationExtended data is available for this paper at https://doi.org/10.1038/s41586-019-1173-8.Supplementary information is available for this paper at https://doi.org/ 10.1038/s41586-019-1173-8.Reprints and permissions information is available at http://www.nature.com/reprints.Correspondence and requests for materials should be addressed to E.L.Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature Limited 2019
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