Letter https://doi.org/10.1038/s41586-019-1173-8

Proteomics reveals NNMT as a master metabolic regulator of cancer-associated fibroblasts Mark 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 all patients were chemotherapy-naive. For each patient and every to its early dissemination throughout the abdominal cavity. anatomic site, both tumour and stromal compartments were micro- Genomic and proteomic approaches have provided snapshots of the dissected and proteins extracted using an optimized high-sensitivity, proteogenomics of ovarian cancer1,2, but a systematic examination label-free proteomic workflow for low-input samples (Methods). In of both the tumour and stromal compartments is critical in total, we quantified 6,944 unique protein groups from 107 analysed understanding ovarian cancer metastasis. Here we develop a label- samples, both tumour and benign, at a protein and peptide false- free proteomic workflow to analyse as few as 5,000 formalin-fixed, discovery rate (FDR) of less than 1%. A median of 4,942 and 4,428 paraffin-embedded cells microdissected from each compartment. proteins were quantified per tumour or stromal sample, respectively, The tumour proteome was stable during progression from in situ at similar dynamic ranges and with excellent reproducibility (Pearson lesions to metastatic disease; however, the metastasis-associated r = 0.98; Fig. 1b, Extended Data Fig. 1a, b, Supplementary Table 2). stroma was characterized by a highly conserved proteomic signature, Unsupervised hierarchical clustering robustly segregated tumour and prominently including the methyltransferase nicotinamide stromal proteomes (Extended Data Fig. 1c). Tumour compartments N-methyltransferase (NNMT) and several of the proteins that were enriched for known markers of HGSC (PAX8, MSLN, MUC16 it regulates. Stromal NNMT expression was necessary and (CA-125), EPCAM) and DNA replication and repair pathways, whereas sufficient for functional aspects of the cancer-associated fibroblast stromal compartments were characterized by expression of extracel- (CAF) phenotype, including the expression of CAF markers and lular matrix components and pathways that included activated fibro- the secretion of cytokines and oncogenic extracellular matrix. blast markers (for example, collagens, vimentin, versican, tenascins and Stromal NNMT expression supported ovarian cancer migration, myosins) (Fig. 1c, Extended Data Fig. 1c–e, Supplementary Tables 3, 4). proliferation and in vivo growth and metastasis. Expression of Pairwise proteomic comparison of primary (invasive fallopian tube NNMT in CAFs led to depletion of S-adenosyl methionine and and ovarian lesions) and metastatic tumour compartments revealed reduction in histone methylation associated with widespread only one protein, FABP4—previously reported to be upregulated in gene expression changes in the tumour stroma. This work supports omental metastases and expressed at the tumour–stromal interface— the use of ultra-low-input proteomics to identify candidate drivers as significantly higher in omental tumour metastases11 (FDR < 0.01, of disease phenotypes. NNMT is a central, metabolic regulator of Fig. 1d, Extended Data Fig. 2a). The tumour compartment was instead CAF differentiation and cancer progression in the stroma that may characterized by patient-specific protein signatures (analysis of variance be therapeutically targeted. (ANOVA) FDR < 0.01, Extended Data Fig. 2b–e) that probably reflect All high-grade serous ovarian cancers contain TP53 mutations3 but the molecular heterogeneity of HGSC between patients1,2. By contrast, other recurrent mutations are rare and serous cancers are generally stromal proteomes clearly clustered by anatomic site, which reveals characterized by copy-number variants1,4. High-grade serous carci- a conserved stromal response to metastasis that is shared between noma (HGSC) has a high proportion of stroma, but little is known patients (Extended Data Fig. 2b–e). Differential expression analysis about how interactions between the cancer cells and the surrounding between the stromal compartments at the four anatomic sites (STIC, extracellular microenvironment affect tumour growth. Several reports fallopian tube, ovary and omentum) identified 128 differentially expressed describe the proteome of human ovarian cancer2,5,6 but none have dif- protein groups, with most contributed by the omental-metastasis- ferentiated between proteins in the stroma and the epithelial tumour associated stroma (FDR < 0.05, Extended Data Fig. 2d). Among compartment. Given that the stroma has a tumour-supporting role and these, we identified a set of 62 protein groups that were universally co-evolves with the epithelial compartment during progression and up- or downregulated in all omental metastases compared to all pri- metastasis7,8, we set out to evaluate the proteome of both compartments mary (invasive fallopian tube or ovarian lesion) stromal sites (FDR in a systematic method. We combined developments in ultra-high- < 0.01, Fig. 1d, Extended Data Fig. 2f). sensitivity mass-spectrometry-based proteomics9,10 with microdissection The stromal signature consisted of 21 up- and 41 downregulated technology to obtain an integrated picture of cancer progression. proteins, which included proteins known to have tumour-supporting To elucidate the proteomic changes that underlie ovarian cancer roles in the stroma such as FAP, LOX, TNC and VCAN and had con- progression in both the tumour and stroma, we identified a cohort siderable overlap with The Cancer Genome Atlas (TCGA) mesenchy- of 11 patients with HGSC with tissue from serous tubal in situ carci- mal subtype (Fig. 1e, Extended Data Fig. 2g, Supplementary Table 5). noma (STIC), invasive fallopian tube lesions, invasive ovarian lesions Downregulated proteins included negative regulators of TGF-β and omental metastases (Fig. 1a, Supplementary Table 1). All tissues signalling such as LTBP4 and SDPR. Other proteins, such as ENPP1 were collected prospectively during the initial debulking surgery and 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 9 Care Campus, Haifa, Israel. These authors contributed equally: Mark A. Eckert, Fabian Coscia. *e-mail: [email protected]

30 MAY 2019 | VOL 569 | NATURE | 723 RESEARCH Letter

a HGSC progression series STIC FT

1. Laser capture microdissection 2. Mass spectrometric analysis3. Label-free quanti cation

pare Com

p53 Stroma e Tim

Intensity Stroma

(m Tumour /z) Tumour Before LCM After LCM Ov Om Tumour Stroma b c 6 COL14A1 d 10 MYH11 10 Total = 6,944 proteins CAV1 6 MFAP5 ) 4 ) ACTA2 10 8 FABP4 8 10 COMP FN1 1,000)

× 2 VIM SDPR FABP4 Stroma 6 6 SDC1 4 LTBP4 CILP 0 -value (–log TNXB

P 4 4 -value (−log THBS2

P MYH11

2 Component 1 –2 Tumour MXRA5 Tumour PAX8 NNMT -test

t 2 2 Stroma EPCAM -test

t TNC –4 KRT7 MUC16 Proteins quanti ed ( 0 FOLR1 0 Primary Metastasis 0 Primary Metastasis STIC FT Ov Om 0 1,000 2,000 3,000 4,000 5,000 –10–50 510 –10–50 510

Proteins (ranked) Fold change metastasis/primary (log2) Fold change metastasis/primary (log2) e STIC Fallopian tube Ovary Omentum Proteinlevel (log2) 32

28

24 Patients 20

Not detected 1 1 1 1 LOX LOX LOX LOX TNC TNC TNC TNC CILP CILP CILP CILP OMD OMD OMD OMD SDC1 SDC1 SDC1 SDC1 NPNT NPNT NPNT NPNT VCAN VCAN VCAN VCAN CNN2 CNN2 CNN2 CNN2 ITGB5 ITGB5 ITGB5 ITGB5 NNMT NNMT NNMT NNMT COMP COMP COMP COMP FBLN2 FBLN2 FBLN2 FBLN2 FABP4 SFRP4 FABP4 SFRP4 FABP4 SFRP4 FABP4 SFRP4 ENPP1 THBS2 ENPP1 THBS2 ENPP1 THBS2 ENPP1 THBS2 COPZ2 COPZ2 COPZ2 COPZ2 MXRA5 MXRA5 MXRA5 MXRA5 CTHRC1 CTHRC1 CTHRC1 CTHRC1 LEPREL2 LEPREL2 LEPREL2 LEPREL2 COL11A COL11A COL11A COL11A Fig. 1 | Compartment-resolved proteomics of ovarian cancer d, Volcano plots comparing primary sites (fallopian tube and ovary) to progression reveals a stromal signature of HGSC metastasis. a, Tumour omental metastases in tumour (left) and stromal (right) compartments. and stromal compartments were microdissected from different anatomic Significantly differentially expressed proteins are highlighted in green sites (STIC, invasive fallopian tube (FT) lesions, ovarian (Ov) lesions (tumour) or purple (stroma). Two-sided t-test FDR < 0.01, n = 11 and omental (Om) metastases) and label-free, quantitative shotgun patients. e, Heat map of proteins upregulated in omental stromal signature proteomics performed to identify proteins differentially expressed across of metastasis across all patients (rows) and anatomic sites (STIC, fallopian all anatomic sites. b, Number of unique proteins quantified by MaxLFQ tube, ovary and omentum). Undetected values are black; missing samples in each anatomic compartment (n = 11 patients). c, Ranking of proteins are white. The box plots in b define the range of the data (whiskers), 25th by expression in tumour compartment (green; n = 43 samples) versus and 75th percentiles (box), and medians (solid line). stromal compartment (purple; n = 42 samples) shows known markers. or the biology of CAFs. Owing to its biochemical activity and roles in (SMA) expression, increased ability to contract collagenous matrices epigenetic regulation12, upregulation of NNMT in the omental stroma and expression of CAF markers8. Knockdown of NNMT in CAFs led was an interesting target. NNMT transfers a reactive methyl group from to a reversion of cell morphology to one that more-closely resembled S-adenosyl methionine (SAM) to nicotinamide to generate S-adenosyl normal omental fibroblasts (Fig. 2d, Extended Data Fig. 4a–c). homocysteine (SAH) and the metabolically inert product 1-methyl Knockdown or overexpression of NNMT led to a significant pertur- nicotinamide (1-MNA). SAM is the universal methyl donor for histones, bation of its enzymatic activity, as assessed by 1-MNA production non-histone proteins, DNA, RNA, lipids and other metabolites. This using mass spectrometry (Fig. 2e). CAF markers, including SMA and activity generates a methyl sink in the form of 1-MNA, which leads to fibronectin, were decreased upon NNMT knockdown and increased depletion of SAM and reduces the global methylation potential of the with overexpression (Fig. 2f, Extended Data Fig. 4d, e). cell13,14. NNMT-mediated SAM depletion regulates gene expression by NNMT was associated with expression of epithelial–mesenchymal attenuating histone methylation in cancer cells, adipocytes and embry- transition markers, and transcriptional regulators of the epithelial– onic stem cells14–17 (Fig. 2a). mesenchymal transition and inhibition of NNMT attenuated the The proteomic analysis revealed that NNMT expression was acquisition of CAF markers by normal stromal cells in response to increased in the stroma of peritoneal and omental metastases com- TGF-β (Extended Data Fig. 4f). NNMT was necessary and sufficient to pared to the benign omental, fallopian tube and ovarian stroma, induce collagen contractility (Fig. 2g) and globally regulated expression including early micrometastases, which was also confirmed in a of thousands of genes, including pro-tumorigenic cytokines (Fig. 2h, tissue microarray containing both primary and metastatic ovarian Extended Data Fig. 4g, Supplementary Tables 6, 7). In further support cancer samples (Fig. 2b, c, Extended Data Fig. 3a–d). Tumour expres- of NNMT as a central regulator of CAF gene expression, genes differ- sion of NNMT did not vary significantly by anatomic site (Extended entially expressed by NNMT in fibroblasts or CAFs were significantly Data Fig. 3e). In both syngeneic and autochthonous18 mouse models enriched for gene signatures associated with the epithelial– of HGSC, NNMT was highly expressed in the stroma of metastases mesenchymal transition, TCGA mesenchymal subtype, and proteins (Extended Data Fig. 3f). High stromal NNMT was also observed in that were highly expressed in the stroma of omental metastases com- breast and colon cancer stroma, which suggests that NNMT expres- pared to primary sites (Extended Data Fig. 4h–k). Functionally, CAFs sion is a feature of CAFs in multiple cancer types (Extended Data support and accelerate tumour growth, progression and metastasis8,19,20. Fig. 3g). Overexpression of NNMT in normal fibroblasts promoted cancer cell CAFs are differentiated from normal fibroblasts by production proliferation; conversely, knockdown of NNMT in CAFs attenuated of oncogenic extracellular matrix components, cytokine secretion, cancer cell proliferation and chemotaxis (Fig. 2i, j, Extended Data cytoskeletal rearrangements associated with smooth muscle actin Fig. 4l, m).

724 | NATURE | VOL 569 | 30 MAY 2019 Letter RESEARCH

a b c Stromal NNMT expression STIC Invasive FT Invasive Ov Om metastases Nicotinamide NNMT high NNMT low P < 0.0001 SAM 1.0 NNMT SAH

1-Methylnicotinamide 0.5 (1-MNA) NNMT IHC Proportion of samples ‘Methyl sink’ 0 Ov Om Periton Normal FT Normal Ov Normal Om Om metastases 4×

dgOvarian CAFs ef CAFs Fibroblasts CAFs Fibroblasts 150 shCtrl shNNMTCtrl NNMT P < 0.0001 8 P = 0.003 P = 0.0017 P = 0.0105 1.0 Fn1 )

6 2 100 shCtrl

4 SMA 0.5

Area (mm 50 2 NNMT

0 0 Relative 1-MNA concentration GAPDH 0 shNNMT Ctrl shCtrl shNNMTCtrl NNMT shCtrl NNMT shNNMT CAFs Fibroblasts

h CAFs Fibroblasts ijCAF medium Fibroblast medium CAF medium 10 2.0 12 shCtrl Ctrl shCtrl ) ) shNNMT 1.5 NNMT shNNMT 10 10 1.5 8 10 P = 0.04 P = 0.02 P = 0.03 P = 0.04 P = 0.04 P = 0.03 1.5 6 8 6 1.0 1.0 4 1.0 4 2 t -test P -value (–log t -test P -value (–log 2 0.5 0.5 0.5 Relative migration Ctrl NNMT

shNNMT shCtrl Relative doubling time Relative doubling time 0 0 –10 –5 0 510 –10 –5 0 510 Fold change Fold change 0 0 0 HeyA8 TYK-nu shCtrl/shNNMT (log2) NNMT/Ctrl (log2) HeyA8 TYK-nu HeyA8 TYK-nu Fig. 2 | NNMT is upregulated in the stroma of HGSC metastases and CAF markers (fibronectin, Fn1; SMA) upon knockdown or overexpression regulates the CAF phenotype. a, NNMT catalyses the transfer of a of NNMT. g, Effect of NNMT overexpression or knockdown on collagen reactive methyl group from SAM to nicotinamide, generating SAH and contractility. Two-sided t-test, n = 3 biological replicates. h, Gene the metabolically inert product 1-MNA, thus depleting SAM and reducing expression analysis identified thousands of genes that were significantly global cellular methylation potential. b, NNMT immunohistochemistry differentially expressed (red or blue) upon knockdown (left) or (IHC) finds increased expression in the stroma of omental metastases. overexpression (right) of NNMT. FDR ANOVA < 0.01, n = 3 biological NNMT is not expressed in the stroma of normal fallopian tube, ovary replicates. i, Proliferation (doubling time) of HeyA8 and TYK-nu or omentum. Scale bars, 100 μm (25 μm for omental metastases 4×). ovarian cancer cells following treatment with the indicated conditioned c, Stromal NNMT expression is increased in omental and peritoneal medium. Proliferation rate increases (doubling time decreases) with metastases compared to ovarian sites (chi-squared test). d, Knockdown of NNMT overexpression and decreases (doubling time increases) upon NNMT in CAFs leads to a more elongated morphology resembling normal knockdown. Two-sided t-test, n = 3 biological replicates. j, Quantification omental fibroblasts (GFP). Scale bar, 10 μm. e, Production of 1-MNA of chemotaxis in response to conditioned medium from CAFs expressing is attenuated upon knockdown and enhanced upon overexpression of shCtrl or shNNMT constructs. Two-sided t-test, n = 3 biological NNMT. Two-sided t-test, n = 3 biological replicates. f, Immunoblot of replicates. All bar graphs represent mean of data and error bars are s.e.m.

We hypothesized that high stromal NNMT expression drives gene contractility, which supports a direct role for DNA hypomethylation expression changes and acquisition of the CAF phenotype through in regulating the CAF phenotype (Extended Data Fig. 7c, d). hypomethylation of DNA, RNA or histones through attenuation of the To understand how NNMT affects histone methylation, we per- SAM:SAH ratio (that is, methylation potential of the cell)14. Indeed, formed targeted histone proteomic analyses, using a multi-reaction methylation potential was directly regulated by NNMT expression, monitoring approach to quantify relative levels of histone lysine as assessed by metabolite profiling with mass spectrometry. NNMT and arginine methylation23. NNMT knockdown increased histone knockdown increased the SAM:SAH ratio, whereas NNMT overex- methylation at residues associated with transcriptional regulation, pression led to a decrease (Fig. 3a). Nicotinamide levels were negatively including an increase in H3K4 and H3K27 trimethylation (me3; associated with NNMT expression (Fig. 3a). Knockdown of NNMT Fig. 3c). Immunoblotting confirmed that H3K27 and H3K4 trimeth- led to increased levels of NAD+(H) and was associated with increased ylation was perturbed upon knockdown or overexpression of NNMT expression of NAD-dependent sirtuin target genes such as catalase (Fig. 3d). Histone methylation was metabolically sensitive to cellular and CD36, and decreased acetylation of H3K9 and α-tubulin15,21 methylation potential and extracellular methionine concentration (Extended Data Fig. 5). Targeted metabolomics identified conserved (Extended Data Fig. 7e, f). Genes regulated by NNMT overexpres- metabolic changes upon both knockdown and overexpression of sion were associated with gene sets regulated by H3K27 demethylation NNMT (Extended Data Fig. 6a, Supplementary Table 8). In particular, (Extended Data Fig. 8a). Chromatin immunoprecipitation sequenc- NNMT regulated the polyamine pathway and increased levels of ing (ChIP-seq) of cells that overexpress NNMT found that NNMT 5-methylthioadenosine, an inhibitor of SAH hydrolase that contrib- activity reduced global H3K27 occupancy, including at the promot- utes to global hypomethylation22 (Extended Data Fig. 6b–d). NNMT ers of NNMT-regulated genes (Fig. 3e, f, Extended Data Fig. 8b, c, expression induced genome-wide DNA-methylation changes that Supplementary Table 11). These data suggest that NNMT mediates significantly altered methylation status in the promoter regions of genome-wide epigenetic and transcriptional changes through hypo- genes, and was enriched for genes involved in collagen production methylation of repressive chromatin marks (Extended Data Fig. 8d). and myosin-driven contractility (Fig. 3b, Extended Data Fig. 7a, b, Cartilage oligomeric matrix protein (COMP)—an extracellular matrix Supplementary Tables 9, 10). Inhibition of DNA methylation with protein24, which was the most upregulated protein in all metastatic 5-azacytidine increased expression of CAF markers and collagen stroma samples (Fig. 1d)—is highly expressed in the stroma of omental

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abcdFold change shNNMT/shCtrl CAFs CAFs Fibroblasts shCtrl shNNMT (log2) shCtrl Ctrl H3K56me1 H3K4me3 shNNMT NNMT H3Q52me1 3 4 DNA methylation H3R42me2 2.0 H1K25me2 Histone H3 n CAFs shCtrl H3R49me2 2 io H3Q19me1

at CAFs shNNMT H3K4me3 P = 0.0014 tr H3K23me1 H3K27me3 3 1.5 en P = 0.0.0296 H3K27me3 2 P = 0.003 6 H3K18me1 nc H4K20me1 Histone H3 co H1K25me3 1.0 H3K36me3

li te Fibroblasts 2 H3K36me2 Density 0 bo H3K4me2 Ctrl NNMT ta P = 0.0001 H3K4me1

P = 0.0.0008 0.5 1 P < 0.0.0001 H3K9me3 H3K4me3 me H4K20me2 H1K25me1 ve 1

ti H4K20me3 Histone H3

la 0 H3K79me3

Relative metabolite concentration H3K9me2 Re 0 0.20.4 0.60.8 1.0 H3K27me1 E-value H3K9me1 –2 H3K27me3 0 0 H3K79me1 M H A M H A H3K27me2 Histone H3 A A N A A N H3K36me1 S S S S H3K79me2

e Ctrl NNMT f g h CAFs shNNMT H3K27me3 ChIP 7 3 3 P = 0.0034 Ctrl Ctrl 6 NNMT CAFs shNNMT 150 NNMT Ctrl DZNep P = 0.0279 5 H3K27me3 2 2 ) 4 2 100 Total H3 3 1 SMA 50 2 1 Area (m m

Upregulated gene s NS 1 GAPDH Normalized read densit y 0 0 0 0 Ctrl

Percentage of input, Comp promoter H3K27me3 IgG DZNep TSS TSS 5 kb TSS 5 kb 5.0 kb –5 kb –5 kb –5.0 kb Fig. 3 | NNMT regulates DNA and histone methylation to drive the in primary fibroblasts overexpressing NNMT. e, H3K27me3 ChIP-seq of CAF phenotype. a, Quantification of SAM, SAH and nicotinamide (NA) normal fibroblasts overexpressing NNMT reveals that NNMT expression upon knockdown or overexpression of NNMT, n = 3 biological replicates. reduces H3K27me3 occupancy adjacent to transcriptional start sites (TSS) b, Distribution of significantly different β-values (Benjamini–Hochberg- of genes significantly upregulated upon NNMT expression (Benjamini– adjusted P value < 0.01) corresponding to the degree of DNA methylation Hochberg-adjusted P value < 0.05, n = 3 biological replicates). within 1,500 bp of transcriptional start sites in CAFs as assessed by global f, Enrichment of H3K27me3 at the Comp promoter as determined by qPCR DNA methylation arrays. n = 2 biological replicates. c, Quantitative in fibroblasts overexpressing NNMT. ANOVA, n = 3 biological replicates. histone methylation proteomics in CAFs expressing shCtrl or shNNMT g, h, Immunoblot of fibroblast markers (g) and collagen contractility constructs following chromatin extraction. me1, mono-methylation; of CAFs expressing shNNMT and treated with the EZH2 histone me2, dimethylation; me3, trimethylation. d, Immunoblotting of H3K4me3 methyltransferase inhibitor DZNep (h). Two-sided t-test, n = 3 biological and H3K27me3 in primary human CAFs when NNMT is inhibited (sh) or replicates. All bar graphs represent mean of data and error bars are s.e.m. metastases, and its transcription is tightly regulated by NNMT expres- NNMT in primary sites (Figs. 1e, 2c). The tissue microarray used sion (Extended Data Fig. 8e–g). Histone methylation at the COMP to validate stromal NNMT expression was also used to evaluate promoter was increased upon knockdown of NNMT, which supports the prognostic role of NNMT in chemotherapy-naive HGSC25. We a central role for NNMT in functionally regulating histone methyla- found that high stromal NNMT protein expression in primary tion of genes that are differentially expressed (Extended Data Fig. 8g). sites was associated with a significantly worse recurrence-free Treatment of CAFs that express short-hairpin RNA (shRNA) targeting and overall survival and platinum resistance (median survival NNMT (shNNMT) with the EZH2 histone methyltransferase inhibitor of 349 versus 598 days and 737 versus 1,489 days, respectively; DZNep, the general histone methyltransferase inhibitor 3DZA Supplementary Table 12, Fig. 4e, Extended Data Fig. 10n, o). or knockdown of EZH2 restored expression of CAF markers and By contrast, expression of NNMT in the tumour compartment was promoted collagen contractility8 (Fig. 3g, h, Extended Data Fig. 9). not predictive of survival or recurrence (Extended Data Fig. 10p, q), In a syngeneic model of HGSC metastasis, cancer cell metastasis which highlights the importance of compartment-resolved studies. to the omentum was significantly increased when ID8 cells were pre- Using laser-capture microdissection combined with an optimized treated with conditioned medium from fibroblasts overexpressing high-sensitivity proteomic pipeline, we quantified up to 5,000 unique NNMT (Fig. 4a). The co-injection of HGSC cells with CAFs expressing protein groups per sample from as little as 5,000 cancer cells. This control shRNA (shCtrl) or shNNMT constructs found that knockdown approach enabled compartment-resolved proteomic analysis of both of stromal NNMT also reduced in vivo proliferation and overall tumour tumour and stromal compartments across the HGSC progression burden (Fig. 4b, Extended Data Fig. 10a). Recently, a small molecule series from STIC to metastatic tumours and revealed a metastatic inhibitor of NNMT was used to treat high-fat-diet-induced obesity stromal signature. Our results emphasize the molecular heterogeneity in mouse models21. The inhibitor, 5-amino-1-methylquinolin-1-ium of ovarian cancer, and reveal that tumour proteomes within individual (NNMTi), was effective at inhibiting NNMT at micromolar concentra- patients are relatively stable during progression—as was also recently tions and demonstrated target engagement as assessed by the cellular observed in breast cancer26. The absence of a proteomic signature that thermal shift assay (Extended Data Fig. 10b–d). Treatment of human differentiates STIC from advanced cancers suggests that STIC likewise CAFs with the NNMTi increased histone methylation, decreased tubu- already possesses the molecular aberrations at both the genomic and lin acetylation and was specific to cells expressing NNMT (Fig. 4c, proteomic levels associated with a fully developed cancer, as sug- Extended Data Fig. 10e, f). NNMT inhibition did not affect the viability gested by recent genomic studies27,28. Despite the marked genetic and of CAFs or ovarian cancer cells (Extended Data Fig. 10g–i). In vivo proteomic heterogeneity of epithelial ovarian cancer across patients, the treatment with the NNMTi decreased tumour burden in an orthotopic metastatic stromal proteome was notably uniform and characterized intraperitoneal model of ovarian cancer metastasis (Fig. 4d), reduced by high NNMT expression and NNMT-regulated gene and protein tumour cell proliferation and increased stromal H3K27 trimethylation expression. NNMT metabolically reprograms the epigenome of the (Extended Data Fig. 10j–m). stroma to co-opt NNMT-dependent processes that occur during stem Although NNMT was primarily expressed in the stroma of omen- cell priming17, metabolic syndrome15 and tumour aggressiveness14 tal metastases, a subset of patients had high stromal expression of (Fig. 4f). Whereas CAF gene expression can be regulated by chromatin

726 | NATURE | VOL 569 | 30 MAY 2019 Letter RESEARCH

abConditioned broblast medium Proliferation Tumour burden 48 h Injection (ip) Ctrl NNMT C57BL/6 P = 0.0016 Mouse ID8 ovarian cancer cells Normal mouse broblasts 6 P = 0.0463 10 GFP/luciferase P = 0.0124 Omental metastases 200

4 150 5

HeyA8 in vivo 100 2 doubling time (days)

ID8 GFP

Tumour mass (mg) 50 Relative ID8 metastasis (AU ) Control media NNMT media 0 0 0 Ctrl NNMT shCtrl shNNMT shCtrl shNNMT Medium

c d 1.5 e f NNMTi P < 0.0001 Overall survival CAFs Cancer cells

Ctrl 10 μM 100 Stromal NNMT low (n = 57) Stromal NNMT high (n = 39) H3K4me3 1.0 NNMT HR 2.015 (95% CI: 1.241–3.272) Cytokines Histone H3 P = 0.0012 (log-rank) and SAM oncogenic 50 SAH ECM H3K27me3 0.5 Survival (%) DNA and histone Tumour mass (g) Histone H3 hypomethylation Proliferation Migration 0 0 0 2,000 4,000 6,000 8,000 Metastasis Ctrl NNMTi Time (days) Fig. 4 | Stromal NNMT supports HGSC progression and is associated histone methylation. d, Tumour burden of nude mice intraperitoneally with a poor prognosis. a, Schematic of experimental design (top). injected with HeyA8 Ovarian cancer cells after 10 days of treatment with Representative images and quantification of omental adhesion following vehicle control (ctrl; PBS; n = 9) or NNMTi (n = 10). Two-sided t-test. intraperitoneal injection of luciferase- or GFP-labelled ID8 mouse ovarian e, Kaplan–Meier survival curves for patients with low (black) or high cancer cells treated with conditioned medium from fibroblasts expressing (red) stromal expression of NNMT in ovarian sites, as assessed by IHC. the indicated constructs, n = 7 mice per group. Two-sided t-test. Scale Two-tailed test. f, Stromal NNMT drives ovarian cancer progression by bar, 500 μm. AU, arbitrary unit. b, In vivo proliferation and total tumour metabolic regulation of histone methylation which causes epigenetic burden of luciferase-labelled ovarian cancer cells co-injected with CAFs and transcriptional changes in stromal cells promoting cancer cell expressing shCtrl or shNNMT constructs, n = 9 tumours per group. proliferation, migration and metastasis. ECM, extracellular matrix. All bar Scale bar, 1 cm. c, Treatment of CAFs with the NNMTi led to increased graphs represent mean of data and error bars are s.e.m. modifiers and DNA methylation29,30, we find that metabolically defined 13. Aksoy, S., Szumlanski, C. L. & Weinshilboum, R. M. Human liver nicotinamide N-methyltransferase. cDNA cloning, expression, and biochemical histone methylation has a central role in defining the pro-tumorigenic characterization. J. Biol. Chem. 269, 14835–14840 (1994). role of the stroma. Inhibition of NNMT activity led to a reversion of the 14. Ulanovskaya, O. A., Zuhl, A. M. & Cravatt, B. F. NNMT promotes epigenetic CAF phenotype, which suggests that stromal methyltransferase activ- remodeling in cancer by creating a metabolic methylation sink. Nat. Chem. Biol. 9, 300–306 (2013). ities can be targeted to normalize the metastatic stroma and should be 15. Kraus, D. et al. 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Acknowledgements We thank H. A. Kenny, K. Watters and A. Mukherjee from F.C. and J.W.C. Human tissues for isolation of primary cells and proteomic the University of Chicago ovarian cancer laboratory for helpful discussions; analysis were identified and collected by S.D.Y. and E.L. Global metabolomics and G. Isenberg, University of Chicago, for editing the manuscript. This work were performed, analysed and interpreted by I.B. under the supervision of O.F. was supported by a Marsha Rivkin Foundation award (M.A.E.), National Cancer Bioinformatics analyses of sequencing data were performed by K.M.H. under Institute (NCI) grants CA111882 and CA211916 (E.L.), the Ludwig Institute for the supervision of J.A. Autochthonous mice were maintained and tissues Cancer Research (E.L.), the Arthur L. and Lee G. Herbst Professorship (E.L.), collected and provided by R.P. Tissue microarrays were interpreted and funding support from S. and J. Harris, M. Field, J. Kane and A. Gerry (M.A.E. scored by S.M.M. and R.R.L. Figures were prepared by M.A.E., F.C. and K.M.H. and S.D.Y.), NIH grant CA175399 and DP2GM128199 (R.E.M.), V Foundation The paper was written by M.A.E. and E.L. The paper was edited by M.A.E., for Cancer Research (V2016-020 to R.E.M.), the Körber Foundation/Körber F.C., M.M., R.E.M. and E.L. All authors reviewed and provided feedback on the European Science Prize (M.M.), the Max-Planck Society for the Advancement manuscript. of Science (M.M.), the Novo Nordisk Foundation (grant agreement NNF14CC0001 and NNF15CC0001; F.C. and M.M.), and University of Chicago Competing interests The authors declare no competing interests. Cancer Center Support Grant P30CA014599. Additional information Reviewer information Nature thanks Amina Qutub and the other anonymous Extended data is available for this paper at https://doi.org/10.1038/s41586- reviewer(s) for their contribution to the peer review of this work. 019-1173-8. Supplementary information is available for this paper at https://doi.org/ Author contributions The study was conceived by M.A.E. and E.L. Proteomic 10.1038/s41586-019-1173-8. sample preparation, analysis and interpretation were performed by F.C. under Reprints and permissions information is available at http://www.nature.com/ supervision of M.M. Experiments were designed by M.A.E., F.C., R.E.M., M.M. reprints. and E.L. Tissues were microdissected by M.A.E. and M.C. Experiments with Correspondence and requests for materials should be addressed to E.L. primary and cancer cells were performed by M.A.E., S.P., A.C., D.A.N. and Publisher’s note: Springer Nature remains neutral with regard to jurisdictional S.M.T. Animal experiments were performed by M.A.E., S.P., S.M.T. and A.C. claims in published maps and institutional affiliations. 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., © The Author(s), under exclusive licence to Springer Nature Limited 2019

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