All chemical reagents were purchased from Sigma-Aldrich unless otherwise noted. Nicotinamide-2,4,5,6-d4 (d4-NA) and S-adenosyl-L-methionine-d3 (S-methyl-d3) (d3-SAM) were purchased from C/D/N isotopes (D-3457 and D-4093, respectively). L-methionine-(methyl-13C,d3) (13Cd3-methionine) was from Sigma-Aldrich (#299154). Serine-2,3,3-d3 (d3-serine) was purchased from Cambridge Isotope Laboratories (DLM-1073-1). S-(5′-deoxyadenosin-5′-yl)-L-homocysteine-d4 (d4-SAH) was purchased from Cayman Chemical Company.
General synthetic methods
1H NMR and 13C NMR spectra were recorded on Bruker DRX-600 spectrometers using residual solvent peak as an internal standard. NMR chemical shifts are reported in ppm using residual solvent peak as internal standard, and J values are reported in Hz. High resolution mass spectra were recorded on an Agilent 6520 mass spectrometer using ESI-TOF.
1-Methylnicotinamide was synthesized using a modification of a protocol reported by French et. al51
. Briefly, nicotinamide (1 g, 8.2 mmol) was charged into flame-dried RBF under N2
and diluted in methanol (10 mL, 0.8 M). Methyl iodide (1.52 mL, 16.4 mmol) was then added dropwise at room temperature and the reaction allowed to stir overnight at room temperature. The resulting yellow precipitate was isolated by filtration, rinsed with methanol, and dried in vacuo
. Recrystallization from methanol afforded a product (51% yield) whose spectral properties were consistent with previously reported values52
(600 MHz, D2
O) δ 9.30 (s, 1H), 8.98 (d, J
= 6.0, 1H), 8.90 (d, J
= 8.4, 1H), 8.19 (t, J
= 7.2, 1H), 4.49 (s, 3H); 13C-NMR
(600 MHz, D2
O) δ 166.7, 148.1, 146.0, 144.4, 134.4, 128.8, 49.4. HRMS
): [M+] calculated for C7
, 137.070939; found, m/z
The same procedure as above was followed to produce d4-1MNAin 71% yield: 1H-NMR (600 MHz, D2O) δ 4.47 (s, 3H); 13C-NMR (600 MHz, D2O) δ 166.7, 147.8 (t, J = 119), 145.7 (t, J = 120), 144.1 (t, J = 110), 128.4 (t, J = 102), 49.4. HRMS (ESI) (m/z): [M+] calculated for C7H5D4N2O+, 141.096046; found, 141.097323.
SKOV3 and OVCAR3 cell lines were obtained from NCI’s Developmental Therapeutics Program. 786O, 769P, H266 and H522 lines were purchased from ATCC. C8161 and MUM2C were provided by Mary Hendrix. All cell lines were cultured in RPMI-1640 medium (Cellgro) supplemented with 10% FBS, 2 mM glutamine and 10 mM HEPES buffer (complete RPMI-1640, 100 μM methionine). Unless otherwise noted, cells were seeded at 4 × 106 cells/150 mm dish in complete RPMI-1640 and allowed to proliferate for 16–24 h before experiments were performed. Low methionine medium was prepared by adding 10 and 20 μM methionine to a methionine-free RPMI-1640 (A14517-01, GIBCO), supplemented with 10% dialyzed FBS, 2 mM glutamine and 10 mM HEPES buffer. See overexpression and RNA interference studies for low methionine culture conditions.
Retroviral overexpression of wildtype and mutant NNMT in human cancer cell lines
The cDNA clone of human NNMT (BC000234, Open BioSystems) in pOTB7 was subcloned into pET-45b (+) vector for further manipulations. Catalytically inactive NNMT-Y20A mutant was generated by QuikChange site-directed mutagenesis using the primer 5′-atctaagccattttaaccctcgggatgc
cctagaaaaatattacaagtttg-3′ and its complement. Wildtype and mutant NNMT were cloned into modified pCLNCX retroviral vector 53
. Retrovirus was prepared by taking 1.5 μg of both pCLNCX and pCL-Ampho vectors and 20 μl of FuGENE HD reagent (Roche) to transfect 80% confluent HEK293T cells. Virus containing supernatant from day 2 was collected and, in the presence of 8 μg/ml polybrene, used to stably infect cells for 72 h. Infection was followed by 7–14 days of selection in medium containing hygromycin B (100 μg/ml). Cells were expanded and cultured in complete RPMI-1640.
For all experiments except for studies in low methionine medium, cells were seeded at 4 × 106 cells/150 mm dish and were allowed to proliferate in complete RPMI for 16–24 h. For studies performed in low (10 μM and 20 μM) methionine medium, cells were washed with PBS, seeded at a concentration of 2 × 106 cells/150 mm dish, and cultured for 48h in methionine-free RPMI supplemented with indicated amounts of methionine.
NNMT activity assay
Cell pellets were resuspended in either 50 mM Tris-HCl, pH 8.0 or PBS (lung carcinoma lines), followed by sonication and centrifugation at 16,000 g for 10 min. Lysates (70–120 μg) were incubated with a reaction mixture (200 μM d4-NA, 50 μM S-adenosyl-L-methionine (SAM) and 2 mM DTT) at room temperature for 15–30 min in a volume of 20 μl. Reactions were quenched with equal amount of methanol, followed by a 10 min centrifugation at 16,000 g. Formation of d4-1MNA was followed by targeted LC-MS analysis. Briefly, 1MNA-d4 was separated with a Luna-NH2 column (5 μm, 100A, 50 × 4.6 mm, Phenomenex) together with a pre-column (NH2, 4 × 3.0 mm). Mobile phase A was composed of 100% CH3CN containing 0.1% formic acid, and mobile phase B was composed of 95:5 v/v H2O:CH3CN supplemented with 50 mM NH4OAc and 0.2% NH4OH. The flow rate started at 0.1 ml/min and the gradient consisted of 5 min 0% B, a linear increase to 100% B over 15 min at a flow rate of 0.4 ml/min, followed by an isocratic gradient of 100% B for 15 min at 0.5 ml/ml before equilibrating for 5 min at 0% B at 0.4 ml/min (40 min total). For each run the ejection volume was 20 μl. MS analysis was performed on an Agilent G6410B tandem mass spectrometer with ESI source. The dwell time for d4-1MNA was set to 100 ms. The capillary was set to 4 kV, the fragmentor was set to 100 V. The drying gas temperature was 350 °C, the drying gas flow rate was 11 l/min, and the nebulizer pressure was 35 psi. The mass spectrometer was run in MRM mode, monitoring the transition of m/z from 141 to 98 for d4-1MNA (positive ionization mode).
RNA interference studies in human cancer cell lines
Hs-NNMT-8 (si-NNMT, cagctactacatgattggtga) and Ctrl-AllStars-1 (si-Control, siRNA that has no homology to any known mammalian gene) were purchased from QIAGEN as FlexiTube siRNA premix. Cells were seeded at 0.25 × 106 cells/100 mm dish followed by treatment with siRNA premix reagent. Cells were cultured in complete RPMI for 72 h and tested for the loss of NNMT activity.
For studies performed in high and low methionine medium, 72 h after transfection complete RPMI medium was exchanged with RPMI containing indicated amount of methionine and cells were allowed to proliferate for additional 24 h. NNMT knockdown was confirmed in each experiment.
Untargeted metabolomic analysis of cancer cell lines
GFP-OE and NNMT-OE human cell lines were seeded at 4 × 106
cells/150 mm dish and cultured in complete RPMI medium for 24 h followed by 4 h serum starvation. Cells were scraped into ice-cold PBS and isolated by centrifugation at 1,400 × g at 4 °C. Water soluble cellular metabolites were extracted using methanol-water extraction protocol, essentially as previously described17
. In brief, cell pellets were re-suspended in 100 μl of a 80:20 mixture of MeOH:H2
O. For some experiments, internal deuterated standards, including 1 nmol d4-
NA, 0.1 nmol d4-
1MNA, 0.1 nmol 13Cd3
-methionine, 0.1 nmol d3-
SAM, 0.1 nmol d4-
SAH, and 10 nmol d3-
serine, were added to the extraction solution, for absolute quantification and sample normalization. The mixture was sonicated for 5 s followed by a 10 min centrifugation at 16,000 × g. The supernatant was collected and stored at −80° C or injected directly into mass spectrometer (30 μl). Metabolites were also extracted by an alternative protocol involving direct scrapping into organic solvent54
for the data shown in Supplementary Fig. 7
. GFP-OE and NNMT-OE samples (3–4 replicates/line) were run sequentially. Water soluble cellular metabolites were separated by hydrophilic interaction chromatography17
with a Luna-NH2
column together with a pre-column. Mobile phase A was composed of 100% CH3
CN, and mobile phase B was composed of 95:5 v/v H2
CN. Both solvents were supplemented with 0.1% formic acid to assist ion formation in a positive mode. For negative mode analysis, mobile phase B was supplemented with 50 mM NH4
OAc and 0.2% NH4
OH. The flow rate started at 0.1 ml/min for 5 min. The gradient started with 0% B for 5 min and increased linearly to 100% B over 40 min with a flow rate of 0.4 ml/min, followed by an isocratic gradient of 100% B for 10 min at 0.5 ml/min. Then column was equilibrated with 0% B for 5 min at 0.4 ml/min. MS analysis, scanning from m/z
= 50–1200, was performed on Agilent 6520 Accurate Mass Q-TOF with ESI source. Untargeted LC-MS analysis was performed in both positive and negative ionization mode. The capillary was set to 4 kV. The drying gas temperature was 350 °C, the drying gas flow rate was 11 l/min, and the nebulizer pressure was 45 psi.
To identify metabolites with differential levels in NNMT-OE versus GFP-OE cells, we employed XCMS analyte profiling software. In brief, XCMS identifies features whose relative intensity varies between sample groups (group 1: NNMT-OE replicates; group2: GFP-OE replicates) and calculates fold changes, as well as P-
values. XCMS software allows quick access to the quality of each feature by generating extracted ion chromatograms display panels (see and Supplementary Fig. 5
). Obtained data sets were first filtered based on P-
< 0.01) and fold change (fold > 2). Significant peak changes between samples were confirmed by manually extracting MS1 signals and by calculating the area under the peak from MS1 chromatograms. We next clustered the changing metabolites into groups based on whether they appear in one, two, or all three (769P, MUM2C and OVCAR3) pairs of NNMT-OE/GFP-OE. Prioritization was given to those metabolites that were found to change in all three cancer cell line sets. Two metabolites identified as 1MNA and SAH (see ) were consistently deregulated across all three cell lines (Supplementary Dataset 1
In addition to ions corresponding to endogenous 1MNA and SAH, an ion with m/z value of 152.074417 was also consistently elevated in all NNMT-OE lines compared to GFP-OE lines. This ion was identified as 3-methoxycarbonyl-1-methylpyridinium by using a combination of high-resolution MS (observed m/z = 152.074417, calculated m/z = 152.070605) and co-elution with authentic sample of 3-methoxycarbonyl-1-methylpyridinium. Elevated levels of 3-methoxycarbonyl-1-methylpyridinium in NNMT-OE metabolomes compared to GFP-OE metabolomes are likely due to the slow alcoholysis of NNMT product 1MNA in the methanolic extracts, as no 152.074417 ion was detected in metabolomes from GFP-OE and NNMT-OE cells when cell pellets were extracted with a 50:50 mixture of CH3CN:H2O. In addition, formation of this ester from synthetic 1MNA was also observed in methanolic solutions containing residual amounts of PBS without cellular extracts, confirming the non-metabolic origin of the compound. Interestingly, this ester is not formed in pure 80:20 MeOH:H2O mixture, suggesting that certain components of residual PBS catalyze the solvolysis. Considering the non-metabolic nature of this compound, it was excluded from the list of deregulated metabolites identified by metabolomic analysis.
LC-MS co-migration studies
Metabolomes from GFP-OE and NNMT-OE 769P cells were prepared and analyzed as described above. The identity of endogenous m/z = 137.07 was confirmed by overlapping its extracted ion chromatogram with MS1 ion chromatogram of d4-1MNAinternal standard (m/z = 141.10). Similarly, the identity of endogenous m/z = 385.13 was confirmed by overlapping its extracted ion chromatogram with MS1 ion chromatogram of d4-SAHinternal standard (m/z = 389.16).
MS/MS fragmentation studies
LC-MS/MS analysis was performed on an Agilent 6520 as just described in positive ionization mode. MS and MS/MS data were collected in scanning mode from m/z = 50–2000 and m/z = 50–2500, and a rate of 1.03 spectra/s. The capillary voltage was set to 4 kV, and the fragmentor voltage was set to 100 V. The drying gas temperature was 350 °C, the drying gas flow rate was 11 l/min, and the nebulizer pressure was 45 psi. The collision energy for 1MNA and SAH was 20 V and 5 V, respectively.
Metabolic labeling studies
769P cells were seeded at 1.5 × 106 cells/150 mm dish and were cultured overnight in complete RPMI medium. The next day, the medium was replaced with serum-free medium containing either d4-1MNA (100 μM) or d4-NA (100 μM). Control samples were prepared by incubating cells with the same concentration of authentic compound (either 1MNA or NA). After an additional 4 or 24 h, cellular metabolomes were prepared and analyzed in the untargeted scanning mode as described above. The resultant chromatograms were analyzed by extracting relative m/z values and quantified by calculating the area under the peak. The following deuterated metabolites were detected: d4-1MNA (m/z = 141.10, RT = 5.6 min, pos. mode), d4-NA (m/z = 127.08, RT = 6.1 min, pos. mode), d3-NA (m/z = 126.07, RT = 6.1 min, pos. mode), d3-NMN(m/z = 338.08, RT = 28.2 min, pos. mode), d3-NAD+ (m/z = 665.12, RT = 28.1 min, neg. mode), and d3-NADH(m/z = 667.14, RT = 33.9 min, neg. mode). These metabolites were absent in control cells and their identity was confirmed by co-elution with corresponding authentic metabolites in control cells and authentic standards (1MNA, m/z = 137.07, RT = 5.6 min, pos. mode; NA, m/z = 123.06, RT = 6.1 min, pos. mode; NMN, m/z = 335.06, RT = 28.2 min, pos. mode; NAD+, m/z = 662.10, RT = 28.1 min, neg. mode). The formation of d3-labeled metabolites in NAD+ biosynthetic pathway could be explained by the oxidation/reduction of NAD(H) which would result in the loss of deuterium. Flux of metabolites through the NAD+ pathway would then generate the steady state levels of d3-labeled metabolites that are measured in this assay. Consistent with this model, we observed the time-dependent increase in the ratio of d3-NA/d4-NA in our metabolic labeling studies.
Targeted MRM measurements of intracellular metabolites
Cells were collected by scraping into ice-cold PBS followed by centrifugation at 1,400 × g. Cellular metabolites were extracted with 100 μl of a 80:20 mixture of MeOH:H2O, containing the following deuterated standards: 0.1 – 1 nmol d4-NA, 0.1 nmol d4-1MNA, 0.1 nmol 13Cd3-methionine, 0.1 nmol d3-SAM, 0.1 nmol d4-SAH, and 10 nmol d3-serine. The mixture was sonicated for 5 s followed by a 10 min centrifugation at 16,000 × g. Supernatant was collected, and 30 μl was subjected to LC-MS analysis. LC separation was performed as described above for metabolomics experiments. MS analysis was performed on Agilent G6410B tandem mass spectrometer with ESI source as described in NNMT activity assay. Mass spectrometer was running in MRM mode. Metabolites were quantified by measuring the area under the peak in comparison to the deuterated standards. Cellular SAH levels were calculated by subtracting the residual amount of SAH present in internal standard d3-SAM from total SAH levels. Amount of SAH coming from d3-SAM constituted less than 30% of total SAH. The following MS transitions and retention time (RT) were used to measure the indicated metabolites: 1MNA (m/z 137 →94, RT = 5.6 min), d4-1MNA (m/z 141 →98, RT = 5.6 min), SAM (m/z 399 →250, RT = 16.2 min), d3-SAM (m/z 402→250, RT = 16.2 min), SAH (m/z 385→136, RT = 20.1 min), d4-SAH (m/z 389→136, RT = 20.1 min), methionine (m/z 150→133, RT = 17.8 min), and 13Cd3-methionine (m/z 154 →137, RT = 17.8 min). For easy comparison between cell lines, absolute concentrations of cellular metabolites are normalized to 4 × 106 cells.
Cell pellets were resuspended in lysis buffer followed by sonication and 10 min centrifugation at 16,000 g. Lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane and blocked in 5% milk in TBST. The primary antibodies used were: anti-NNMT (Abcam, ab58743), anti-H3K9-me1 (Abcam, ab8896), anti-H3K9-me2 (Cell Signaling, 9753), anti-H3K9-me3 (Abcam, ab8898), anti-H3-total (Abcam, ab1791), anti-H3K4-me1 (Abcam, ab8895), anti-H3K4-me2 (Abcam, ab32356), anti-H3K4-me3 (Abcam, ab8580), anti-H3K27-me2 (Abcam, ab24684), anti-H3K27-me3 (Millipore, 07-449), anti-H3K79-me2 (Abcam, ab3594), anti-H4K20-me2 (Cell Signaling, 9759), anti-PP2A-total (Millipore, 07-324), anti-PP2A-me (Millipore, 04-1479), anti-PP2A-deme (Millipore, 05-577), anti-dimethyl-arginine, asymmetric or ASYM25 (Millipore, 09-814), anti-H3R17-me2a (Abcam, ab8284).
Migration and invasion studies
Migration and invasion assays were performed as described previously8, 15
. For 1MNA treatment studies, 0.5 mM 1MNA was added to GFP-OE 769P cells during serum starvation and to the upper and bottom chamber during migration assay.
DNA methylation assay
Genomic DNA was isolated from 769P cells using DNeasy blood & tissue kit from QIAGEN. 1 μg of DNA was degraded into nucleosides using DNA Degradase Plus (Zymo Research). LC separation was achieved with a Sinergy Fusion-RP column (4 μm, 80A, 50 × 4.6 mm, Phenomenex) together with a pre-column (Fusion-RP, 4 × 3.0 mm). Mobile phase A was composed of 100% H2
O, and mobile phase B was composed of 100% MeOH. Both solvents were supplemented with 0.1% formic acid to assist ion formation in a positive mode. The flow rate started at 0.1 ml/min for 5 min. The gradient started with 0% B for 5 min and increased linearly to 100% B over 20 min with a flow rate of 0.4 ml/min, followed by an isocratic gradient of 100% B for 2 min at 0.5 ml/min. Then column was equilibrated with 0% B for 3 min at 0.4 ml/min. MS analysis, scanning from m/z
= 50–1200, was performed on Agilent 6520 Accurate Mass Q-TOF. 5-Methyl-2′-deoxycytidine (5mdC) content was calculated as [5mdC]/[dG] using external calibration curve as described55
mRNAs were isolated (RNeasy Mini Kit, QIAGEN) from NNMT-OE and Y20A-OE 769P cells, reversed transcribed, and hybridized to Affymetrix Human 1.0 ST microarray. Data were then filtered for genes that were upregulated or downregulated (>1.4-fold) in NNMT-OE versus Y20A-OE cells for further analysis.
Real-time (RT)-PCR analysis
mRNAs were isolated using RNeasy Mini Kit (QIAGEN). The cDNAs were synthesized by reverse transcription using the SABiosciences RT2 kit. Obtained cDNAs were added to RT2 SYBR Green mastermix followed by RT-PCR using custom RT2 Profiler PCR Arrays (SABiosciences). RT-PCR was performed on ABI 7900HT cycler (384-well block, Applied Biosystems). Results were normalized to the average of three housekeeping genes, including ACTB, GAPDH and HPRT1. Gene list is shown in table below.
|Gene||Catalog #||Refseq #||Official Full Name|
|SNAI2||PPH02475||NM_003068||Snail homolog 2 (Drosophila)|
|Transforming growth factor, beta 2|
|ADAMTS6||PPH15788||NM_197941||ADAM metallopeptidase with thrombospondin type 1 motif, 6|
|Laminin, beta 3|
|HPRT1||PPH01018||NM_000194||Hypoxanthine phosphoribosyltransferase 1|
Data are shown as mean ± SEM. P-values were calculated using unpaired, two-tailed Student’s t-test. A P-value of < 0.05 was considered significant.