Research ArticleExperimental Studies

Nicotinamide N-Methyltransferase and Its Precursor Substrate Methionine Directly and Indirectly Control Malignant Metabolism During Progression of Renal Cell Carcinoma

SINA HOLSTEIN, SIMONE VENZ, HEIKE JUNKER, REINHARD WALTHER, MATTHIAS B. STOPE and UWE ZIMMERMANN
Anticancer Research October 2019, 39 (10) 5427-5436; DOI: https://doi.org/10.21873/anticanres.13736

Abstract

Background/Aim: Renal cell carcinoma (RCC) is one of the most common tumor diseases in adults, and new specific biomarkers are urgently needed to define diagnosis and prognosis of patients with RCC as well as monitor the outcome of therapeutic interventions. The enzyme nicotinamide N-methyltransferase (NNMT) is believed to represent such a marker molecule in RCC therapy. Materials and Methods: NNMT expression was examined by western blotting in samples from patients with RCC and in RCC cell lines. Effects of NNMT on cell growth and metabolism were assessed using the Hoechst 33342 reagent assay and Vita-Orange cell viability assay. Incubation experiments were performed to study the influence of methionine and interleukin-6 (IL6) on expression of NNMT. Results: In patient samples, NNMT was up-regulated depending on the stage of progression. Investigations in an RCC cell culture model showed that after modulation of NNMT expression, cellular metabolism, but not cell growth was affected. This regulatory function was also dependent on the presence of the NNMT precursor substrate methionine and IL6. Conclusion: The metabolism-regulatory activity of NNMT depends on the precursor substrate methionine and the presence of IL6. The function of methionine appears to be dependent on the stage of progression, since in individual RCC cell lines, opposing effects on metabolism were demonstrated. This, in turn, reflects the thoroughly complex situation in the clinic.

Renal cell carcinoma (RCC) is one of the most common tumor diseases in adults and to a large extent occurs as the clear-cell histological type. Due to the lack of early symptoms and despite the development of modern imaging methods, RCC is usually diagnosed late, often only in the metastatic stage (1). By characterizing molecular pathogenesis mechanisms, new targets for targeted therapy of RCC have been identified, e.g. inhibitors of tyrosine kinases (sunitinib, sorafenib), mechanistic target of rapamycin (temsirolimus, everolimus), and vascular endothelial growth factor (bevacizumab). In addition, new specific biomarkers are needed to define diagnosis and prognosis of patients with RCC and monitor the outcome of therapeutic interventions.

The protein nicotinamide N-methyltransferase (NNMT) might be such a marker molecule in RCC. Under physiological conditions, this enzyme is mainly located in the liver and catalyzes the biotransformation of low-molecular weight compounds. In RCC, NNMT appears to be up-regulated in cancer tissue and may represent a biomarker (2-4). However, the cellular function of NNMT during RCC initiation and progression is still largely unknown. First data correlated NNMT activity with cell mobility and metastasis (5, 6). Furthermore, whether NNMT participates in methylatory processes of epigenetic-regulatory mechanisms has been discussed. The enzyme is involved in the NAD metabolism via its substrate nicotinamide and in methyl metabolism through the consumption of S-adenosyl methionine (7, 8).

The aim of the study presented here was to characterize NNMT involvement in progression of RCC. Since enzyme functionality is linked to energy and methionine metabolism, NNMT properties in cell growth and metabolism are primarily addressed. Moreover, the expression levels of NNMT in patient samples were determined in order to evaluate its suitability as an RCC biomarker.

Materials and Methods

Patient samples and protein preparation. Tissue samples and related clinical data were collected based on approval from the Ethics Committee at the Medical Faculty of the University of Greifswald (III UV 12/03). For western blot analyses, tissue samples from patients with clear cell RCC were preserved at −80°C immediately after resection. After histological evaluation, eight samples each were classified into three subgroups pT1, pT2 and pT3, as well as the non-malignant control group. Protein preparation was performed using peqGOLD TriFast™ (Peqlab, Erlangen, Germany) according to the manufacturer's instruction. Tissue (50-100 mg) was homogenized with 1 ml peqGOLD TriFast™. To ensure the dissociation of nucleotide complexes, samples were incubated at room temperature for 5 min. After addition of 0.2 ml chloroform, samples were shaken vigorously for 15 s. After a 20-min incubation at room temperature, the samples were centrifuged (5 min at 12,000 × g) to achieve phase separation. To remove genomic DNA, the interphase and the phenolic phase were incubated with 300 μl of ethanol for 3 min and centrifuged at 2,000 × g for 15 min. The supernatant was used to precipitate the proteins by adding 1.5 ml isopropanol. After centrifugation at 12,000 × g for 10 min, pellets were washed twice with 0.3 M guanidinium chloride in 95% ethanol and finally twice with 100% ethanol each for 20 min. Protein samples were dried, resuspended in sample buffer (8 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 40 mM Tris and 50 mM dithiothreitol and the total amount of protein was determined by modified Bradford assay.

Cell culture and treatment. Human RCC cell lines 769-P, 786-O, and Caki-2, as well as the human embryonic kidney cell line HEK-293, were obtained from the Leibniz Institute DSMZ–German Collection of Microorgansims and Cell Cultures (Braunschweig, Germany) and propagated in RPMI 1640 medium supplemented with 20 μM methionine (Carl Roth, Karlsruhe, Germany), 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 11 mM glucose (B. Braun Melsungen, Melsungen, Germany), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (PAN-Biotech, Aidenbach, Germany) at 37°C in a humified atmosphere with 5% CO2. In the case of Caki-2 cells, 16.5 mM glucose (B. Braun Melsungen) was used.

The amino acid methionine is essential for cell growth. Since the plasma concentration of methionine in humans is 20-40 μM, RCC cells were propagated with a minimum concentration of 20 μM methionine. In studies on the influence of methionine, the concentration in cell culture media was increased to 100 μM (7). The specified concentrations of methionine were obtained by supplementing an appropriate amount of methionine solution (Carl Roth).

Cloning of pcDNA3.1-NNMT. The vector pcDNA3.1+ (Invitrogen AG, Carlsbad, USA) was linearised with HindIII and XhoI. The human NMMT encoding DNA (position 159-953, accession number NM_006169) was amplified by polymerase chain reaction (PCR) using Caki-1 complementary DNA (cDNA) as template and primer with internal HindIII/XhoI sites as well as a Kozak sequence in front of the ATG start codon. The PCR product was ligated into the HindIII/XhoI sites of pcDNA3.1(+) vector. All purification steps were carried out by QIAquick PCR purification kit (QIAGEN GmbH, Hilden, Germany) following the supplier's instructions. All required enzymes (restriction endonucleases, Phusion® High Fidelity DNA Polymerase, T4 DNA Ligase) were purchased from New England Biolabs (Frankfurt a.M., Germany). Finally, the cloning product was confirmed by sequencing.

Transfection experiments. Transient transfection was performed with jetPRIME® transfection reagent (Polyplus Transfection, Illkirch, France). The plasmid pcDNA3.1_NNMT was used to overexpress NNMT in HEK-293, Caki-2 and 769-P cells. The knockdown of NNMT was performed by application of siNNMT (5’-CGUCG UCACUGACUACUCdTdT-3’) in Caki-1 and 786-O cells. As controls, the plasmid pcDNA3.1 or scrambled siRNA (5’-GCUCCC UCCUUCGAUUUAUdTdT-3’) were used in mock transfections.

For transfection, cells were seeded 24 hours prior to treatment and used according to the manufacturer's instructions. In detail, 2 μg nucleic acid was diluted into 200 μl jetPRIME® buffer, and mixed by vortexing for 10 s. Then 4 μl jetPRIME® was added and the mixture vortexed for 10 s, and spun down briefly. After an incubation of 10 min at room temperature, 200 μl of transfection mix were added per well dropwise onto the cells in serum-containing medium, and distributed evenly before incubation at 37°C. When necessary, the transfection medium was replaced after 4 h by cell growth medium and the plates were returned to the incubator.

Cell viability assay. To determine cellular viability, different cell numbers were used for analyses of treatment with variable methionine concentrations (20 μM and 100 μM methionine) or with 50 ng/ml IL6 (1.5×103 cells for HEK-293, 786-O and 769-P; 1×103 cells for Caki-2) or analyses after transfection of cells (5×103 cells for HEK-293, 6×103 cells for 786-O and 769-P; 4×103 cells for Caki-2). The cells were seeded in a 96-well cell culture plate with 100 μl of media, incubated for 24 h and 72 h, and analyzed according to the manufacturer's instructions using Vita-Orange Cell Viability Assay (Biotool, Houston, TX, USA). Briefly, to the medium, 11 μl of Vita-Orange solution were added and evenly distributed by panning. After 1 h of incubation at 37°C, absorption was measured with an Infinite 200 Plate Reader (Tecan, Männedorf, Switzerland) at 450 nm.

Cell growth assay. Cell growth was determined utilizing Hoechst 33342 Reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were treated as described in the cell viability assay, frozen, and incubated after thawing for 10 min with 10 μl Hoechst solution. After dilution with 100 μl of PBS, fluorescence was detected using an Infinite 200 Plate Reader (Tecan) at 350/480 nm.

Preparation of protein from cell lines. After washing with 5 ml PBS, cells were detached from the cell culture plate with a cell scraper and transferred to a 1.5 ml Eppendorf tube and centrifuged at 500×g for 5 min. The supernatant was discarded. Cells were lysed with 200 μl lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5% NP-40, 0.5% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM NaF, 1 mM Na3VO4, and complete protease inhibitor cocktail from Roche Applied Science (Mannheim, Germany), frozen at −80°C and then centrifuged after thawing (for 10 min at 17,000 × g and 4°C). Total protein concentration was determined by Bradford assay.

Western blot analysis. Equal amounts of protein were separated by Mini-Protean system (BioRad, Munich, Germany), transferred onto a Protran nitrocellulose membrane (70 min at 1 mA/cm2; Whatman, Dassel, Germany), and subsequently blocked with Roti-Block (Carl Roth) for 1 h. Proteins of interest were detected by primary antibodies (4°C for 16 h, diluted in 5% BSA in TBST with NaN3) directed against NNMT (1:5000, 15123 1 AP; Proteintech, Rosemont, IL, USA), IL6 receptor (IL6R) (1:1000, 23457 1 AP; Proteintech), α-tubulin (1:50000, ab7291; Abcam, Cambridge, UK) as control, and peroxidase-coupled secondary antibodies (30 min, 1:30000, #7074, #7076; Cell Signaling Technology, Frankfurt, Germany). After incubation with 2 ml Enhanced Chemiluminescence Detection Substrate (Thermo Fisher Scientific) for 3 min, signals were visualized by X-ray films and subsequently quantified in digital form (ImageJ Software, https://imagej.nih.gov/ij/index.html). The quantification of the respective proteins was relative to the associated total protein amount of the lane.

Statistics. Statistical analyses were performed using the nonparametric Mann–Whitney test. Results with p≤0.05 were considered significant. Data are given as median values and were prepared using Prism 8 Software (GraphPad Software, La Jolla, CA, USA).

Results

NNMT expression in samples from patients with RCC and in RCC cell lines. Little is known about the cellular functionality of NNMT during the formation and progression of RCC. Therefore, the protein expression of NNMT in samples from 24 patients with RCC of different stages (eight samples each of stages pT1, pT2 and pT3) were evaluated and compared to eight samples from healthy tissues (Figure 1). Analysis demonstrated an increase in NNMT expression during progression from pT1 to pT3 compared to the non-malignant samples. This increase was statistically significant in stages pT2 (p=0.0006) and pT3 (p=0.0002) and may indicate a biological function of the protein in tumor. For further experimental investigations, an RCC model system was established consisting of the non-malignant renal cell line HEK-293 and the RCC cell lines Caki-2, 786-O and 769-P. In all cell lines, NNMT was detected at different amounts (Figure 2A), whereby non-malignant HEK-293 cells had the lowest NNMT protein content compared to RCC cells (compared to HEK-293 with median=1.0): Caki-2: 6.55-fold; 786-O: 52.1-fold; 769-P: 4.82-fold).

NNMT-dependent modulation of cellular metabolism and cell growth in RCC cells. Enhanced cell growth and cancer-specific alterations in cellular metabolism are among the most significant malignant events during cancer progression. A possible involvement of NNMT was investigated applying a metabolic activity assay and a proliferation assay. Owing to the low expression of NNMT in HEK-293, Caki-2, and 769-P cells, transient overexpression was established for further experiments. After transfection, overexpression of NNMT protein was achieved by a factor of 7.0 in HEK-293 and 14.5 in Caki-2 and 769-P cells (Figure 2B).

Figure 1.
Figure 1.

Relative protein expression of nicotinamide N-methyltransferase (NNMT) in samples from patients with renal cell carcinoma (RCC). Quantitative analysis of western blots from samples from 24 patients' different stages of RCC (eight samples each) compared to eight samples from healthy donors. Total protein staining of a membrane is shown as loading control. Data are presented as scatter dot plot with median (lines) and interquartile range (bars). ***Significantly different at p≤0.001.

Overexpression experiments revealed a significant decrease in metabolic activity in NNMT-transfected HEK-293 (p<0.0001), Caki-2 (p<0.0001), and 769-P (p<0.0001) cells (Figure 3A-C, left panel). However, this had an inhibitory effect on cell growth only in the case of non-malignant HEK-293 cells (p=0.0006) (Figure 3A, right panel); no changes were observed in Caki-2 and 769-P RCC cells (Figure 3B and C, right panel). The knockdown of NNMT in transfected 786-O cells confirmed these observations. Here, siRNA-mediated reduction of NNMT protein expression led to an increase in metabolic activity (p<0.0001), which had no effect on the cell growth rate (Figure 3D).

NNMT functionality is controlled by the NNMT precursor substrate methionine. For the methylation of substrates, NNMT requires the precursor substrate methionine which is converted by methionine adenosyltransferase to the methyl donor S-adenosyl methionine. Alterations in concentrations of methionine to 100 μM did not lead to a significant change in NNMT expression (Figure 4). However, incubation experiments over 3 days showed a significant increase in metabolic activity in HEK-293 (p<0.0001) 786-O (p<0.0001) and 769-P (p<0.0001) cells on the third day in the presence of 100 μM methionine (Figure 5A, C and D, left panel), with the exception of Caki-2 (Figure 5B). In contrast to NNMT overexpression and knockdown approaches (Figure 3), effects on cell growth were also observed during incubation with the higher concentration of methionine. The increased metabolic activity in the presence of 100 μM methionine led to a reduction in growth of HEK-293 (Figure 5A, right panel; p<0.0001) and 786-O (Figure 5C, right panel; p=0.0096) cells. For Caki-2 and 769-P cells, on the other hand, no differences were detectable between growth at the two methionine concentrations used (Figure 5B and D, right panel).

Figure 2.
Figure 2.

Protein expression of nicotinamide N-methyltransferase (NNMT) in different renal cell carcinoma (RCC) cell lines. Quantitative analysis of western blots from Caki-2, 786-O, and 769-P RCC cells and human embryonic kidney cell line HEK-293. A: NNMT expression was evaluated relative to that of HEK-293 cells. B: Relative protein expression after overexpression of NNMT in HEK-293, Caki-2, and 769-P cells, compared to mock transfection. B: Relative protein expression after knockdown of NNMT in 786-O cells compared to scrambled siRNA (sisc) transfection. Representative western blots for NNMT detection are shown (n=4). Total protein staining of membranes are given as loading control. Data are presented as mean values with standard deviation. *Significantly different at p≤0.05.

Figure 3.
Figure 3.

Metabolic activity and proliferation of different renal cell carcinoma (RCC) cell lines after transfection with nicotinamide N-methyltransferase (NNMT) overexpression vector and NNMT-specific small interfering RNA (siNNMT). Left panel: Relative viability of cell lines with low expression of NNMT human embryonic kidney cell line HEK-293 (n=18) (A), and Caki-2 (n=12) (B) and 769-P (n=18) (C) RCC cells after forced overexpression of NNMT and high-level NNMT-expressing 786-O cells after knockdown of NNMT (n=18) (D). Right panel: Relative cell proliferation of corresponding cells (n=18). Data are presented as box blots with median (lines) and interquartile range (bars). Significantly different at ***p≤0.001 and ****p≤0.0001.

Figure 4.
Figure 4.

Relative expression of nicotinamide N-methyltransferase (NNMT) protein in different renal cell carcinoma (RCC) cell lines – influence of methionine concentration. Quantitative analysis of western blots from HEK-293, Caki-2, 786-O, and 769-P cells. Expression was evaluated compared to that of HEK-293 cells at the specified methionine concentration (n=4). Data are presented as box blots with median (lines) and interquartile range (bars).

NNMT-dependent modulation of the metabolic activity is mediated by IL6. The activity of NNMT is partially regulated by interleukins and is integrated into cellular signal transduction pathways. All four renal cell lines used expressed IL6R, however, IL6R expression was only dependent on methionine concentration in the case of 769-P cells (Figure 6A). Incubation experiments with 50 ng/ml IL6 confirmed the hypothesis that the IL6 signaling pathway is involved in the regulatory activity of NNMT. While metabolic activity was regulated by IL6 (Figure 6B), in the presence of the interleukin there was no influence on cell growth (Figure 6C). It was highly noteworthy that incubation with IL6 led to a significant decrease in metabolic activity of Caki-2 cells, but that under comparable conditions, it led to an increase in metabolic activity in 786-O cells.

Discussion

NNMT expression in tumor tissue is up-regulated in some entities such as prostate, bladder, pancreatic, and gastric cancer (9-13). In bladder cancer, the NNMT concentration in urine even correlates with tumor progression and may be used as a diagnostic biomarker in the future (14). In RCC, the biomarker function of NNMT has already been discussed (2, 15, 16), but the underlying studies are mainly based on proteomic or transcriptomic approaches. The cellular and molecular role of NNMT in RCC, however, is still largely unknown.

The analysis of patient material in this study demonstrated significant up-regulation of NNMT expression in RCC and confirms the findings of the only study to date on NNMT expression in patients with RCC (17). This fact was reflected in the in vitro model of the non-malignant cell line HEK-293 and the three RCC lines Caki-2, 769-P and 786-O. NNMT expression was many times higher in the three malignant cell lines than in HEK-293 cells. Furthermore, NNMT seemed to have a greater influence on cell growth in non-malignant HEK-293 cells than in the malignant cell lines. Both after overexpression of NNMT and at higher concentrations of the NNMT precursor substrate methionine, the influence was more pronounced in HEK-293 cells than in RCC cells. Notably, in the in vitro RCC model, antiproliferative properties of NNMT were demonstrated. These observations are in contrast to current data attributing oncogenic and proliferative properties to NNMT in breast, pancreatic, lung, and oral squamous cell carcinoma (18-22). On the other hand, the antiproliferative properties of NNMT shown in this study correspond to its function in metabolism. The overexpression of NNMT led to a reduction in metabolic activity and this would be expected to result in reduced cell growth. Thus, NNMT might possibly play a different role in RCC cells than in cells of other entities. Interestingly, overexpression and knock-down experiments demonstrated that metabolic activity did not always correlate with cell growth. This fact sheds critical light on colorimetric assays such as WST or 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assays that determine enzyme substrate turnover and are interpreted as indirect detection of biomass and thus cell growth.

Figure 5.
Figure 5.

Metabolic activity and proliferation in different renal cell carcinoma (RCC) cell lines due to the influence of methionine concentration. Left panel: Relative viability of human embryonic kidney cell line HEK-293 (A), and Caki-2 (B) and 769-P (C) RCC cells after 1 or 3 days applying methionine concentrations of 20 μM or 100 μM in the medium (n=18). Right panel: Relative cell proliferation of corresponding cells (n=12). Data are presented as box blots with median (lines) and interquartile range (bars). Significantly different at *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.

Figure 6.
Figure 6.

Influence of interleukin-6 (IL6) treatment on the metabolic activity and cell proliferation of different RCC cell lines. A: Protein expression of the IL6 receptor (IL6R) in an human embryonic kidney cell line HEK-293, and Caki-2, 786-O, and 769-P RCC cells at methionine concentration of 20 μM or 100 μM in the medium. Relative expression was evaluated compared to that of HEK-293 cells at the specified methionine concentration. A representative western blot for IL6R detection is shown (n=4). Total protein staining of a membrane is given as a loading control. B: Relative viability of HEK-293, Caki-2, 786-O, and 769-P cells after treatment with 50 ng/ml IL6 compared to controls (co; n=18). C: Relative proliferation of HEK-293, Caki-2, 786-O and 769-P cells after treatment with 50 ng/ml IL6 compared to controls (co; n=18). Data are presented as box blots with median (lines) and interquartile range (bars). Significantly different at *p≤0.05, ***p≤0.001 and ****p≤0.0001.

Besides the expression level of NNMT, the availability of the precursor substrate methionine also plays a role in protein function. With the exception of Caki-2 cells, an increase in available methionine concentration also led to an increase in metabolic activity. The methionine product S-adenosyl methionine is a universal methyl donor in numerous enzyme-catalyzed reactions and is being tested for the treatment of various diseases, including cancer (23, 24). In colorectal cancer, S-adenosyl methionine has been shown to control cell growth in a concentration- and time-dependent manner. Higher S-adenosyl methionine concentrations and longer incubation times led to the inhibition of cell growth, while lower concentrations and short incubation times stimulated proliferation (25). Thus, the experimental conditions used are crucial in the assessment of the proliferative effects of methionine. Furthermore, we cannot exclude from the present study that methionine- and S-adenosyl methionine-mediated but NNMT-independent mechanisms led to an effect on the demonstrated metabolic activity and cell growth. Both the expression level of NNMT and the availability of the S-adenosyl methionine precursor methionine seem to be important for the malignant activity of RCC cells.

NNMT is an important factor in energy metabolism (26). Alterations in NNMT activity due to modulation of protein expression or concentration of the precursor substrate methionine therefore also have an effect on cell growth. IL6 is a key factor in the progression and proliferation of RCC (27-29) and we therefore analyzed whether this factor is also involved in the cellular functionality of NNMT. The data showed both methionine-dependent expression of IL6R and IL6 induced modulation of metabolic activity. However, this was not detectable in all cell lines, so that the function of IL6 in NNMT-regulated RCC progression remains unclear. It is remarkable, however, that IL6 had pro-metabolic and anti-metabolic activity depending on the cell line. Thus, the hypothetical signal axis NNMT–methionine–IL6 can be considered to be unique in different patients, which makes the use of NNMT as a diagnostic or prognostic biomarker unlikely.

In summary, NNMT plays a multifunctional role in RCC cells. Its up-regulation during tumor progression suggests an important oncogenic function. This does not affect tumor growth, but cellular metabolism. In contrast to other cancer entities, increased intracellular concentration of NNMT leads to reduced metabolism in RCC. Additionally, the concentration of the precursor substrate methionine seems to be of importance for NNMT activity. With increasing methionine concentration, the metabolic activity of RCC cells also increased. The regulatory function of NNMT in RCC appears to be progression-dependent, depends on the precursor substrate methionine and appears very individual in the clinical context. Furthermore, cellular function is mediated by additional factors, possibly IL6.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Received July 24, 2019.
  • Revision received September 5, 2019.
  • Accepted September 10, 2019.

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Nicotinamide N-Methyltransferase and Its Precursor Substrate Methionine Directly and Indirectly Control Malignant Metabolism During Progression of Renal Cell Carcinoma
SINA HOLSTEIN, SIMONE VENZ, HEIKE JUNKER, REINHARD WALTHER, MATTHIAS B. STOPE, UWE ZIMMERMANN
Anticancer Research Oct 2019, 39 (10) 5427-5436; DOI: 10.21873/anticanres.13736
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