Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells
- Stacey L. Borrego†1,
- Johannes Fahrmann†2, 8,
- Rupsa Datta3,
- Chiara Stringari3, 9,
- Dmitry Grapov4, 10,
- Michael Zeller5, 6,
- Yumay Chen1,
- Ping Wang1,
- Pierre Baldi5, 6,
- Enrico Gratton3,
- Oliver Fiehn2, 7 and
- Peter Kaiser1, 6Email author
© Borrego et al. 2016
Received: 16 November 2015
Accepted: 16 March 2016
Published: 2 May 2016
The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as “methionine dependence” or “methionine stress sensitivity” in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.
Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential.
This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions.
KeywordsCancer metabolism S-adenosylmethionine SAM Methionine Homocysteine Methionine stress
Methionine is an essential amino acid necessary for normal growth and cell function. It contributes to protein synthesis and is the precursor to S-adenosylmethionine (SAM), the principal methyl donor in the cell. SAM is a versatile molecule required for methylation of DNA, RNA, proteins, and lipids by a variety of methyltransferases. In addition, SAM is critical for the formation of 1-methylnicotinamide, a primary factor involved in stem cell pluripotency , polyamine biosynthesis , and the methionine salvage pathway . As SAM donates its activated methyl group in methylation reactions, it is converted to S-adenosylhomocysteine, which is further hydrolyzed to homocysteine in a reversible reaction (Fig. 1a) . Homocysteine is a junction metabolite, and its metabolism can be either directed toward the remethylation pathway to regenerate methionine by receiving a methyl group from betaine or N5-methyltetrahydrofolic acid or toward cysteine and glutathione synthesis in the transsulfuration pathway . Homocysteine inhabits a critical position where, depending on demand, metabolic flux can be redirected to increase methylation potential or produce antioxidants.
Although initial studies suggested methionine limitation to be responsible for the “methionine dependence phenotype,” limited availability of SAM is the actual culprit. Work by Coalson and colleagues has shown that methionine dependent cells endogenously synthesize methionine at normal levels in homocysteine media (Met-Hcy+) but show reduced SAM synthesis . Accordingly, by supplementing homocysteine growth medium with SAM, cell proliferation of methionine-dependent breast cancer cells can be restored, suggesting SAM limitation as the cause for methionine dependence . Furthermore, SAM limitation induced by knockdown of methionine adenosyltransferase (MAT), the enzyme responsible for synthesis of SAM from methionine and ATP, mimics the cell cycle arrest induced by replacing methionine with homocysteine in the growth media [10, 11]. The specific cell cycle arrest in the G1 phase induced by homocysteine medium or MAT knockdown is reminiscent of an evolutionary conserved metabolite responsive cell cycle checkpoint first described in yeast. This “SAM-checkpoint” was proposed to protect cellular integrity and maintain epigenetic stability as it halts cell cycle progression before intracellular SAM concentrations get too low to support the various methylation reactions necessary for normal cell physiology. Cancer cells have a highly responsive SAM-checkpoint, likely because of their higher demand on SAM . Details of why cancer cells depend on high levels of SAM remain to be discovered, but their increased proliferation rate requires constant duplication of chromatin methylation marks, methylation of RNAs, and SAM-dependent synthesis of membrane lipids. In addition, many cancer cells are characterized by hyperactive polyamine synthesis , which consumes SAM. A decarboxylated form of SAM reacts with the polyamine putrescine to form spermidine and spermine, generating the by-product 5′-deoxy-5′-methylthioadenosine (MTA). Further processing of MTA generates adenine and replenishes the methionine pool . Although polyamine synthesis clearly contributes to SAM usage in cancer cells and modified polyamines, specifically diacetylspermine, have been shown as clear biomarkers in lung  and colon cancer , SAM limitation or growth in homocysteine media do not imitate effects of polyamine depletion and cannot be rescued by spermidine supplementation [11, 16].
While some progress has been made by linking methionine dependency of cancer to SAM limitation and induction of a specific cell cycle arrest, our understanding of this striking metabolic dependence of cancer remains minimal. Here, we characterized the dynamics of metabolic changes in breast cancer cells when they are shifted to homocysteine-based growth media, using untargeted metabolomics as well as stable isotope-labeled tracer studies to follow the metabolic fate of homocysteine.
Cell lines and growth conditions
MDA-MB-468 were maintained in DMEM (Sigma-Aldrich, D0422) supplemented with 10 % dialyzed FBS (Gemini Bio-Products), 1.5 μM cyanocobalmin (vitamin B12), 4 mM l-glutamine, 100 μM l-cysteine (Fisher Scientific), and 100 μM l-methionine (Sigma-Aldrich). In the case of methionine-free media, 370 μM DL-homocysteine (Sigma-Aldrich) or 370 μM DL-homocysteine-2H4 (13C Molecular, 12714-158) was added in the absence of methionine.
Resistant cell lines were isolated as described in Hoffman et al. . Briefly, MDA-MB-468 resistant clones were isolated after prolonged culturing in methionine-free media. The majority of MDA-MB-468 cells detach; however, resistant clones begin to appear after 2 weeks. Clones were isolated, and proliferation rates were measured using CellTiter-Glo luminescent cell viability assay (Promega).
Both MD468 and MB468res-R8 cell lines were tested for authentication via STR profiling in January 2016 by Genetica DNA Laboratories (a LabCorp brand; Burlington, NC) using the commercially available PowerPlex® 16HS amplification kit (Promega Corporation) and GeneMapper ID v3.2.1 software (Applied Biosystems). Authentication of each cell line was confirmed by a 100 % match to the reference STR profile of MDA-MB-468 (ATCC HTB-132) cells from ATCC.
Anchorage independence assay
To detect anchorage independence of transformed cells, MDA-MB-468 and resistant cell lines were cultured in soft agar for 30 days. Each assay was conducted in a 35-mm Petri dish with a 0.5 % agar base layer and 0.35 % agar top layer prepared using low-gelling-temperature agarose (Sigma, A9045) in methionine growth media. The top layer of each assay contained 5000 cells except for control plates. Assays were maintained at 37 °C in a humidified incubator, and 0.5 ml methionine growth media was added to each plate twice weekly. Colonies were stained with 0.005 % crystal violet for 1 h, washed once with PBS, and manually counted.
Preparing cells for metabolite extraction
Cells were harvested by first washing attached cells with 10 ml PBS before adding 0.5 % trypsin (Life Technologies, 25300-054) for 10 min and incubating at 37 °C. Cells were collected in complete DMEM (4 °C) without methionine or homocysteine and centrifuged (300×g, 5 min, 4 °C); from this point on, cells were kept on ice during all processing steps. Cell pellets were washed with 10 ml PBS (4 °C) and counted. The appropriate number of cells was aliquoted and centrifuged (300×g, 5 min, 4 °C). Cell pellets were resuspended in 1 ml PBS (4 °C), transferred to a pre-weighed microcentrifuge tube, and centrifuged (300×g, 5 min, 4 °C). PBS was aspirated, and cell pellets were weighed before flash freezing in liquid nitrogen. Samples were stored at −80 °C until shipped on dry ice. This protocol was performed as described in T.W.-M. Fan et al. .
MDA-MB-468 and MDA-MB-468res-R8 cells were cultured in methionine or homocysteine media for 24, 48, 72, and 96 h. Lysates were prepared in 8 M urea buffer (8 M urea, 200 mM NaCl, 0.2 % SDS, 100 mM Tris (pH 7.5), 1 mM PMSF, 1 ng/ml aprotinin) and resolved by 10 % SDS-PAGE. Protein levels were detected by immunoblot using MAT2A (Abcam, ab77471) and MAT2B (Abcam, ab86506) antibodies.
FLIM acquisition and phasor data analysis
MDA-MB-468 and MDA-MB-468res-R8 cells were cultured in methionine or homocysteine media. To inhibit oxidative phosphorylation, cells were treated with 4 mM potassium cyanide (KCN) for 5 min before imaging.
Fluorescence lifetime imaging microscopy (FLIM) was performed on Zeiss LSM 710 microscope (Carl Zeiss, Jena, Germany) using a ×40 water immersion objective, 1.2 N.A. (Carl Zeiss, Oberkochen, Germany) coupled with two-photon excitation of 740 nm (titanium:sapphire MaiTai laser from Spectra-Physics, Mountain View, CA). Image scan speed was 25.21 μs/pixel, and image size is 256 × 256 pixels. The emission signal was collected using a 460/80-nm bandpass filter and photomultiplier tube (H7422P-40, Hamamatsu, Japan) was used for detection. FLIM data was acquired using A320 FastFLIM FLIMbox (ISS, Champaign, IL). SimFCS software, developed at the Laboratory for Fluorescence Dynamics (LFD, UC Irvine), was employed for both FLIM data acquisition and analysis. FLIM-phasor approach was used for phasor analysis, as described previously [19, 20]. Briefly, lifetime information from every pixel of the image is transformed into a phasor and plotted on a phasor plot. This can be mapped back onto the image to create the FLIM map.
Extracellular flux (XF) analyses
All XF analyses were performed on the Seahorse Bioscience XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA) using the manufacturer’s protocol. Cells were plated in 24-well Seahorse XF-24 assay plates at a density of 90,000 cells per well in Met+ media (as described above). After 6 h, eight wells were washed with pre-warmed PBS before Met+ media and Met-Hcy+ media were added in four wells each. This process was repeated for the 8, 4, and 2 h time points. One hour before metabolic flux analysis, cells were washed once with un-buffered, serum-free media (Caisson Labs, DMP39-10XLT) containing 100 uM methionine or 370 μM DL-homocysteine and incubated with un-buffered media at 37 °C in a non-CO2 incubator for 1 h. Four baseline oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements were taken before sequential injection of the following mitochondrial inhibitors: oligomycin (1 μg/ml), carbonyl cyanide-4-phenylhydrazone (FCCP) (0.4 μM), and rotenone (0.1 μM). Four measurements were taken after the addition of each inhibitor, and OCR and ECAR values were automatically calculated and recorded by the Seahorse XF-24 software. Basal respiration was calculated by averaging the four measurements of OCR before injection of inhibitors. Basal ECAR values were calculated in the same manner. Spare respiration capacity was calculated by subtracting basal OCR values from OCR values after FCCP treatment.
The MiniX database  was used as a Laboratory Information Management System (LIMS) and for sample randomization prior to all analytical procedures.
For analysis of biogenic amines, cell lysates, stored at −80 °C, were thawed on ice and extracted with ice-cold “degassed” 3:3:2 acetonitrile/isopropanol/ultra-pure water. Supernatant-containing extracted metabolites were dried to completeness under reduced pressure and resuspended in 60 μL of 80:20 ACN/H2O containing an internal Val-Tyr-Val (Sigma-Aldrich). Resuspended samples were analyzed on an Agilent 1290A Infinity Ultra High Performance Liquid Chromatography system with an Agilent Accurate Mass-6550-QTOF mass spectrometer. The column (45 °C) was a Waters Acquity UPLC BEH (150-mm length × 2.1-mm internal diameter; 1.7-μM particles) coupled with a Waters Acquity VanGuard BEH C18 Pre-column (50-mm length × 2.1-mm internal diameter; 1.7-μM particles). The solvent system included (A) 100 % water (LCMS grade) containing 10 mM ammonium formate and 0.125 % formic acid and (B) 95:5 v/v acetonitrile to water containing 10 mM ammonium formate and 0.125 % formic acid. The gradient started from 0 min 100 % (B), 0–2 min 100 % (B), 2–7.7 min 70 % (B), 7.7–9.5 min 40 % (B), 9.5–10.25 min 30 % (B), 10.25–12.75 min 100 % (B), and 12.75–16.75 min 100 % (B). The flow rate was 0.4 mL/min and with an injection volume of 5 μL. ESI capillary voltage was +3.5 kV with collision energies of 20 eV MSMS collection in positive acquisition mode. Data was collected at a mass range of m/z 60–1700 Da with a spectral acquisition speed of 4 spectra per second.
Data was processed using MZmine 2.10 software. Metabolites were identified by searching against a precursor accurate mass and retention time library in conjunction with matching tandem mass spectra against the LipidBlast virtual MS/MS database . Data are reported as peak heights for the quantification ion (m/z) at the specific retention time for each annotated and unknown metabolite.
Stable isotope tracer studies
Evaluation of methionine, homocysteine, and cystathionine enrichment
Cells were extracted with ice-cold 1 ml of degassed 3:3:2 acetonitrile/isopropanol/ultra-pure water, the supernatant removed, and solvents evaporated to dryness under reduced pressure. To remove membrane lipids and triacylglycerides, dried samples were reconstituted with acetonitrile/water (1:1), decanted, and taken to dryness under reduced pressure. Samples were derivatized with methoxyamine hydrochloride in pyridine and subsequently by MTBSTFA (Sigma-Aldrich) and analyzed by gas chromatography mass spectrometry.
An Agilent 7890A gas chromatograph (Santa Clara, CA) was used with a 30 m × 0.25-mm i.d. (internal diameter) × 0.25 μM HP-5 MS Column (Agilent J&W GC Columns). An Agilent 7693 auto-sampler was used to eliminate cross-contamination during GCMS analysis. One microliter (1 μl) of sample was injected at 60 °C (ramped to 250 °C) in splitless mode with a 30-s purge time. The chromatographic gradient consisted of a constant flow of 1 mL/min, ramping the oven temperature from 60 to 350 °C over 37 min. Mass spectrometry was done using an Agilent 5977A MSD spectrometer, 290 °C transfer line temperature, electron ionization at −70 eV, and an ion source temperate of 230 °C. Mass spectra were acquired at 1555 V at m/z 50–600 with 2.7 spectra/s.
Acquired spectra were converted to netCDF files using vendor (Agilent) software and submitted for non-targeted enrichment analysis using the Non-targeted Fate Detection Software version 1.1 . Spectrum of enriched peaks was manually compared against reference spectrum-derived authentic standard and from parallel experiments using non-labeled homocysteine.
Enrichment due to presence of deuterium was validated using a secondary independent mass isotopomer distribution (MID) analyzer developed at the West Coast Metabolomics Center. MID calculations were determined using a modified least-squares linear regression matrix modeled after Jennings et al. ; corrected for natural abundance; and reduced isotopic probability with increasing deuterium enrichment. MID values are reported as fractional proportions (1 = 100 % enriched) that the respective mass isotopologue contributes to the overall abundance of the respective analyte.
Evaluation of SAM and SAH enrichment
SAM and S-adenosylhomocysteine (SAH) levels were determined by hydrophilic interaction liquid chromatography ultra high pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry (HILIC-UHPLC-qTOFMS) as described above. Enrichment due to presence of deuterium was determined using the MID analyzer as described above. Spectrum of enriched peaks was manually compared against reference spectrum-derived authentic standard and from parallel experiments using non-labeled homocysteine.
Results and discussion
A methionine-dependent and methionine-independent breast cancer cell line pair
The majority of malignant cells exhibit the methionine-dependent phenotype as indicated by cell cycle arrest and apoptosis when cultured in media where methionine (methionine media (Met+)) is replaced with the immediate metabolic precursor homocysteine (homocysteine media, Met-HCY+) [3, 28, 29]. Although methionine dependence is specific for cancer cells and non-tumorigenic cells proliferate well in homocysteine medium, long-term culturing of malignant cell lines in Met-Hcy+ can select methionine-independent, methionine stress resistant clones [10, 30]. The data presented in this study focuses on the triple negative breast cancer cell line MDA-MB-468 (shortened to MB468) due to our ability to derive multiple methionine stress resistant clones (MB468res) as controls . These methionine stress resistant cells propagate at similar rates as the parental MB468 cell line when cultured in methionine growth medium; however, MB468res cells continue to proliferate in Met-Hcy+ medium unlike MB468 cells (Fig. 1c, d). Spontaneous reversion to methionine independence has been associated with loss of transformation-specific properties such as anchorage independence . We therefore tested whether phenotypes associated with tumorigenicity are also lost in MB468res cells. When cultured in soft agar with Met+ growth media, MB468 cells efficiently formed colonies consistent with their tumorigenic phenotype. In contrast, MB468res-R8 cells formed only few or very small colonies (Fig. 1e), indicating a loss of the transformation phenotype. Similar results were obtained with the MB468res-R21 and R28 lines. However, whereas R8 and R21 did not form colonies, the R28 line formed numerous tiny colonies scarcely visible with the naked eye. We continued our studies with the MB468res-R8 cell line as their proliferation rate in Met+ medium is closest to the parental MB468 cell line, which eliminates potential effects on metabolite demand due to growth rate differences. MB468res-R8 cells provide an excellent control for our investigation of methionine dependence because cell morphology, proliferation rate, growth medium requirements, and genetic background resemble those of methionine stress sensitive MB468 cells. The molecular mechanism of reversion to methionine independence is unknown. Reversion is an extremely rare event and stable for several generations. However, after several generations, MB468res-R8 cultures can revert back to methionine dependence, suggesting epigenetic mechanisms behind this phenomenon.
Homocysteine media induces a metabolic down-regulation of oxidative phosphorylation
We compared fluorescence lifetimes of NADH from both MB468 and MB468res-R8 cells cultured in Met+ and Met-Hcy+ media. We used phasor cluster analysis to quantify the signals of higher and lower free/bound NADH ratios of both cell lines (Fig. 2d). In Met+ media, both cell lines had similar profiles with approximately 80 % of the signal representing a lower free/bound ratio providing a baseline for oxidative phosphorylation activity in these cell types (Fig. 2e). After 48 h, cells were transferred to Met-Hcy+ media; 70 % of the MB468 and 64 % of the MB468res-R8 signal represented the lower free/bound NADH ratio and thus a reduction in cellular respiration. Importantly, the addition of potassium cyanide, a potent inhibitor of mitochondrial respiration, resulted in a decrease in the signal of the lower free/bound ratio, similar to culturing in Met-Hcy+ media alone (Fig. 2a, e). The similar metabolic trajectories of potassium cyanide-treated cells and Met-Hcy+ cultures suggest that methionine stress may affect mitochondrial function or force cells to reduce oxidative phosphorylation.
We further analyzed mitochondrial function using an extracellular flux (XF) analyzer, which measures OCR as an indicator for mitochondrial respiration. Both MB468 and MB468res-R8 cells were cultured in either Met+ media for 12 h or Met-Hcy+ media for 2, 4, 8, and 12 h prior to measuring basal levels of OCR (Fig. 2f). Within 4 h in Met-Hcy+ media, both MB468 and MB468res-R8 cell lines respond by transiently increasing OCR. However, after 8 h, both cell lines showed decreased respiration, which continued to decline. Interestingly, we observed the MB468res-R8 cells to have a faster decline in respiration as compared to MB468 cells. In additional analyses with the XF analyzer, spare respiration capacity was measured in MB468 and MB468res-R8 cells cultured in Met+ media and treated with the electron transport chain accelerator FCCP. MB468 cells can maximize OCR 1.9 times that of basal levels when stressed with FCCP, whereas MB468res-R8 cells have a maximum 1.5-fold increase of OCR under the same conditions. We hypothesize that the faster decline in mitochondrial respiration in Met-Hcy+ media (Fig. 2f) and the reduced effect of KCN in the FLIM analysis (Fig. 2e) as compared to MB468 cells may be due to an innate lower capacity for respiration. With a smaller reserved respiration capacity, MB468res-R8 cells may feel the effects of dysfunctional mitochondria sooner but may not be as reliant on oxidative phosphorylation resulting in improved tolerance of metabolic stress as compared to MB468 cells. Regardless of these differences, the general response of both cell lines to Met-Hcy+ media and KCN is comparable, indicating reduced mitochondrial respiration during culturing in Met-Hcy+ media as determined by both FLIM and XF analyses.
Metabolite changes associated with culturing in homocysteine media
Metabolites connected to methionine
The differences in the levels of MTA may contribute to the difference in methionine levels between the two cell lines. MB468 cells have a slight decrease in methionine starting at 2 h, staying around 0.7-fold abundance until 24 h when the levels decrease to half the starting quantity (Fig. 4b). MB468res-R8 cells increase in methionine levels as early as 2 h but return to normal after 8 h. It is important to note that the MB468res-R8 cells continue to proliferate in Met-Hcy+ media unlike the MB468 cells, which may affect the use of metabolites in each cell line after 24 h, the approximate doubling time of these cells. However, the early changes we observed can be attributed to a metabolic response to the switch from methionine to homocysteine in the growth medium.
Purines and pyrimidines
Both adenine and adenosine are by-products of the methionine salvage and remethylation pathways and are essential components in purine synthesis. With few exceptions, changes in purine as well as pyrimidine concentrations were not evident until 12 to 24 h after cells were shifted (Fig. 4c). In MB468res-R8 cells, only adenine increased to approximately 1.5-fold of the starting quantity within 2 h. After 12 and 24 h, inosine, inosine monophosphate, and hypoxanthine were significantly increased (Fig. 4d). In contrast, MB468 cells respond to Met-Hcy+ medium with a delayed decrease in inosine, guanine, and guanosine, as well as an immediate steep decrease in adenine levels as described above. Unlike the overall increase in purines in MB468res-R8 cells, many purines in MB468 cells decrease to half the starting quantity by 24 h. Interestingly, the changes in components of the purine synthesis pathway are not observed for the pyrimidines thymine and cytosine. In both MB468 and MB468res-R8 cells, thymine and cytosine abundance indicate a minimal response to methionine stress. Uracil, the pyrimidine derivative found in RNA, showed a comparable trend to the purine response in both cell lines as levels decrease in MB468 cells and increase in MB468res-R8 cells over the course of 24 h (Fig. 4d).
Glycolysis and citric acid cycle
Similar to our earlier observations of decreased mitochondrial respiration in Met-Hcy+ media (Fig. 2), the tricarboxylic acid (TCA)-related metabolites succinate, fumarate, and malate indicate general reductions in MB468 cells. In contrast, MB468res-R8 cells show a twofold increase in malate and fumarate with less striking increases in other TCA cycle components including citrate, succinate, and aconitate (Fig. 5b). The reduction in MB468-dependent TCA intermediates corroborates with decreased oxidative phosphorylation; however, the difference in response of MB468res-R8 may be a result of their lower respiration capacity and thus their reduced dependence on respiration in these cells.
Homocysteine metabolism is redirected during HCY culturing
It is interesting to note that while MB468res-R8 cells process homocysteine forward through the remethylation pathway, MB468 cells process an average of 6 % more homocysteine through the reversible hydrolytic activity of S-adenosylhomocysteine hydrolase (Fig. 6g). SAH is the by-product of all biological transmethylation reactions requiring SAM and is also generated from homocysteine and adenosine. This reverse processing of homocysteine in MB468 cells may partially contribute to the decrease in adenosine abundance (Fig. 6h). Comparing the relative concentration of SAM and SAH can indicate the methylation potential of the cell. However, it has been shown that a reduced ratio bares little consequence without a corresponding increase in SAH [42, 43]. In both MB468 and MB468res-R8 cells, SAM and SAH were measured over a 24-h period in Met-Hcy+ media (Fig. 6i). For both cell lines, the SAM to SAH ratio decreased within 2 h of the media switch. In MB468 cells, this decrease in SAM to SAH is a result of a simultaneous decrease in SAM to less than one fifth starting quantity and increase in SAH to 1.5 times the starting quantity. After 12 h, however, both SAH and SAM begin to recover toward starting levels. In contrast, the SAM to SAH decrease observed in MB468res-R8 cells is a result of SAM levels declining to half the starting quantity with minimal perturbations in SAH. Considering SAM levels are twofold greater in MB468 cells in Met+ media as compared to MB468res-R8 cells, the consequences of an altered SAM to SAH ratio and elevated SAH levels in MB468 cells on the methylation potential of the cell may play an important role in methionine sensitivity. Clearly, both cell lines experience limited methylation potential as indicated by feedback up-regulation of methionine adenosyltransferase (MAT), the enzyme that produces SAM from methionine and ATP (Fig. 6j). Although labeled methionine levels are higher in MB468res-R8 cells, in both cell lines, it appears that the catalytic MAT2A subunit is, for unknown reasons, unable to use the available methionine for SAM synthesis and therefore protein levels increase to compensate this deficiency. Interestingly, MAT2B, the regulatory subunit of the enzyme, is unaffected by methionine perturbations.
Homocysteine and its relationship to ROS, mitochondria, and oxidative stress
Transsulfuration is critical to maintain the cellular redox balance as this metabolic pathway leads directly to glutathione synthesis (Fig. 6a). Interestingly, signaling pathways in yeast that are activated by methionine or SAM limitation induce a massive increase in glutathione synthesis suggesting a common physiological response to methionine stress in yeast and mammals [44–47]. Accordingly, the redirection of homocysteine metabolism to the transsulfuration pathway suggests the activation of a protective mechanism in response to Met-Hcy+ media. Under normal culturing conditions, the majority of cellular glutathione exists in its reduced form (GSH). As an antioxidant, glutathione protects the cell from oxidative stress by reducing the disulfide bonds of cytoplasmic proteins and in the process is oxidized to form glutathione disulfide (GSSG) . Interestingly, in Met+ media the abundance of GSH is twice as much in MB468 as compared to MB468res-R8 despite similar levels of GSSG (Fig. 6k). After culturing in Met-Hcy+ media, both cell lines experience oxidative stress as indicated by increasing levels of GSSG and a steep increase in GSH, presumably to combat oxidative pressure. The timing of this transition to the transsulfuration pathway and glutathione synthesis occurred within 4 h post media switch in MB468res-R8 cells and by 12 h in MB468 cells. This timing mirrors the increases of labeled cystathionine in both cell lines (Fig. 6d), supporting the hypothesis that redirection of homocysteine metabolism to transsulfuration is due to the cellular need to balance redox potentials. Cysteine abundances could not be analyzed by stable isotope tracer studies because metabolic processing of cystathionine results in an unlabeled molecule of cysteine and a deuterium-labeled alpha-ketobutyrate molecule. However, using an untargeted approach, cysteine levels indicate a similar increase as compared to both glutathione and cystathionine with peak abundances at 8 h in MB468 and 4 h in MB468res-R8 cells post media switch (Fig. 6l).
The oxidative stress associated with Met-Hcy+ media is not unexpected as studies on atherosclerosis have identified a relationship between elevated homocysteine levels in plasma and oxidative stress in tissue . The observed stress may be attributed to auto-oxidation of homocysteine, which generates hydrogen peroxide and reactive oxygen species (ROS). Alternatively, the source of stress may be related to inhibition of ATP synthesis in the mitochondria. N-methyl-d-aspartate (NMDA) receptors are glutamate and glycine-activated calcium channels that are largely found in neurons. Recent studies have identified active NMDA receptors in human neuroblastoma cells, small-cell lung cancer cells, and breast cancer cells and tumors, but not normal tissues [50, 51]. Free and reduced homocysteine molecules can bind to NMDA receptors at the glutamate binding location and activate an influx of calcium ions resulting in reduced ATP synthesis, reduced oxidative phosphorylation, and an accumulation of ROS . Although this is a suggested mechanism for Met-Hcy+-induced oxidative stress, the loss of oxidative phosphorylation activity is evident in both cell lines (Fig. 2) and the response to counter an oxidative insult by increasing glutathione abundance supports this scenario (Fig. 6k).
In conclusion, the present study reveals dynamic changes in the metabolic phenotype associated with methionine dependency of breast cancer and further highlights the importance of SAM as a major regulator of tumorigenesis. Importantly, using a labeled precursor, we discovered that in both MB468 and MB468res-R8 cell types, homocysteine does not significantly contribute to formation of methionine and ultimately S-adenosylmethionine. Our study indicates that SAM metabolism, which is required for all methylation reactions, depends on availability of methionine in the cancer cell lines investigated and cannot be substituted by homocysteine-forming pathways. Moreover, these findings shed light on the metabolic reprogramming, which accompanies methionine stress beyond methylation potential. Such knowledge is instrumental for elucidating novel targets of therapeutic intervention, which can be exploited in future studies. Given the importance of SAM in lipid biogenesis and our observed changes in LLS, assessing the impact of methionine stress on lipid composition and synthesis is of considerable interest and warrants further investigation.
extracellular acidification rate
fluorescence lifetime imaging microscopy
gas chromatography time-of-flight mass spectrometry
glutathione disulfide, oxidized glutathione
hydrophilic interaction liquid chromatography ultra high-pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry
long lifetime species
methionine adenosyltransferase II, alpha
methionine adenosyltransferase II, beta
MDA-MB-468 breast cancer cell line
MDA-MB-468res-R8 methionine stress resistant breast cancer derived cell line
homocysteine media without methionine
mass isotopomer distribution
nicotinamide adenine dinucleotide
oxygen consumption rate
This work was funded by NSF IGERT DGE 0549479, NIH F31 GM105321-01 to Stacey Borrego, NIH GM-066164-10, and pilot project funding by the West Coast Metabolomics Center as part of NIH-DK097154. The work of MZ and PB was supported by grants NSF IIS-1321053 and NIH LM010235 to PB.
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