Methionine‑MAT2A‑SAM axis controls lipid levels by regulating ACSL4

Introduction

Metabolism serves as the fundamental basis of cellular life, providing energy for biological processes, enabling the biosynthesis of macromolecules and maintaining redox homeostasis (1). The cellular metabolic network, comprising the four core pathways, amino acid, glucose, lipid and nucleotide metabolism, forms the structural and functional foundation of cellular biochemistry (2). Amino acid metabolism occupies a particularly pivotal position within this network due to its notable diversity in molecular species and metabolic routes, enabling its critical involvement in multiple cellular processes (3–8). Emerging evidence has revealed that deficiencies in essential amino acids, including tryptophan, tyrosine, branched-chain amino acids (such as isoleucine, leucine and valine), methionine, phenylalanine and threonine, can disrupt lipid homeostasis, with potential implications for neurodevelopmental disorders (9). In diabetic murine models, depletion of serine and glycine has been demonstrated to exacerbate neuropathy progression (10). A mechanistic study has further shown that essential amino acid deficiency in hepatocytes leads to functional impairment of ubiquitin protein ligase E3 component n-recognin 1, causing aberrant ubiquitination and subsequent degradation of the lipid droplet-stabilizing protein, perilipin 2. This molecular cascade ultimately suppresses hepatic lipid catabolism and promotes fatty liver pathogenesis (11).

To sustain their rapid proliferation, tumor cells must overcome three fundamental metabolic demands: i) Rapid energy production; ii) biosynthesis of macromolecules; and iii) maintenance of redox homeostasis (1). Amino acids serve as essential providers of both carbon and nitrogen sources, playing pivotal roles in meeting these metabolic challenges. Consequently, amino acid restriction therapies have emerged as a promising therapeutic strategy against tumors (12–17). Notably, the sulfur-containing amino acid, cysteine, assumes particular importance in tumor metabolism, serving as a critical precursor for glutathione (GSH) synthesis and providing cellular protection against ferroptosis (18,19). Ferroptosis is an iron-dependent form of regulated cell death characterized by three key metabolic features: i) The glutathione peroxidase 4/GSH antioxidant system (dependent on cysteine availability); ii) Fe2+-mediated Fenton reactions; and iii) acyl-CoA synthetase long chain family member 4 (ACSL4)-catalyzed biosynthesis of polyunsaturated fatty acid-containing phospholipids (PUFA-PLs) (20). Notably, cellular cysteine can be acquired through two distinct pathways: Exogenous uptake via the solute carrier family 7 member 11 (SLC7A11) transporter or endogenous synthesis from methionine via the methionine adenosyltransferase 2A (MAT2A)-initiated transsulfuration pathway. Both SLC7A11 and MAT2A are frequently upregulated in tumors, making them attractive therapeutic targets (21–25). Notably, ACSL4 plays a contradictory role in the occurrence and development of tumors. On the one hand, ACSL4 can provide biomacromolecules for tumor proliferation by regulating the synthesis of PUFA-PLs (26). On the other hand, an increase in PUFA-PLs levels also increases the sensitivity of tumor cells to ferroptosis (27). Beyond their canonical roles in protein biosynthesis and cellular metabolism, amino acids serve as pivotal allosteric modulators that fine-tune metabolic flux through the direct regulation of rate-limiting enzymes (28). While the crosstalk between amino acid metabolism and glucose homeostasis has been extensively characterized, particularly through the dynamic interconversion of non-essential amino acid networks, the unidirectional nature of amino acid-lipid metabolic interactions has resulted in comparatively limited understanding of the amino acid-mediated regulation of lipid metabolism.

The present study aimed to systematically investigate the metabolic consequences of essential amino acid deprivation, with particular emphasis on delineating the mechanistic role of methionine in regulating intracellular lipid homeostasis. To assess alterations in lipid metabolism, flow cytometry coupled with fluorescent lipid probes were employed to evaluate intracellular lipid accumulation and cellular fatty acid uptake efficiency following essential amino acid deprivation. Comprehensive analyses were performed to elucidate the underlying molecular mechanisms of methionine-mediated lipid metabolic regulation. Western blotting and reverse transcription-quantitative PCR (RT-qPCR) were conducted to examine methionine restriction-induced modulation of ACSL4 expression, a critical regulator of fatty acid metabolism (27). Furthermore, the feedback regulation within the methionine-MAT2A-S-adenosylmethionine (SAM) metabolic axis was systematically investigated through combined western blotting and RT-qPCR analyses, with a specific focus on the SAM-dependent regulation of MAT2A protein stability and its consequent impact on lipid metabolic homeostasis.

Materials and methods

Cell culture

Mouse embryonic fibroblasts (MEFs) (CRL-2991; ATCC) and the HT1080, DU145 and H1299 cell lines were obtained from the American Tissue Culture Collection (ATCC) and cultured following ATCC recommendations. These cells were also subjected to STR identification and mycoplasma testing. The cells were sustained in Dulbecco's modified Eagle's medium (DMEM) with high glucose, sodium pyruvate (1 mM), glutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and 10% (v/v) fetal bovine serum (FBS; MeisenCTCC; Zhejiang Meisen Cell Technology Co., Ltd.) at 37°C and 5% CO2. In addition, the cultivation conditions for methionine deficiency are based on DMEM (cat. no. D0422; MilliporeSigma) with high glucose, sodium pyruvate (1 mM), glutamine (2 mM), penicillin (100 U/ml), streptomycin (0.1 mg/ml) and 10% (v/v) fetal bovine serum (FBS) (MeisenCTCC) at 37°C and 5% CO2. Alternatively, S-adenosylmethionine (SAM; 0.4 mM), S-adenosylhomocysteine (SAH; 0.4 mM), homocysteine (Hcy; 0.4 mM) or adenosine dialdehyde (ADOX; 0.4 mM) were added separately to methionine deficient culture medium to treat cells. Cells were also treated with a constructed culture medium lacking essential amino acids (isoleucine, 0.4 mM; isoleucine, 0.4 mM; lysine, 0.4 mM; methionine, 0.4 mM; phenylalanine, 0.4 mM; threonine, 0.4 mM; tryptophan, 0.4 mM; valine, 0.4 mM; histidine, 0.4 mM; and arginine, 0.4 mM).

Reagents

The reagents used included propidium iodide (PI; cat. no. P4170; MilliporeSigma), BODIPY581/591 C11 (cat. no. D3861; Thermo Fisher Scientific, Inc.), BODIPY (cat. no. HY-D1614; MedChemExpress), BODIPY™ FL C12 (cat. no. D3822; Thermo Fisher Scientific, Inc.), SAM (cat. no. A7007; MilliporeSigma), S-Adenosylhomocysteine (SAH; cat. no. A9384; MilliporeSigma), homocysteine (cat. no. H4628; MilliporeSigma), anti-MAT2A (cat. no. A19272; ABclonal Biotech Co., Ltd.), anti-ACSL4 (cat. no. A20414; ABclonal Biotech Co., Ltd.) and DMEM with high glucose (cat. no. D0422-100 ml; MilliporeSigma). In addition, all the components of the D777 culture medium (with 4,500 mg/l glucose, L-glutamine and sodium pyruvate, without sodium bicarbonate, powder, suitable for cell culture) were purchased to construct an amino acid-deficient culture medium. The medium mainly included methionine (cat. no. M0960000), leucine (cat. no. L8000), isoleucine (cat. no. I2752), lysine (cat. no. L5501), phenylalanine (cat. no. P17008), tyrosine (cat. no. T1145), tryptophan (cat. no. T0254), valine (cat. no. V0500), histidine (cat. no. H8125) and arginine (cat. no. A5006), all from MilliporeSigma.

Fatty acid uptake and intracellular lipid detection

Fatty acid uptake was measured by flow cytometry. Briefly, HT1080 and H1299 cells were plated at a density of 5×104 cells per well in a 24-well dish and cultured for 12 h in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin at 37°C with 5% CO2. Subsequently, 2 µM BODIPY-C12 was added to the cell culture medium and incubated for 10 h alongside the indicated treatment. Following this, the cells were trypsinized, washed with PBS containing 5% FBS and resuspended in 0.3 ml of PBS with 5% FBS for flow cytometry analysis (Attune NxT; Thermo Fisher Scientific, Inc.). Acquired data were analyzed using FlowJo version 10 software (FlowJo LLC). A minimum of 10,000 cells were analyzed per condition. Intracellular lipids were also measured by flow cytometry (Attune NxT.). Acquired data were analyzed using FlowJo version 10 software. The protocol was the same as that aforementioned except 1 µg/ml BODIPY was added to the cell culture medium and incubated for 30 min after the indicated treatment.

Lipid reactive oxygen species (ROS) detection

Lipid ROS were analyzed by flow cytometry. Briefly, cells were plated at a density of 1×105 cells per well in a 24-well dish and cultured overnight in DMEM. Subsequently, 4 µM BODIPY581/591 C11 was added to the cell culture medium and incubated for 30 min after the indicated treatment. Excess BODIPY581/591 C11 was then removed by washing the cells with PBS twice. Labeled cells were trypsinized and resuspended in PBS with 5% FBS. Oxidation of BODIPY581/591 C11 resulted in a shift of the fluorescence emission peak from 590 to 510 nm proportional to the lipid ROS generation, which was detected using a flow cytometer (Attune NxT). Acquired data were analyzed using FlowJo version 10 software.

Cell death detection

Cell death was analyzed by flow cytometry. Briefly, cells were plated at a density of 2×105 cells per well in a 12-well dish for 12 h in fresh medium. After the indicated treatment, 1 µg/ml PI was added to the cell culture medium and incubated for 30 min. Then, cells were trypsinized, washed with PBS containing 5% FBS and resuspended in 0.3 ml of PBS with 5% FBS for flow cytometry (Attune NxT). Acquired data were analyzed using FlowJo software.

Apoptosis detection

Apoptosis was analyzed by flow cytometry. Briefly, cells were plated in 12-well culture plates at a density of 1×105 cells per well and cultured in DMEM for 12 h. Cells were then trypsinized, washed with PBS containing 5% FBS and resuspended in 0.3 ml of PBS. Subsequently, PI and Annexin V-FITC were added to the cells and incubated for 30 min, and cell apoptosis was detected by flow cytometry (Attune NxT). Acquired data were analyzed using FlowJo.

Transfections

Transfections were conducted using Lipofectamine™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. HT1080 cells seeded in a 6-well dish were transfected with 40 ng of the following small interfering RNAs (Suzhou GenePharma Co., Ltd.): NC (scrambled), sense: 5′-UUCUCCGAACGUGUCACGUUTT-3′, antisense: 5′-AACGUGACACGUUCGGAGAATT-3′; MAT2A-1, sense: 5′-GGAUCGAGGUGCUGUGCUUTT-3′, antisense: 5′AAGCACAGCACCUCGAUCCTT-3′; MAT2A-2, sense: 5′-GGGAUGCCCUAAAGGAGAATT-3′, antisense: 5′-UUCUCCUUUAGGGCAUCCCTT-3′ and MAT2A-3, sense: 5′-GCCUAUGGCCACUUUGGUATT-3′, antisense: 5′-UACCAAAAGUGGCCAUAGGCTT-3′. After transfection, the cells were placed in a 37°C cell culture incubator and the medium was changed after 8 h. After 48 h, the cells were lysed and the interference efficiency was measured using western blotting.

Western blotting

Cell lysate preparation, SDS-PAGE and electrophoretic transference were accomplished as previously described (19). Briefly, the protein sample was quantified using a NanoPhotometer® N30 Touch and each lane of the 8% SDS gel was loaded with 10 µg protein sample. Membranes were blocked with 5% non-fat dried milk in TBS (pH 7.2) containing 0.1% Tween 20 (TBST), incubated with the appropriate primary antibodies in 5% non-fat dried milk in TBST overnight at 4°C. Then, the membrane was incubated with the appropriate secondary antibodies in 5% non-fat dried milk in TBST at room temperature for 50 min. The following primary antibodies were used: Anti-MAT2A, anti-ACSL4 and β-actin (cat. no. HC201-01; TransGen Biotech Co., Ltd.). The following secondary antibodies were used: HRP-conjugated goat anti-rabbit (cat. no. AS014; ABclonal Biotech Co., Ltd.) and HRP-conjugated goat anti-mouse (cat. no. AS003; ABclonal Biotech Co., Ltd.). Chemiluminescence was detected using the ChampChemi 610 (SinSage Technology Co., Ltd.), and the software used for densitometry was ImageJ (National Institutes of Health).

RT-qPCR

Total RNA was extracted from cells using TRNzol (cat. no. DP424; Tiangen Biotech Co., Ltd.) and reverse transcribed with the PrimeScript™ RT reagent Kit with gDNA Eraser (cat. no. RR047A; Takara Bio, Inc.) according to the manufacturer's protocol. The resulting cDNA was then subjected to qPCR using the TB Green Premix Ex Taq™ (Tli RNase H Plus) (cat. no. RR820A; Takara Bio, Inc.). The detection and analysis were accomplished as previously described (19). The sequences of the primers used for qPCR were as follows: ACSL4 (human) forward, 5′-CATCCCTGGAGCAGATACTCT-3′ and reverse, 5′-TCACTTAGGATTTCCCTGGTCC-3′; MAT2A (human) forward, 5′-ACCAGAAAGTGGTTCGTGAAG-3′ and reverse, 5′-CAAGGCTACCAGCACGTTACA-3′; MAT2B (human) forward, 5′-TTCACTGGTCTGGCAATGAAC-3′ and reverse, 5′-AGGGCTGTCAGTAATAGGTCTT-3′; MAT2A (mouse) forward, 5′-GCTTCCACGAGGCGTTCAT-3′ and reverse, 5′-AGCATCACTGATTTGGTCACAA-3′; MAT2B (mouse) forward, 5′-AGGGAACCTTTCACTGGTCTG-3′ and reverse 5′-ATTTGGAGCAATCGAGCTGAG-3′; β-actin (human) forward, 5′-GTTGTCGACGACGAGCG-3′ and reverse, 5′-GCACAGAGCCTCGCCTT-3′; and β-actin (mouse) forward, 5′-GGCTGTATTCCCCTCCATCG-3′ and reverse, 5′-CCAGTTGGTAACAATGCCATGT-3′.

Public database analyses

The expression levels of ACSL4 in tumor and normal tissues were analyzed using the Expression DIY module on the GEPIA 2 website (http://gepia2.cancer-pku.cn/#index). The analysis parameter settings were as follows: Log2 FC cut-off (1); q-value cut-off (0.01); log scale (yes); jitter size (0.4); and matched normal data (match TCGA normal and GTEx data).

Overall survival

The survival analysis module on the GEPIA 2 website was used to analyze the relationship between ACSL4 expression and patient survival. The analysis parameter settings were as follows: Methods (overall survival); group cut-off (median); cut-off high (%) (50); cut-off low (%) (50); hazard ratio (yes); 95% confidence interval (yes); and axis units (months).

Statistical analysis

GraphPad Prism (v.10.4.0; Dotmatics) software was used to calculate the P-values using two-sided unpaired t-test or one-way ANOVA followed by Tukey's Honestly Significant Difference. All data presented in the figures represent the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Results

Methionine restriction downregulates cellular lipid levels

To systematically evaluate the role of essential amino acids in lipid metabolism regulation, an essential amino acid-deficient culture system was established by excluding leucine, isoleucine, lysine, methionine, phenylalanine, tyrosine, tryptophan, valine, histidine and arginine from the medium. HT1080 cells cultured in this modified medium were subsequently analyzed for intracellular lipid accumulation using BODIPY staining. Quantitative analysis revealed that methionine deprivation specifically caused a significant reduction in cellular lipid content (Fig. 1A). Next, the functional consequences of essential amino acid deficiency on fatty acid uptake capacity were examined using BODIPY-C12 fluorescence assays. Notably, methionine-deficient conditions produced a notable impairment in fatty acid internalization among all tested amino acid deprivations (Fig. 1B).

Figure 1. Methionine restriction downregulates cellular lipid levels. (A) HT1080 cells were cultured without leucine, isoleucine, lysine, methionine, phenylalanine, tyrosine, tryptophan, valine, histidine or arginine as indicated for 24 h, and the intracellular lipids were determined by BODIPY staining coupled with flow cytometry (n=3). (B) HT1080 cells were cultured without leucine, isoleucine, lysine, methionine, phenylalanine, tyrosine, tryptophan, valine, histidine, or arginine as indicated for 24 h, and fatty acid uptake capacity was determined by BODIPY-C12 staining coupled with flow cytometry (n=3). (C) Western blot analysis of ACSL4 expression in HT1080 cells treated with medium without leucine, isoleucine, lysine, methionine, phenylalanine, tyrosine, tryptophan, valine, histidine or arginine as indicated for 24 h. (D) Western blot analysis of ACSL4 expression in MEFs cells treated with medium without methionine for 12 h. Data are presented as the mean ± SD with P-values determined by one-way ANOVA followed by Tukey's Honestly Significant Difference. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AA, all amino acids; L, isoleucine; I, isoleucine; K, lysine; M, methionine; F, phenylalanine; T, threonine; W, tryptophan; V, valine; H, histidine; R, arginine; ACSL4, acyl-CoA synthetase long chain family member 4; MEFs, mouse embryonic fibroblasts; Met, methionine.

Given the established role of ACSL4 in facilitating fatty acid uptake through its dual function of converting fatty acids to acyl-CoA derivatives and inhibiting fatty acid efflux, ACSL4 protein expression in HT1080 cells under essential amino acid deprivation conditions was first examined. Western blot analysis revealed that methionine deficiency reduced the ACSL4 protein levels (Fig. 1C). To validate this finding across different cellular contexts, the investigation was extended to MEFs under methionine-restricted conditions. Consistent with the initial observation, methionine deprivation significantly decreased ACSL4 expression in MEFs cells (Fig. 1D). Taken together, the results suggest that methionine deficiency attenuates cellular lipid accumulation and the fatty acid uptake capacity, potentially through downregulation of ACSL4 protein expression, revealing a novel mechanistic link between methionine availability and lipid metabolic regulation.

Methionine regulates ACSL4 through SAM

As the sulfur-containing amino acid methionine functions as a vital cellular methyl donor primarily through its metabolic derivative SAM, it was next determined whether methionine regulates ACSL4 expression and fatty acid uptake capacity via SAM-dependent mechanisms. To test this hypothesis, methionine deprivation experiments in HT1080 cells were performed followed by SAM supplementation. Western blot analysis demonstrated that SAM administration rescued the methionine deficiency-induced reduction in ACSL4 protein levels (Fig. 2A). Complementary RT-qPCR analysis further revealed that SAM supplementation similarly restored the suppressed ACSL4 mRNA expression under methionine-depleted conditions (Fig. 2B). To elucidate whether SAM regulates ACSL4 expression through methylation-dependent mechanisms, HT1080 cells were treated with the methylation inhibitor, adenosine dialdehyde (ADOX), and the ACSL4 transcript levels were analyzed. RT-qPCR analysis revealed that ADOX treatment significantly suppressed ACSL4 mRNA expression (Fig. 2C), indicating methylation-dependent regulation. This regulatory mechanism in H1299 cells was further validated, as western blot analysis confirmed that the methionine-SAM axis controls ACSL4 protein abundance possibly through methylation modifications (Fig. 2D). Functional assays demonstrated that this methylation-dependent regulation directly impacts cellular fatty acid uptake capacity (Fig. 2E). Collectively, these results establish that SAM-mediated methylation may represent a critical epigenetic mechanism through which methionine regulates ACSL4 expression and function in lipid metabolism.

Figure 2. Methionine regulates ACSL4 through SAM. (A) Western blot analysis of ACSL4 expression in HT1080 cells cultured ± methionine or ± SAM as indicated for 24 h. The ACSL4 mRNA levels in HT1080 cells cultured (B) ± methionine and ± SAM (C) or ± ADOX as indicated for 24 h were analyzed using reverse transcription-quantitative PCR (n=3). (D) Western blot analysis of ACSL4 expression in H1299 cells cultured ± methionine, ± SAM or ± ADOX as indicated for 48 h. (E) H1299 cells were cultured ± methionine, ± SAM or ± ADOX as indicated for 48 h, and fatty acid uptake capacity was determined by BODIPY-C12 staining coupled with flow cytometry (n=3). Data are presented as mean ± SD with P-values determined by one-way ANOVA followed by Tukey's Honestly Significant Difference. *P<0.05, **P<0.01, ****P<0.0001. ACSL4, acyl-CoA synthetase long chain family member 4; SAM, S-adenosylmethionine; ADOX, adenosine dialdehyde; Met, methionine.

MAT2A can be dynamically regulated by the level of SAM

As the key enzyme catalyzing methionine-to-SAM conversion, MAT2A occupies a central position in the methionine-SAM metabolic axis (24). To investigate whether MAT2A serves as a critical regulator of SAM homeostasis and lipid metabolism, MAT2A protein expression in HT1080 cells under essential amino acid deprivation was first examined. Western blot analysis revealed that the MAT2A levels were specifically upregulated by methionine deficiency (Fig. 3A). Using a dose-response approach, it was demonstrated that MAT2A expression exhibits sensitivity to extracellular methionine concentrations in DU145 cells (Fig. 3B). Notably, SAM supplementation reversed the observed methionine deprivation-induced MAT2A upregulation (Fig. 3C), suggesting a feedback regulatory mechanism. Further mechanistic studies in H1299 cells showed that MAT2A protein levels remained unchanged following ADOX-mediated methylation inhibition, compared with the methionine deprivation only group (Fig. 3D). Consistent with this result, transcriptional analysis across multiple cell lines (MEFs, HT1080 and H1299) revealed that, while the methionine-SAM axis regulates MAT2A mRNA expression, this control occurs independently of the methyltransferase activity of SAM (Fig. 3E). Notably, the regulatory subunit, MAT2B, was unaffected by these metabolic perturbations (Fig. 3F). These results establish that MAT2A is subject to SAM-mediated feedback regulation through a novel methylation-independent mechanism, highlighting the sophisticated control of methionine metabolism.

Figure 3. MAT2A can be dynamically regulated by the level of SAM. (A) Western blot analysis of MAT2A expression in HT1080 cells treated with medium without leucine, isoleucine, lysine, methionine, phenylalanine, tyrosine, tryptophan, valine, histidine, or arginine as indicated for 24 h. (B) Western blot analysis of MAT2A expression in DU145 cells treated with medium without methionine as indicated for 0, 3, 6, 9, 12, 24, 30 and 36 h. (C) Western blot analysis of MAT2A expression in DU145 cells cultured ± methionine, ± SAM, ± SAH or ± Hcy as indicated for 30 h. (D) Western blot analysis of MAT2A expression in H1299 cells cultured ± methionine, ± SAM or ± ADOX, as indicated for 48 h. The (E) MAT2A and (F) MAT2B mRNA levels in MEFs and HT1080 and H1299 cells cultured ± methionine, ± SAM or ± ADOX as indicated for 12 h, were analyzed using reverse transcription-quantitative PCR (n=3). Data are presented as mean ± SD with P-values determined by one-way ANOVA followed by Tukey's Honestly Significant Difference. **P<0.01, ***P<0.001, ****P<0.0001. ns, non-significant; AA, all amino acids; L, isoleucine; I, isoleucine; K, lysine; M, methionine; F, phenylalanine; T, threonine; W, tryptophan; V, valine; H, histidine; R, arginine; MEFs, mouse embryonic fibroblasts; SAM, S-adenosylmethionine; ADOX, adenosine dialdehyde; SAH, S-adenosylhomocysteine; Hcy, homocysteine; MAT2A/B, methionine adenosyltransferase 2A/B; Met, methionine.

Knocking down MAT2A reduces intracellular lipid levels

To systematically validate the regulatory role of methionine in lipid metabolism, complementary experimental approaches were performed using HT1080 cells. First, methionine deprivation (using commercial methionine deficient medium) led to significant reductions in both intracellular lipid accumulation (BODIPY staining) and fatty acid uptake capacity (BODIPY-C12 assay) (Fig. 4A and B). To directly assess the involvement of MAT2A in these metabolic processes, RNA interference-mediated knockdown was employed. MAT2A downregulation similarly reduced the intracellular lipid content (Fig. 4C and D) and impaired fatty acid internalization (Fig. 4E), phenocopying the effects of methionine deprivation. These consistent findings across orthogonal experimental approaches establish MAT2A as a key metabolic regulator and potential therapeutic target for modulating cellular lipid homeostasis.

Figure 4. Knocking down MAT2A expression reduces the intracellular lipid levels. (A) HT1080 cells were cultured ± methionine as indicated for 20 h, and the intracellular lipids were determined by BODIPY staining coupled with flow cytometry (n=3). (B) HT1080 cells were cultured ± methionine as indicated for 12 h, and fatty acid uptake capacity was determined by BODIPY-C12 staining coupled with flow cytometry (n=3). (C) Western blot analysis of MAT2A expression in HT1080 cells transfected with siMAT2A or siNC. (D) The intracellular lipids in HT1080 cells transfected with siMAT2A or siNC were determined by BODIPY staining coupled with flow cytometry (n=3). (E) Fatty acid uptake capacity in HT1080 cells transfected with siMAT2A or siNC was determined by BODIPY-C12 staining coupled with flow cytometry (n=3). Data are presented as mean ± SD, with P-values determined by one-way ANOVA followed by Tukey's Honestly Significant Difference. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. MAT2A, methionine adenosyltransferase 2A; si, small interfering (RNA); NC, negative control; Met, methionine.

Methionine restriction suppresses tumor cell proliferation by inhibiting ACLS4

Lipids serve as fundamental structural components of cellular membranes and play a critical role in supporting the rapid proliferation of tumor cells. The aforementioned results demonstrated that methionine functions as a positive regulator of intracellular lipid homeostasis. Analysis of The Cancer Genome Atlas datasets revealed elevated ACSL4 expression in multiple malignancies, including esophageal carcinoma, liver hepatocellular carcinoma, pancreatic adenocarcinoma and stomach adenocarcinoma (Fig. 5A). Notably, high ACSL4 expression was significantly associated with poorer overall survival in patients with cancer, including cholangiocarcinoma, esophageal carcinoma and colon adenocarcinoma (Fig. 5B), suggesting its clinical relevance as a potential prognostic marker. Functional studies in HT1080 cells showed that methionine deprivation simultaneously suppressed ACSL4 expression (Fig. 5C) and impaired HT1080 cell proliferation (Fig. 5D), while not inducing ferroptosis (Fig. 5E and F); however, it has a certain inducing effect on apoptosis (Fig. 5G). Taken together, these results establish methionine as an essential metabolic requirement for tumor cell proliferation, operating through ACSL4-mediated lipid metabolic pathways.

Figure 5. Methionine restriction suppresses tumor cell proliferation by inhibiting ACLS4. (A) Analysis of ACSL4 expression in tumor and adjacent tissues based on TCGA database. (B) Analysis of the correlation between ACSL4 expression and overall survival based on TCGA database through GEPIA2 (http://gepia2.cancer-pku.cn/). (C) Western blot analysis of ACSL4 expression in HT1080 cells treated with medium without methionine as indicated for 12 h. (D) The number of HT1080 cells after methionine deprivation as indicated for 36 h were counted using a microscope and cell counter (n=3). (E) HT1080 cells were cultured ± methionine as indicated for 36 h and the lipid ROS level was determined by BODIPY-C11 staining coupled with flow cytometry (n=3). (F) HT1080 cells were cultured ± methionine as indicated for 36 h and cell death was determined by PI staining coupled with flow cytometry (n=3). (G) HT1080 cells were cultured ± methionine as indicated for 36 h and cell apoptosis was determined by PI staining coupled with flow cytometry (n=3). Data are presented as mean ± SD, with P-values determined by one-way ANOVA followed by Tukey's Honestly Significant Difference. *P<0.05, **P<0.01, ***P<0.001. ns, non-significant; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; ESCA, esophageal carcinoma; LIHC, liver hepatocellular carcinoma; PAAD, pancreatic adenocarcinoma; STAD, stomach adenocarcinoma; T, tumor; N, normal; HR, hazard ratio; ACSL4, acyl-CoA synthetase long chain family member 4; SSC, side Scatter; ROS, reactive oxygen species; PI, propidium iodide; Met, methionine.

Discussion

Amino acid metabolism serves as a critical metabolic hub supporting the biosynthetic and energetic demands of rapidly proliferating tumor cells. Among the essential amino acids, methionine, a sulfur-containing metabolite with pleiotropic functions, has emerged as a promising therapeutic target in oncology (14,29). The present study identified a unique role for methionine in governing lipid homeostasis, demonstrating its superior capacity to modulate intracellular lipid accumulation and fatty acid uptake compared with other essential amino acids. Mechanistically, it was established that methionine exerts this control through SAM-dependent regulation of ACSL4, a master regulator of fatty acid metabolism (27). Downregulation of MAT2A, the rate-limiting enzyme in methionine-to-SAM conversion (24), phenocopied methionine deprivation by reducing lipid stores and impairing fatty acid internalization. Notably, these metabolic perturbations translated to significant anti-proliferative effects in tumor cells, highlighting the therapeutic potential of targeting methionine metabolism. Furthermore, methionine restriction has emerged as a promising therapeutic strategy in oncology, primarily through its depletion of SAM, the universal methyl donor critical for numerous cellular processes (30–32). The present study extends this paradigm by revealing an additional mechanism whereby methionine restriction impairs tumor proliferation through ACSL4 downregulation-mediated inhibition of fatty acid uptake. The therapeutic potential of targeting methionine metabolism is further underscored by the frequent upregulation of MAT2A across diverse malignancies (33,34). In view of this, MAT2A has become an important target for cancer treatment, and inhibitors developed around it are constantly emerging (24,35,36). Notably, the MAT2A inhibitor, AG-270, has entered phase I clinical trials as a potential therapeutic agent for malignant tumors (24). Furthermore, ACSL4, a crucial enzyme in lipid metabolism regulation, has been demonstrated to facilitate metastatic extravasation in ovarian cancer through its enhancement of membrane fluidity and cellular invasiveness (37). Consequently, targeted disruption of the methionine-MAT2A-ACSL4 axis represents a potential therapeutic strategy to simultaneously suppress tumor proliferation and metastasis.

MAT2A is essential for maintaining intracellular SAM homeostasis. Emerging evidence indicates that the regulatory subunit, MAT2B, mediates SAM-dependent feedback inhibition of MAT2A activity (38–41). In addition, the present study extended these findings by demonstrating that: i) Methionine deprivation upregulates MAT2A expression; ii) SAM supplementation suppresses this induction; and iii) this regulatory mechanism operates independently of the methyltransferase activity of SAM. We hypothesize that mTORC1 may serve as a key mediator in SAM-dependent regulation of MAT2A expression. This speculation is supported by existing evidence demonstrating that mTORC1 can modulate MAT2A expression through its downstream effector, MYC (42). Furthermore, a study has suggested that SAM may exert feedback control on MAT2A through the SAM sensor, SAMTOR, which interfaces with both the mTORC1 and MYC signaling pathways (43). Therefore, the feedback regulation of MAT2A by SAM contributes to the steady-state of methionine-MAT2A-SAM.

ACSL4, a key regulator of lipid metabolism, catalyzes the ATP-dependent conversion of fatty acids to acyl-CoA esters. Extensive studies have established that ACSL4 governs cellular fatty acid uptake capacity by maintaining the intracellular acyl-CoA pool (44,45). The present study revealed that methionine deprivation decreased ACSL4 protein abundance, leading to impaired fatty acid internalization. In future research, the construction of isogenic ACSL4-knockout cell lines will be critical for characterizing ACSL4-dependent lipid metabolic pathways and for determining whether methionine restriction can further modulate intracellular lipid dynamics in ACSL4-deficient contexts. Notably, this lipid metabolic perturbation can be rescued by SAM supplementation, suggesting SAM-dependent regulation of ACSL4. However, the molecular mechanisms underlying SAM-mediated control of ACSL4 expression and activity require further investigation. Previous evidence provides important mechanistic insights into this regulatory process. A previous study demonstrated that SAM modulates mTORC1 activity through its sensor protein, SAMTOR (43). As a central nutrient-sensing hub, mTORC1 plays a pivotal role in coordinating cellular anabolism and metabolic reprogramming. Notably, mTORC1 has been shown to directly regulate ACSL4 expression (46). Based on these established relationships, we propose a model wherein SAM regulates ACSL4 levels through SAMTOR-mediated modulation of mTORC1 signaling activity.

As the primary product of the methionine-MAT2A-SAM metabolic axis, SAM serves as the universal methyl donor for diverse biological methylation reactions, including protein, DNA, RNA and small molecule modifications (47). Given this extensive substrate diversity, we hypothesize that the regulatory mechanisms by which the methionine-MAT2A-SAM axis influences ACSL4 and lipid metabolism remain incompletely characterized. Furthermore, the additional role of SAM as a precursor for polyamine biosynthesis (48) suggests potential cross-regulation between polyamine metabolism and ACSL4-mediated lipid pathways. Collectively, these observations highlight the multifaceted regulatory capacity of the methionine-MAT2A-SAM axis in cellular metabolism.

The present study has certain limitations. Notably, the BODIPY probe used in the present study functions as a broad-spectrum lipid stain, lacking molecular specificity. Consequently, while this method effectively detects overall lipid uptake, it does not discriminate between distinct lipid species. This represents a limitation of the current approach, as it precludes detailed characterization of the specific lipid subtypes involved in the observed processes.

In conclusion, the present study elucidated a novel regulatory mechanism by which methionine governs cellular lipid homeostasis through modulation of fatty acid uptake. These findings establish the methionine-MAT2A-SAM axis as a central metabolic regulator and reveal its therapeutic potential for targeted cancer interventions.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Zhejiang Sci-Tech University Research Start-up Fund (grant no. 24042191-Y).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

CX conceived the project, designed the research and led the entire experiment, from design to execution. JM assisted with the experiments. CX analyzed the data and wrote the manuscript. CX and JM confirm the authenticity of all the raw data. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References