Novel MAFG-METTL14-SCD1 axis regulates lipid metabolism mediating choroidal melanoma distant metastasis

Abstract

Background: Tumor invasion and metastasis are strongly influenced by cell membrane fluidity, regulated by lipid metabolism. In choroidal melanoma (CM), a highly metastatic cancer, the relationship between lipid metabolism, membrane fluidity, and metastatic mechanisms remains unclear.

Methods: We examined m⁶A methylation in CM patient samples. Lipidomic profiling was performed in control, METTL14-silenced, or SCD1-silenced CM cells. Transcriptomics were analyzed after METTL14 manipulation. Transmission electron microscopy assessed ultrastructural changes, while multiplex immunohistochemistry validated the clinical relevance of the MAFG-METTL14-SCD1 axis. The anti-metastatic effect of combining the SCD1 inhibitor aramchol with a stearate-rich diet (S-HFD) was tested in nude mouse CM metastasis models.

Results: Lipidomics revealed that SCD1 promotes CM progression via cardiolipin and fatty acid metabolism pathways. Silencing SCD1 reduced membrane fluidity, while its upregulation in CM was driven by METTL14-mediated m⁶A methylation at the 2492 mRNA site. Elevated MAFG expression further activated METTL14. Mechanistically, this MAFG-METTL14-SCD1 axis enhanced CM invasiveness. In preclinical models, aramchol combined with S-HFD markedly suppressed distant metastasis.

Conclusions: Our study identifies SCD1-mediated lipid remodeling as a key driver of enhanced membrane fluidity and metastatic potential in CM. Inhibition of SCD1 increases lipid saturation, reduces membrane fluidity, induces oxidative stress, and suppresses liver and lung metastasis. The MAFG-METTL14-SCD1 axis thus represents a critical regulator of CM progression, and combined therapeutic targeting with aramchol and S-HFD offers promising translational potential.

Keywords: Choroidal melanoma; High-fat diet; Lipid metabolism; Metastasis; SCD1.

Fig. 1

METTL14 promotes choroidal melanoma invasion and metastasis by mediating lipid metabolic pathways to facilitate membrane fluidity. a, m⁶A dot blot assays demonstrating the upregulation of m⁶A modification levels in tissues of patients with CM. b, Differential expression of METTL14 in normal individuals and patients with UVM, as represented by the GTEx and TCGA databases. (GTEx: Normal, n = 53; TCGA: UVM, n = 80). c, Representative multiplexed immunohistochemistry staining images of normal choroidal samples and CM samples. METTL14 (yellow), nuclei stained with DAPI (blue). Circular regions are shown at higher magnification on the right. d, IHC assay detecting METTL14 protein expression in normal choroid tissues (Normal: n = 10) and CM tissues (CM: n = 34). e, Transmission electron microscopy (TEM) analysis showing lipid droplet reduction and mitochondrial contraction after silencing METTL14 in MUM-2B cells. LD, lipid droplet; M, mitochondria. Scale bars: 1 μm. f, Non-targeted metabolomics analysis of shNC and METTL14 knockdown MUM-2B stable cells. Volcano plot showing 83 downregulated and 296 upregulated differential metabolites in METTL14-treated CM cells (MUM-2B) (FC > 1.5, P < 0.05) compared to the control group. KEGG enrichment analysis of metabolic pathways related to the differential metabolites. g, Peak area values of Behenic acid, Stearic acid, and Nervonic acid in the unsaturated fatty acid biosynthesis pathway, comparing the shNC and shM14 groups. h, Free fatty acid colorimetry (NEFA/FFA) revealing increased free fatty acid content after silencing METTL14. i, Flow cytometry assays detecting ROS levels after interfering with METTL14 expression in CM cells. j, Membrane fluidity test showing changes in cell membrane fluidity after interfering with METTL14 expression in C918 and MUM-2B cells. k-l, Representative coronal and axial sectional PET images showing the uptake of ¹⁸F-FDG in the liver of mice in two different groups (shNC and shM14, n = 5 for each). m, Representative images of livers excised from mice injected with METTL14-silencing and negative control cells (scale bar: 1 cm). n, Representative Oil Red O (ORO) staining images of mouse lung and liver tissues in two different groups (shNC and shM14, n = 5 for each)

Fig. 2

METTL14 mediates lipid metabolism component alterations through SCD1 to promote the characteristics of choroidal melanoma metastasis. a, Venn diagram and pathway enrichment analysis identifying SCD1, HSD17B8, ELOVL3, ACSL6, ACSBG1, ACADL, and ACAA2 as downstream target candidates of METTL14. b, RM2target (http://rm2target.canceromics.org/) showing the correlation between SCD1 and METTL14 in various cancers. c, Volcano plot showing 315 downregulated and 259 upregulated differential metabolites in SCD1-treated CM cells (MUM-2B) (FC > 1.5, P < 0.05) compared to the control group. d, KEGG enrichment analysis of metabolism pathways associated with differential metabolites. e, Peak area values of Behenic acid and Stearic acid in the unsaturated fatty acid biosynthesis pathway, comparing the shNC group vs. shSCD1 group. f, Free fatty acid colorimetry (NEFA/FFA) showing an increase in free fatty acid content after silencing SCD1. g, Flow cytometry showing ROS levels after silencing SCD1. h-i, Representative coronal and axial sectional PET images showing the uptake of ¹⁸F-FDG in the lung and liver of mice in two different groups (shNC and shSCD1, n = 5 for each). j-k, Representative images of excised lungs and livers from mice injected with the indicated cells. (Scale bar, 1 cm). l, Representative hematoxylin and eosin (H&E) staining images of lung and liver tissues. m, Immunohistochemical analysis of mouse lung and liver tissues with an anti-SCD1 antibody in two different groups (shNC and shSCD1, n = 5 for each). Scale bar, 50 μm. n, Representative ORO staining images of frozen mouse lung and liver tissues

Fig. 3

METTL14 mediates choroidal melanoma lipid metabolism by inducing SCD1 m⁶A methylation modification. a, Schematic diagram illustrating the METTL14-SCD1 regulatory axis via a specific m⁶A modification-dependent mechanism. b, The SRAMP (http://www.cuilab.cn/sramp) predicting the distribution of high-confidence m⁶A methylation sites and scores evaluation for SCD1 mRNA. c, Linear correlation between METTL14 expression and SCD1 expression. d, qRT-PCR analysis showing alterations in SCD1 mRNA levels after METTL14 depletion. e, FISH assay showing the expression and co-localization of METTL14 (green) with SCD1 (red) in CM cells. DAPI (blue) is used as the nuclear marker. Scale bar, 5 μm. f, m⁶A-MeRIP-qPCR assay showing enrichment of SCD1 by m⁶A-specific antibodies. g, ENCORI-CLIP database (http://starbase.sysu.edu.cn/) illustrating the RBP-mRNA interactions between METTL14 and SCD1. h, Double luciferase assay confirming METTL14 directly binds to the 2492 site of SCD1 3’UTR, with mutations at this site. i, Representative western blot images showing SCD1 expression in normal choroidal tissues and CM tissues. j, Immunohistochemical analysis of human normal choroidal and CM tissue samples with the indicated antibodies. Representative images shown. Scale bar, 100 μm. (Normal: n = 10, CM: n = 34). k, Membrane fluidity assay showing changes in cell membrane fluidity after interfering with SCD1 expression in C918 and MUM-2B cells. l, Transmission electron microscopy (TEM) showing lipid droplet reduction and mitochondrial contraction following the silencing of SCD1 in MUM-2B cells. LD, lipid droplet; M, mitochondria. Scale bars, 1 μm

Fig. 4

The METTL14-SCD1 signaling axis regulates lipid metabolism to promote CM invasion and metastasis. a, Radar chart illustrating the top 10 downregulated differential metabolic products obtained from the shNC + shMETTL14 group vs. shNC + shSCD1 group (P < 0.05). b, Rescue experiments determining cell membrane fluidity by overexpressing SCD1 upon silencing METTL14 in C918 and MUM-2B cells. c, Rescue experiments revealing free fatty acid content treated by overexpression of SCD1 upon silencing METTL14 in MUM-2B cells. d, Flow cytometry detecting ROS levels in the recovery experiment groups. e, Rescue experiments determining the number of lipid droplets after overexpression of SCD1 upon silencing METTL14 in MUM-2B cells. LD, lipid droplet. Scale bars, 5 μm and 1 μm. f-g, Representative coronal and axial sectional PET images showing the uptake of ¹⁸F-FDG in the lung and liver of mice in three different groups (shNC + pcDH, shM14 + pcDH, and shM14 + SCD1). h, Representative images of excised lungs and livers from mice injected with the indicated cells. Scale bar, 1 cm. i, Representative images of H&E staining of lung and liver tissues. j, Immunohistochemical analysis of mouse lung and liver tissues performed with an anti-METTL14 antibody in three different groups (shNC + pcDH, shM14 + pcDH, and shM14 + SCD1). Scale bars, 50 μm. k, Representative ORO staining images of lung and liver tissues

Fig. 5

METTL14 in CM is induced by MAFG through the activation of the response element. a, Schematic diagram illustrating how MAFG transcriptionally activates METTL14 to regulate CM cell invasion properties. b, UCSC (https://xena.ucsc.edu/) and TCGA (https://portal.gdc.cancer.gov/) predicting MAFG as the transcriptional activator of METTL14. c, GTEx (https://www.genome.gov/Funded-Programs-Projects/Genotype-Tissue-Expression-Project) and TCGA (https://portal.gdc.cancer.gov/) databases showing high expression of MAFG in patients with UVM (Normal: n = 53, UVM: n = 80). d, Immunohistochemical analysis of human normal choroidal and CM tissue samples with the indicated antibodies. Representative images shown. Scale bar, 100 μm. (Normal: n = 10, CM: n = 34). e, Linear correlation between METTL14 expression and MAFG expression. f, qRT-PCR analysis of METTL14 mRNA levels after MAFG inhibition. g, Representative western blot images showing METTL14 expression in CM cells after interfering with MAFG expression. h, Double luciferase assay confirming specific binding sites of MAFG to the METTL14 promoter after mutations at the METTL14 promoter. i-j, Representative coronal and axial sectional PET images showing the uptake of ¹⁸F-FDG in the lung and liver of mice in three different groups (shNC + pcDH, shMAFG + pcDH, and shMAFG + M14). k, Representative images of excised lungs and livers from mice injected with the indicated cells. Scale bar, 1 cm. l, IHC assay revealing the expression level of MAFG after silencing MAFG or combined with overexpressing METTL14 in MUM-2B cells. m, Representative ORO images of lung and liver tissues

Fig. 6

SCD1 inhibitor aramchol exhibits diet-dependent anti-metastatic effects. a, Representative multiplexed immunohistochemistry staining images of human normal choroidal tissue and CM tissue samples showing METTL14 (yellow), MAFG (purple), SCD1 (green), and DAPI (blue). b, Schematic representation of the therapy schedule. Mice were acclimated to the cages for 3 days, during which they were all fed normal chow (brown-color pellets). Following acclimation, the mice were divided into three dietary groups: normal-fat diet (NFD), high-fat diet rich in stearate (S-HFD), and high-fat diet rich in oleic acid (O-HFD). The interventions began 3 days prior to injection and continued throughout the study (n = 5). c, Representative coronal and axial sectional PET images showing the uptake of ¹⁸F-FDG in the lung of mice in four different groups. d, Representative images of excised lungs from mice injected with the indicated cells. Scale bar, 1 cm. e, Representative H&E staining images of lung tissues

Fig. 7

Proposed model showing that the newly identified MAFG-METTL14-SCD1 axis regulates lipid metabolism to mediate distant metastasis of choroidal melanoma