Canonical Wnt is inhibited by targeting one-carbon metabolism through methotrexate or methionine deprivation

Abstract

The nutrient-sensing metabolite S-adenosylmethionine (SAM) controls one-carbon metabolism by donating methyl groups to biochemical building blocks, DNA, RNA, and protein. Our recent work uncovered a requirement for cytoplasmic arginine methylation during Wnt signaling through the activity of protein arginine methyltransferase 1 (PRMT1), which transfers one-carbon groups from SAM to many protein substrates. Here, we report that treatments that decrease levels of the universal methyl donor SAM were potent inhibitors of Wnt signaling and of Wnt-induced digestion of extracellular proteins in endolysosomes. Thus, arginine methylation provides the canonical Wnt pathway with metabolic sensing properties through SAM. The rapid accumulation of Wnt-induced endolysosomes within 30 minutes was inhibited by the depletion of methionine, an essential amino acid that serves as the direct substrate for SAM production. We also found that methionine is required for GSK3 sequestration into multivesicular bodies through microautophagy, an essential step in Wnt signaling activity. Methionine starvation greatly reduced Wnt-induced endolysosomal degradation of extracellular serum proteins. Similar results were observed by addition of nicotinamide (vitamin B3), which serves as a methyl group sink. Methotrexate, a pillar in the treatment of cancer since 1948, decreases SAM levels. We show here that methotrexate blocked Wnt-induced endocytic lysosomal activity and reduced canonical Wnt signaling. Importantly, the addition of SAM during methionine depletion or methotrexate treatment was sufficient to rescue endolysosomal function and Wnt signaling. Inhibiting the Wnt signaling pathway by decreasing one-carbon metabolism provides a platform for designing interventions in Wnt-driven disease.

Keywords: S-adenosylmethionine; Wnt signaling; arginine methylation; methotrexate; nicotinamide.

Fig. 1.

Methionine depletion inhibits Wnt signaling. (A) The universal cofactor for methylation SAM is generated through a biochemical mechanism known as the methionine cycle. Methionine is converted into SAM through an ATP-driven reaction. Methyltransferases use SAM as a cofactor to transfer methyl groups (in red) to a myriad of substrates, generating the byproduct SAH. SAH is rapidly converted to homocysteine, which can become remethylated to form methionine, completing the cycle. (B) Experimental design used for testing the effect of methionine deprivation on Wnt-induced endocytosis. Cultured cells were preincubated in methionine-depleted medium for 30 min and then treated with Wnt3a for 30 min (in the case of HeLa cell phase microscopy analyses or in situ protease protection assays) or 12 h (for HEK293T BAR/β-catenin luciferase assays). (C–F) Treatment with Wnt3a for 30 min triggered the formation of large puncta visualized by phase microscopy in HeLa cells; methionine depletion for 30 min strikingly inhibited vesicle formation in the presence of Wnt3a (compare D to F); the indicated stippled rectangles were enlarged. (G) Enlargements of Wnt3a-induced vesicles (2–5 μm in diameter) which were absent minus methionine. (H) Quantification of the diameters of Wnt-induced vesicles using ImageJ. (I) Time-course analysis of methionine depletion for 30, 60, 120, or 240 min followed by 30 min of Wnt3a signaling; note the significant decrease in vesicle formation after only 30 min (despite using nondialyzed serum in this case). (J–M) Wnt-induced GSK3 sequestration inside vesicles was inhibited by methionine starvation for 30 min, as assessed by in situ proteinase K protection assay (compare K to M). (N) Methionine depletion inhibited Wnt signaling in BAR luciferase reporter assays, while methionine readdition in column 4, rescued Wnt inhibition. Wnt luciferase reporter (BAR) was normalized using Renilla (49). (Scale bars: 10 μm.) **P < 0.01; n < 3.

Fig. 2.

Wnt-induced endocytosis and lysosomal activity require methionine and PRMT1. (A–D) Methionine depletion inhibited PRMT1 sequestration into GSK3-containing vesicles by Wnt3a treatment in in situ protease protection analyses (compare B to D). (E) PRMT1 and GSK3 colocalization in vesicles quantified by Pearson’s correlation coefficient using ImageJ. (F–H) Methionine depletion decreased Wnt-induced endolysosomal activity, assessed by the endocytosis and digestion of BSA-DQ. (I–K) PRMT1 depletion with siRNA (siPRMT1) decreased Wnt3a-induced lysosomal activity compared with siScrambled control (siCtr) cells (compare J to K), as assessed by endocytosis of BSA-DQ. (L) Diagram of the BSA-DQ assay, which is endocytosed and fluoresces only after the protein is digested in lysosomes. (M) Lysosomal activity quantification of BSA-DQ fluorescence per cell after 30 min of methionine depletion. (N) Wnt-induced degradation of BSA-DQ in lysosomes requires the PRMT1 enzyme (ImageJ quantification per cell). (Scale bars: 10 μm.) **P < 0.01; n < 4.

Fig. 3.

SAM addition to the culture medium rescues inhibition of Wnt signaling. (A–L) Methionine depletion decreased Wnt-induced vesicle formation and inhibited the Wnt-induced sequestration of PRMT1 and GSK3 after 30 min of Wnt3a treatment, and this effect was rescued by the addition of 1 mM SAM to the culture medium. (M) Quantification of the rescue of PRMT1 and GSK3 protease protection in vesicles by Pearson’s correlation coefficient. (N) Addition of 1 mM SAM increased Wnt-induced BAR luciferase activity when methionine was depleted. (O) Schematic depiction of the effect of addition of nicotinamide (vitamin B3) to the culture medium. Nicotinamide becomes readily methylated (generating 1-methylnicotinamide) and serves as a sink to store methyl groups and decrease SAM levels (59, 60). (P) Addition of 8 mM nicotinamide to the culture medium inhibited BAR/β-catenin luciferase activity. (Scale bars: 10 μm.) **P < 0.01; n < 3.

Fig. 4.

Methotrexate inhibits Wnt signaling, as well as Wnt-induced endocytosis, lysosomal activity, and GSK3 and PRMT1 sequestration through the folate cycle. (A) One-carbon metabolism is composed of the methionine and folate cycles; methotrexate (MTX) inhibits DHF from entering the cycle. (B) Methotrexate (20 μM) inhibited Wnt signaling (12 h), as assessed by BAR-Renilla luciferase assays. Note that Wnt inhibition was rescued by the addition of SAM (lane 4). (C) Methotrexate induced significant cellular toxicity only at concentrations above 30 μM in HEK293 cells stably expressing Renilla (12-h treatment with MTX). (D–F) Methotrexate treatment inhibited Wnt-induced lysosomal activity (assessed by the fluorescence of endocytosed BSA-DQ after 30 min of Wnt3a treatment). (G) Quantification of the inhibition of Wnt-induced endolysosomal activity by MTX using ImageJ. (H–W) MTX (20 μM) inhibited the formation of vesicles by Wnt3a (by phase contrast microscopy) and decreased PRMT1 and GSK3 sequestration (by in situ proteinase K protection). Note that the addition of 1 mM SAM rescued the effects of MTX (arrowheads). (X) Quantification of the inhibition by MTX, and rescue by SAM, of Wnt-induced endolysosomal vesicle formation assessed by phase contrast microscopy. (Y) Quantification of GSK3 and PRMT1 colocalization in endolysosomes/MVBs after 30 min of Wnt3a treatment by Pearson’s correlation coefficient. (Scale bars: 10 μm.) *P < 0.05; **P < 0.01; n < 3.