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PHGDH arginine methylation by PRMT1 promotes serine synthesis and represents a therapeutic vulnerabi

Issuing time:2023-03-06 12:47Author:Enlibio

Abstract

Serine synthesis is crucial for tumor growth and survival, but its regulatory mechanism in cancer remains elusive. Here, using integrative metabolomics and transcriptomics analyses, we show a heterogeneity between metabolite and transcript profiles. Specifically, the level of serine in hepatocellular carcinoma (HCC) tissues is increased, whereas the expression of phosphoglycerate dehydrogenase (PHGDH), the first rate-limiting enzyme in serine biosynthesis pathway, is markedly downregulated. Interestingly, the increased serine level is obtained by enhanced PHGDH catalytic activity due to protein arginine methyltransferase 1 (PRMT1)-mediated methylation of PHGDH at arginine 236. PRMT1-mediated PHGDH methylation and activation potentiates serine synthesis, ameliorates oxidative stress, and promotes HCC growth in vitro and in vivo. Furthermore, PRMT1-mediated PHGDH methylation correlates with PHGDH hyperactivation and serine accumulation in human HCC tissues, and is predictive of poor prognosis of HCC patients. Notably, blocking PHGDH methylation with a TAT-tagged nonmethylated peptide inhibits serine synthesis and restrains HCC growth in an HCC patient-derived xenograft (PDX) model and subcutaneous HCC cell-derived xenograft model. Overall, our findings reveal a regulatory mechanism of PHGDH activity and serine synthesis, and suggest PHGDH methylation as a potential therapeutic vulnerability in HCC.

Introduction

Cancer cells commonly reprogram metabolic patterns to meet their biomass demands for rapid and sustainable growth1,2,3. Serine metabolism is activated in diverse cancer types to support tumor growth and metastasis, by providing precursors for macromolecule (such as proteins, nucleotides, and lipids) synthesis and once-carbon units for methylation reactions. Moreover, serine synthesis coupling one-carbon metabolism produces glutathione (GSH) and NADPH, two main intrinsic antioxidant agents, to maintain redox balance in cancer cells4,5,6,7. The intracellular serine pool required for serine metabolism in cancer cells can be replenished by extracellular import or de novo biosynthesis6,8. Accordingly, serine metabolism-targeting strategies, including dietary serine/glycine restriction, serine synthesis inhibition, or a combination of each, have been recently explored and exhibit favorable effects for cancer treatment5,9,10,11,12,13. However, the underlying mechanism by which serine synthesis is regulated in cancer remains largely unknown.

Phosphoglycerate dehydrogenase (PHGDH) is the first and rate-limiting enzyme in the de novo serine biosynthesis pathway, catalyzing 3-phosphoglycerate (3-PG) to form 3-phosphohydroxypyruvate14,15. Recent studies reveal that PHGDH is frequently overexpressed to activate serine synthesis and promote tumor growth in several types of cancer, including breast cancer, non-small-cell lung cancer (NSCLC), and melanoma16,17,18. PHGDH knockdown or inhibitor markedly restrains the growth of these PHGDH-overexpressing cancers, suggesting PHGDH is a promising target for cancer therapy16,17,18,19,20,21,22. PHGDH upregulation in cancer has been reported to be a consequence of gene amplification or transcriptional upregulation mediated by activating transcription factor 4, nuclear factor erythroid-2-related factor 2, or MYC18,23,24,25. Moreover, the downregulation of E3 ubiquitin ligases (Parkin and RNF5), or upregulation of deubiquitinating enzyme JOSD2, has been reported as crucial mechanism for PHGDH overexpression in breast cancer or lung adenocarcinoma26,27,28. Although the mechanisms underlying PHGDH overexpression in cancer have been well illustrated, the role and regulatory mechanism of PHGDH activity in cancer remains poorly understood.

Protein arginine methylation, a ubiquitous post-translational modification, is catalyzed by protein arginine methyltransferases (PRMTs)29,30. Currently, nine PRMTs have been characterized in mammals, which are classified into three different types according to their catalytic specificities. The type I PRMTs (PRMT1-4, PRMT6, and PRMT8) catalyze the formation of mono-methylarginine (me1) and asymmetrical di-methylarginine (me2a), the type II PRMTs (PRMT5 and PRMT9) generate me1 and symmetrical di-methylarginine (me2s), while the type III PRMT (PRMT7) catalyzes the production of me130,31. Emerging evidence has shown that protein arginine methylation plays a crucial role in a wide range of biological processes, such as mRNA splicing, DNA damage, transcription and signal transduction31,32. Notably, recent studies demonstrate that PRMTs-mediated protein arginine methylation regulates cancer metabolism, including glucose metabolism, glutamine metabolism, and lipid metabolism33,34,35,36,37. However, the role of protein arginine methylation in serine metabolism of cancer cells remains largely unknown.

In this study, we show the essential role of PHGDH methylation for serine synthesis and HCC growth. Although downregulated at mRNA and protein levels, PHGDH is activated by PRMT1-mediated R236 methylation, thereby promoting serine synthesis, redox homeostasis, and HCC growth. Blocking PHGDH methylation by a TAT-tagged nonmethylated peptide inhibits HCC growth with no obvious toxicity, suggesting PHGDH methylation might be a therapeutic vulnerability for HCC treatment.

Results

Discordance exists between HCC metabolomic and transcriptomic profiles

Emerging evidence reveals heterogeneity between metabolomic and transcriptomic profiles in some cancers, such as clear cell renal cell carcinoma (ccRCC)38. To investigate the relationship between metabolite and gene expression features in HCC, 29 paired HCC and adjacent normal tissues (cohort 1) were subjected to untargeted metabolomics analysis, and 27 paired tissues from the same cohort were used for RNA-seq analysis (Supplementary Table 1). Unsupervised principal component analysis of both metabolomic and transcriptomic profiles revealed almost completed separation of tumor versus normal samples (Supplementary Fig. 1a, b). A total of 169 metabolites (78 increased and 91 decreased, Supplementary Data 1) and 2798 genes (1605 upregulated and 1193 downregulated) with differential abundance in tumor tissues compared with normal tissues were identified by metabolomics and transcriptomics analyses, respectively (Supplementary Fig. 1c, d). In agreement with previous metabolomics studies39,40, we observed decreased levels of succinate, fumarate and malate in tricarboxylic acid cycle (TCA), as well as increased levels of D-phenyllactic acid and L-tryptophan in HCC tissues relative to normal tissues. KEGG pathway enrichment analysis of the differential metabolites revealed alterations in glycine, serine and threonine metabolism, TCA cycle, arginine biosynthesis, as well as alanine, aspartate, and glutamate metabolism (Supplementary Fig. 1e). Besides, enrichment of differential abundance scores was obtained to display the tendency for metabolites in a given pathway to be upregulated or downregulated in HCC tissues compared with normal tissues (Supplementary Fig. 1f)38. The differential abundance scores of related metabolic genes were also calculated, and subsequently compared with the metabolite scores. To our surprise, we observed a lack of linear correlation between metabolomics and transcriptomics data. Specifically, five out of ten metabolic pathways showed increased metabolite abundance but reduced gene expression levels (Fig. 1a). These results demonstrate discordance between metabolomic and transcriptomic profiles in HCC.

a The differential abundance scores of metabolites and genes based on KEGG pathway were plotted against each other. A score of 1 denotes all metabolites in a given pathway increase, while a score of −1 denotes all metabolites in a given pathway decrease. b GO analysis of differential metabolites between HCC tissues and normal tissues. Statistical analysis was performed using the two-sided hypergeometric test with Bonferroni correction. c Serine levels in human HCC tissues and paired normal tissues (n = 20 samples, cohort 2). Statistical analysis was performed using the paired two-tailed Student’s t-test. d, e Representative images (d) and quantitative analysis (e) of IHC staining using PHGDH antibody (PA5-27578, ThermoFisher) in HCC tissues and paired normal tissues (n = 42 samples, cohort 3). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t-test. f PHGDH activity in human HCC tissues and paired normal tissues (n = 20 samples, cohort 2). Statistical analysis was performed using the paired two-tailed Student’s t-test. g Growth rates of Huh7 and PLC/PRF/5 cells grown in complete (CM) or serine- and glycine-depleted (-SG) medium treated with or without NCT-503 (20 μM). Data are presented as the mean ± SD (n = 4 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. h Schematic of U-[13C]-glucose incorporation into serine and glycine in cells. i, j Incorporation of U-[13C]-glucose carbon into serine (i) and glycine (j) in parental and PHGDH KO cells. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. k Growth rates of parental and PHGDH KO cells grown in CM or -SG medium. Data are presented as the mean ± SD (n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. l–n Huh7 parental and PHGDH KO cells were subcutaneously inoculated into nude mice fed with a control (+SG) or serine- and glycine-free diet (-SG diet). Tumor images (l), weight (m), and volume (n) were presented. Data are presented as the mean ± SD (n = 6 mice). Statistical analysis in m was performed using the two-tailed Student’s t-test, and statistical analysis in n was performed using the two-way ANOVA with Bonferroni correction. o Representative images of IHC staining for Ki-67 in tumor xenografts. Scale bars, 50 μm. Source data are provided as a Source Data file.

Elevated PHGDH catalytic activity is essential for HCC growth

Functional enrichment analysis of differential metabolites revealed two main pathways: urea cycle, and glycine and serine metabolism (Fig. 1b). Given the essential role of the urea cycle in liver for detoxification41,42, we had a particular interest in investigating the deregulation of glycine and serine metabolism in HCC development. Among the differential metabolites in this pathway, the level of serine, an oncogenesis-supportive metabolite4, was significantly elevated in HCC tissues relative to normal tissues (Supplementary Fig. 2a), which was consistent with previous metabolomics studies39,43,44. The increased serine level was further confirmed by serine detection kit in 20 paired HCC tissues and adjacent normal liver tissues (Fig. 1c, cohort 2). We then determined whether the increase in serine level is attributable to the upregulation of PHGDH, the rate-limiting enzyme catalyzing the first committed step of the de novo serine synthesis pathway4,45. However, RNA-seq analysis showed a marked decrease in the mRNA level of PHGDH in HCC tissues (Supplementary Fig. 2b). In support of this observation, immunohistochemistry (IHC) analysis of 42 paired HCC samples (cohort 3) using two different commercial PHGDH antibodies demonstrated that the PHGDH protein level was profoundly downregulated in HCC tissues compare with normal tissues (Fig. 1d, e, Supplementary Fig. 2c, d). The decreases in both mRNA and protein levels of PHGDH in HCC tissues were consistent with previous transcriptomics and proteomics data41,46. To our surprise, we observed an obvious increase in the catalytic activity of PHGDH in 20 HCC tissues relative to their normal counterparts (Fig. 1f). Together, these data suggest that serine synthesis is activated in HCC due to increased PHGDH activity, although the mRNA and protein levels of PHGDH are downregulated.

Given that serine synthesis promotes tumor growth and survival4,8, we next determined whether increased PHGDH activity favors the growth of HCC cells. Treatment with PHGDH inhibitor NCT-503 inhibited the growth and proliferation in two HCC cell lines, Huh7 and PLC/PRF/5 (Fig. 1g, Supplementary Fig. 2e, f). Because serine can be imported extracellularly via its transporters or de novo synthesized through the PHGDH-mediated serine synthesis pathway6,8, we combined NCT-503 with serine/glycine starvation (-SG) to deplete serine in HCC cells. Interestingly, serine/glycine deprivation alone led to a decrease in cell growth and proliferation, and further sensitized HCC cells to NCT-503 treatment (Fig. 1g, Supplementary Fig. 2e, f). These results suggest that PHGDH activity is essential for HCC growth. To confirm these observations, we knocked out PHGDH expression (PHGDH KO) in Huh7 and PLC/PRF/5 cells using CRIPSR/Cas9 technology (Supplementary Fig. 2g). Using U-[13C]-glucose tracing (Fig. 1h), we found that 30–40% of the total serine pool and 40–50% of the total glycine pool were labeled by U-[13C]-glucose at 24 h of tracing in control parental cells, whereas the incorporation of 13C into serine and glycine was totally abrogated when PHGDH was knocked out (Fig. 1i, j), which was consistent with a previous report26. The PHGDH KO cells, theoretically with no PHGDH activity, exhibited reduced growth and proliferation compared with parental cells. In addition, the inhibition of growth and proliferation caused by PHGDH KO was further exaggerated when cells were grown in -SG medium (Fig. 1k, Supplementary Fig. 2h, i). These findings were further supported by a mouse xenograft model, in which marked decreases in the size, weight, growth rate, and Ki-67 staining intensity of PHGDH KO tumors were observed compared with that of parental cells. Notably, the growth and proliferation of PHGDH KO tumors were almost completely blocked when the mice were fed with a -SG diet (Fig. 1l, o, Supplementary Fig. 2j). Collectively, these results indicate that the enhanced PHGDH activity is essential for HCC growth, although PHGDH is downregulated at both mRNA and protein levels in HCC.

Arginine methylation at R236 elevates PHGDH activity by increasing substrate affinity

To investigate the regulatory mechanism of PHGDH activity, we analyzed immunoprecipitated PHGDH from human HEK293T cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition to CHCHD3 which was reported in another study27, the protein arginine methyltransferase PRMT1 was identified with top score as a potential PHGDH-interacting protein (Supplementary Fig. 3a, b). Because the activity of metabolic enzymes can be regulated by post-translational modifications, we, therefore, hypothesized that PHGDH might be methylated by PRMT1, thereby altering its activity. Immunoblotting analysis using antibodies against me1, me2a, and me2s showed that immunoprecipitated GFP-tagged PHGDH indeed underwent mono-methylation and asymmetrical di-methylation (Supplementary Fig. 3c). The mono-methylation of endogenous PHGDH was further confirmed in HEK293T, Huh7, and PLC/PRF/5 cells (Fig. 2a). Of note, the arginine methylation of PHGDH was markedly reduced by PRMT inhibitors, AMI-1 or adenosine dialdehyde (AdOx), with a concomitant decrease in PHGDH activity (Fig. 2a, Supplementary Fig. 3c, d). These data indicate that arginine methylation enhances PHGDH catalytic activity.

a Endogenous PHGDH was immunoprecipitated in cells treated with AMI-1 or AdOx for 24 h. The mono-methylation (me1) level of PHGDH was determined by immunoblotting. PHGDH activity was measured and normalized to PHGDH protein. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. b MS identification of R236 mono-methylation of FLAG-PHGDH immunopurified by FLAG beads. c Sequence conservation of PHGDH protein using the Consurf coloring scheme. Maroon and cyan indicate high and low conservation, respectively. d HEK293T cells expressing GFP-PHGDH WT, R236K or V83A were treated with AdOx for 24 h. The mono-methylation (me1) level of immunoprecipitated GFP-PHGDH was determined by immunoblotting. e FLAG-PHGDH WT, R236K or V83A was expressed in HEK293T cells. The mono-methylation of immunopurified FLAG-PHGDH was measured using a site-specific antibody against R236 mono-methylation (mePHGDH (R236me1)), which was pre-incubated with the R236 mono-methylated (R236me1) peptide or the unmodified peptide before use. f Endogenous PHGDH was immunoprecipitated in cells treated with AMI-1 or AdOx for 24 h. The R236 mono-methylation level of PHGDH was determined by immunoblotting using mePHGDH (R236me1) antibody. g HEK293T cells stably expressing FLAG-PHGDH WT, R236K or V83A were treated with AMI-1 or AdOx for 24 h. The R236 mono-methylation level of immunopurified FLAG-PHGDH was determined by immunoblotting using mePHGDH (R236me1) antibody. The activity of immunopurified FLAG-PHGDH was measured and normalized to FLAG-PHGDH protein. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. h, i FLAG-PHGDH WT, R236K or V83A was expressed in HEK293T cells and immunopurified by FLAG beads. The Km value of FLAG-PHGDH for 3-PG (h) or NAD+ (i) was determined. Data are presented as the mean ± SD (n = 3 independent experiments). Source data are provided as a Source Data file.

The sites of PHGDH arginine methylation were then predicted using the GPS-MSP tool47, with R236 showing the highest methylation score (Supplementary Fig. 3e). MS analysis of immunoprecipitated PHGDH confirmed the mono-methylation of PHGDH at R236 (Fig. 2b). Moreover, R236 and its surrounding amino acids (aa) were highly conserved among diverse species (Fig. 2c, Supplementary Fig. 3f), and well matched the consensus RGG/RG methylation motif of PRMT132. To investigate whether R236 is the main methylation site in PHGDH, we mutated R236 to lysine (R236K), which preserved its positive charge but was resistant to PRMTs-mediated methylation. Compared with wild-type (WT), the PHGDH R236K mutant revealed an obvious decreased mono-methylation level. AdOx treatment could attenuate the mono-methylation level of PHGDH WT, but not the R236K mutant (Fig. 2d). These results suggest that R236 is a major methylation site of PHGDH.

To better understand R236 methylation of PHGDH, we generated a site-specific antibody against mono-methylated R236 (mePHGDH (R236me1)), the specificity of which was verified by dot blot and peptide competition analyses (Fig. 2e, Supplementary Fig. 3g). This antibody recognized R236 mono-methylation of both exogenous and endogenous PHGDH, the level of which was markedly attenuated by AMI-1 or AdOx treatment (Fig. 2f, g). Moreover, the R236K mutant showed obvious reduced level of R236 mono-methylation in comparison with WT, accompanied by a significant decrease in PHGDH activity (Fig. 2g). Consistent with our observations in Fig. 2a, AMI-1 or AdOx treatment suppressed the activity of PHGDH WT, but did not alter the activity of R236K mutant (Fig. 2g). Together, these data demonstrate that R236 methylation increases PHGDH activity.

We next asked how arginine methylation enhances PHGDH activity by examining the affinity of PHGDH for its substrate 3-PG and coenzyme NAD+. Interestingly, the Km value of PHGDH for 3-PG, but not NAD+, was elevated by 2- to 3-fold following AMI-1 or AdOx treatment (Supplementary Fig. 3h, i). In agreement with this observation, R236K mutation resulted in ~5-fold increase in the Km value for 3-PG, whereas the Km value for NAD+ remained unchanged (Fig. 2h, i). These results indicate that R236 methylation elevates PHGDH activity by increasing its affinity for the 3-PG substrate.

PRMT1 methylates PHGDH at R236 and elevates its catalytic activity

Protein arginine methylation in mammals is catalyzed by protein arginine methyltransferases which comprise nine members, PRMT1-930. To identify the writer of PHGDH R236 methylation, we co-expressed FLAG-PHGDH with different GFP-PRMTs in HEK293T cells and measured their interactions by co-immunoprecipitation (co-IP) assay. PRMT1, PRMT5, and PRMT8 were found to potentially interact with PHGDH (Supplementary Fig. 4a). We selected PRMT1 for further study for the following reasons: (1) LC-MS/MS analysis identified PRMT1 with top score from immunoprecipitated PHGDH (Supplementary Fig. 3a, b); (2) The mono-methylation and asymmetrical di-methylation of PHGDH was consistent with PRMT1’s catalytic properties; (3) The surrounding aa sequence of R236 matched the RGG/RG methylation motif of PRMT132; (4) PRMT5 was recognized as a nonspecific binding protein for FLAG M2 agarose48; (5) PRMT8 is mainly expressed in human brain49,50. Reciprocal co-IP analysis showed that endogenous PRMT1 interacted with PHGDH in HEK293T, Huh7, and PLC/PRF/5 cells (Fig. 3a, b). To determine whether PRMT1 directly binds PHGDH, GST pulldown assay was conducted using recombinant human GST-PRMT1 and His-PHGDH proteins. Of note, a direct interaction between PRMT1 and PHGDH was observed (Fig. 3c).

a, b Reciprocal co-IP analysis of PHGDH and PRMT1 in HEK293T, Huh7 and PLC/PRF/5 cells. IgG was used as a negative control. c Recombinant GST-PRMT1 was incubated with recombinant His-PHGDH, followed by GST pulldown and immunoblotting analysis with GST and His antibodies. d FLAG-PHGDH WT or different point mutants were co-expressed with HA-PRMT1 in HEK293T cells. FLAG-PHGDH was immunopurified with FLAG beads, followed by immunoblotting using HA antibody. e Endogenous PHGDH was immunoprecipitated in cells transfected with siScramble or siPRMT1. Immunoblotting was performed with indicated antibodies. PHGDH activity was measured and normalized to PHGDH protein. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t test. f Endogenous PHGDH was immunoprecipitated in PRMT1 KD cells rescued with HA-PRMT1 WT or SGT/AAA mutant. Immunoblotting was performed with indicated antibodies. PHGDH activity was measured and normalized to PHGDH protein. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t test. g Recombinant GST-PRMT1, His-PHGDH, and His-PSAT1 were incubated with or without S-adenosyl methionine (SAM). The reaction mixture from the in vitro methylation was then subjected to Coomassie Brilliant Blue staining, immunoblotting analysis with mePHGDH (R236me1) antibody, and PHGDH activity assay. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. h, i The Km value of immunoprecipitated PHGDH for 3-PG (h) or NAD+ (i) from cells transfected with siScramble or siPRMT1. Data are presented as the mean ± SD (n = 3 independent experiments). j HEK293T cells expressing FLAG-PHGDH WT, R236K or V83A were transfected with siScramble or siPRMT1, followed by IP with FLAG beads. Immunoblotting was performed with indicated antibodies. The activity of PHGDH was measured and normalized to FLAG-PHGDH protein. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. Source data are provided as a Source Data file.

We next mapped the domain of PHGDH that binds PRMT1 by co-expressing different truncated GFP-PHGDH with HA-PRMT1 in HEK293T cells for co-IP assay (Supplementary Fig. 4b). The substrate binding domain 1 (SBD1, 1-102aa) of PHGDH was found to be required for PRMT1 binding (Supplementary Fig. 4c). This binding region was further narrowed down to 70-102aa of PHGDH (Supplementary Fig. 4d). We then mutated the amino acids (L70, T78, T79, V83, D84, A87, G92, V95, N97, and P99), which were evolutionally conserved and located on the surface of PHGDH protein in the 70-102aa region (Supplementary Fig. 4e, f). Among them, V83A mutation markedly disrupted the interaction between PHGDH and PRMT1 (Fig. 3d), suggesting that V83 is critical for the binding of PHGDH with PRMT1.

Given that PRMT1 interacts with PHGDH, we determined whether PRMT1 methylates PHGDH, thereby promoting its catalytic activity. siRNA-mediated depletion of PRMT1 significantly attenuated the methylation level of PHGDH in HEK293T, Huh7, and PLC/PRF/5 cells, concomitant with an obvious reduction in the activity of both endogenous and exogenous PHGDH (Fig. 3e, Supplementary Fig. 5a–c). Similar results were observed upon treatment with the PRMT1 inhibitor GSK3368715 (Fig. 5d)30. In contrast, PRMT1 overexpression led to significant increased PHGDH methylation and activity (Supplementary Fig. 5e, f). To further validate these findings, we used a short hairpin RNA (shRNA) targeting the 3′UTR of PRMT1 mRNA to suppress PRMT1 expression in Huh7 and PLC/PRF/5 cells, and then re-expressed PRMT1 WT or the enzymatically inactive PRMT1 mutant (SGT/AAA)51. Reconstituted expression of PRMT1 WT, but not the SGT/AAA mutant, effectively rescued the methylation level and catalytic activity of PHGDH in PRMT1 knockdown (KD) cells (Fig. 3f). These results suggest that PRMT1 promotes R236 methylation of PHGDH. To investigate whether PRMT1 directly methylates PHGDH, an in vitro methylation assay using recombinant human GST-PRMT1 and His-PHGDH proteins were performed. The R236 methylation of His-PHGDH was detectable in the presence of PRMT1 WT accompanied by an obvious increase of PHGDH activity, whereas incubation of PRMT1 SGT/AAA mutant failed to induce PHGDH methylation and increase PHGDH activity (Fig. 3g). Notably, PRMT1 KD led to a 3- to 4-fold increase in the Km value of PHGDH for 3-PG, but had no obvious effect on the Km value for NAD+ (Fig. 3h, i). Taken together, these data demonstrate that PRMT1 directly binds and methylates PHGDH, resulting in elevated PHGDH catalytic activity by promoting its affinity for 3-PG.

Given our findings that V83 of PHGDH is critical for PRMT1 binding, we presumed that mutation of V83 might reduce PHGDH methylation and inhibit PHGDH activity. Indeed, V83A mutation led to an appreciable decrease in PHGDH methylation and activity, to a similar level observed with R236K mutation (Figs. 2d, e, 2g, 3j). Compared with PHGDH WT, treatment with AMI or AdOx, or PRMT1 KD resulted in a negligible change in R236 methylation and activity of both R236K and V83A mutants (Figs. 2d, 2g, 3j). Moreover, V83A mutation of PHGDH showed a ~5-fold elevation in the Km value of PHGDH for 3-PG, but not NAD+, akin to what we have observed for the R236K mutation (Fig. 2h, i). It has been reported that R236 is located at the catalytic center and is critical for PHGDH activity52. To exclude the possibility that R236K mutation blocks PHGDH activity regardless of reduced R236 methylation, we also included the V83A mutant, a methylation-defective surrogate, in the following functional studies.

PHGDH methylation mediated by PRMT1 promotes serine synthesis and ameliorates oxidative stress

Given that PHGDH is the first rate-limiting enzyme for de novo serine synthesis4,45, we next sought to assess whether R236 methylation of PHGDH influences serine synthesis. PHGDH WT, R236K, or V83A mutant was re-expressed approximately equal to physiological levels in PHGDH KO cells. As expected, R236 mono-methylation was not observed in PHGDH KO cells, while re-introducing PHGDH WT, but not R236K or V83A mutant, restored R236 methylation level (Fig. 4a). U-[13C]-glucose tracing showed that re-introducing PHGDH WT markedly rescued 13C-glucose-derived serine and glycine levels in PHGDH KO cells, but not the R236K or V83A mutant (Fig. 4b, c). These observations were recapitulated by detecting serine and glycine levels using serine and glycine detection kits (Supplementary Fig. 6a, b). These data demonstrate that PHGDH methylation promotes serine synthesis in HCC cells.

a–e PHGDH was knocked out (PHGDH KO) using CRIPSR/Cas9 technology, followed by re-expression of PHGDH WT, R236K (RK) or V83A (VA). Immunoblotting was performed with indicated antibodies (a). Incorporation of U-[13C]-glucose carbon into serine (b) and glycine (c) was detected by LC-MS/MS. GSH (d) and ROS (e) levels were measured in these cells grown in CM or -SG medium. Data in b–e are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. f–j PHGDH KO plus PRMT1 KD cells were rescued with PHGDH WT, R236K (RK) or V83A (VA), combining with or without PRMT1 re-expression. Immunoblotting was performed with indicated antibodies (f). Incorporation of U-[13C]-glucose carbon into serine (g) and glycine (h) was detected by LC-MS/MS. GSH (i) and ROS (j) levels were measured in cells grown in CM or -SG medium. Data in g–j are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t test. Source data are provided as a Source Data file.

Serine synthesis supports tumor cell growth by maintaining intracellular redox homeostasis, promoting macromolecule synthesis, and providing once-carbon units for methylation reactions45. We and others previously reported that HCC progression is closely associated with oxidative stress, accompanied with a higher antioxidant capacity53,54,55. Therefore, we postulated that the activated serine synthesis by PHGDH methylation might increase the antioxidant capacity, thereby preventing HCC cells from oxidative stress-induced growth inhibition. To test this hypothesis, we examined the levels of GSH and NADPH, which are two major cellular reductants and downstream products of serine synthesis pathway15. The GSH level, GSH/GSSG and NADPH/NADP+ ratios were profoundly reduced in PHGDH KO cells grown in complete medium (CM), which was consistent with a previous study56, and were rescued by reconstituted expression of PHGDH WT, but not the R236K or V83A mutant (Fig. 4d, Supplementary Fig. 6c, d). Interestingly, serine/glycine deprivation diminished the GSH level, GSH/GSSG ratio, and NADPH/NADP+ ratio in parental cells, and resulted in a further decrease in PHGDH KO cells. This decrease was restored by re-expressing PHGDH WT in a manner similar to that of cells grown in CM medium, but not when R236K or V83A mutant was re-expressed (Fig. 4d, Supplementary Fig. 6c, d). The reduction in GSH and NADPH production caused a prominent accumulation of reactive oxygen species (ROS) levels (Fig. 4e). Collectively, these results illustrate that PHGDH methylation prevents ROS accumulation and maintains redox balance by activating serine synthesis in HCC cells.

To determine the role of PRMT1 in PHGDH methylation-mediated stimulation of serine synthesis and elevation of antioxidant capacity, we knocked down PRMT1 using a shRNA targeting the 3′UTR of PRMT1 mRNA in PHGDH KO cells, followed by re-expression of PHGDH WT, R236K or V83A mutant in combination with or without PRMT1 re-expression. R236 methylation of PHGDH was only observed by re-expressing both PHGDH WT and PRMT1 in PHGDH KO plus PRMT1 KD cells (Fig. 4f). In the absence of PRMT1 expression, rescuing with PHGDH WT, R236K, or V83A mutant failed to restore the reduced serine and glycine levels resulted from PHGDH KO. A combination of re-expressing PRMT1 and PHGDH WT, but not its R236K or V83A mutant, appreciably restored serine and glycine levels in PHGDH KO plus PRMT1 KD cells (Fig. 4g, h, Supplementary Fig. 6e, f), suggesting PRMT1 promotes serine synthesis by methylating PHGDH at R236. Of note, the GSH level, GSH/GSSG ratio, and NADPH/NADP+ ratio in PHGDH KO plus PRMT1 KD cells could be elevated only when PHGDH WT and PRMT1 were simultaneously re-introduced (Fig. 4i, Supplementary Fig. 6g, h). In support of this observation, the rescue of PHGDH KO-induced ROS accumulation was observed by PHGDH WT re-expression in the presence, but not the absence, of PRMT1. Re-expressing R236K or V83A mutant has negligible effect on the increased ROS levels regardless of the presence or absence of PRMT1 (Fig. 4J). Together, these results demonstrate that PRMT1-mediated PHGDH R236 methylation promotes serine synthesis and ameliorates oxidative stress in HCC.

PHGDH methylation mediated by PRMT1 promotes HCC growth

We next investigated whether R236 methylation of PHGDH plays a role in regulating HCC cell growth. The reduction of cell growth and proliferation, resulting from PHGDH KO observed in Fig. 1k and Supplementary Fig. 2h, i, was greatly restored by re-expressing PHGDH WT in cells grown in either CM or -SG medium. The decreased cell growth remained marginally affected when re-introducing the R236K or V83A mutant of PHGDH (Fig. 5a–c, Supplementary Fig. 7a–c). To confirm these observations, we generated a mouse xenograft model by subcutaneously inoculating PHGDH KO Huh7 cells rescued with PHGDH WT, R236K, or V83A mutant into BALB/c nude mice fed with a control (+SG) or -SG diet. By examining the size, weight, and growth rate of tumors, we found that rescued expression of R236K or V83A significantly inhibited the growth of tumors, as compared with rescue of PHGDH WT. It is noteworthy that when the mice were fed with a -SG diet, R236K or V83A re-expression almost completely blocked tumor growth (Fig. 5d–f). Moreover, the growth inhibition of tumors was accompanied by a marked decrease in the proliferation of tumor cells, as reflected by Ki-67 staining (Fig. 5g, h). These data demonstrate that PHGDH methylation at R236 facilitates the growth and proliferation of HCC cells in vitro and in vivo.

a–c PHGDH KO Huh7 cells were re-expressed with PHGDH WT, R236K (RK), or V83A (VA). Growth rates (a), colony formation assay (b), and quantification of clone numbers (c) of these cells grown in CM or -SG medium. Data in a are presented as the mean ± SD (n = 5 independent experiments), and statistical analysis was performed using the two-way ANOVA with Bonferroni correction. Data in c are presented as the mean ± SD (n = 3 independent experiments), and statistical analysis was performed using the two-tailed Student’s t-test. d–f Cells in a–c were subcutaneously inoculated into nude mice fed with a +SG or -SG diet. Tumor images (d), weight (e), and volume (f) were shown. Data are presented as the mean ± SD (n = 6 mice). Statistical analysis in d was performed using the two-tailed Student’s t-test, and statistical analysis in f was performed using the two-way ANOVA with Bonferroni correction. g, h Representative images (g) and quantitative analysis (h) of IHC staining for Ki-67 in tumor xenografts in d–f. Scale bars, 50 μm. Data in h are presented as the mean ± SD (n = 6 mice). Statistical analysis was performed using the two-tailed Student’s t test. i PHGDH KO plus PRMT1 KD Huh7 cells were rescued with PHGDH WT, R236K (RK) or V83A (VA), combining with or without PRMT1 re-expression. Growth rates of these cells grown in CM or -SG medium. Data are presented as the mean ± SD (n = 5 independent experiments), and statistical analysis was performed using the two-way ANOVA with Bonferroni correction. j–l Cells in i were subcutaneously inoculated into nude mice fed with a +SG or -SG diet. Tumor images (j), weight (k), and volume (l) were presented. Data are presented as the mean ± SD (n = 5 mice). Statistical analysis in k was performed using the two-tailed Student’s t-test, and statistical analysis in l was performed using the two-way ANOVA with Bonferroni correction. m, n Representative images (m) and quantitative analysis (n) of IHC staining for Ki-67 in tumor xenografts in j–l. Scale bars, 50 μm. Data in n are presented as the mean ± SD (n = 5 mice). Statistical analysis was performed using the two-tailed Student’s t test. Source data are provided as a Source Data file.

We then determined the role of PRMT1 in PHGDH methylation-induced HCC growth by re-expressing PHGDH WT, R236K mutant, V83A mutant, or PRMT1 in PHGDH KO plus PRMT1 KD cells. Interestingly, the impeded growth of Huh7 and PLC/PRF/5 cells caused by PHGDH KO plus PRMT1 KD was significantly restored only when PHGDH WT and PRMT1 were both re-expressed. Compare with cells grown in CM medium, this restoration was more prominent when serine and glycine in the medium were deprived (Fig. 5i, Supplementary Fig. 7d–h). We further verified these results by implanting PHGDH KO plus PRMT1 KD Huh7 cells rescued with PHGDH WT, R236K mutant, V83A mutant, or PRMT1 into nude mice fed with a control or -SG diet. In line with the data obtained in vitro, the size, weight, and growth rate of tumors, as well as the ratio of Ki-67-positive cells in tumors, were substantially elevated by rescued expression of PHGDH WT plus PRMT1, but not by rescuing with PHGDH WT alone, or rescuing with PHGDH mutants plus PRMT1 (Fig. 5j–n). Furthermore, the growth of HCC cells was markedly reduced by the treatment of PRMT1 inhibitor GSK3368715 both in vitro (Supplementary Fig. 7i–k) and in vivo (Supplementary Fig. 7l–n) in response to serine/glycine starvation. Overall, these results indicate that R236 methylation of PHGDH mediated by PRMT1 promotes the growth and proliferation of HCC cells in vitro and in vivo.

PHGDH is hypermethylated at R236 by PRMT1 in human HCC tissues

To evaluate the clinical relevance of PRMT1-mediated PHGDH methylation, IHC analysis was performed on 42 paired HCC samples (cohort 3), the same cohort used for detecting PHGDH protein level (Fig. 1d, e, Supplementary Fig. 2c, d). IHC results revealed that the levels of PHGDH R236 mono-methylation and PRMT1 were both markedly enhanced in HCC tissues relative to adjacent normal liver tissues (Fig. 6a–d). To confirm these observations, we performed co-IP or immunoblotting analysis in 20 paired HCC tissues and adjacent normal liver tissues (cohort 2), the same cohort used for measuring serine level and PHGDH activity (Fig. 1c, f). Consistent with the observations of IHC staining in Fig. 1d, e and Supplementary Fig. 2c, d, PHGDH protein was expressed at lower level in HCC tissues (T) compared with normal tissues (N) (Fig. 6e, f, Supplementary Fig. 8a). Interestingly, HCC tissues displayed an obvious increase in the level of methylated PHGDH compared with normal tissues (Fig. 6e, f, and Supplementary Fig. 8b). The decreased level of PHGDH protein and increased level of PHGDH methylation were also observed in another cohort with 16 paired HCC tissues and adjacent normal liver tissues (cohort 4, Supplementary Fig. 8c–f). In parallel, PRMT1 protein level was found to be upregulated in HCC tissues (cohort 2, Fig. 6e, f, and Supplementary Fig. 8g). Moreover, quantification of the staining intensity showed that PRMT1 protein level in HCC tissues was positively correlated with the amount of methylated PHGDH and PHGDH catalytic activity (Fig. 6g–i, Supplementary Fig. 8h). The R236 methylation level of PHGDH also displayed a positive relationship with PHGDH activity (Fig. 6j). In addition, we compared the overall survival duration of HCC patients, and found that higher levels of both PHGDH methylation and PRMT1 protein were correlated with worse HCC patient prognosis (Fig. 6k, l). Taken together, these results exemplify the requirement for a high level of PRMT1-mediated PHGDH methylation for HCC development.

a, b Representative images (a) and quantitative analysis (b) of IHC staining using mePHGDH (R236me1) antibody in HCC tissues and paired normal tissues (n = 42 samples, cohort 3). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t test. c, d Representative images (c) and quantitative analysis (d) of IHC staining for PRMT1 in HCC tissues and paired normal tissues (n = 42 samples, cohort 3). Scale bars, 50 μm. Statistical analysis was performed using the paired two-tailed Student’s t-test. e, f IP and immunoblotting analysis with indicated antibodies in 20 HCC tissues (T) and paired normal tissues (N). WCL, whole cell lysates. R236 mono-methylation of immunoprecipitated PHGDH was determined and normalized to PHGDH protein (n = 20, cohort 2). g Representative images of IHC staining with mePHGDH (R236me1) and PRMT1 antibodies in HCC tissues (n = 42 samples, cohort 3). Scale bars, 50 μm. h Pearson correlation test analyzing the relationship between the IHC staining intensity of mePHGDH (R236me1) and PRMT1 in HCC tissues (n = 42 samples, cohort 3). i Pearson correlation test analyzing the relationship between PHGDH activity and PRMT1 staining intensity in HCC tissues (n = 20 samples, cohort 2). j Pearson correlation test analyzing the relationship between PHGDH activity and the staining intensity of mePHGDH (R236me1) in HCC tissues (n = 20 samples, cohort 2). k, l Overall survival of HCC patients based on the IHC staining intensity of mePHGDH (R236me1) (k) or PRMT1 (l) (n = 30 samples). Statistical analysis was performed using the two-sided log-rank test. Source data are provided as a Source Data file.

Blocking PHGDH methylation with a synthesized peptide suppresses HCC growth

Given that PRMT1-mediated PHGDH R236 methylation is necessary for HCC development, we investigated whether PHGDH methylation can be therapeutically targeted. We synthesized a competitive nonmethylated PHGDH peptide containing the R236 methylation site (230-241aa) with a trans-activator of transcription (TAT) peptide tagged at its N-terminal region. A corresponding mono-methylated peptide at R236 and untreated group were used as negative controls (Fig. 7a). Reciprocal co-IP analysis showed that both the methylated and nonmethylated peptides had no obvious effect on the interaction between PHGDH and PRMT1 (Supplementary Fig. 9a–c). In addition, endogenous PRMT1 could be immunoprecipitated with the biotin-conjugated nonmethylated peptide, but not the biotin-conjugated TAT or methylated peptide (Supplementary Fig. 9d). Interestingly, pretreatment with PRMT1 inhibitor GSK3368715 blocked the binding of nonmethylated peptide with PRMT1 (Supplementary Fig. 9e). We then examined the effect of the synthesized peptide on PHGDH methylation and activity. The nonmethylated peptide, but not methylated peptide, markedly inhibited the R236 methylation and enzymatic activity of PHGDH (Fig. 7b, c), concomitant with an obvious decrease in serine and glycine levels (Fig. 7d, e), although the levels of phosphoserine aminotransferase 1 (PSAT1) and phosphoserine phosphatase (PSPH), another two enzymes in serine biosynthesis pathway, were compensatively increased (Supplementary Fig. 9f). These data suggest that the nonmethylated peptide inhibits PHGDH R236 methylation and catalytic activity by binding the active site of PRMT1, but has no obvious effect on the interaction of PHGDH with PRMT1. Moreover, compared with the methylated peptide treatment, cells treated with the nonmethylated peptide displayed repressed GSH level, GSH/GSSG ratio, and NADPH/NADP+ ratio, as well as enhanced ROS levels. These alterations were further exacerbated when combining the nonmethylated peptide treatment with serine/glycine depletion (Supplementary Fig. 9g–j). Reconciling with these observations, nonmethylated peptide treatment or serine/glycine deprivation alone inhibited the growth and proliferation of HCC cells, while combined treatment showed a further reduction in HCC cell growth and proliferation (Fig. 7f, Supplementary Fig. 9k, i).

a The aa sequence of the TAT-methylated and TAT-nonmethylated peptides. b, c Endogenous PHGDH was immunoprecipitated in Huh7 and PLC/PRF/5 cells treated with the methylated or nonmethylated peptides (20 μM) for 24 h. Immunoblotting was performed with indicated antibodies (b). PHGDH activity was measured and normalized to PHGDH protein (c). Data in c are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t test. d, e Total serine (d) and glycine (e) levels in cells grown in -SG medium with treatment of the methylated or nonmethylated peptides (20 μM) for 24 h. Data are presented as the mean ± SD (n = 3 independent experiments). Statistical analysis was performed using the two-tailed Student’s t-test. f Growth rates of Huh7 and PLC/PRF/5 cells grown in CM or -SG medium with treatment of the methylated or nonmethylated peptides (20 μM). Data are presented as the mean ± SD (n = 5 independent experiments). Statistical analysis was performed using the two-way ANOVA with Bonferroni correction. g–i Images (g), weight (h), and volume (i) of HCC PDX tumors from mice fed with a -SG diet and treated with the methylated or nonmethylated peptides. Data are presented as the mean ± SD (n = 6 mice). Statistical analysis in h was performed using the two-tailed Student’s t test, and statistical analysis in i was performed using the two-way ANOVA with Bonferroni correction. j Representative images of IHC staining for Ki-67 in PDX tumors in g–i. Scale bars, 50 μm. k–m Images (k), weight (l), and volume (m) of Huh7 and PLC/PRF/5 tumor xenografts from mice fed with a -SG diet and treated with the methylated or nonmethylated peptides. Data are presented as the mean ± SD (n = 5 mice). Statistical analysis in l was performed using the two-tailed Student’s t-test, and statistical analysis in m was performed using the two-way ANOVA with Bonferroni correction. n Representative images of IHC staining for Ki-67 in Huh7 and PLC/PRF/5 tumor xenografts in k-m. Scale bars, 50 μm. Source data are provided as a Source Data file.

To explore the antitumor efficacy of the nonmethylated peptide in vivo, an HCC patient-derived xenograft (PDX) model was established. The growth of PDX tumors in mice fed with a -SG diet was almost completely prevented by treatment with the nonmethylated peptide, but not the methylated peptide (Fig. 7g–j, Supplementary Fig. 9m). The antitumor effect of the nonmethylated peptide was further confirmed in a subcutaneous mouse xenograft model of Huh7 and PLC/PRF/5 cells (Supplementary Figs. 7k–n, 9n). Importantly, by measuring mice body weight and evaluating the pathological features of major organs, we found treatment with the nonmethylated peptide showed no obvious toxic effects in mice (Supplementary Fig. 10a, b), although a slight loss of weight (<11%) was observed, probably due to dietary serine/glycine restriction as reported previously10. Thus, these findings indicate that targeting PHGDH methylation in cooperation with dietary seine/glycine restriction may be a potential therapeutic strategy for HCC treatment.

Discussion

This study reveals that discordance exists between metabolite abundance and gene expression in HCC. Specifically, the level of serine in HCC is increased, but the expression of PHGDH, a rate-liming enzyme for serine synthesis, is downregulated. The elevated serine level in HCC results from enhanced PHGDH catalytic activity, which is due to PRMT1-mediated arginine methylation of PHGDH at R236. PRMT1-mediated PHGDH methylation and activation promotes serine synthesis and maintains redox homeostasis, supporting HCC growth. Blocking PHGDH methylation using a TAT-tagged nonmethylated peptide combined with dietary serine/glycine restriction synergistically suppresses HCC growth. Overall, our findings provide mechanistic insights into the regulation of PHGDH activity and serine synthesis by PRMT1-mediated arginine methylation, and suggest that inhibition of PHGDH methylation has potential therapeutic applications in HCC.

Metabolic deregulation is a hallmark of cancer. During the past decade, metabolomics, transcriptomics, or integrative analysis of metabolomic and transcriptomic profiles, have been widely conducted to identify potential metabolic biomarkers in HCC39,41,57,58,59. However, these studies mainly focused on the metabolic genes or metabolites with consistent differential abundance between gene expression and metabolite patterns. Here, by combining gene expression and metabolic profile analyses, we find a heterogeneity between metabolomics and transcriptomics data in HCC. Similar discordance has also been observed and reported in other cancer types, such as ccRCC38. The heterogeneity between metabolomic profile and gene expression pattern in HCC is not only at the level of metabolic pathways, but also the individual metabolites and metabolic genes. Specifically, we show serine level is significantly elevated in HCC tissues compared with normal tissues, but PHGDH, the rate-limiting enzyme of serine synthesis, is significantly downregulated both at mRNA and protein levels. Therefore, expression of metabolic genes does not always substitute for metabolite abundance. The discordance between metabolomic and transcriptomic profiles may arise from the complex metabolic diversion, post-transcriptional or post-translational regulation of metabolic enzymes, and the presence of enzyme isoforms. In addition, a broader coverage of the metabolome and a larger HCC cohort are needed for future better understanding of such heterogeneity.

Previous studies have reported that PHGDH is frequently overexpressed in a number of different cancer types including breast cancer, NSCLC, and melanoma, due to gene amplification, transcription, or post-translational modification16,17,18,23,24,25,26,27,28. However, we show an obvious decrease of PHGDH mRNA and protein levels in HCC tissues. Recent transcriptomics and proteomics studies also displayed reduced mRNA and protein levels of PHGDH respectively in HCC tissues41,46. Despite the downregulation in mRNA and protein levels, it is worth noting that the catalytic activity of PHGDH is greatly increased in HCC tissues. Indeed, a very early study reported that PHGDH activity was enhanced in tumors from a rat liver cancer model60. The increased PHGDH activity enables the stimulation of serine synthesis and resultant serine accumulation to support HCC growth, which is consistent with previous reports showing that PHGDH-mediated serine synthesis is essential for HCC development and sorafenib resistance23,56,61. It should be noted that we cannot rule out the contribution of serine transporters to the increased serine level in HCC. Interestingly, a recent study demonstrated that although high PHGDH activity supports cancer cell proliferation, cells with low PHGDH expression exhibit higher metastatic potential in breast cancer62. Therefore, the low PHGDH expression may enable HCC metastasis, while the elevated PHGDH activity promotes cell survival and growth. Moreover, in addition to serine, α-ketoglutaric acid (α-KG) is generated as an intermediate, which can replenish TCA cycle and regulate histone demethylation to support cancer cell growth4,16,17. Therefore, the role and function of α-KG in HCC is worth further investigation.

We demonstrate that R236 methylation of PHGDH promotes its activity and stimulates serine synthesis. It has been reported that R236 is located in the catalytic center of PHGDH and is responsible for PHGDH activity, likely due to its positive charge property that anchors the carboxyl group of 3-PG52. To this end, mutation of arginine to a negatively charged glutamate (R236E), which indeed disrupts the enzyme function of PHGDH, is commonly used as an enzymatically inactive PHGDH mutant52,63,64. Interestingly, we show that R236K mutation, which retains its positive charge, also largely suppresses the affinity of PHGDH for 3-PG to abrogate PHGDH activity. Therefore, the positive charge property of R236 does not seem to be a key factor for determining PHGDH activity, thus providing a rationale for the role of R236 methylation in PHGDH activation. This is further supported by using a V83A mutant in parallel in our functional studies, which disrupts the interaction of PHGDH with PRMT1 and exhibits decreased PHGDH methylation level and catalytic activity comparable to that of R236K mutant.

Protein arginine methylation is increasingly associated with cancer development by, at least in part, regulating metabolic reprogramming. For example, PRMT5 mediates R321 methylation of the sterol regulatory element-binding protein and prevents its ubiquitin-proteosome degradation, thereby increasing de novo lipogenesis and accelerating HCC growth37. In addition, pyruvate kinase M2 isoform is methylated at R445, R447, and R455 by PRMT4, through which aerobic glycolysis is switched to oxidative phosphorylation to enable the proliferation and metastasis of breast cancer cells33. PRMT4 has also been shown to suppress glutamine metabolism in pancreatic ductal adenocarcinoma by methylating malate dehydrogenase 1 (MDH1), or inhibits HCC glycolysis by methylating glyceraldehyde-3-phosphate dehydrogenase, both leading to reduced growth and proliferation of cancer cells34,35. However, whether PRMTs are involved in regulating serine metabolism in cancer is unknown. Besides, the role of PRMT1 in metabolic reprogramming of cancer cells remains poorly understood. We demonstrate that PRMT1 promotes serine synthesis in HCC by methylating and subsequently activating PHGDH. This finding reveals a mechanistic link between serine metabolism and protein arginine methylation. PRMT1 has been reported to be upregulated in HCC to promote cell growth and metastasis65,66, but the underlying mechanism, especially its direct protein target in HCC, is largely unclear. We show that PRMT1 directly interacts, methylates, and activates PHGDH, thereby promoting serine biosynthesis, maintaining redox homeostasis, and supporting HCC growth.

Dietary serine and glycine limitation has recently attracted much attention for restraining tumor growth both in cultured cancer cells and tumor-bearing mice9,12,67,68, highlighting a crucial role of serine metabolism in cancer progression. We found that inhibition of PHGDH activity by either PHGDH inhibitors or blocking PHGDH methylation renders HCC cells more susceptible to serine and glycine deprivation, suggesting both extracellularly imported serine and de novo synthesized serine are crucial for HCC growth. Indeed, it has been reported that depletion of PSAT1 in combination with dietary serine/glycine restriction synergistically suppresses tumorigenesis in c-MYC-driven liver cancer11. Different from other studies using PHGDH inhibitors or depleting PHGDH in PHGDH-overexpressing cancers, we surprisingly found that PHGDH inhibition also works for HCC in which PHGDH is downregulated, although the mechanism underlying PHGDH downregulation in HCC is unclear and requires further investigation. This finding supports the notion that PHGDH activity is the key factor in determining the anticancer effect of PHGDH inhibition. Our study thus may broaden the use of PHGDH targeting strategies for cancer therapy, not only in PHGDH-overexpressing cancers, but also in cancer types or subtypes with PHGDH hyperactivation although PHGDH is not overexpressed.

Given the crucial role of PRMT1-mediated PHGDH methylation in HCC growth, we develop a therapeutic peptide to block PHGDH methylation. Through targeting PHGDH methylation, this peptide effectively inhibits serine synthesis and suppresses HCC growth in cooperation with serine and glycine restriction, with no obvious side effects. Together, our study reveals a mechanism by which PHGDH activity and serine metabolism are regulated, and suggests PRMT1-mediated PHGDH methylation as a potential therapeutic target for HCC.

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