SHMT2 Expression Research Articles

RUNX2 as a novel biomarker for early identification of patients progressing to advanced-stage mycosis fungoides.

The majority of patients with mycosis fungoides (MF) have an indolent disease course, but a substantial fraction (20-30%) of patients progress to advanced stages - usually with a grave prognosis. Early differentiation between indolent and aggressive types of MF is important for the choice of treatment regimen and monitoring of the individual patient. Good biomarkers are therefore desired. Here, we used spatial transcriptomics on skin samples at time-of-diagnosis to enable prediction of patients who later progressed to advanced stages of MF. Formalin-fixed, paraffin-embedded skin biopsies at time of diagnosis from six patients with MF who progressed to advanced stages of disease within 4 months to 12 years after diagnosis, and nine patients who remained in early-stage disease over 9 to 27 years were analyzed using the GeoMx Digital Spatial Profiler to capture spatially resolved high-plex RNA gene expression data. Five different regions of interest (the epidermis, the basal layer of epidermis, CD4+ T-cells and neighboring cells, and Pautrier's microabscesses) were profiled for further assessment. Interestingly, RUNX2, SHMT2, and MCM7 were upregulated in the enriched population of malignant T-cells in Pautrier's microabscesses in patients who later developed advanced stages of disease. Expression of RUNX2, SHMT2 and MCM7 in malignant T-cells was confirmed in a subset of patients in MF skin using scRNA-seq datasets across multiple studies and correlating with stage of disease. Taken together, we provide first evidence that RUNX2 has potential as a biomarker to identify MF patients progressing to advanced stage disease. As RUNX2 has not previously been linked to MF, our data also shows the analytical strength of combining spatial transcriptomics with scRNA-seq analysis.

Knockdown of ERN1 disturbs the expression of phosphoserine aminotransferase 1 and related genes in glioblastoma cells

BackgroundEndoplasmic reticulum stress and synthesis of serine are essential for tumor growth, but the mechanism of their interaction is not clarified yet. The overarching goal of this work was to investigate the impact of ERN1 (endoplasmic reticulum to nucleus signaling 1) inhibition on the expression of serine synthesis genes in U87MG glioblastoma cells concerning the suppression of cell proliferation. MethodsWild type U87MG glioblastoma cells and their clones with overexpression of transgenes dnERN1 (without cytoplasmic domain of ERN1) and dnrERN1 (with mutation in endoribonuclease of ERN1), and empty vector (as control) were used. The silencing of ERN1 and XBP1 was also used to inhibition of ERN1 and its function. Gene expression was measured by qPCR. ResultsWe show that the expression of PSAT1 and several other related to serine synthesis genes is suppressed in cells with ERN1 inhibition by dissimilar mechanisms: PHGDH gene through ERN1 protein kinase, because its expression was resistant to inhibition of ERN1 endoribonuclease, but ATF4 gene via endoribonuclease of ERN1. However, in the control of PSAT1 and PSPH genes both enzymatic activities of ERN1 signaling protein are involved. At the same time, ERN1 knockdown strongly increased SHMT1 expression, which controls serine metabolism and enhances the proliferation and invasiveness of glioma cells. The level of microRNAs, which have binding sites in PSAT1, SHMT1, and PSPH mRNAs, was also changed in cells harboring dnERN1 transgene. Inhibition of ERN1 suppressed cell proliferation and enzymatic activity of PHGDH, a rate-limiting enzyme for serine synthesis. ConclusionChanges in the expression of phosphoserine aminotransferase 1 and other genes related to serine synthesis are mediated by diverse ERN1-dependent mechanisms and contributed to suppressed proliferation and enhanced invasiveness of ERN1 knockdown glioblastoma cell.

Uncovering SIRT3 and SHMT2-dependent pathways as novel targets for apigenin in modulating colorectal cancer: In vitro and in vivo studies

Despite significant advances in the treatment of colorectal cancer (CRC), identification of novel targets and treatment options are imperative for improving its prognosis and survival rates. The mitochondrial SIRT3 and SHMT2 have key roles in metabolic reprogramming and cell proliferation. This study investigated the potential use of the natural product apigenin in CRC treatment employing both in vivo and in vitro models and explored the role of SIRT3 and SHMT2 in apigenin-induced CRC apoptosis. The role of SHMT2 in CRC patients’ survival was verified using TCGA database. In vivo, apigenin treatment restored the normal colon appearance. On the molecular level, apigenin augmented the immunohistochemical expression of cleaved caspase-3 and attenuated SIRT3 and SHMT2 mRNA expression CRC patients with decreased SHMT2 expression had improved overall and disease-free survival rates. In vitro, apigenin reduced the cell viability in a time-dependent manner, induced G0/G1 cell cycle arrest, and increased the apoptotic cell population compared to the untreated control. Mechanistically, apigenin treatment mitigated the expression of SHMT2, SIRT3, and its upstream long intergenic noncoding RNA LINC01234 in CRC cells. Conclusively, apigenin induces caspase-3-dependent apoptosis in CRC through modulation of SIRT3-triggered mitochondrial pathway suggesting it as a promising therapeutic agent to improve patient outcomes.

A study on the significance of serine hydroxymethyl transferase expression and its role in bladder cancer

Bladder cancer (BLCA) is a common malignant tumor in urinary system all over the world. However, due to its high recurrence rate and complex causes, clinicians often have limited options for surgical and drug treatments. Recent researchs on the molecular mechanism of BLCA have reveals its biological progress and potential for early diagnosis. Serine hydroxymethyltransferase 1/2 (SHMT1/2) is a crucial enzyme in the one-carbon metabolism of tumor cells, and the expression levels of these isozymes have been found to be associated with the biological progression of various malignant tumors. However, the impact of SHMT1/2 on the biological progression of bladder cancer and its molecular regulation mechanism remain unclear. In this research utilizes BLCA clinical sample data, the TCGA database, and in vitro cell experiments to predict the expression levels of SHMT1/2 in BLCA. The findings indicate that SHMT1 remained unchanged, while SHMT2 expression is increased in BLCA, which was related to poor prognosis. Additionally, SHMT2 affects the growth, migration, and apoptosis of bladder cancer cells in vitro. It also influences the expression levels of E-cadherin and N-cadherin, ultimately impacting the malignant biological progression of bladder tumors. These results establish a correlation between SHMT2 and the malignant biological progression of BLCA, providing a theoretical basis for the early diagnosis and treatment of bladder cancer.

HOXD8 suppresses renal cell carcinoma growth by upregulating SHMT1 expression.

Amplification of amino acids synthesis is reported to promote tumorigenesis. The serine/glycine biosynthesis pathway is a reversible conversion of serine and glycine catalyzed by cytoplasmic serine hydroxymethyltransferase (SHMT)1 and mitochondrial SHMT2; however, the role of SHTM1 in renal cell carcinoma (RCC) is still unclear. We found that low SHMT1 expression is correlated with poor survival of RCC patients. The in vitro study showed that overexpression of SHMT1 suppressed RCC proliferation and migration. In the mouse tumor model, SHMT1 significantly retarded RCC tumor growth. Furthermore, by gene network analysis, we found several SHMT1-related genes, among which homeobox D8 (HOXD8) was identified as the SHMT1 regulator. Knockdown of HOXD8 decreased SHMT1 expression, resulting in faster RCC growth, and rescued the SHMT1 overexpression-induced cell migration defects. Additionally, ChIP assay found the binding site of HOXD8 to SHMT1 promoter was at the -456~-254 bp region. Taken together, SHMT1 functions as a tumor suppressor in RCC. The transcription factor HOXD8 can promote SHMT1 expression and suppress RCC cell proliferation and migration, which provides new mechanisms of SHMT1 in RCC tumor growth and might be used as a potential therapeutic target candidate for clinical treatment.

SHMT as a Potential Therapeutic Target for Renal Cell Carcinoma.

Serine hydroxymethyltransferase (SHMT) is a serine-glycine-one-carbon metabolic enzyme in which SHMT1 and SHMT2 encode the cytoplasmic and mitochondrial isoenzymes, respectively. SHMT1 and SHMT2 are key players in cancer metabolic reprogramming, and thus are attractive targets for cancer therapy. However, the role of SHMT in patients with renal cell carcinoma (RCC) has not been fully elucidated. We aimed to systematically analyze the expression, gene regulatory network, prognostic value, and target prediction of SHMT1 and SHMT2 in patients with kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), and kidney renal papillary cell carcinoma (KIRP); elucidate the association between SHMT expression and RCC; and identify potential new targets for clinical RCC treatment. Several online databases were used for the analysis, including cBioPortal, TRRUST, GeneMANIA, GEPIA, Metascape, UALCAN, LinkedOmics, and TIMER. SHMT1 and SHMT2 transcript levels were significantly down- and upregulated, respectively, in patients with KICH, KIRC, and KIRP, based on sample type, individual cancer stage, sex, and patient age. Compared to men, women with KIRC and KIRP showed significantly up- and downregulated SHMT1 transcript levels, respectively. However, SHMT2 transcript levels were significantly upregulated in the patients mentioned above. KIRC and KIRP patients with high SHMT1 expression had longer survival periods than those with low SHMT1 expression. In patients with KIRC, the findings were similar to those mentioned above. However, in KICH patients, the findings were the opposite regarding SHMT2 expression. SHMT1 versus SHMT2 were altered by 9% versus 3% (n = 66 KICH patients), 4% versus 4% (n = 446 KIRC patients), and 6% versus 7% (n = 280 KIRP patients). SHMT1 versus SHMT2 promoter methylation levels were significantly up- and downregulated in patients with KIRP versus KIRC and KIRP, respectively. SHMT1, SHMT2, and their neighboring genes (NG) formed a complex network of interactions. The molecular functions of SHMT1 and its NG in patients with KICH, KIRC, and KIRP, included clathrin adaptor, metalloendopeptidase, and GTPase regulator activities; lipid binding, active transmembrane transporter activity, and lipid transporter activity; and type I interferon receptor binding, integrin binding, and protein heterodimerization, respectively. Their respective Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were involved in lysosome activity, human immunodeficiency virus 1 infection, and endocytosis; coronavirus disease 2019 and neurodegeneration pathways (multiple diseases); and RIG-I-like receptor signaling pathway, cell cycle, and actin cytoskeleton regulation. The molecular functions of SHMT2 and its NG in patients with KICH, KIRC, and KIRP included cell adhesion molecule binding and phospholipid binding; protein domain-specific binding, enzyme inhibitor activity, and endopeptidase activity; and hormone activity, integrin binding, and protein kinase regulator activity, respectively. For patients with KIRC versus KIRP, the KEGG pathways were involved in cAMP and calcium signaling pathways versus microRNAs (MiRNAs) in cancer cells and neuroactive ligand-receptor interactions, respectively. We identified the key transcription factors of SHMT1 and its NG. SHMT1 and SHMT2 expression levels were different in patients with RCC. SHMT1 and SHMT2 may be potential therapeutic and prognostic biomarkers in these patients. Transcription factor (MYC, STAT1, PPARG, AR, SREBF2, and SP3) and miRNA (miR-17-5P, miR-422, miR-492, miR-137, miR-30A-3P, and miR-493) regulations may be important strategies for RCC treatment.

Two antisense RNAs—AFAP1-AS1 and MLK7-AS1—promote colorectal cancer progression by sponging miR-149-5p and miR-485-5p

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Antisense RNAs (asRNAs) are closely associated with cancer malignancy. This study aimed to identify the action mechanism of asRNAs in controlling CRC malignancy. Analysis of the RNA sequencing data revealed that AFAP1-AS1 and MLK7-AS1 were upregulated in CRC patients and cell lines. High levels of both asRNAs were associated with poor prognosis in patients with CRC. Both invitro and invivo experiments revealed that the knockdown of the two asRNAs decreased the proliferative and metastatic abilities of CRC cells. Mechanistically, AFAP1-AS1 and MLK7-AS1 decreased the levels of miR-149-5p and miR-485-5p by functioning as ceRNAs. Overexpression of miRNAs by introducing miRNA mimics suppressed the expression of SHMT2 and IGFBP5 by directly binding to the 3' UTR of their mRNA. Knockdown of both asRNAs decreased the expression of SHMT2 and IGFBP5, which was reversed by inhibition of both miRNAs by miRNA inhibitors. Invivo pharmacological targeting of both asRNAs by small interfering RNA-loaded nanoparticles showed that knockdown of asRNAs significantly reduced tumor growth and metastasis. Our findings demonstrate that AFAP1-AS1 and MLK7-AS1 promote CRC progression by sponging the tumor-suppressing miRNAs miR-149-5p and miR-485-5p, thus upregulating SHMT2 and IGFBP5.

Abstract 695: Serine Hydroxymethyltransferase 2 Regulates Mitochondrial Function And Ceramide Metabolism In Macrophages During Atherosclerosis

Increasing evidence suggests a critical role for amino acid metabolism in cardiovascular disease, highlighting potential therapeutic targets and our need for further understanding. Plasma serine has been found to be a biomarker for cardiovascular disease, while glycine is negatively correlated with coronary artery disease. Furthermore, attenuated plasma glycine:serine is associated with unstable atherosclerotic plaques. Mitochondrial Serine hydroxymethyl transferase (SHMT) catalyzes the bulk formation of glycine from serine in most cell types and drives one-carbon metabolism in the cytosol (SHMT1) or mitochondria (SHMT2). SHMT2 is necessary for mitochondrial protein translation and critical in development since global knockout mice are nonviable. We found LPS-treated macrophages have reduced SHMT2 expression concomitant with attenuated intracellular glycine:serine. Deletion of macrophage SHMT2 in vitro enhanced intracellular serine, which contributes to the rate-limiting step of ceramide de novo biosynthesis. C16:0 ceramide is highly abundant in atherosclerotic plaques and is positively associated with plaque severity, and SHMT2-deleted macrophages treated with C16:0 fatty acid enhances intracellular ceramide. Furthermore, deletion of SHMT2 reduced mitochondrial respiration and enhanced glycolysis similar to LPS-treated macrophages. Moreover, we found SHMT2 is highly expressed in plaque-associated macrophages in both human and mouse atherosclerotic plaques. We therefore hypothesized that SHMT2 regulates macrophage inflammatory phenotype in atherosclerosis through alterations in mitochondrial function and ceramide biosynthesis. To determine whether macrophage-expressed SHMT2 contributes to atherosclerosis, we developed a novel Shmt2 -floxed mouse crossed with LysM-Cre ( Shmt2 MKO ) and Apoe -/- mice. After 12 weeks on Western diet, we observed no differences in plasma glycine:serine or circulating lipids. Nevertheless, Shmt2 MKO / Apoe -/- mice had significantly enhanced plaque area compared to littermate controls. Our findings, representing the first investigation of conditional SHMT2 knockout mice, suggest a novel immunometabolic role for SHMT2 in macrophages in atherosclerosis.

Understanding The Regulatory Role of USP32 and SHMT2 in The Progression of Gastric Cancer.

Gastric cancer is the fifth most common neoplasm and the fourth reason for mortality globally. Incidence rates are highly variable and dependent on risk factors, epidemiologic and carcinogenesis patterns. Previous studies reported that Helicobacter pylori (H. pylori) infection is one the strongest known risk factor for gastric cancer. USP32 is a deubiquitinating enzyme identified as a potential factor associated with tumor progression and a key player in cancer development. On the other hand, SHMT2 is involved in serine-glycine metabolism to support cancer cell proliferation. Both USP32 and SHMT2 are reported to be upregulated in many cancer types, including gastric cancer, but its complete mechanism is not fully explored yet. The present study explored possible mechanism of action of USP32 and SHMT2 in the progression of gastric cancer. In this experimental study, Capsaicin (0.3 g/kg/day) and H. pylori infection combination was used to successfully initiate gastric cancer conditions in mice. It was followed by 40 and 70 days of treatment to establish initial and advanced conditions of gastric cancer. Histopathology confirmed formation of signet ring cell and initiation of cellular proliferation in the initial gastric cancer. More proliferative cells were also observed. In addition, tissue hardening was confirmed in the advanced stage of gastric cancer. USP32 and SHMT2 showed progressive upregulated expression, as gastric cancer progress. Immunohistologically, it showed signals in abnormal cells and high-intensity signals in the advanced stage of cancer. In USP32 silenced tissue, expression of SHMT2 was completely blocked and reverted cancer development as evident with less abnormal cell in initial gastric cancer. Reduction of SHMT2 level to one-fourth was observed in the advanced gastric cancer stages of USP32 silenced tissue. USP32 had a direct role in regulating SHMT2 expression, which attracted therapeutic target for future treatment.

Impairments in SHMT2 expression or cellular folate availability reduce oxidative phosphorylation and pyruvate kinase activity

BackgroundSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine-producing THF-conjugated one-carbon units and glycine in the mitochondria. Biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. SHMT2 expression has also been shown to decrease with aging in human fibroblasts. Immortalized cell models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels, suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. This study examined the role of SHMT2 and folate availability in regulating mitochondrial function, energy metabolism, and cellular proliferative capacity in both heterozygous and homozygous cell models of reduced SHMT2 expression. In this study, primary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6J dam crossed with a heterozygous Shmt2+/− male to generate Shmt2+/+ (wild-type) or Shmt2+/− (HET) MEF cells. In addition, haploid chronic myeloid leukemia cells (HAP1, wild-type) or HAP1 cells lacking SHMT2 expression (ΔSHMT2) were cultured for 4 doublings in either low-folate or folate-sufficient culture media. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, mitochondrial membrane potential, and mitochondrial function.ResultsHomozygous loss of SHMT2 in HAP1 cells impaired cellular folate accumulation and altered mitochondrial DNA content, formate production, membrane potential, and basal respiration. Formate rescued proliferation in HAP1, but not ΔSHMT2, cells cultured in low-folate medium. Pyruvate kinase activity and protein levels were impaired in ΔSHMT2 cells and in MEF cells exposed to low-folate medium. Mitochondrial biogenesis protein levels were elevated in Shmt2+/− MEF cells, while mitochondrial mass was increased in both homozygous and heterozygous models of SHMT2 loss.ConclusionsThe results from this study indicate disrupted mitochondrial FOCM impairs mitochondrial folate accumulation and respiration, mitochondrial formate production, glycolytic activity, and cellular proliferation. These changes persist even after a potentially compensatory increase in mitochondrial biogenesis as a result of decreased SHMT2 levels.

SHMT2 is Associated with Tumor Purity, CD8+ T Immune Cells Infiltration, and a Novel Therapeutic Target in Four Different Human Cancers.

This study was launched to identify the SHMT2 associated Human Cancer subtypes. Cancer is the 2nd leading cause of death worldwide. Previous reports revealed the limited involvement of SHMT2 in human cancer. In the current study, we comprehensively analyzed the role of SHMT2 in 24 major subtypes of human cancers using in silico approach and identified a few subtypes that are mainly associated with SHMT2. We aim to comprehensively analyze the role of SHMT2 in 24 major subtypes of human cancers using in silico approach and identified a few subtypes that are mainly associated with SHMT2. Earlier, limited knowledge exists in the medical literature regarding the involvement of Serine Hydroxymethyltransferase 2 (SHMT2) in human cancer. In the current study, we comprehensively analyzed the role of SHMT2 in 24 major subtypes of human cancers using in silico approach and identified a few subtypes that are mainly associated with SHMT2. Pan-cancer transcriptional expression profiling of SHMT2 was done using UALCAN while further validation was performed using GENT2. For translational profiling of SHMT2, we utilized Human Protein Atlas (HPA) platform. Promoter methylation, genetic alteration, and copy number variations (CNVs) profiles were analyzed through MEXPRESS and cBioPortal. Survival analysis was carried out through Kaplan-Meier (KM) plotter platform. Pathway enrichment analysis of SHMT2 was performed using DAVID, while the gene-drug network was drawn through CTD and Cytoscape. Furthermore, in the tumor microenvironment, a correlation between tumor purity, CD8+ T immune cells infiltration, and SHMT2 expression was accessed using TIMER. SHMT2 was found overexpressed in 24 different subtypes of human cancers and its overexpression was significantly associated with the reduced Overall survival (OS) and Relapse-free survival durations of Breast cancer (BRCA), Kidney renal papillary cell carcinoma (KIRP), Liver hepatocellular carcinoma (LIHC), and Lung adenocarcinoma (LUAD) patients. This implies that SHMT2 plays a significant role in the development and progression of these cancers. We further noticed that SHMT2 was also up-regulated in BRCA, KIRP, LIHC, and LUAD patients of different clinicopathological features. Pathways enrichment analysis revealed the involvement of SHMT2 enriched genes in five diverse pathways. Furthermore, we also explored some interesting correlations between SHMT2 expression and promoter methylation, genetic alterations, CNVs, tumor purity, and CD8+ T immune cell infiltrates. Our results suggested that overexpressed SHMT2 is correlated with the reduced OS and RFS of the BRCA, KIRP, LIHC, and LUAD patients and can be a potential diagnostic and prognostic biomarker for these cancers.

Growth Hormone Alters Circulating Levels of Glycine and Hydroxyproline in Mice

Growth hormone (GH) has established effects on protein metabolism, such as increasing protein synthesis and decreasing amino acid degradation, but its effects on circulating amino acid levels are less studied. To investigate this relationship, metabolomic analyses were used to measure amino acid concentrations in plasma and feces of mice with alterations to the GH axis, namely bovine GH transgenic (bGH; increased GH action) and GH receptor knockout (GHRKO; GH resistant) mice. To determine the effects of acute GH treatment, GH-injected GH knockout (GHKO) mice were used to measure serum glycine. Furthermore, liver gene expression of glycine metabolism genes was assessed in bGH, GHRKO, and GH-injected GHKO mice. bGH mice had significantly decreased plasma glycine and increased hydroxyproline in both sexes, while GHRKO mice had increased plasma glycine in both sexes and decreased hydroxyproline in males. Glycine synthesis gene expression was decreased in bGH mice (Shmt1 in females and Shmt2 in males) and increased in GHRKO mice (Shmt2 in males). Acute GH treatment of GHKO mice caused decreased liver Shmt1 and Shmt2 expression and decreased serum glycine. In conclusion, GH alters circulating glycine and hydroxyproline levels in opposing directions, with the glycine changes at least partially driven by decreased glycine synthesis.

SHMT2 promotes the tumorigenesis of renal cell carcinoma by regulating the m6A modification of PPAT

ObjectiveSerine hydroxymethyltransferase 2 (SHMT2) is the first rate-limiting enzyme for serine/glycine biosynthesis and one carbon metabolism. Here, we explore the underlying mechanism of how SHMT2 functions in renal cell carcinoma (RCC) initiation. MethodsIn this study, SHMT2 expression was assessed in RCC tissues. In vitro experiments were performed to investigate the functional role of SHMT2. The detailed mechanisms of SHMT2-mediated PPAT were addressed. ResultsIncreased SHMT2 facilitated RCC cell proliferation by inducing the G1/S phase transition. And SHMT2 promoted the expression of PPAT. Mechanism dissection revealed that SHMT2 enhanced the m6A modification through the endogenous methyl donor SAM mediated by SHMT2 via serine/glycine one carbon metabolic networks. SHMT2-catalyzed serine/glycine conversion regulated PPAT expression in an m6A-IGF2BP2-dependent manner. SHMT2 promoted RCC cell proliferation by upregulating PPAT expression. ConclusionsSHMT2 promotes RCC tumorigenesis by increasing PPAT expression. Thus, SHMT2 may be a novel potential therapeutic target for RCC.

Impaired Mitochondrial Folate Metabolism Reduces Mitochondrial Respiration in Mouse Muscle Progenitor Cells

ObjectivesMitochondrial dysfunction is a central contributor to aging. Mitochondrial DNA mutations increase with aging and are associated with low serum folate. Furthermore, both increased age and low serum folate are suggested to increase skeletal muscle fiber loss and impair muscle function. However, no research has focused on the metabolic pathway linking the causal relationship. Data from our lab indicate that folate-mediated one-carbon metabolism may play a role, as loss of the mitochondrial specific isozyme (serine hydroxymethyltransferase 2, SHMT2) and folate depletion both result in mitochondrial DNA instability and impaired mitochondrial function. Furthermore, serum folate and SHMT2 gene expression levels decrease with aging, potentially providing a causal mechanism for age-associated mitochondrial DNA mutations and skeletal muscle impairments. To date, little is known about the relationship between loss of SHMT2 enzyme expression and folate status in skeletal muscles.MethodsTibialis anterior muscles were harvested from 10-week-old wild-type (Shmt2+/+) and heterozygous (Shmt2+/−) mice that were weaned onto either a folate-sufficient diet or a modified diet lacking folic acid. Skeletal muscles used to isolate primary muscle progenitor cells (MPCs) were isolated from 20-week-old Shmt2+/+ and Shmt2+/− mouse hindlimbs. MPCs were cultured to 80–90% confluency and then differentiated in low-serum medium for 7–10 days. Skeletal muscles were examined for mitochondrial DNA content and MPCs were examined for mitochondrial function.ResultsHeterozygous loss of Shmt2 resulted in an 80% reduction in SHMT2 protein levels in mouse tibialis anterior muscle. Mice fed a folate-depleted diet for 7 weeks exhibited a 25% reduction in mitochondrial DNA content compared to the folate-sufficient group. In MPCs, reduced Shmt2 expression impaired basal and maximal respiration. Furthermore, protein levels of mitochondrial NADH dehydrogenase 3 were reduced and protein levels of PGC1α and mitofusin 2 were elevated with Shmt2 heterozygosity in MPCs.ConclusionsOur results demonstrate that folate depletion reduced mitochondrial DNA content in tibialis anterior skeletal muscle and loss of SHMT2 impairs MPCs mitochondrial respiration and may influence mitochondrial biogenesis and dynamics.Funding SourcesPresident's Council of Cornell Women Award.

Abstract 436: Loss Of Serine Hydroxymethyltransferase 2 In Macrophages Enhances Atherosclerosis While Reducing Macrophage Mitochondrial Respiration

Despite significant advancements in therapeutics and intervention, cardiovascular diseases remain the leading cause of death worldwide. Atherosclerosis, the accumulation of lipid-laden plaques in large arteries, is the underlying cause of most cardiovascular diseases. While the role of lipids is well-established in atherosclerotic cardiovascular disease, the contribution of amino acids is becoming increasingly recognized. Indeed, lower circulating glycine is consistently reported in patients with cardiovascular diseases. The mitochondrial serine hydroxymethyltransferase (SHMT2) is responsible for the bulk of glycine biosynthesis in most cell types. SHMT2 catalyzes the formation of glycine from serine and drives one-carbon metabolism necessary for mitochondrial protein translation. SHMT2 is critical in development since global knockout mice are nonviable. Previous studies showed patients with significant coronary artery disease exhibit a reduced glycine:serine ratio compared with healthy individuals. We discovered LPS-treated macrophages similarly attenuated glycine:serine concomitant with reduced SHMT2 expression. Moreover, we found SHMT2 is highly expressed in plaque-associated macrophages in both human and mouse atherosclerotic plaques. We hypothesized that SHMT2 regulates macrophage inflammatory phenotype in atherosclerosis through alterations in mitochondrial function. Deletion of SHMT2 reduced mitochondrial respiration while enhancing glycolysis similar to LPS-treated macrophages. In addition, SHMT2 deletion augmented oxidized-LDL accumulation in macrophages. To determine whether macrophage-expressed SHMT2 contributes to atherosclerosis, we applied CRISPR/Cas9 and Cre-lox recombination system to generate myeloid-specific SHMT2 knockout ( Shmt2 MKO ) mice. We further crossed Shmt2 MKO mice with Apoe knockout ( Apoe -/- ) mice to study atherosclerosis following Western diet feeding. Compared to littermate controls ( Shmt2 fl / Apoe -/- ), Shmt2 MKO / Apoe -/- mice had significantly enhanced plaque area. Our findings, representing the first investigation of conditional SHMT2 knockout mice, suggest a novel immunometabolic role for SHMT2 in macrophages in atherosclerosis.

Serine hydroxymethyltransferase 2 predicts unfavorable outcomes in multiple cancer: a systematic review and meta-analysis.

BackgroundSerine hydroxymethyltransferase (SHMT) is critical for one-carbon unit metabolism and is increasingly reported to be associated with tumor patients’ outcomes. Thus, we designed and performed this meta-analysis to reveal its prognostic role and relationship with clinicopathological characteristics in human cancer.MethodsA systematic search of PubMed, Embase, Web of Science and Cochrane Library (CENTRAL) was carried out. Two reviewers independently screened all references for eligibility according to the inclusion criteria. The Newcastle-Ottawa Quality Assessment Scale was used to assess the quality and data was extracted for the meta-analysis.ResultsTen studies, composed of 1,942 patients in total, were included in this meta-analysis. Higher expression of SHMT2 means an unfavorable prognosis [overall survival: hazard ratio (HR) =2.14, 95% confidence interval (CI): 1.53 to 2.99; progression-free survival (PFS)/disease-free survival (DFS)/recurrence-free survival (RFS): HR =1.90, 95% CI: 1.31 to 2.76]. Furthermore, higher SHMT2 expression is associated with larger tumor size [odds ratio (OR) =2.09, 95% CI: 1.58 to 2.77], more lymph node invasions [OR =2.67, 95% CI: 1.78 to 4.00), and higher tumor node metastasis classification (TNM) stage (OR =2.23, 95% CI: 1.55 to 3.21). Higher expression of SHMT2 is also related to higher histopathological grade (OR =3.46, 95% CI: 1.46 to 8.27) and distant metastasis (OR =1.25, 95% CI: 0.32 to 4.90), however, with significant heterogeneity (I2=61%, P=0.08 for distant metastasis; I2=82%, P<0.001 for histopathological grade). The prognostic clinical role of SHMT1 in clinical patients has not been directly investigated yet.DiscussionSHMT2 may serve as a promising prognostic biomarker in various cancer, especially in the alimentary system. Further large-scale studies are warranted to verify the possible effect.

Abstract P5-05-05: Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells

Abstract Introduction. With the goal of defining the in-breast environment that facilitates the development of estrogen receptor negative (ER-) breast cancer (BC), we have previously identified a set of genes involved in lipid metabolism with increased expression in the contralateral unaffected breast of women with ER- BC (PMID: 28263391). MCF-10A cells exposed to lipids in vitro display profound changes in gene expression, chromatin packing density, chromatin accessibility and histone posttranslational modifications (PTMs). In addition, flux through thirty-eight metabolic reactions was significantly increased by this exposure, including those of the electron transport chain (ETC). However, the mechanisms by which lipids induce molecular changes that result in epigenomic reprogramming, and potentially promote malignant transformation remain to be elucidated. We hypothesized that dysfunction of the ETC and increased serine, glycine, one carbon (SGOC) metabolism pathway activity play a role in the modulation of epigenetics. Methods. To interrogate potential mechanisms that link lipids to ER- BC genesis, estrogen and progesterone receptor negative MCF-10A cells were cultured in MEBM/MEGM and exposed to vehicle, octanoic acid (C8) or linoleic acid (C18) for 24 hours. Histone PTMs were assayed by liquid chromatography-mass spectrometry. Gene expression was measured by RNA-seq and differentially expressed genes between vehicle and lipid treated groups were identified using the DESeq2 R package. ATAC-seq was performed to study chromatin accessibility. MCF-10A cells were exposed to C8 in presence or absence of inhibitors of Complex I (Metformin) and Complex III (Antimycin A) of the ETC. Real-time qPCR was performed to measure epigenetic modulators, Unfolded Protein Response (UPR) pathway and SGOC metabolism genes. Results. Histone PTMs changed significantly following 24 hours exposure to C8 or C18. Exposure to C8 resulted in the modulation of the expression of genes involved in the UPR/recognition of mitochondrial dysfunction (ATF4), de novo serine biosynthesis (PHGDH, PSAT1, SPH) and one-carbon metabolism (SHMT2, MTHFD2) pathways as well as numerous genes coding for epigenetic modulators. Upon C18 exposure, the binding motif for ATF4 was among the most significantly enriched motifs in the open chromatin regions and p53 was among the motifs with significantly decreased accessibility. Interestingly ATF4 actives PHGDH expression while p53 suppresses it. Exposure to C8 in presence of ETC inhibitors affected gene expression in the same direction as lipid exposure and co-treatment had an additive effect. Fatty acid oxidation and inhibition of the ETC increased the expression of PHGDH: C8, 2.6-fold (p-value = 0.005); Metformin, 2.3-fold (p-value = 0.0052); Antimycin A, 5.1-fold (p-value &amp;lt; 0.0001); C8 plus Metformin, 5.3-fold (p-value &amp;lt; 0.0001), and C8 plus Antimycin A, 5.2-fold (p-value &amp;lt; 0.0001). Conclusions. The modulation of epigenetic effectors does not appear to be a consequence of increased flux through the ETC but rather a consequence of increased SGOC metabolism, and the methionine cycle. Our in vitro model shows that lipid-induced stress increases ATF4 expression. ATF4 controls the expression of PHGDH, PSAT1, SPH, SHMT2 and MTHFD2 which influences epigenetics through S-adenosyl methionine production, the major methyl donor. PHGDH overexpression appears to be the key in our model as it has been associated with ER- BC and its engineered overexpression in non-transformed breast cells causes alterations associated with malignant transformation. The increase in PDGDH expression is hypothesized to compensate for mitochondrial dysfunction and to restore redox homeostasis by producing precursors that can be used for the production of antioxidants, such as glutathione. Citation Format: Mariana Bustamante Eduardo, Shivangi Yadav, Seema Khan, Susan Clare. Unraveling the mechanism of Lipid-induced malignant transformation in non-transformed breast epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-05.

Reduced Shmt2 Expression Impairs Mitochondrial Folate Accumulation and Respiration, and Leads to Uracil Accumulation in Mouse Mitochondrial DNA

BackgroundAdequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in nuclear dTMP synthesis leads to increased uracil misincorporation into DNA, affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in nontransformed tissues have been understudied. ObjectivesThis study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA (mtDNA) integrity and mitochondrial function in mouse tissues. MethodsLiver mtDNA content, and uracil content in liver mtDNA, were measured in Shmt2+/− and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid; C) or a modified diet lacking folic acid (0 mg/kg folic acid) for 7 wk. Shmt2+/− and Shmt2+/+ mouse embryonic fibroblast (MEF) cells were cultured in defined culture medium containing either 0 or 25 nM folate (6S-5-formyl-tetrahydrofolate, folinate) to assess proliferative capacity and mitochondrial function. Chi-square tests, linear mixed models, and 2-factor ANOVA with Tukey post hoc analyses were used to analyze data. ResultsShmt2+/− mice exhibited a 48%–67% reduction in SHMT2 protein concentrations in tissues. Interestingly, Shmt2+/− mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/− mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/− mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. ConclusionsThis study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis in mice, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.

Overexpression of SHMT2 Predicts a Poor Prognosis and Promotes Tumor Cell Growth in Bladder Cancer.

SHMT2 was overexpressed in many tumors, however, the role of SHMT2 in bladder cancer (BLCA) remains unclear. We first analyzed the expression pattern of SHMT2 in BLCA using the TNMplot, Oncomine, the Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) databases. Next, the association between SHMT2 expression and overall survival (OS)/disease-free survival (DFS) in BLCA patients were analyzed using TCGA and PrognoScan database. The correlation between SHMT2 expression and clinicopathology was determined using TCGA database. Furthermore, the genes co-expressed with SHMT2 and their underlying molecular function in BLCA were explored based on the Oncomine database, Metascape and gene set enrichment analysis (GSEA). Finally, the effects of SHMT2 on cell proliferation, cell cycle, and apoptosis were assessed using in vitro experiments. As a results, SHMT2 was significantly overexpressed in BLCA tissues and cells compared to normal bladder tissues and cells. A high SHMT2 expression predicts a poor OS of BLCA patients. In addition, SHMT2 expression was higher in patients with a high tumor grade and in those who were older than 60 years. However, the expression of SHMT2 was not correlated with gender, tumor stage, lymph node stage, and distant metastasis stage. Finally, overexpression of SHMT2 promoted BLCA cell proliferation and suppressed apoptosis, the silencing of SHMT2 significantly inhibited BLCA cell proliferation by impairing the cell cycle, and promoting apoptosis. SHMT2 mediates BLCA cells growth by regulating STAT3 signaling. In summary, SHMT2 regulates the proliferation, cell cycle and apoptosis of BLCA cells, and may act as a candidate therapeutic target for BLCA.

Loss of SHMT2 and Folate Deficiency Impair Energy Metabolism in Mouse Embryonic Fibroblasts Cells

ObjectivesSerine hydroxymethyltransferase 2 (SHMT2) catalyzes the reversible conversion of tetrahydrofolate (THF) and serine producing THF-conjugated one-carbon units and glycine in the mitochondria. Recently biallelic SHMT2 variants were identified in humans and suggested to alter the protein’s active site, potentially disrupting enzymatic function. Indeed, patient fibroblasts exhibited impaired oxidative capacity, however, with no changes in oxidative phosphorylation protein levels. Furthermore, immortalized cells models of total SHMT2 loss or folate deficiency exhibit decreased oxidative capacity and impaired mitochondrial complex I assembly and protein levels suggesting folate-mediated one-carbon metabolism (FOCM) and the oxidative phosphorylation system are functionally coordinated. We have developed a mouse model of reduced Shmt2 expression that provides an appropriate reference model to further study the deleterious human SHMT2 variants and their connection to folate metabolism. MethodsPrimary mouse embryonic fibroblasts (MEF) were isolated from a C57Bl/6 dam crossed with a heterozygous Shmt2+/– (HET) male. MEF embryos were genotyped, and Shmt2+/+ (wild type) or Shmt2+/– MEF cells were cultured for 4 doubling in 2 nM (deficient) or 25 nM folate (sufficient) medium. Cells were examined for proliferation, total folate levels, mtDNA content, protein levels of pyruvate kinase and PGC1α, pyruvate kinase enzyme activity, and mitochondrial membrane potential and function. ResultsLoss of Shmt2 and folate deficiency significantly reduced cell proliferation and oxygen consumption rate. Total cellular folate levels and pyruvate kinase enzyme activity and protein levels were reduced with folate deficiency. While there were no changes in mitochondrial DNA content or complex 1 proteins NDUFA9 or ND3, mitochondrial membrane potential was impaired and PGC1α protein levels were elevated in Shmt2+/– MEF cells. ConclusionsOur results demonstrate that loss of SHMT2 impairs cellular proliferation and that SHMT2 expression and folate status impact cellular energy metabolism and mitochondrial health. Funding SourcesThis study was financially supported by a President’s Council of Cornell Women Award and Cornell University Division of Nutritional Sciences funds.