Serine Hydroxymethyltransferase 2 Research Articles

Abstract 4902: Mitochondrial and cytosolic folylpolyglutamate synthetase in one-carbon metabolism and anti-tumor efficacy of mitochondrial-targeted antifolates

Abstract Folates mediate one-carbon (C1) transfers, which are essential for cellular homeostasis and survival. C1 metabolism encompasses distinct cytosol (Cyto) and mitochondria (Mito) pathways connected by an interchange between serine, glycine and formate. Mito C1 metabolism provides cellular glycine and C1 units (as formate) for de novo synthesis of thymidylate and purine nucleotides in the Cyto. Polyglutamyl folates are the predominant folate forms in cells and are generally preferred for C1 transfers. Thus, folate polyglutamylation is essential for cellular homeostasis. Folate polyglutamylation is catalyzed by folypolyglutamate synthetase (FPGS), including Cyto (cFPGS) and Mito (mFPGS) isoforms. C1 metabolism is critical for tumor growth and thus offers a plethora of therapeutic targets for cancer. Of particular interest are Mito C1 inhibitors including “non-classical” pyrazolopyran compounds SHIN1 and SHIN2, and “classical” pyrrolo[3,2-d]pyrimidine antifolate compounds typified by AGF347, all targeting serine hydroxymethyltransferase 2 (SHMT2). Both SHIN1 and AGF347 effected in vitro anti-tumor efficacy, and SHIN2 and AGF347 showed in vivo efficacy toward tumor xenografts. We systematically explored the roles of cFPGS and mFPGS levels as critical determinants of C1 inhibitor target engagement and net Cyto versus Mito C1 flux, resulting in anti-tumor efficacy. We found that FPGS transcript levels significantly correlated with the effects of AGF347 toward a panel of human pancreatic cancer cell lines including MIA PaCa-2. We engineered MIA PaCa-2 cells with FPGS gene knockout to stably express inducible mFPGS or cFPGS form. FPGS in mFPGS-transfected cells (mFPGS#32) was expressed mainly in Mito over Cyto, whereas FPGS in cFPGS-transfected cells (cFPGS#3) was expressed exclusively in Cyto. Accumulation of radiolabeled folate and AGF347 increased with increasing FPGS in both Cyto and Mito for mFPGS#32, but only in Cyto for cFPGS#3. Metabolomics with [2,3,3-2H]serine in cFPGS#3 and mFPGS#32 established FPGS levels in Mito versus Cyto as important determinants of C1 fluxes. Increased FPGS levels in mFPGS#32 enhanced C1 flux in Mito far greater than in Cyto, and greater than for the increased FPGS in cFPGS#3. As a result, Cyto-targeted antifolates pemetrexed and AGF94 were only modestly impacted by increasing FPGS levels in both mFPGS#32 and cFPGS#3 (~3x). In contrast, increasing FPGS levels dramatically enhanced the inhibitory effects of Mito-targeted antifolates such as AGF347 in mFPGS#32 (~8-25x), and to a greater extent than in cFPGS#3 (~6-8x). Conversely, increasing FPGS levels substantially attenuated the cytotoxic effect of SHIN1 in mFPGS#32 (~19x). In summary, FPGS levels are an important determinant of C1 fluxes, particularly in Mito where they contribute to cytotoxic potencies of Mito-targeted C1 inhibitors at SHMT2. Citation Format: Carrie O’Connor, Jade Katinas, Mathew Schneider, Md. Junayed Nayeen, Xun Bao, Jing Li, Charles Dann, Aleem Gangjee, Larry H. Matherly, Zhanjun Hou. Mitochondrial and cytosolic folylpolyglutamate synthetase in one-carbon metabolism and anti-tumor efficacy of mitochondrial-targeted antifolates. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4902.

Serine and one-carbon metabolism sustain non-melanoma skin cancer progression

Non-melanoma skin cancer (NMSC) is a tumor that arises from human keratinocytes, showing abnormal control of cell proliferation and aberrant stratification. Cutaneous basal cell carcinoma (cBCC) and cutaneous squamous cell carcinoma (cSCC) are the most common sub-types of NMSC. From a molecular point of view, we are still far from fully understanding the molecular mechanisms behind the onset and progression of NMSC and to unravel targetable vulnerabilities to leverage for their treatment, which is still essentially based on surgery. Under this assumption, it is still not elucidated how the central cellular metabolism, a potential therapeutical target, is involved in NMSC progression. Therefore, our work is based on the characterization of the serine anabolism/catabolism and/or one-carbon metabolism (OCM) role in NMSC pathogenesis. Expression and protein analysis of normal skin and NMSC samples show the alteration of the expression of two enzymes involved in the serine metabolism and OCM, the Serine Hydroxy-Methyl Transferase 2 (SHMT2) and Methylen-ThetraHydroFolate dehydrogenase/cyclohydrolase 2 (MTHFD2). Tissues analysis shows that these two enzymes are mainly expressed in the proliferative areas of cBCC and in the poorly differentiated areas of cSCC, suggesting their role in tumor proliferation maintenance. Moreover, in vitro silencing of SHMT2 and MTHFD2 impairs the proliferation of epidermoid cancer cell line. Taken together these data allow us to link the central cellular metabolism (serine and/or OCM) and NMSC proliferation and progression, offering the opportunity to modulate pharmacologically the involved enzymes activity against this type of human cancer.

Serine hydroxymethyltransferase 2 knockdown induces apoptosis in ccRCC by causing lysosomal membrane permeabilization via metabolic reprogramming

Serine hydroxymethyltransferase 2 (SHMT2) plays an important role in converting serine to glycine and supplying carbon to one-carbon metabolism to sustain cancer cell proliferation. However, the expression, function, and underlying mechanisms of SHMT2 in clear cell renal cell carcinoma (ccRCC) remain largely unknown. In this study, we demonstrated that SHMT2 was upregulated in ccRCC tissues compared with controls and associated with patient survival. SHMT2 knockdown inhibited proliferation, migration, and invasion in ccRCC cells. Overexpression of SHMT2 promoted tumor progression. Mechanistically, SHMT2 depletion disrupted one-carbon metabolism, increased reactive oxygen species (ROS) levels, and decreased ATP levels via metabolic reprogramming, which destroyed cell homeostasis. The SHMT2 knockdown-induced stress activated autophagy. A mass of autophagosomes fused with lysosomes, resulting in lysosomal membrane permeabilization (LMP) and leakage of lysosomal contents into the cytoplasm, which eventually led to apoptosis. Our work reveals that SHMT2 functions as an oncogenic gene to promote ccRCC progression. SHMT2 depletion induces apoptosis by causing LMP through excessive activation of the autophagy-lysosome pathway via metabolic reprogramming.

Metformin Treatment Abrogates the Competitive Advantage of Dnmt3a R878H HSPCs By Enhancing DNA Methylation Activity

Clonal hematopoiesis of indeterminate potential (CHIP) refers to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) carrying leukemogenic mutations in individuals without evidence of a blood disorder. CHIP carriers are at an increased risk of blood cancers and cardiovascular diseases. Targeting the clonal expansion of mutant HSPCs has the potential to mitigate CHIP-related illnesses. The most commonly mutated gene in CHIP is DNMT3A, which encodes an enzyme that catalyzes the methyl transfer from S-adenosylmethionine (SAM) to cytosines in DNA. The heterozygous DNMT3A hotspot mutation at R882 exerts a dominant negative effect on the wild-type (WT) allele, resulting in decreased DNA methylation activity. Prior studies have shown that murine HSPCs carrying mutant Dnmt3a R878H (equivalent to R882 in humans) have an advantage over WT cells in competition assays. Here, we aimed to first identify differences in the functional properties between Dnmt3a-mutated and WT HSPCs. Given the crosstalk between mitochondrial metabolism and DNA methylation, we investigated potential differences in mitochondrial activity and found that the level of mitochondrial respiration was higher in Dnmt3aR878H/+ HSPCs than in WT cells. To determine if this translated to differential dependencies, we silenced the expression of critical subunits in the each of five electron transport chain (ETC) complexes. Knockdown of NDUFV1 in Complex I was the most effective in reducing the competitive advantage of Dnmt3aR878H/+ cells in vitro. To explore translational relevance of this finding, we tested the impact of metformin, a pharmacologic inhibitor of Complex I, on Dnmt3a-mutated HSPCs. Treatment with metformin at clinically relevant concentrations suppressed the competitive advantage of mutant cells in vitro. Importantly, this effect was rescued by expression of NDI1, a metformin-resistant yeast analog of Complex I. To extend these findings, we conducted a competitive repopulation assay between CD45.2+ Dnmt3aR878H/+ and CD45.1+ Dnmt3a+/+ HSPCs. Metformin treatment abrogated the in vivo competitive advantage of mutant cells over an 8-month period based on peripheral blood chimerism. To decipher the mechanism of action, we performed transcriptome and metabolome profiling of mutant HSPCs treated with or without metformin for 1 month. GSEA analysis of the transcriptome data revealed a downregulation of genes associated with stemness in metformin-treated cells. Intriguingly, we found that metformin increased the expression of genes involved in one-carbon metabolism, which generates SAM. Consistent with these findings, metabolomics analysis showed that the levels of several key metabolites in one-carbon metabolism, including SAM, were higher in metformin-treated cells compared with controls. Genetic and pharmacologic inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism, as well as the addition of exogeneous S-adenosylhomocysteine (SAH), an inhibitor of SAM-dependent methyltransferases, effectively rescued the effect of metformin on mutant cells in vitro. Based on the above findings, we hypothesized that metformin treatment might enhance the residual DNA methylation activity found in Dnmt3aR878H/+ mutated cells by increasing SAM concentration. In support of this hypothesis, we found that exogeneous SAM reproduced the effect of metformin, and HSPCs with homozygous Dnmt3aR878H mutations were insensitive to metformin. Furthermore, we performed DNA methylome profiling of untreated and metformin-treated mutant and WT HSPCs using RRBS. This analysis revealed that the hypomethylated regions (HMRs) in untreated Dnmt3aR878H/+ HSPCs relative to WT controls were enriched for regions of repressed chromatin with H3K27 methylation. Importantly, metformin treatment increased the level of DNA methylation at these regions in mutated HSPCs. In summary, our findings demonstrate that Dnmt3aR878H/+ HSPCs depend on ETC activity and particularly complex I activity to maintain their competitive advantage over WT cells. We propose a model in which inhibition of complex I activity with metformin increases one-carbon metabolism and SAM level, thereby restoring DNA methylation at HMRs and the differentiation of mutant HSPCs. Our findings have important implications for the development of preventive interventions against DNMT3A-mutated CHIP.

Exosome-mediated transfer of circ_0063526 enhances cisplatin resistance in gastric cancer cells via regulating miR-449a/SHMT2 axis.

Exosomes are critical mediators of intercellular communication. Exosomal circular RNAs (circRNAs) have been reported to play critical roles in the development of chemoresistance in various tumors, including gastric cancer. However, the role of exosomal circ_0063526 in cisplatin (CDDP) resistance of gastric cancer is still unclear. The expression of circ_0063526, microRNA-449a (miR-449a) and serine hydroxymethyltransferase 2 (SHMT2) mRNA was determined by quantitative real-time PCR (qRT-PCR). Cell viability was assessed by the Cell Counting Kit-8 assay. Cell migration and invasion were evaluated by the transwell assay and wound healing assay. Western blot assay was used to measure the protein expression of light Chain 3 (LC3) I/II, p62 and SHMT2. Exosomes were detected using transmission electron microscopy. The size distribution of exosomes was analyzed by nanoparticle tracking analysis. The interaction between miR-449a and circ_0063526 or SHMT2 was confirmed by a dual-luciferase reporter and RNA pull-down assays. Circ_0063526 expression was increased in gastric cancer tissues and cells and CDDP-resistant cells. Extracellular circ_0063526 could be packaged into exosomes and transmitted to sensitive cells, thus disseminating CDDP resistance. Knockdown of exosomal circ_0063526 inhibited CDDP resistance via suppressing migration, invasion and autophagy in gastric cancer cells. Moreover, circ_0063526 was identified as a molecular sponge of miR-449a to upregulate SHMT2 expression. Further, exosomal circ_0063526 regulated SHMT2 expression to enhance CDDP resistance of gastric cancer cells. Additionally, high expression of exosomal circ_0063526 in serum was associated with poor response to CDDP treatment in gastric cancer patients. Exosomal circ_0063526 facilitated CDDP resistance in gastric cancer via regulating the miR-449a/SHMT2 axis.

Knockdown of SHMT2 enhances the sensitivity of gastric cancer cells to radiotherapy through the Wnt/β-catenin pathway.

Gastric cancer (GC) is one of the most common malignant tumors. The mechanism of GC radioresistance and new radiosensitizers must be revealed and developed to treat GC. Serine hydroxymethyltransferase 2 (SHMT2) is responsible for encoding the mitochondrial form of the pyridoxal phosphate-dependent enzyme. SHMT2 plays a critical role in several types of cancers, while its possible effect on the radiological resistance in GC is still unclear. In this study, we investigated the role of SHMT2 in the radiological resistance of GC. Our data confirmed that SHMT2 was highly expressed in radiation-resistant GC cells. SHMT2 reduced the radiosensitivity of GC cells. In addition, SHMT2 is involved in radiation-induced GC cell apoptosis. Further, SHMT2 regulated the Wnt/β-catenin pathway, therefore reducing the radiosensitivity of GC cells in vivo. In conclusion, we revealed that depletion of SHMT2 enhanced the sensitivity of GC cells to interventional radiotherapy through the Wnt/β-catenin pathway.

SHMT2 Induces Stemness and Progression of Head and Neck Cancer.

Various enzymes in the one-carbon metabolic pathway are closely related to the development of tumors, and they can all be potential targets for cancer therapy. Serine hydroxymethyltransferase2 (SHMT2), a key metabolic enzyme, is very important for the proliferation and growth of cancer cells. However, the function and mechanism of SHMT2 in head and neck cancer (HNC) are not clear. An analysis of The Cancer Genome Atlas (TCGA) data showed that the expression of SHMT2 was higher in tumor tissue than in normal tissue, and its expression was significantly associated with male sex, aggressive histological grade, lymph node metastasis, distant metastasis, advanced TNM stage, and lymphovascular invasion in HNC. SHMT2 knockdown in FADU and SNU1041 cell lines significantly inhibited cell proliferation, colony formation, migration, and invasion. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using TCGA data revealed that SHMT2 was closely related to cancer stem cell regulation and maintenance. Furthermore, we found that silencing SHMT2 inhibited the expression of stemness markers and tumor spheroid formation compared with a control group. On the contrary, stemness markers were significantly increased after SHMT2 overexpression in HEP-2 cells. Interestingly, we found that knocking down SHMT2 reduced the expression of genes related to the Notch and Wnt pathways. Finally, silencing SHMT2 significantly reduced tumor growth and decreased stemness markers in a xenograft model. Taken together, our study suggests that targeting SHMT2 may play an important role in inhibiting HNC progression.

SHMT2-mediated mitochondrial serine metabolism drives 5-FU resistance by fueling nucleotide biosynthesis.

5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in invitro and invivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches.

Inhibition of SHMT2 mRNA translation increases embryonic mortality in sheep†.

The one-carbon metabolism (OCM) pathway provides purines and thymidine for synthesis of nucleic acids required for cell division, and S-adenosyl methionine for polyamine and creatine syntheses and the epigenetic regulation of gene expression. This study aimed to determine if serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in the OCM pathway, is critical for ovine trophectoderm (oTr) cell function and conceptus development by inhibiting translation of SHMT2 mRNA using a morpholino antisense oligonucleotide (MAO). In vitro treatment of oTr cells with MAO-SHMT2 decreased expression of SHMT2 protein, which was accompanied by reduced proliferation (P = 0.053) and migration (P < 0.05) of those cells. Intrauterine injection of MAO-SHMT2 in ewes on Day 11 post-breeding tended to decrease the overall pregnancy rate (on Days 16 and 18) compared with MAO-control (3/10 vs. 7/10, P = 0.07). The three viable conceptuses (n = 2 on Day 16 and n = 1 on Day 18) recovered from MAO-SHMT2 ewes had only partial inhibition of SHMT2 mRNA translation. Conceptuses from the three pregnant MAO-SHMT2 ewes had similar levels of expression of mRNAs and proteins involved in OCM as compared with conceptuses from MAO-control ewes. These results indicate that knockdown of SHMT2 protein reduces proliferation and migration of oTr cells (in vitro) to decrease elongation of blastocysts from spherical to elongated forms. These in vitro effects suggest that increased embryonic deaths in ewes treated with MAO-SHMT2 are the result of decreased SHMT2-mediated trophectoderm cell proliferation and migration supporting a role for the OCM pathway in survival and development of ovine conceptuses.

Glycyrrhetinic acid restricts mitochondrial energy metabolism by targeting SHMT2

SummaryGlycyrrhetinic acid (GA) is a natural product of licorice with mitochondria targeting properties and shows broad anticancer activities, but its targets and underlying mechanisms remain elusive. Here, we identified the mitochondrial enzyme serine hydroxymethyltransferase 2 (SHMT2) as a target of GA by using chemical proteomics. Binding to and inhibiting the activity of SHMT2 by GA were validated in vitro and in vivo. Knockout of SHMT2 or inhibiting SHMT2 with GA restricts mitochondrial energy supplies by downregulating mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid β-oxidation, and consequently suppresses cancer cell proliferation and tumor growth. Crystal structures of GA derivatives indicate that GA occupies SHMT2 folate-binding pocket and regulates SHMT2 activity. Modifications at GA carboxylic group with diamines significantly improved its anticancer potency, demonstrating GA as a decent structural template for SHMT2 inhibitor development.

Downregulation of SHMT2 promotes the prostate cancer proliferation and metastasis by inducing epithelial-mesenchymal transition

Serine hydroxymethyltransferase 2 (SHMT2) is a key enzyme that regulates serine/glycine transition; however, its specific function and molecular mechanisms in tumors remain controversial. In this study, we aimed to enhance the understanding in this regard. Through in vitro and in vivo experiments, as well as data analyses using public databases, we investigated the effect of SHMT2 in prostate cancer. Our results indicated that SHMT2 acts as a prostate cancer tumor proliferation suppressor and negatively regulates the aggressive behavior of prostate cancer through activation of epithelial–mesenchymal transition. Additionally, downregulated SHMT2 expression was observed in more advanced prostate cancer phenotypes, and further analysis showed that its depletion promoted proliferation and migration in prostate cancer cell lines. Taken together, our results revealed the function of SHMT2 in prostate cancer and may potentially play a role in the exploration of new therapeutic strategies.

Serine hydroxymethyltransferase 2 (SHMT2) potentiates the aggressive process of oral squamous cell carcinoma by binding to interleukin enhancer-binding factor 2 (ILF2)

ABSTRACT Oral squamous cell carcinoma (OSCC) is a frequent threatening head and neck malignancy. Serine hydroxymethyltransferase 2 (SHMT2) was identified to be upregulated in OSCC and its high expression was associated with poor patient prognosis. This paper set out to assess the influence of SHMT2 on OSCC progression and the potential mechanisms related to interleukin enhancer-binding factor 2 (ILF2). First of all, reverse transcription-quantitative PCR (RT-qPCR) and western blot examined the expression of SHMT2 and ILF2 in OSCC cells. Cell Counting Kit-8 (CCK-8) and colony formation assays appraised cell proliferation. Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) staining was to estimate the apoptotic rate of cells. Further, wound healing and transwell assays verified the migration and invasion of cells. Western blot was adopted to detect the expression of factors related to apoptosis, migration, and epithelial–mesenchymal transition (EMT). The possible interaction of SHMT2 and ILF2 was predicted by a Molecular INTeraction (MINT) and BioGRID databases and determined using co-immunoprecipitation (IP) assay. Subsequently, ILF2 was overexpressed to investigate whether SHMT2 regulated OSCC progression by binding to ILF2. Results implied that SHMT2 possessed increased expression in OSCC cells, and OSCC cell viability, migration, invasion, EMT were inhibited and apoptosis was potentiated after its silencing. ILF2 bound to SHMT2 and ILF2 expression was downregulated after SHMT2 silencing in OSCC cells. Importantly, ILF2 overexpression abolished the suppressive role of SHMT2 interference in the progression of OSCC. Collectively, SHMT2 could promote the progression of OSCC by binding to ILF2.

Upregulation of Endothelial DKK1 (Dickkopf 1) Promotes the Development of Pulmonary Hypertension Through the Sp1 (Specificity Protein 1)/SHMT2 (Serine Hydroxymethyltransferase 2) Pathway.

Pulmonary hypertension (PH) is a cancer-like proliferative disease, which has no curative treatment options. The dysfunction of pulmonary artery endothelial cells plays a key role in PH. DKK1 (Dickkopf 1) is a secretory glycoprotein that exerts proproliferative effects on tumor cells. In the present study, we aimed to identify the role and underlying mechanism of DKK1 in the development of PH, which still remain unclear. We found endothelial DKK1 expression was upregulated in serum and lung tissues obtained from patients with PH, mice with hypoxia-induced PH, and human pulmonary artery endothelial cells cultured under hypoxic conditions. Endothelium-specific DKK1-knockout (DKK1ECKO) mice significantly ameliorated hypoxia+Sugen5416 and hypoxia-induced PH. More importantly, neutralizing anti-DKK1 antibody treatment significantly attenuated established hypoxia+Sugen5416 PH. Results of proteome analysis of control and DKK1-knockdown human pulmonary artery endothelial cells identified a significantly differentially expressed protein, SHMT2 (serine hydroxymethyltransferase 2), a key metabolic enzyme in one-carbon metabolism, as a novel DKK1 target. DKK1 knockdown in human pulmonary artery endothelial cells cultured under hypoxic conditions decreased the cellular NADPH/NADP+ ratio, increased reactive oxygen species levels and the extent of mitochondrial DNA damage, and inhibited mitochondrial membrane hyperpolarization. In the context of this altered redox defense and mitochondrial disorder, DKK1 induced a proproliferative and antiapoptotic phenotype in endothelial cells. Furthermore, we confirmed that DKK1 regulated SHMT2 transcription through the AKT-Sp1 (specificity protein 1) signaling axis. Our data provide robust evidence and molecular explanations for the associations between DKK1, redox defense, mitochondrial disorders, and PH and reveal a novel target for PH treatment.

The role of SHMT2 in modulating lipid metabolism in hepatocytes via glycine-mediated mTOR activation.

Serine hydroxymethyltransferase 2 (SHMT2) converts serine into glycine in the mitochondrial matrix, transferring a methyl group to tetrahydrofolate. SHMT2 plays an important role in the maintenance of one-carbon metabolism. Previously, we found a negative correlation between the serine concentration and the progression of fatty liver disease (FLD). However, little is known about the role of SHMT2 in hepatic lipid metabolism. We established SHMT2 knockdown (KD) mouse primary hepatocytes using RNA interference to investigate the role of SHMT2 in lipid metabolism. SHMT2 KD hepatocytes showed decreased lipid accumulation with reduced glycine levels compared to the scramble cells, which was restored upon reintroducing SHMT2. SHMT2 KD hepatocytes showed downregulation of the mTOR/PPARɣ pathway with decreased gene expression related to lipogenesis and fatty acid uptake. Pharmacological activation of mTOR or PPARɣ overexpression blocked the inhibitory effect of SHMT2 KD on lipid accumulation. We also showed that glycine activated mTOR/PPARɣ signaling and identified glycine as a mediator of SHMT2-responsive lipid accumulation in hepatocytes. In conclusion, silencing SHMT2 in hepatocytes ameliorates lipid accumulation via the glycine-mediated mTOR/PPARɣ pathway. Our findings underscore the possibility of SHMT2 as a therapeutic target of FLD.

SHMT2 Drives the Progression of Colorectal Cancer by Regulating UHRF1 Expression.

Introduction Serine hydroxymethyltransferase 2 (SHMT2) has a critical role in serine-glycine metabolism to drive cancer cell proliferation. Yet, the function of SHMT2 in tumorigenesis, especially in human colorectal cancer (CRC) progression, remains largely unclear. Materials and Methods CRC and paired normal samples were collected in the Department of Colorectal Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, and assessed by real-time polymerase chain reaction (qPCR) analysis, western blot (WB), and immunohistochemistry (IHC). Moreover, SHMT2 expression in human CRC cells was identified by qPCR and WB. The CRC cell proliferation, migration, and invasion after SHMT2 knockdown were explored through in vitro and in vivo assays. mRNA-seq assays were used to investigate the underlying mechanisms behind the SHMT2 function. Results It was found that SHMT2 mRNA and protein were overexpressed in CRC tissue compared to the levels in normal mucosa. Positive expression of SHMT2 was significantly correlated with TNM stage and lymph node metastasis, and elevated expression of SHMT2 resulted as an independent prognostic factor in patients with CRC. SHMT2 knockdown impaired the proliferation of CRC in vitro and in vivo and induced cell cycle arrest by regulating UHRF1 expression. Conclusion Taken together, our findings reveal that UHRF1 is a novel target gene of SHMT2, which can be used as a potential therapeutic strategy for CRC therapy.

Induction of serine hydroxymethyltransferase 2 promotes tumorigenesis and metastasis in neuroblastoma.

High-risk neuroblastoma (NB) remains an extremely difficult subgroup to cure and is associated with MYCN amplification. Serine hydroxymethyltransferase 2 (SHMT2) regulates serine metabolism in a myc-dependent manner; it is upregulated in several cancers and is associated with tumor aggressiveness. Akt-2, an important regulator of MYCN via the PI3K/Akt pathway, induces metastatic potential in NB. The association between SHMT2 and PI3K/Akt in hepatocyte regeneration has been well established but its mechanistic interaction in cancer has yet to be clearly elucidated. Herein, we evaluated the exact role of SHMT2 on the PI3K/Akt pathway, in addition to NB tumorigenesis and metastatic potential in vitro. SHMT2 gene expression and overall survival (OS) were assessed. Two human NB cell lines were examined. SHMT2 silencing and overexpression were performed. The downstream effects were analyzed with immunoblotting, RT-qPCR and functional assays were performed. We found SHMT2 gene expression is associated with decreased OS and MYCN amplification. SHMT2 protein and mRNA expression are increased in MYCN-amplified cells. SHMT2 expression has a direct interaction with Akt-2 and MYCN. Induction of SHMT2 increased cellular proliferation, colony formation and cellular migration and SHMT2 expression was increased in metastatic NB cells. We conclude that SHMT2 regulates N-Myc via phosphorylation of Akt-2 and plays an important role in NB tumorigenesis by contributing to cell growth, migration, colony formation and metastasis in vitro.

Generation of SHMT2 knockout human embryonic stem cell line (WAe009-A-67) using CRISPR/Cas9 technique

Serine hydroxymethyltransferase 2 (SHMT2), a catalytic enzyme playing an important role in aerobic cellular respiration and mitochondrial metabolism, might be pivotal in self-renewal and differentiation of human pluripotent stem cells. Herein, we used the CRISPR/Cas9 editing system to construct a homozygous SHMT2 knockout (SHMT2-KO) human embryonic stem cell (hESC) line, exhibiting a normal karyotype, colony morphology, and high expression levels of pluripotent proteins. Furthermore, SHMT2 knockout did not impact the self-renewal ability or differentiation potential into three germ layers of hESCs. Accordingly, this cell line provides a valuable model for further assessing SHMT2 functions in human embryonic development.

SHMT2 inhibition disrupts the TCF3 transcriptional survival program in Burkitt lymphoma

AbstractBurkitt lymphoma (BL) is an aggressive lymphoma type that is currently treated by intensive chemoimmunotherapy. Despite the favorable clinical outcome for most patients with BL, chemotherapy-related toxicity and disease relapse remain major clinical challenges, emphasizing the need for innovative therapies. Using genome-scale CRISPR-Cas9 screens, we identified B-cell receptor (BCR) signaling, specific transcriptional regulators, and one-carbon metabolism as vulnerabilities in BL. We focused on serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in one-carbon metabolism. Inhibition of SHMT2 by either knockdown or pharmacological compounds induced anti-BL effects in vitro and in vivo. Mechanistically, SHMT2 inhibition led to a significant reduction of intracellular glycine and formate levels, which inhibited the mTOR pathway and thereby triggered autophagic degradation of the oncogenic transcription factor TCF3. Consequently, this led to a collapse of tonic BCR signaling, which is controlled by TCF3 and is essential for BL cell survival. In terms of clinical translation, we also identified drugs such as methotrexate that synergized with SHMT inhibitors. Overall, our study has uncovered the dependency landscape in BL, identified and validated SHMT2 as a drug target, and revealed a mechanistic link between SHMT2 and the transcriptional master regulator TCF3, opening up new perspectives for innovative therapies.

Serine hydroxymethyltransferase 2 expression promotes tumorigenesis in rhabdomyosarcoma with 12q13-q14 amplification.

The 12q13-q14 chromosomal region is recurrently amplified in 25% of fusion-positive (FP) rhabdomyosarcoma (RMS) cases and is associated with a poor prognosis. To identify amplified oncogenes in FP RMS, we compared the size, gene composition, and expression of 12q13-q14 amplicons in FP RMS with those of other cancer categories (glioblastoma multiforme, lung adenocarcinoma, and liposarcoma) in which 12q13-q14 amplification frequently occurs. We uncovered a 0.2 Mb region that is commonly amplified across these cancers and includes CDK4 and 6 other genes that are overexpressed in amplicon-positive samples. Additionally, we identified a 0.5 Mb segment that is only recurrently amplified in FP RMS and includes 4 genes that are overexpressed in amplicon-positive RMS. Among these genes, only serine hydroxymethyltransferase 2 (SHMT2) was overexpressed at the protein level in an amplicon-positive RMS cell line. SHMT2 knockdown in amplicon-positive RMS cells suppressed growth, transformation, and tumorigenesis, whereas overexpression in amplicon-negative RMS cells promoted these phenotypes. High SHMT2 expression reduced sensitivity of FP RMS cells to SHIN1, a direct SHMT2 inhibitor, but sensitized cells to pemetrexed, an inhibitor of the folate cycle. In conclusion, our study demonstrates that SHMT2 contributes to tumorigenesis in FP RMS and that SHMT2 amplification predicts differential response to drugs targeting this metabolic pathway.

Serine hydroxymethyltransferase 2: a novel target for human cancer therapy.

Serine and glycine are the primary sources of one-carbon units that are vital for cell proliferation. Their abnormal metabolism is known to be associated with cancer progression. As the key enzyme of serine metabolism, Serine Hydroxymethyltransferase 2 (SHMT2) has been a research hotspot in recent years. SHMT2 is a PLP-dependent tetrameric enzyme that catalyzes the reversible transition from serine to glycine, thus promoting the production of one-carbon units that are indispensable for cell growth and regulation of the redox and epigenetic states of cells. Under a hypoxic environment, SHMT2 can be upregulated and could promote the generation of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione for maintaining the redox balance. Accumulating evidence confirmed that SHMT2 facilitates cell proliferation and tumor growth and is tightly associated with poor prognosis. In this review, we present insights into the function and research development of SHMT2 and summarize the possible molecular mechanisms of SHMT2 in promoting tumor growth,in the hope that it could provide clues to more effective clinical treatment of cancer.