DNA methylation of sarcosine dehydrogenase (SARDH) loci as a prognosticator for renal cell carcinoma.

DNA methylation plays an important role in the genesis and progression of tumor diseases. To identify new DNA methylation markers possibly associated with the clinical characteristics of renal cell carcinoma (RCC), we investigated loci in the sarcosine dehydrogenase (SARDH) gene. SARDH is involved in the metabolism of the glycine­derivative sarcosine and is closely linked through a functional control loop. Statistical evaluation of methylation data and clinical characteristics of patients showed that kidney tumors with clinically aggressive features such as a high tumor stage, positive lymph nodes, distant metastases or a previously advanced tumor status exhibited significantly lower methylation of a locus in the SARDH gene. Moreover, SARDH methylation was found to be a significant prognostic factor for recurrence­free survival in RCC patients showing statistical independence from the clinical prognosticators, grade, stage and state of metastasis. In conclusion, the methylation status of the SARDH­CGI was identified as an independent prognostic candidate marker for RCC.

Alteration of the tumor suppressor SARDH in sporadic colorectal cancer: A functional and transcriptome profiling-based study.

Sporadic colorectal cancer (sCRC) is one of the leading causes of cancer death worldwide. As a highly heterogeneous complex disease, the currently reported classical genetic markers for sCRC, including APC, KRAS, BRAF, and TP53 gene mutations and epigenetic alterations, can explain only some sCRC patients. Here, we first reported a deleterious c.551C>T mutation in SARDH in sCRC. SARDH was identified as a novel tumor suppressor gene and was abnormally decreased in sCRC at both the transcriptional and the translational level. SARDH mRNA levels were also down-regulated in oesophageal cancer, lung cancer, liver cancer, and pancreatic cancer in the TCGA database. SARDH overexpression inhibited the proliferation, migration, and invasion of CRC cell lines, whereas its depletion improved these processes. SARDH overexpression was down-regulated in multiple pathways, especially in the chemokine pathway. The SARDH transcript level was positively correlated with the methylation states of CXCL1 and CCL20. Therefore, we concluded that SARDH depletion is involved in the development of sCRC.

Analysis of polymorphisms of genes associated with folate-mediated one-carbon metabolism and neural tube defects in Chinese Han Population.

BACKGROUND: The polymorphism of genes involved in folate-mediated one-carbon metabolism may be a risk factor for neural tube defects (NTDs). In the present study, we aimed to investigate the single nucleotide polymorphisms (SNPs) of the genes BHMT, CUBN, FTCD, GAMT, GART, SARDH, SHMT1, and MUT, and their effect on NTDs in the Chinese Han population. METHODS: A total of 270 NTDs cases and 192 controls were enrolled in this study. The SNPs were analyzed with the next-generation sequencing method. The folate levels of brain tissues from 113 available NTDs cases and 123 available controls were measured. RESULTS: Next-generation sequencing identified 818 single nucleotide variants, including 214 SNPs used for further analysis. Statistical analysis showed that two independent SNP loci, rs2797840 and rs2073817 in SARDH, may be associated with the susceptibility of NTDs. Specifically, the minor allele G of rs2797840 was significantly associated with NTDs risk in spina bifida subgroup (p value = 0.0348). For subjects whose folate content was measured, the protective allele G of rs2797840 was significantly associated with increased folate content of brain. rs2797840 is within several ENCODE regulatory regions, indicating this SNPs may influence expression of SARDH. CONCLUSION: The SNPs rs2797840 and rs2073817 in SARDH may serve as an indicator for the occurrence of NTDs in the Chinese Han population, and rs2797840 may also be an indicator for folate content of brain.

TMEFF2 and SARDH cooperate to modulate one-carbon metabolism and invasion of prostate cancer cells.

BACKGROUND: The transmembrane protein with epidermal growth factor and two follistatin motifs, TMEFF2, has been implicated in prostate cancer but its role in this disease is unclear. We recently demonstrated that the tumor suppressor role of TMEFF2 correlates, in part, with its ability to interact with sarcosine dehydrogenase (SARDH) and modulate sarcosine level. TMEFF2 overexpression inhibits sarcosine-induced invasion. Here, we further characterize the functional interaction between TMEFF2 and SARDH and their link with one-carbon (1-C) metabolism and invasion. METHODS: RNA interference was used to study the effect of SARDH and/or TMEFF2 knockdown (KD) in invasion, evaluated using Boyden chambers. The dependence of invasion on 1-C metabolism was determined by examining sensitivity to methotrexate. Real-time PCR and Western blot of subcellular fractions were used to study the effect of SARDH KD or TMEFF2 KD on expression of enzymes involved in one-carbon (1-C) metabolism and on TMEFF2 expression and localization. Protein interactions were analyzed by mass spectrometry. Cell viability and proliferation were measured by cell counting and MTT analysis. RESULTS: While knocking down SARDH affects TMEFF2 subcellular localization, this effect is not responsible for the increased invasion observed in SARDH KD cells. Importantly, SARDH and/or TMEFF2 KD promote increased cellular invasion, sensitize the cell to methotrexate, render the cell resistant to invasion induced by sarcosine, a metabolite from the folate-mediated 1-C metabolism pathway, and affect the expression level of enzymes involved in that pathway. CONCLUSIONS: Our findings define a role for TMEFF2 and the folate-mediated 1-C metabolism pathway in modulating cellular invasion.

The role of sarcosine metabolism in prostate cancer progression.

Metabolomic profiling of prostate cancer (PCa) progression identified markedly elevated levels of sarcosine (N-methyl glycine) in metastatic PCa and modest but significant elevation of the metabolite in PCa urine. Here, we examine the role of key enzymes associated with sarcosine metabolism in PCa progression. Consistent with our earlier report, sarcosine levels were significantly elevated in PCa urine sediments compared to controls, with a modest area under the receiver operating characteristic curve of 0.71. In addition, the expression of sarcosine biosynthetic enzyme, glycine N-methyltransferase (GNMT), was elevated in PCa tissues, while sarcosine dehydrogenase (SARDH) and pipecolic acid oxidase (PIPOX), which metabolize sarcosine, were reduced in prostate tumors. Consistent with this, GNMT promoted the oncogenic potential of prostate cells by facilitating sarcosine production, while SARDH and PIPOX reduced the oncogenic potential of prostate cells by metabolizing sarcosine. Accordingly, addition of sarcosine, but not glycine or alanine, induced invasion and intravasation in an in vivo PCa model. In contrast, GNMT knockdown or SARDH overexpression in PCa xenografts inhibited tumor growth. Taken together, these studies substantiate the role of sarcosine in PCa progression.

Mutations in the sarcosine dehydrogenase gene in patients with sarcosinemia.

Sarcosinemia is an autosomal recessive metabolic trait manifested by relatively high concentrations of sarcosine in blood and urine. Sarcosine is a key intermediate in 1-carbon metabolism and under normal circumstances is converted to glycine by the enzyme sarcosine dehydrogenase. We encountered six families from two different descents (French and Arab), each with at least one individual with elevated levels of sarcosine in blood and urine. Using the "candidate gene approach" we sequenced the gene encoding sarcosine dehydrogenase (SARDH), which plays an important role in the conversion of sarcosine to glycine, and found four different mutations (P287L, V71F, R723X, R514X) in three patients. In an additional patient, we found a uniparental disomy in the region of SARDH gene. In two other patients, we did not find any mutations in this gene. We have shown for the first time that mutations in the SARDH gene are associated with sarcosinemia. In addition, our results indicate that other genes are most probably involved in the pathogenesis of this condition.

Folate network genetic variation, plasma homocysteine, and global genomic methylation content: a genetic association study.

BACKGROUND: Sequence variants in genes functioning in folate-mediated one-carbon metabolism are hypothesized to lead to changes in levels of homocysteine and DNA methylation, which, in turn, are associated with risk of cardiovascular disease. METHODS: 330 SNPs in 52 genes were studied in relation to plasma homocysteine and global genomic DNA methylation. SNPs were selected based on functional effects and gene coverage, and assays were completed on the Illumina Goldengate platform. Age-, smoking-, and nutrient-adjusted genotype--phenotype associations were estimated in regression models. RESULTS: Using a nominal P ≤ 0.005 threshold for statistical significance, 20 SNPs were associated with plasma homocysteine, 8 with Alu methylation, and 1 with LINE-1 methylation. Using a more stringent false discovery rate threshold, SNPs in FTCD, SLC19A1, and SLC19A3 genes remained associated with plasma homocysteine. Gene by vitamin B-6 interactions were identified for both Alu and LINE-1 methylation, and epistatic interactions with the MTHFR rs1801133 SNP were identified for the plasma homocysteine phenotype. Pleiotropy involving the MTHFD1L and SARDH genes for both plasma homocysteine and Alu methylation phenotypes was identified. CONCLUSIONS: No single gene was associated with all three phenotypes, and the set of the most statistically significant SNPs predictive of homocysteine or Alu or LINE-1 methylation was unique to each phenotype. Genetic variation in folate-mediated one-carbon metabolism, other than the well-known effects of the MTHFR c.665C>T (known as c.677 C>T, rs1801133, p.Ala222Val), is predictive of cardiovascular disease biomarkers.

The tumor suppressor activity of the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) correlates with its ability to modulate sarcosine levels.

The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed in brain and prostate and overexpressed in prostate cancer, but its role in this disease is unclear. Several studies have suggested that TMEFF2 plays a role in suppressing the growth and invasive potential of human cancer cells, whereas others suggest that the shed portion of TMEFF2, which lacks the cytoplasmic region, has a growth-promoting activity. Here we show that TMEFF2 has a dual mode of action. Ectopic expression of wild-type full-length TMEFF2 inhibits soft agar colony formation, cellular invasion, and migration and increases cellular sensitivity to apoptosis. However, expression of the ectodomain portion of TMEFF2 increases cell proliferation. Using affinity chromatography and mass spectrometry, we identify sarcosine dehydrogenase (SARDH), the enzyme that converts sarcosine to glycine, as a TMEFF2-interacting protein. Co-immunoprecipitation and immunofluorescence analysis confirms the interaction of SARDH with full-length TMEFF2. The ectodomain does not bind to SARDH. Moreover, expression of the full-length TMEFF2 but not the ectodomain results in a decreased level of sarcosine in the cells. These results suggest that the tumor suppressor activity of TMEFF2 requires the cytoplasmic/transmembrane portion of the protein and correlates with its ability to bind to SARDH and to modulate the level of sarcosine.