Cells in the Polyaneuploid Cancer Cell State are Pro-Metastatic.

There remains a large need for a greater understanding of the metastatic process within the prostate cancer field. Our research aims to understand the adaptive - ergo potentially metastatic - responses of cancer to changing microenvironments. Emerging evidence has implicated a role of the Polyaneuploid Cancer Cell (PACC) state in metastasis, positing the PACC state as capable of conferring metastatic competency. Mounting in vitro evidence supports increased metastatic potential of cells in the PACC state. Additionally, our recent retrospective study of prostate cancer patients revealed that PACC presence in the prostate at the time of radical prostatectomy was predictive of future metastatic progression. To test for a causative relationship between PACC state biology and metastasis, we leveraged a novel method designed for flow-cytometric detection of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in subcutaneous, caudal artery, and intracardiac mouse models of metastasis. This approach provides both quantitative and qualitative information about the number and PACC-status of recovered CTCs and DTCs. Collating data from all models, we found that 74% of recovered CTCs and DTCs were in the PACC state. In vivo colonization assays proved PACC populations can regain proliferative capacity at metastatic sites following dormancy. Additional direct and indirect mechanistic in vitro analyses revealed a PACC-specific partial Epithelial-to-Mesenchymal-Transition phenotype and a pro-metastatic secretory profile, together providing preliminary evidence that PACCs are mechanistically linked to metastasis.

Epithelial-to-mesenchymal transition and NF-kB pathways are promoted by a mutant form of DDB2, unable to bind PCNA, in UV-damaged human cells.

BACKGROUND: DNA-Damaged Binding protein 2 (DDB2) is a protein involved in the early step of Nucleotide Excision Repair. Recently, it has been reported that DDB2 is involved in epithelial-to-mesenchymal transition (EMT), key process in tumour invasiveness and metastasis formation. However, its role is not completely known. METHODS: Boyden chamber and cell adhesion assays, and ICELLigence analysis were performed to detect HEK293 adhesion and invasion. Western blotting and gelatine zymography techniques were employed to assess the EMT protein levels and MMP enzymatic activity. Immunofluorescence analysis and pull-down assays facilitated the detection of NF-kB sub-cellular localization and interaction. RESULTS: We have previously demonstrated that the loss of DDB2-PCNA binding favours genome instability, and increases cell proliferation and motility. Here, we have investigated the phenotypic and molecular EMT-like changes after UV DNA damage, in HEK293 clones stably expressing DDB2Wt protein or a mutant form unable to interact with PCNA (DDB2PCNA-), as well as in HeLa cells transiently expressing the same DDB2 constructs. Cells expressing DDB2PCNA- showed morphological modifications along with a reduced expression of E-cadherin, an increased activity of MMP-9 and an improved ability to migrate, in concomitance with a significant upregulation of EMT-associated Transcription Factors (TFs), whose expression has been reported to favour tumour invasion. We observed a higher expression of c-Myc oncogene, NF-kB, both regulating cell proliferation and metastatic process, as well as ZEB1, a TF significantly associated with tumorigenic potential and cell migratory ability. Interestingly, a novel interaction of DDB2 with NF-kB was detected and found to be increased in cells expressing the DDB2PCNA-, suggesting a direct modulation of NF-kB by DDB2. CONCLUSION: These results highlight the role of DDB2-PCNA interaction in counteracting EMT since DDB2PCNA- protein induces in HEK293 transformed cells a gain of function contributing to the acquisition of a more aggressive phenotype.

The Role of Phosphatidylethanolamine N-Methyltransferase (PEMT) and Its Waist-Hip-Ratio-Associated Locus rs4646404 in Obesity-Related Metabolic Traits and Liver Disease.

In previous genome-wide association studies (GWAS), genetic loci associated with obesity and impaired fat distribution (FD) have been identified. In the present study, we elucidated the role of the PEMT gene, including the waist-hip-ratio-associated single nucleotide polymorphism rs4646404, and its influence on obesity-related metabolic traits. DNA from 2926 metabolically well-characterized subjects was used for genotyping. PEMT expression was analyzed in paired visceral (vis) and subcutaneous (sc) adipose tissue (AT) from a subset of 574 individuals. Additionally, PEMT expression was examined in vis, sc AT and liver tissue in a separate cohort of 64 patients with morbid obesity and liver disease. An in vitro Pemt knockdown was conducted in murine epididymal and inguinal adipocytes. Our findings highlight tissue-specific variations in PEMT mRNA expression across the three studied tissues. Specifically, vis PEMT mRNA levels correlated significantly with T2D and were implicated in the progression of non-alcoholic steatohepatitis (NASH), in contrast to liver tissue, where no significant associations were found. Moreover, sc PEMT expression showed significant correlations with several anthropometric- and metabolic-related parameters. The rs4646404 was associated with vis AT PEMT expression and also with diabetes-related traits. Our in vitro experiments supported the influence of PEMT on adipogenesis, emphasizing its role in AT biology. In summary, our data suggest that PEMT plays a role in regulating FD and has implications in metabolic diseases.

PEMT rs7946 Polymorphism and Sex Modify the Effect of Adequate Dietary Choline Intake on the Risk of Hepatic Steatosis in Older Patients with Metabolic Disorders.

In humans, PEMT rs7946 polymorphism exerts sex-specific effects on choline requirement and hepatic steatosis (HS) risk. Few studies have explored the interaction effect of the PEMT rs7946 polymorphism and sex on the effect of adequate choline intake on HS risk. In this cross-sectional study, we investigated the association between PEMT polymorphism and adequate choline intake on HS risk. We enrolled 250 older patients with metabolic disorders with (n = 152) or without (n = 98; control) ultrasonically diagnosed HS. An elevated PEMT rs7946 A allele level was associated with a lower HS risk and body mass index in both men and women. Dietary choline intake-assessed using a semiquantitative food frequency questionnaire-was associated with reduced obesity in men only (p for trend < 0.05). ROC curve analysis revealed that the cutoff value of energy-adjusted choline intake for HS diagnosis was 448 mg/day in women (AUC: 0.62; 95% CI: 0.57-0.77) and 424 mg/day in men (AUC: 0.63, 95% CI: 0.57-0.76). In women, GG genotype and high choline intake (>448 mg/day) were associated with a 79% reduction in HS risk (adjusted OR: 0.21; 95% CI: 0.05-0.82); notably, GA or AA genotype was associated with a reduced HS risk regardless of choline intake (p < 0.05). In men, GG genotype and high choline intake (>424 mg/day) were associated with a 3.7-fold increase in HS risk (OR: 3.7; 95% CI: 1.19-11.9). Further adjustments for a high-density lipoprotein level and body mass index mitigated the effect of choline intake on HS risk. Current dietary choline intake may be inadequate for minimizing HS risk in postmenopausal Taiwanese women carrying the PEMT rs7946 GG genotype. Older men consuming more than the recommended amount of choline may have an increased risk of nonalcoholic fatty liver disease; this risk is mediated by a high-density lipoprotein level and obesity.

PEMT Mediates Hepatitis C Virus-Induced Steatosis, Explains Genotype-Specific Phenotypes and Supports Virus Replication.

The hepatitis C virus (HCV) relies on cellular lipid pathways for virus replication and also induces liver steatosis, but the mechanisms involved are not clear. We performed a quantitative lipidomics analysis of virus-infected cells by combining high-performance thin-layer chromatography (HPTLC) and mass spectrometry, using an established HCV cell culture model and subcellular fractionation. Neutral lipid and phospholipids were increased in the HCV-infected cells; in the endoplasmic reticulum there was an ~four-fold increase in free cholesterol and an ~three-fold increase in phosphatidyl choline (p < 0.05). The increase in phosphatidyl choline was due to the induction of a non-canonical synthesis pathway involving phosphatidyl ethanolamine transferase (PEMT). An HCV infection induced expression of PEMT while knocking down PEMT with siRNA inhibited virus replication. As well as supporting virus replication, PEMT mediates steatosis. Consistently, HCV induced the expression of the pro-lipogenic genes SREBP 1c and DGAT1 while inhibiting the expression of MTP, promoting lipid accumulation. Knocking down PEMT reversed these changes and reduced the lipid content in virus-infected cells. Interestingly, PEMT expression was over 50% higher in liver biopsies from people infected with the HCV genotype 3 than 1, and three times higher than in people with chronic hepatitis B, suggesting that this may account for genotype-dependent differences in the prevalence of hepatic steatosis. PEMT is a key enzyme for promoting the accumulation of lipids in HCV-infected cells and supports virus replication. The induction of PEMT may account for virus genotype specific differences in hepatic steatosis.

Nutritional Genomics in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic condition associated with genetic and environmental factors in which fat abnormally accumulates in the liver. NAFLD is epidemiologically associated with obesity, type 2 diabetes, and dyslipidemia. Environmental factors, such as physical inactivity and an unbalanced diet, interact with genetic factors, such as epigenetic mechanisms and polymorphisms for the genesis and development of the condition. Different genetic polymorphisms seem to be involved in this context, including variants in PNPLA3, TM6SF2, PEMT, and CHDH genes, playing a role in the disease's susceptibility, development, and severity. From carbohydrate intake and weight loss to omega-3 supplementation and caloric restriction, different dietary and nutritional factors appear to be involved in controlling the onset and progression of NAFLD conditions influencing metabolism, gene, and protein expression. The polygenic risk score represents a sum of trait-associated alleles carried by an individual and seems to be associated with NAFLD outcomes depending on the dietary context. Understanding the exact extent to which lifestyle interventions and genetic predispositions can play a role in the prevention and management of NAFLD can be crucial for the establishment of a personalized and integrative approach to patients.

Genetic Variants in One-Carbon Metabolism and Their Effects on DHA Biomarkers in Pregnant Women: A Post-Hoc Analysis.

The delivery of docosahexanoic acid (DHA) to the fetus is dependent on maternal one-carbon metabolism, as the latter supports the hepatic synthesis and export of a DHA-enriched phosphatidylcholine molecule via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. The following is a post-hoc analysis of a choline intervention study that sought to investigate whether common variants in one-carbon metabolizing genes associate with maternal and/or fetal blood biomarkers of DHA status. Pregnant women entering their second trimester were randomized to consume, until delivery, either 25 (n = 15) or 550 (n = 15) mg choline/d, and the effects of genetic variants in the PEMT, BHMT, MTHFD1, and MTHFR genes on DHA status were examined. Variant (vs. non-variant) maternal PEMT rs4646343 genotypes tended to have lower maternal RBC DHA (% total fatty acids) throughout gestation (6.9% vs. 7.4%; main effect, p = 0.08) and lower cord RBC DHA at delivery (7.6% vs. 8.4%; main effect, p = 0.09). Conversely, variant (vs. non-variant) maternal MTHFD1 rs2235226 genotypes exhibited higher cord RBC DHA (8.3% vs. 7.3%; main effect, p = 0.0003) and higher cord plasma DHA (55 vs. 41 µg/mL; main effect, p = 0.05). Genotype tended to interact with maternal choline intake (p < 0.1) to influence newborn DHA status for PEMT rs4646343 and PEMT rs7946. These data support the need to consider variants in one-carbon metabolic genes in studies assessing DHA status and requirements during pregnancy.

Phosphatidylethanolamine N-Methyltransferase Knockout Modulates Metabolic Changes in Aging Mice.

Phospholipid metabolism, including phosphatidylcholine (PC) biosynthesis, is crucial for various biological functions and is associated with longevity. Phosphatidylethanolamine N-methyltransferase (PEMT) is a protein that catalyzes the biosynthesis of PC, the levels of which change in various organs such as the brain and kidneys during aging. However, the role of PEMT for systemic PC supply is not fully understood. To address how PEMT affects aging-associated energy metabolism in tissues responsible for nutrient absorption, lipid storage, and energy consumption, we employed NMR-based metabolomics to study the liver, plasma, intestine (duodenum, jejunum, and ileum), brown/white adipose tissues (BAT and WAT), and skeletal muscle of young (9-10 weeks) and old (91-132 weeks) wild-type (WT) and PEMT knockout (KO) mice. We found that the effect of PEMT-knockout was tissue-specific and age-dependent. A deficiency of PEMT affected the metabolome of all tissues examined, among which the metabolome of BAT from both young and aged KO mice was dramatically changed in comparison to the WT mice, whereas the metabolome of the jejunum was only slightly affected. As for aging, the absence of PEMT increased the divergence of the metabolome during the aging of the liver, WAT, duodenum, and ileum and decreased the impact on skeletal muscle. Overall, our results suggest that PEMT plays a previously underexplored, critical role in both aging and energy metabolism.

PEMT variants are associated with nonsyndromic cleft lip with or without cleft palate in Chile.

Aim: To assess the association between PEMT variants and nonsyndromic cleft lip with or without cleft palate in Chile and the effects of these variants on global DNA methylation. Subjects & methods: The authors obtained genotypes for nine variants from 247 cases and 453 controls for genotype-phenotype associations. The effect of significant polymorphisms on global DNA methylation (percentage of long interspersed element-1 methylation) was evaluated in a subsample of 95 controls. Results: After multiple comparison corrections, variants rs7649 and rs4646409 were associated with nonsyndromic cleft lip with or without cleft palate. Carriers of risk alleles presented lower DNA methylation levels than noncarriers. Conclusion: According to functional analysis for risk variants from previous reports, the authors infer that a decrease of methyl group availability is occurring in affected subjects.

This study evaluated if variants in the gene named PEMT confers an increased risk for nonsyndromic cleft lip with or without cleft palate in Chile and its possible effects on methylation of DNA, a variable linked to gene expression modulation. The study found that the variants recognized as rs7649 and rs4646409 increase the risk of nonsyndromic cleft lip with or without cleft palate in the Chilean population and decrease DNA methylation. The authors conclude that this gene may be involved in this birth defect. New studies are needed to confirm the relation between this condition and DNA methylation mediated by these genetic variants.

Associations of plasma betaine, plasma choline, choline intake, and MTHFR polymorphism (rs1801133) with anthropometric parameters of healthy adults are sex-dependent.

BACKGROUND: Choline and its metabolites apppear to have relationships with body mass index (BMI), body fat, and body weight, but the research results have proved inconsistent. We thus investigated the associations of plasma levels of trimethylamine N-oxide (TMAO), choline, and betaine with anthropometric measurements, including modulatory effects of genetics and diet. METHODS: The study was performed on a group of 421 adults, aged 20-40 years, who had been recruited in Poland. Plasma concentrations of choline, betaine, and TMAO were determined using reverse-phase ultra-high-performance liquid chromatography electrospray ionisation mass spectrometry. The following polymorphisms were genotyped using TaqMan probes: rs180113 (MTHFR), rs70991108 (DHFR), rs2236225 (MTHFD1), and rs7946 and rs12325817 (PEMT). We employed multivariate linear regression to examine the associations between anthropometric measurements, one-carbon metabolism metabolites, and genotypes. RESULTS: Higher plasma choline was associated with higher BMI (ß = 0.17; p < 0.01), body weight (ß = 0.11; p < 0.05), body fat mass (FM) (ß = 0.10; p < 0.05), and waist circumference (WC) (ß = 0.14; p < 0.01), whereas higher choline intake was associated with lower body FM (ß = -0.14; p < 0.01) and lower WC (ß = -0.12; p < 0.01). After stratification by sex, plasma betaine was found to be associated with lower BMI (ß = -0.20; p < 0.05) and body weight (ß = -0.16; p < 0.05) in men only, whereas choline intake was associated with lower body FM (ß = -0.19; p < 0.05) and waist-to-hip ratio (WHR) (ß = -0.19; p < 0.05) and MTHFR CC genotype was associated with WHR (ß = 0.15; p < 0.05) in women only. CONCLUSIONS: Higher plasma betaine and higher dietary choline are associated with lower FM and body weight, whereas higher plasma choline is positively associated with body weight status and adiposity. Moreover, these associations appear to be sex-specific.

Prenatal choline supplementation improves biomarkers of maternal docosahexaenoic acid (DHA) status among pregnant participants consuming supplemental DHA: a randomized controlled trial.

BACKGROUND: Dietary methyl donors (e.g., choline) support the activity of the phosphatidylethanolamine N-methyltransferase (PEMT) pathway, which generates phosphatidylcholine (PC) molecules enriched in DHA that are exported from the liver and made available to extrahepatic tissues. OBJECTIVES: This study investigated the effect of prenatal choline supplementation on biomarkers of DHA status among pregnant participants consuming supplemental DHA. METHODS: Pregnant participants (n = 30) were randomly assigned to receive supplemental choline intakes of 550 mg/d [500 mg/d d0-choline + 50 mg/d deuterium-labeled choline (d9-choline); intervention] or 25 mg/d (25 mg/d d9-choline; control) from gestational week (GW) 12-16 until delivery. All participants received a daily 200-mg DHA supplement and consumed self-selected diets. Fasting blood samples were obtained at baseline, GW 20-24, and GW 28-32; maternal/cord blood was obtained at delivery. Mixed-effects linear models were used to assess the impact of prenatal choline supplementation on maternal and newborn DHA status. RESULTS: Choline supplementation (550 vs. 25 mg/d) did not achieve a statistically significant intervention × time interaction for RBC PC-DHA (P = 0.11); a significant interaction was observed for plasma PC-DHA and RBC total DHA, with choline supplementation yielding higher levels (+32-38% and +8-11%, respectively) at GW 28-32 (P < 0.05) and delivery (P < 0.005). A main effect of choline supplementation on plasma total DHA was also observed (P = 0.018); its interaction with time was not significant (P = 0.068). Compared with controls, the intervention group exhibited higher (P = 0.007; main effect) plasma enrichment of d3-PC (d3-PC/total PC). Moreover, the ratio of d3-PC to d9-PC was higher (+50-67%; P < 0.001) in the choline intervention arm (vs. control) at GW 20-24, GW 28-32, and delivery. CONCLUSIONS: Prenatal choline supplementation improves hepatic DHA export and biomarkers of DHA status by bolstering methyl group supply for PEMT activity among pregnant participants consuming supplemental DHA. This trial is registered at www.clinicaltrials.gov as NCT03194659.

Lower plasma glutathione, choline, and betaine concentrations are associated with fatty liver in postmenopausal women.

Postmenopausal women are at high risk of hepatic steatosis, which may be associated one-carbon metabolism (OCM) abnormalities. We hypothesized that lower folate, choline, betaine, and glutathione (GSH) concentrations but higher total homocysteine and trimethylamine N-oxide concentrations are associated with fatty liver (FL) in postmenopausal women. We aimed to identify relationships between OCM and nonalcoholic fatty liver disease biomarkers in postmenopausal women. A total of 131 postmenopausal women participated in this study and were stratified by the incidence of FL based on the hepatic steatosis index (HSI). Food intake was evaluated using dietary records. Aspartate aminotransferase and alanine aminotransferase concentrations in serum were measured using the colorimetric method. Total homocysteine and GSH concentrations in plasma were measured using high-performance liquid chromatography. Folate and phosphatidylcholine (PC) concentrations were determined in red blood cells using an enzyme-linked immunosorbent assay. Other OCM biomarkers concentrations were measured using the isotope dilution analysis. Women with FL (HSI > 36) had lower GSH, choline, and betaine concentrations than women without FL (HSI < 36). Higher HSI level was negatively correlated with betaine and PC and positively correlated with plasma choline/betaine ratio. Lower GSH and higher carnitine concentrations in the blood are associated with an increased risk of FL. MTHFR (rs180130) T-allele carriers had lower levels of GSH than the CC homozygotes. Postmenopausal women with FL have lower GSH, choline, and betaine concentrations, which may play a role in fat accumulation in the liver. It seems important to consider the dietary intakes of these nutrients in postmenopausal women.

Epithelial plasticity in COPD results in cellular unjamming due to an increase in polymerized actin.

The airway epithelium is subjected to insults such as cigarette smoke (CS), a primary cause of chronic obstructive pulmonary disease (COPD) and serves as an excellent model to study cell plasticity. Here, we show that both CS-exposed and COPD-patient derived epithelia (CHBE) display quantitative evidence of cellular plasticity, with loss of specialized apical features and a transcriptional profile suggestive of partial epithelial-to-mesenchymal transition (pEMT), albeit with distinct cell motion indicative of cellular unjamming. These injured/diseased cells have an increased fraction of polymerized actin, due to loss of the actin-severing protein cofilin-1. We observed that decreasing polymerized actin restores the jammed state in both CHBE and CS-exposed epithelia, indicating that the fraction of polymerized actin is critical in unjamming the epithelia. Our kinetic energy spectral analysis suggests that loss of cofilin-1 results in unjamming, similar to that seen with both CS exposure and in CHBE cells. The findings suggest that in response to chronic injury, although epithelial cells display evidence of pEMT, their movement is more consistent with cellular unjamming. Inhibitors of actin polymerization rectify the unjamming features of the monolayer. This article has an associated First Person interview with the first author of the paper.

SLUG-related partial epithelial-to-mesenchymal transition is a transcriptomic prognosticator of head and neck cancer survival.

Partial epithelial-to-mesenchymal transition (pEMT) contributes to cellular heterogeneity that is associated with nodal metastases and unfavorable clinical parameters in head and neck squamous cell carcinomas (HNSCCs). We developed a single-cell RNA sequencing signature-based pEMT quantification through cell type-dependent deconvolution of bulk RNA sequencing and microarray data combined with single-sample scoring of molecular phenotypes (Singscoring). Clinical pEMT-Singscores served as molecular classifiers in multivariable Cox proportional hazard models and high scores prognosticated poor overall survival and reduced response to irradiation as independent parameters in large HNSCC cohorts [The Cancer Genome Atlas (TCGA), MD Anderson Cancer Centre (MDACC), Fred Hutchinson Cancer Research Center (FHCRC)]. Differentially expressed genes confirmed enhanced cell motility and reduced oxidative phosphorylation and epithelial differentiation in pEMThigh patients. In patients and cell lines, the EMT transcription factor SLUG correlated most strongly with pEMT-Singscores and promoted pEMT, enhanced invasion, and resistance to irradiation in vitro. SLUG protein levels in HNSCC predicted disease-free survival, and its peripheral expression at the interphase to the tumor microenvironment was significantly increased in relapsing patients. Hence, pEMT-Singscores represent a novel risk predictor for HNSCC stratification regarding clinical outcome and therapy response that is partly controlled by SLUG.

Identification of Prognostic Biomarkers Originating From the Tumor Stroma of Betel Quid-Associated Oral Cancer Tissues.

BACKGROUND: Partial epithelial-mesenchymal transition (p-EMT) is a distinct clinicopathological feature prevalent in oral cavity tumors of The Cancer Genome Atlas. Located at the invasion front, p-EMT cells require additional support from the tumor stroma for collective cell migration, including track clearing, extracellular matrix remodeling and immune evasion. The pathological roles of otherwise nonmalignant cancer-associated fibroblasts (CAFs) in cancer progression are emerging. METHODS: Gene set enrichment analysis was used to reveal differentially enriched genes and molecular pathways in OC3 and TW2.6 xenograft tissues, representing mesenchymal and p-EMT tumors, respectively. R packages of genomic data science were executed for statistical evaluations and data visualization. Immunohistochemistry and Alcian blue staining were conducted to validate the bioinformatic results. Univariate and multivariate Cox proportional hazards models were performed to identify covariates significantly associated with overall survival in clinical datasets. Kaplan-Meier curves of estimated overall survival were compared for statistical difference using the log-rank test. RESULTS: Compared to mesenchymal OC3 cells, tumor stroma derived from p-EMT TW2.6 cells was significantly enriched in microvessel density, tumor-excluded macrophages, inflammatory CAFs, and extracellular hyaluronan deposition. By translating these results to clinical transcriptomic datasets of oral cancer specimens, including the Puram single-cell RNA-seq cohort comprising ~6000 cells, we identified the expression of stromal TGFBI and HYAL1 as independent poor and protective biomarkers, respectively, for 40 Taiwanese oral cancer tissues that were all derived from betel quid users. In The Cancer Genome Atlas, TGFBI was a poor marker not only for head and neck cancer but also for additional six cancer types and HYAL1 was a good indicator for four tumor cohorts, suggesting common stromal effects existing in different cancer types. CONCLUSIONS: As the tumor stroma coevolves with cancer progression, the cellular origins of molecular markers identified from conventional whole tissue mRNA-based analyses should be cautiously interpreted. By incorporating disease-matched xenograft tissue and single-cell RNA-seq results, we suggested that TGFBI and HYAL1, primarily expressed by stromal CAFs and endothelial cells, respectively, could serve as robust prognostic biomarkers for oral cancer control.

Reduced Zeb1 Expression in Prostate Cancer Cells Leads to an Aggressive Partial-EMT Phenotype Associated with Altered Global Methylation Patterns.

Prostate cancer is the most common cancer in American men and the second leading cause of cancer-related death. Most of these deaths are associated with metastasis, a process involving the epithelial-to-mesenchymal (EMT) transition. Furthermore, growing evidence suggests that partial-EMT (p-EMT) may lead to more aggressive disease than complete EMT. In this study, the EMT-inducing transcription factor Zeb1 was knocked down in mesenchymal PC-3 prostate cancer cells (Zeb1KD) and resulting changes in cellular phenotype were assessed using protein and RNA analysis, invasion and migration assays, cell morphology assays, and DNA methylation chip analysis. Inducible knockdown of Zeb1 resulted in a p-EMT phenotype including co-expression of epithelial and mesenchymal markers, a mixed epithelial/mesenchymal morphology, increased invasion and migration, and enhanced expression of p-EMT markers relative to PC-3 mesenchymal controls (p ≤ 0.05). Treatment of Zeb1KD cells with the global de-methylating drug 5-azacytidine (5-aza) mitigated the observed aggressive p-EMT phenotype (p ≤ 0.05). DNA methylation chip analysis revealed 10 potential targets for identifying and/or targeting aggressive p-EMT prostate cancer in the future. These findings provide a framework to enhance prognostic and/or therapeutic options for aggressive prostate cancer in the future by identifying new p-EMT biomarkers to classify patients with aggressive disease who may benefit from 5-aza treatment.

Choline metabolome response to prenatal choline supplementation across pregnancy: A randomized controlled trial.

Pregnancy places a unique stress upon choline metabolism, requiring adaptations to support both maternal and fetal requirements. The impact of pregnancy and prenatal choline supplementation on choline and its metabolome in free-living, healthy adults is relatively uncharacterized. This study investigated the effect of prenatal choline supplementation on maternal and fetal biomarkers of choline metabolism among free-living pregnant persons consuming self-selected diets. Participants were randomized to supplemental choline (as choline chloride) intakes of 550 mg/d (500 mg/d d0-choline + 50 mg/d methyl-d9-choline; intervention) or 25 mg/d d9-choline (control) from gestational week (GW) 12-16 until Delivery. Fasting blood and 24-h urine samples were obtained at study Visit 1 (GW 12-16), Visit 2 (GW 20-24), and Visit 3 (GW 28-32). At Delivery, maternal and cord blood and placental tissue samples were collected. Participants randomized to 550 (vs. 25) mg supplemental choline/d achieved higher (p < .05) plasma concentrations of free choline, betaine, dimethylglycine, phosphatidylcholine (PC), and sphingomyelin at one or more study timepoint. Betaine was most responsive to prenatal choline supplementation with increases (p ≤ .001) in maternal plasma observed at Visit 2-Delivery (relative to Visit 1 and control), as well as in the placenta and cord plasma. Notably, greater plasma enrichments of d3-PC and LDL-C were observed in the intervention (vs. control) group, indicating enhanced PC synthesis through the de novo phosphatidylethanolamine N-methyltransferase pathway and lipid export. Overall, these data show that prenatal choline supplementation profoundly alters the choline metabolome, supporting pregnancy-related metabolic adaptations and revealing biomarkers for use in nutritional assessment and monitoring during pregnancy.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Comparison of Associations between One-Carbon Metabolism, Lipid Metabolism, and Fatty Liver Markers in Normal-Weight and Overweight People Aged 20-40 Years.

The aim of the present study was to compare biomarkers of one-carbon metabolism (OCM), lipid metabolism, and fatty liver in people with normal and increased body weight. The study was performed on 421 participants, aged 20-40 years, enrolled in Poznan, Poland, in 2016-2018. Choline and betaine intakes were assessed. DNA samples were genotyped for polymorphisms of phosphatidylethanolamine N-methyltransferase (PEMT; rs7946 and rs12325817), methylene tetrahydrofolate reductase (MTHFR; rs180113), methylenetetrahydrofolate dehydrogenase (MTHFD1; rs2236225), and dihydrofolate reductase (DHFR; rs70991108). To assess the associations between blood metabolites (choline, betaine, folate, L-carnitine, o-acetyl-L-carnitine, and trimethylamine N-oxide]), circulating lipids, and fatty liver indices, multiple logistic regression analyses were performed. Overweight/obese participants had 5.8% higher choline (p < 0.05) and 10% higher L-carnitine (p < 0.001) levels than normal-weight subjects. Serum folate and betaine levels were associated with lower total cholesterol (p < 0.001 and p < 0.05), low-density lipoprotein (LDL) cholesterol (p < 0.001 and p < 0.05, respectively), triacylglycerols (p < 0.01 and p < 0.001), and triglyceride glucose index (p < 0.001 and p < 0.01, respectively), though only in overweight/obese people. The PEMT rs12325817 CC genotype was associated with higher levels of high-density lipoprotein (HDL) cholesterol (p < 0.01) in overweight/obese people. The associations between OCM markers, fatty liver indices, and blood lipids differ in subjects with normal and excessive body weight.

Stable Isotopic Tracer Phospholipidomics Reveals Contributions of Key Phospholipid Biosynthetic Pathways to Low Hepatocyte Phosphatidylcholine to Phosphatidylethanolamine Ratio Induced by Free Fatty Acids.

There is a strong association between hepatocyte phospholipid homeostasis and non-alcoholic fatty liver disease (NAFLD). The phosphatidylcholine to phosphatidylethanolamine ratio (PC/PE) often draws special attention as genetic and dietary disruptions to this ratio can provoke steatohepatitis and other signs of NAFLD. Here we demonstrated that excessive free fatty acid (1:2 mixture of palmitic and oleic acid) alone was able to significantly lower the phosphatidylcholine to phosphatidylethanolamine ratio, along with substantial alterations to phospholipid composition in rat hepatocytes. This involved both a decrease in hepatocyte phosphatidylcholine (less prominent) and an increase in phosphatidylethanolamine, with the latter contributing more to the lowered ratio. Stable isotopic tracer phospholipidomic analysis revealed several previously unidentified changes that were triggered by excessive free fatty acid. Importantly, the enhanced cytidine diphosphate (CDP)-ethanolamine pathway activity appeared to be driven by the increased supply of preferred fatty acid substrates. By contrast, the phosphatidylethanolamine N-methyl transferase (PEMT) pathway was restricted by low endogenous methionine and consequently low S-adenosylmethionine, which resulted in a concomitant decrease in phosphatidylcholine and accumulation of phosphatidylethanolamine. Overall, our study identified several previously unreported links in the relationship between hepatocyte free fatty acid overload, phospholipid homeostasis, and the development of NAFLD.