Synthetase Short-chain Family Member Research Articles

Extracellular Vesicle-Mediated Metastasis Suppressors NME1 and NME2 Modify Lipid Metabolism in Fibroblasts

Simple SummaryCommunication between cancer and stromal cells involves paracrine signalling mediated by extracellular vesicles (EVs). EVs transmit essential factors among cells of the tumour microenvironment. EVs derived from both cancer and stromal cells have been implicated in tumour progression. In this study, we focused on the first identified metastasis suppressor NME1, and on its close homolog NME2, and investigated their function in EVs in the interplay between cancer and stromal cells.Nowadays, extracellular vesicles (EVs) raise a great interest as they are implicated in intercellular communication between cancer and stromal cells. Our aim was to understand how vesicular NME1 and NME2 released by breast cancer cells influence the tumour microenvironment. As a model, we used human invasive breast carcinoma cells overexpressing NME1 or NME2, and first analysed in detail the presence of both isoforms in EV subtypes by capillary Western immunoassay (WES) and immunoelectron microscopy. Data obtained by both methods showed that NME1 was present in medium-sized EVs or microvesicles, whereas NME2 was abundant in both microvesicles and small-sized EVs or exosomes. Next, human skin-derived fibroblasts were treated with NME1 or NME2 containing EVs, and subsequently mRNA expression changes in fibroblasts were examined. RNAseq results showed that the expression of fatty acid and cholesterol metabolism-related genes was decreased significantly in response to NME1 or NME2 containing EV treatment. We found that FASN (fatty acid synthase) and ACSS2 (acyl-coenzyme A synthetase short-chain family member 2), related to fatty acid synthesis and oxidation, were underexpressed in NME1/2-EV-treated fibroblasts. Our data show an emerging link between NME-containing EVs and regulation of tumour metabolism.

The mechanistic target of rapamycin complex 1 pathway involved in hepatic gluconeogenesis through peroxisome-proliferator-activated receptor γ coactivator-1α.

Cattle can efficiently perform de novo generation of glucose through hepatic gluconeogenesis to meet post-weaning glucose demand. Substantial evidence points to cattle and non-ruminant animals being characterized by phylogenetic features in terms of their differing capacity for hepatic gluconeogenesis, a process that is highly efficient in cattle yet the underlying mechanism remains unclear. Here we used a variety of transcriptome data, as well as tissue and cell-based methods to uncover the mechanisms of high-efficiency hepatic gluconeogenesis in cattle. We showed that cattle can efficiently convert propionate into pyruvate, at least partly, via high expression of acyl-CoA synthetase short-chain family member 1 (ACSS1), propionyl-CoA carboxylase alpha chain (PCCA), methylmalonyl-CoA epimerase (MCEE), methylmalonyl-CoA mutase (MMUT), and succinate-CoA ligase (SUCLG2) genes in the liver (P < 0.01). Moreover, higher expression of the rate-limiting enzymes of gluconeogenesis, such as phosphoenolpyruvate carboxykinase (PCK) and fructose 1,6-bisphosphatase (FBP), ensures the efficient operation of hepatic gluconeogenesis in cattle (P < 0.01). Mechanistically, we found that cattle liver exhibits highly active mechanistic target of rapamycin complex 1 (mTORC1), and the expressions of PCCA, MMUT, SUCLG2, PCK, and FBP genes are regulated by the activation of mTORC1 (P < 0.001). Finally, our results showed that mTORC1 promotes hepatic gluconeogenesis in a peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) dependent manner. Collectively, our results not only revealed an important mechanism responsible for the quantitative differences in the efficiency of hepatic gluconeogenesis in cattle versus non-ruminant animals, but also established that mTORC1 is indeed involved in the regulation of hepatic gluconeogenesis through PGC-1α. These results provide a novel potential insight into promoting hepatic gluconeogenesis through activated mTORC1 in both ruminants and mammals.

Acetyl-Coenzyme A Synthetase 2 Potentiates Macropinocytosis and Muscle Wasting Through Metabolic Reprogramming in Pancreatic Cancer

Rapid deconditioning, also called cachexia, and metabolic reprogramming are two hallmarks of pancreatic cancer. Acetyl-coenzyme A synthetase short-chain family member 2 (ACSS2) is an acetyl-enzyme A synthetase that contributes to lipid synthesis and epigenetic reprogramming. However, the role of ACSS2 on the nonselective macropinocytosis and cancer cachexia in pancreatic cancer remains elusive. In this study, we demonstrate that ACSS2 potentiates macropinocytosis and muscle wasting through metabolic reprogramming in pancreatic cancer. Clinical significance of ACSS2 was analyzed using samples from patients with pancreatic cancer. ACSS2-knockout cells were established using the clustered regularly interspaced short palindromic repeats-associated protein 9 system. Single-cell RNA sequencing data from genetically engineered mouse models was analyzed. The macropinocytotic index was evaluated by dextran uptake assay. Chromatin immunoprecipitation assay was performed to validate transcriptional activation. ACSS2-mediated tumor progression and muscle wasting were examined in orthotopic xenograft models. Metabolic stress induced ACSS2 expression, which is associated with worse prognosis in pancreatic cancer. ACSS2 knockout significantly suppressed cell proliferation in 2-dimensional and 3-dimensional models. Macropinocytosis-associated genes are upregulated in tumor tissues and are correlated with worse prognosis. ACSS2 knockout inhibited macropinocytosis. We identified Zrt- and Irt-like protein 4 (ZIP4) as a downstream target of ACSS2, and knockdown ofZIP4 reversed ACSS2-induced macropinocytosis. ACSS2 upregulated ZIP4 through ETV4-mediated transcriptional activation. ZIP4 induces macropinocytosis through cyclic adenosine monophosphate response element-binding protein-activated syndecan 1 (SDC1) and dynamin 2 (DNM2). Meanwhile, ZIP4 drives muscle wasting and cachexia via glycogen synthase kinase-β (GSK3β)-mediated secretion of tumor necrosis factor superfamily member 10 (TRAIL or TNFSF10). ACSS2 knockout attenuated muscle wasting andextended survival in orthotopic mouse models. ACSS2-mediated metabolic reprogramming activates the ZIP4 pathway, and promotes macropinocytosis via SDC1/DNM2 and drives muscle wasting through the GSK3β/TRAIL axis, which potentially provides additional nutrients for macropinocytosis in pancreatic cancer.

Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.

Overcoming resistance to chemotherapies remains a major unmet need for cancers, such as triple-negative breast cancer (TNBC). Therefore, mechanistic studies to provide insight for drug development are urgently needed to overcome TNBC therapy resistance. Recently, an important role of fatty acid β-oxidation (FAO) in chemoresistance has been shown. But how FAO might mitigate tumor cell apoptosis by chemotherapy is unclear. Here, we show that elevated FAO activates STAT3 by acetylation via elevated acetyl-coenzyme A (CoA). Acetylated STAT3 upregulates expression of long-chain acyl-CoA synthetase 4 (ACSL4), resulting in increased phospholipid synthesis. Elevating phospholipids in mitochondrial membranes leads to heightened mitochondrial integrity, which in turn overcomes chemotherapy-induced tumor cell apoptosis. Conversely, in both cultured tumor cells and xenograft tumors, enhanced cancer cell apoptosis by inhibiting ASCL4 or specifically targeting acetylated-STAT3 is associated with a reduction in phospholipids within mitochondrial membranes. This study demonstrates a critical mechanism underlying tumor cell chemoresistance.

Deoxynivalenol exposure inhibits biosynthesis of milk fat and protein by impairing tight junction in bovine mammary epithelial cells

Deoxynivalenol (DON) is one of the most common feed contaminants, and it poses a serious threat to the health of dairy cows. The existing studies of biological toxicity of DON mainly focus on the proliferation, oxidative stress, and inflammation in bovine mammary epithelial cells, while its toxicity on the biosynthesis of milk components has not been well documented. Hence, we investigated the toxic effects and the underlying mechanism of DON on the bovine mammary alveolar cells (MAC-T). Our results showed that exposure to various concentrations of DON significantly inhibited cell proliferation, induced apoptosis, and altered the cell morphology which was manifested by cell distortion and shrinkage. Moreover, the transepithelial electrical resistance (TEER) values of MAC-T cells exposed to DON were gradually decreased in a time- and concentration- dependent manner, but lactate dehydrogenase (LDH) leakage was significantly increased with the maximum increase of 2.4-fold, indicating the cell membrane and tight junctions were damaged by DON. Importantly, DON significantly reduced the synthesis of β-casein and lipid droplets, along with the significantly decreases of phospho-mTOR, phospho-4EBP1, phospho-JAK2, and phospho-STAT5. Gene expression profiles showed that the expressions of several genes related to lipid synthesis and metabolism were changed, including acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid binding protein 3 (FABP3), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), and insulin-induced gene 1 (INSIG1). GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in ribosome, glutathione metabolism, and lipid biosynthetic process, which play important roles in the toxicological process induced by DON. Taken together, DON affects the proliferation and functional differentiation of MAC-T cells, which might be related to the cell junction disruption and morphological alteration. Our data provide new insights into functional differentiation and transcriptomic alterations of MAC-T cells after DON exposure, which contributes to a comprehensive understanding of DON-induced toxicity mechanism.

ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction.

Propionate is a gut microbial metabolite that has been reported to have controversial effects on metabolic health. Here we show that propionate is activated by acyl‐CoA synthetase short‐chain family member 3 (ACSS3), located on the mitochondrial inner membrane in brown adipocytes. Knockout of Acss3 gene (Acss3–/–) in mice reduces brown adipose tissue (BAT) mass but increases white adipose tissue (WAT) mass, leading to glucose intolerance and insulin resistance that are exacerbated by high‐fat diet (HFD). Intriguingly, Acss3–/– or HFD feeding significantly elevates propionate levels in BAT and serum, and propionate supplementation induces autophagy in cultured brown and white adipocytes. The elevated levels of propionate in Acss3–/– mice similarly drive adipocyte autophagy, and pharmacological inhibition of autophagy using hydroxychloroquine ameliorates obesity, hepatic steatosis and insulin resistance of the Acss3–/– mice. These results establish ACSS3 as the key enzyme for propionate metabolism and demonstrate that accumulation of propionate promotes obesity and Type 2 diabetes through triggering adipocyte autophagy.

Dietary threonine deficiency affects expression of genes involved in lipid metabolism in adipose tissues of Pekin ducks in a genotype-dependent manner

Dietary threonine (Thr) deficiency increases hepatic triglyceride content and reduces sebum and abdominal fat percentages in lean type (LT), but not in fatty type (FT) Pekin ducks. However, the molecular changes regarding the role of Thr in lipid metabolism in LT and FT ducks induced by Thr deficiency remains unknown. This study compared differential expression gene profiles related to lipid metabolism in FT and LT Pekin ducks affected by Thr deficiency. We performed transcriptomic profiling and scanned the gene expression in the liver, sebum, and abdominal fat of Pekin ducks fed either Thr-deficient or Thr-adequate diet for 21 days from 14 to 35 days of age. There were 187, 52, and 50 differentially expressed genes (DEGs) identified in the liver, sebum, and abdominal fat of LT ducks affected by Thr deficiency, of which 12, 9, and 5 genes were involved in lipid metabolism, respectively. Thr deficiency altered the expression of 27, 6, and 3 genes in FT ducks’ liver, sebum, and abdominal fat, respectively. None of the DEGs had a relationship with lipid metabolism in FT ducks. KEGG analysis showed that the DEGs in the LT ducks’ livers were enriched in lipid metabolism pathways (linolenic acid metabolism, glycerophospholipid metabolism, and arachidonic acid metabolism) and amino acid metabolism pathways (biosynthesis of amino acids, phenylalanine metabolism, β-alanine metabolism, and glycine, serine and threonine metabolisms). The DEGs in the sebum and abdominal fat of LT ducks were not enriched in lipid and amino acid metabolic pathways. Additionally, DEGs involved in lipid metabolism were found to be upregulated by Thr deficiency in LT ducks, such as malic enzyme 3 (ME3), acyl-CoA synthetase short-chain family member 2 (ACSS2) in liver, and lipase member M (LIPM) in sebum. In summary, dietary Thr deficiency regulated the gene expression involved in lipid metabolism in the liver, sebum, and abdominal fat of Pekin ducks in a genotype-dependent manner.

Hsa-miR-15b-5p regulates the proliferation and apoptosis of human vascular smooth muscle cells by targeting the ACSS2/PTGS2 axis.

A previous bioinformatic analysis from our group predicted that the interaction of microRNA (miRNA/miR)-15b with the acyl-CoA synthetase short chain family member 2 (ACSS2) gene was important for the development of abdominal aortic aneurysm (AAA). Apoptosis of aortic vascular smooth muscle cells (VSMCs) is a pathological feature of AAA. The present study aimed to explain the roles of miR-15b/ACSS2 in AAA by exploring their effects on the proliferation and apoptosis of aortic VSMCs. Human aortic VSMCs (T/G HA-VSMC cell line) were divided into six groups and were transfected with miR-15b-5p mimics, mimic negative control (NC), miR-15b-5p inhibitors, inhibitor NC, miR-15b-5p mimics+pcDNA3.1 and miR-15b-5p mimics+ACSS2-overexpessing vector. CCK-8 assay was used to determine cell proliferation. Annexin V-FITC/PI staining and flow cytometry assays were used to measure cell apoptosis. Dual-luciferase reporter assays were used to confirm the targeted relationship between miR-15b-5p and ACSS2. Reverse transcription-quantitative PCR and/or western blotting were used to examine the expression levels of miR-15b-5p, ACSS2 and prostaglandin-endoperoxide synthase 2 (PTGS2). Following transfection of T/G HA-VSMCs with mimics and inhibitors to respectively upregulate and downregulate miR-15b-5p, the results demonstrated that overexpression of miR-15b-5p inhibited cell proliferation and promoted cell apoptosis; silencing of miR-15b-5p obtained the opposite results. ACSS2 may be a direct target of miR-15b-5p, since the luciferase activity of a ACSS2 wild-type vector, but not that of a ACSS2 mutant reporter, was significantly inhibited by miR-15b-5p mimics compared with controls. Additionally, the expression levels of ACSS2 and its downstream gene PTGS2 were significantly reduced or increased following transfection with miR-15b-5p mimics or inhibitors, respectively. Furthermore, overexpression of ACSS2 reversed the antiproliferative and proapoptotic effects of miR-15b-5p mimics by blocking the production of PTGS2 protein. In conclusion, miR-15b-5p may promote the apoptosis and inhibit the proliferation of aortic VSMCs via targeting the ACSS2/PTGS2 axis. The present study provided preliminary evidence indicating that the miR-15b-5p/ACSS2/PTGS2 axis may be a potential target for the treatment of AAA.

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate-BCoA-CPT1A axis for the regulation of immune homeostasis.

I ncreased Host Energy Metabolism in the Proximal Colon‐Microbiota Interface Elevates Blood Pressure

Introduction : Hypertensive subjects present with alterations in gut microbiota and short chain fatty acids (SCFAs). SCFAs are the major metabolic products of bacterial fermentation in the intestine. While acetate and propionate are energy sources for peripheral tissues, butyrate is the primary energy source for the host colonic epithelium via β-oxidation or glycolysis. Since butyrate is known to elevate blood pressure (BP), we hypothesized that the mechanism of gut microbial butyrate-mediated elevation in BP is linked with an increased energy metabolism in the proximal colon. Methods : To test this hypothesis, 7 weeks old concomitantly raised male germ-free Sprague Dawley rats (GF, n=5-6) were compared with GF acquiring microbiota acutely for 10 days (germ-free conventionalized rats, GFC, n=6) for (1) BP, (2) cecal butyrate by proton nuclear magnetic resonance (1-H NMR), (3) microbial profiling by 16S RNA sequencing and (4) proximal colonic transcriptomic signatures for energy metabolism by real time qRT-PCR. Results : GFC rats acquired microbiota successfully (Figure 1 C and D) and represented an energy-repleted state with a marked increase of the colonic energy substrate, butyrate (Figure 1E). Interestingly, GFC rats had a significant increase in systolic and diastolic BP compared to GF rats (Figure 1 A and B, p<0.05). This increase in BP was associated with a significant upregulation of all 11 genes tested by qRT-PCR for energy metabolism pathways for β-oxidation and glycolysis in the colon of the GFC rats compared to GF rats (Figure 1F). These genes are: peroxisome proliferator activated receptor γ (Pparγ), free fatty acid receptor 2 (Ffar2), acyl-CoA synthetase short chain family member 1(Acss1), acyl-CoA dehydrogenase long chain(Acadl), carnitine palmitoyltransferase 2(Cpt2), hexokinase 2(Hk2), phosphofructokinase, platelet(Pfkp), phosphoglycerate mutase 1(Pgam1), glycerol-3-phosphate dehydrogenase 2(Gpd2), sirtuin 3(Sirt3)and carnitine O-acetyltransferase(Crat). Conclusion : Collectively our data reported here is the first to provide evidence for a direct relationship between host energy metabolism at the proximal gut-microbiota interface and BP regulation. Further, our studies suggest that butyrate, which is a predominant colonic energy metabolite, contributes to the mechanism governing this relationship between energy metabolism in the proximal colon with BP regulation.

Maturation system affects lipid accumulation in bovine oocytes.

This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

ACSS3 represses prostate cancer progression through downregulating lipid droplet-associated protein PLIN3.

Current endocrine therapy for prostate cancer (PCa) mainly inhibits androgen/androgen receptor (AR) signaling. However, due to increased intratumoural androgen synthesis and AR variation, PCa progresses to castration-resistant prostate cancer (CRPC), which ultimately becomes resistant to endocrine therapy. A search for new therapeutic perspectives is urgently needed.Methods: By screening lipid metabolism-related gene sets and bioinformatics analysis in prostate cancer database, we identified the key lipid metabolism-related genes in PCa. Bisulfite genomic Sequence Polymerase Chain Reaction (PCR) (BSP) and Methylation-Specific Polymerase Chain Reaction (PCR) (MSP) were preformed to detect the promoter methylation of ACSS3. Gene expression was analyzed by qRT-PCR, Western blotting, IHC and co-IP. The function of ACSS3 in PCa was measured by CCK-8, Transwell assays. LC/MS, Oil Red O assays and TG and cholesterol measurement assays were to detect the levels of TG and cholesterol in cells. Resistance to Enzalutamide in C4-2 ENZR cells was examined in a xenograft tumorigenesis model in vivo.Results: We found that acyl-CoA synthetase short chain family member 3 (ACSS3) was downregulated and predicted a poor prognosis in PCa. Loss of ACSS3 expression was due to gene promoter methylation. Restoration of ACSS3 expression in PCa cells significantly reduced LD deposits, thus promoting apoptosis by increasing endoplasmic reticulum (ER) stress, and decreasing de novo intratumoral androgen synthesis, inhibiting CRPC progression and reversing Enzalutamide resistance. Mechanistic investigations demonstrated that ACSS3 reduced LD deposits by regulating the stability of the LD coat protein perilipin 3 (PLIN3).Conclusions: Our study demonstrated that ACSS3 represses prostate cancer progression through downregulating lipid droplet-associated protein PLIN3.

Abstract PO-046: LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma

Abstract Loss of liver kinase B (LKB1, encoded by STK11) has been observed in about one-third of lung adenocarcinomas (ADCs) and can cause the inactivation of its major target AMP-activated protein kinase (AMPK) and subsequently, activation of the mammalian target of rapamycin (mTOR) pathway. In addition to its inhibitory effects on tumor growth and proliferation, AMPK may affect acetyl-CoA homeostasis and induces histone acetylation through regulating the activity and nuclear translocation of acyl-CoA synthetase short-chain family member 2 (ACSS2) and ATP citrate lyase (ACLY). Nuclear and/or cytoplasmic expression of LKB1/AMPK/mTOR pathway markers (LKB1, p-AMPKα, p-S6), enzymes involved in acetate and lipid metabolism (p-ACC, ACSS2, ACLY), acetyl-histone H3 (ac-H3), and immune regulatory proteins (PD-L1, COX-2) was assessed in lung ADCs (N=100) by immunohistochemistry. The expression of mRNAs coding for proteins involved in antitumor immunity was also analyzed in STK11-mutant and wild-type cases with using TCGA data. Associations with the clinicopathological parameters as well as the antiproliferative effects of mTOR and metabolic inhibitors (rapamycin, metformin, phenformin, and ACSS2 inhibitor) and a histone deacetylase (HDAC) inhibitor (valproic acid) were also studied in ADC cell lines harboring KRAS and/or STK11 mutations. In cases with low or no LKB1 expression, we observed significantly lower expression of p-AMPKα, p-ACC, ac-H3, ACSS2, and ACLY (P &amp;lt; .01). PD-L1 expression was also slightly decreased in these samples. The expression of most of the studied mRNAs, particularly those involved in innate immunity, was also lower in STK11 mutant cases. In our in vitro studies, rapamycin had a significant antiproliferative effect on KRAS- and/or STK11-mutant cell lines with high mTOR activity. The ACSS2 inhibitor and valproic acid were not effective as monotherapies, however, valproic acid significantly increased the antitumoral effects of rapamycin in STK11-mutant cells similarly to phenformin. Our results suggest that histone acetylation levels are lower in ADCs with LKB1 loss, perhaps by lower expression and decreased nuclear translocation of ACSS2 and ACLY. HDAC inhibitors may have the benefit to maintain histone acetylation in STK11-mutant ADCs, therefore, restoring the expression of tumor suppressors or proteins involved in antitumor immunity, which are silenced in cancer cells. Simultaneous inhibition of mTOR and HDACs may exhibit significant antitumor properties, especially in cancer cells with STK11 mutation and high mTOR activity. Supported by the Hungarian Respiratory Foundation, National Bionics Program of National Research, Development and Innovation Fund of Hungary (ED_17-1-2017-0009), and grants of the Hungarian National Research, Development and Innovation Office (NKFI-FK-128404). Citation Format: Ildiko Krencz, Daniel Sztankovics, Titanilla Danko, Gabor Petovari, Eniko Vetlenyi, Regina Raffay, Judit Moldvay, Judit Papay, Anna Sebestyen. LKB1 loss-associated changes in histone acetylation are potential therapeutic targets in lung adenocarcinoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-046.

Mir-193b Regulates the Differentiation, Proliferation, and Apoptosis of Bovine Adipose Cells by Targeting the ACSS2/AKT Axis.

Simple SummarySeveral studies have shown that miR-193b plays an important role in preadipocyte differentiation. Herein, we explored the role of bta-miR-193b in adipocyte development, using EdU, flow cytometry, CCK-8, RT-qPCR, Western blotting, and oil red O staining. The result showed that bta-miR-193b could regulate the proliferation, differentiation and apoptosis of adipocytes. The dual-fluorescent reporter vector experiments showed that bta-miR-193b directly targeted ACSS2, and the regulatory function of ACSS2 is opposite to that of miR-193b. Meanwhile, we demonstrated that ACSS2 could significantly promote the expression of AKT and pAKT proteins. In conclusion, our research provides new insights that confirm that bta-miR-193b inhibits bovine adipose cell proliferation, and promotes apoptosis by negative regulation of the ACSS2/AKT pathway.The precise functions and molecular mechanisms of microRNAs (miRNAs) in adipocytes are primarily unknown. Studies have demonstrated that miR-193b plays a pivotal role in the differentiation of preadipocytes. Herein, we evaluated the effects of bta-miR-193b on the growth and development of adipocytes, using the EdU cell proliferation method, flow cytometry analysis, CCK-8 assay, RT-qPCR, Western blotting, and oil red O staining. We observed that the overexpression of bta-miR-193b significantly affected the differentiation, proliferation, and apoptosis of adipocytes. The results of the dual-fluorescent reporter vector experiments demonstrated that bta-miR-193b directly targeted Acyl-CoA synthetase short-chain family member 2 (ACSS2). Additionally, the effects of ACSS2 overexpression on the proliferation and apoptosis in adipose cells were the opposite of those induced by bta-miR-193b. We also demonstrated that ACSS2 can significantly promote the expression of AKT and pAKT proteins. Therefore, this study presents a novel mechanism by which bta-miR-193b regulates adipocyte development by targeting ACSS2.

MiR-30a-GNG2 and miR-15b-ACSS2 Interaction Pairs May Be Potentially Crucial for Development of Abdominal Aortic Aneurysm by Influencing Inflammation.

Abdominal aortic aneurysm (AAA) is a lethal vascular degenerative disease for the elderly, but current therapeutic options are limited. This study was to explore the molecular mechanisms of AAA to screen underlying treatment targets for AAA. The gene and microRNA (miRNA) expression profiles of human AAA were downloaded from Gene Expression Omnibus database under accession number GSE57691, GSE62179, and GSE63541. Differentially expressed genes (DEGs) and microRNAs (miRNAs; DEMs) were identified using the Linear Models for Microarray data method. Protein-protein interaction (PPI) network, module analysis, and miRNA-mRNA regulatory network analyses were performed to screen hub genes and miRNAs that regulated the hub genes. The Database for Annotation, Visualization and Integrated Discovery was used to predict the functions of genes. GEPIA and Tumor-miRNA-Pathway online software were used to validate the expressions of crucial DEMs and DEGs in other cancers, respectively. As a result, in the GSE57691 dataset, a total of 584 DEGs were found to be specific for AAA, 521 of which were used for constructing the PPI network. ACSS2 (acyl-CoA synthetase short-chain family member 2), GNG2 (G protein subunit gamma 2), and CXCL1 (C-X-C motif chemokine ligand 1) and CCR7 (C-C motif chemokine receptor 7) were believed to be hub genes by calculating their topological features in the PPI network. Upregulated GNG2 could interact with CXCL1 and CCR7 to involve in chemokine signaling pathway, while downregulated ACSS2 was associated with lipid biosynthetic process. In the miRNA-mRNA regulatory network, ACSS2 was found to be regulated by hsa-miR-15b; hsa-miR-30a could modulate the expression of GNG2. In line with our analysis in AAA, GNG2, ACSS2, hsa-miR-30a, and hsa-miR-15b were also confirmed to be significantly upregulated or downregulated in several cancer types. In conclusion, hsa-miR-30a-GNG2 and hsa-miR-15b-ACSS2 interaction pairs may represent novel mechanisms for explaining the pathogenesis of AAA. Targeted regulation of them may be potential strategies for treatment of AAA.

Screening of core genes and pathways in breast cancer development via comprehensive analysis of multi gene expression datasets.

Breast cancer has been the leading cause of cancer-associated mortality in women worldwide. Perturbation of oncogene and tumor suppressor gene expression is generally considered as the fundamental cause of cancer initiation and progression. In the present study, three gene expression datasets containing information of breast cancer and adjacent normal tissues that were detected using traditional gene microarrays were downloaded and batch effects were removed with R programming software. The differentially expressed genes between breast cancer and normal tissue groups were closely associated with cancer development pathways. Interestingly, five pathways, including ‘extracellular matrix-receptor interaction’, ‘peroxisome proliferator-activated receptors signaling pathway’, ‘propanoate metabolism’, ‘pyruvate metabolism’ and ‘regulation of lipolysis in adipocytes’, were thoroughly connected by 10 genes. Patients with upregulation of six of these hub genes (acetyl-CoA carboxylase β, acyl-CoA dehydrogenase medium chain, adiponectin, C1Q and collagen domain containing, acyl-CoA synthetase short chain family member 2, phosphoenolpyruvate carboxykinase 1 and perilipin 1) exhibited improved breast cancer prognosis. Additionally, breast cancer-specific network analysis identified several gene-gene interaction modules. These gene clusters had strong interactions according to the scoring in the whole network, which may be important to the development of breast cancer. In conclusion, the present study may improve the understanding of the mechanisms of breast cancer and provide several valuable prognosis and treatment signatures.

Regulated Acyl-CoA Synthetase Short-Chain Family Member 2 Accumulation during Spermatogenesis.

ObjectiveAcyl-CoA synthetase short-chain family member 2 (ACSS2) activity provides a major source of acetyl-CoA to drive histone acetylation. This study aimed to unravel the ACSS2 expression during mouse spermatogenesis, where a dynamic and stage-specific genome-wide histone hyperacetylation occurs before histone eviction.Materials and MethodsIn this experimental study, ACSS2 expression levels during spermatogenesis were verified by Immunodetection. Testis paraffin-embedded sections were used for IHC staining with anti-H4 pan ac and anti-ACSS2. Co-detection of ACSS2 and H4K5ac was performed on testis tubular sections by immunofluorescence. Proteins extracts from fractionated male germ cells were subjected to western-blotting and immunoblot was probed with anti- ACSS2 and anti-actin.ResultsThe resulting data showed that the commitment of progenitor cells into meiotic divisions leads to a robust accumulation of ACSS2 in the cell nucleus, especially in pachytene spermatocytes (P). However, ACSS2 protein drastically declines during post-meiotic stages, when a genome-wide histone hyperacetylation is known to occur.ConclusionThe results of this study are in agreement with the idea that the major function of ACSS2 is to recycle acetate generated after histone deacetylation to regenerate acetyl-CoA which is required to maintain the steady state of histone acetylation. Thus, it is suggested that in spermatogenic cells, nuclear activity of ACSS2 maintains the acetate recycling until histone hyperacetylation, but disappears before the acetylation-dependent histone degradation.

Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation.

Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which EtOH affects stem cell differentiation are unclear. Our goal here is to examine the effects of EtOH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation. We designed ESC lines in which aldehyde dehydrogenase (ALDH2, NCBI#11669) and acyl-CoA synthetase short-chain family member 2 (ACSS2, NCBI#60525) were knocked out by CRISPR-Cas9 technology. We selected these genes because of their key roles in EtOH oxidation in order to dissect the effects of EtOH metabolism on differentiation. By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH2 rather than ALDH1A2. We found increases in mRNAs of differentiation-associated genes (Hoxa1, Cyp26a1, and RARβ2) upon EtOH treatment of WT and Acss2-/- ESCs, but not Aldh2-/- ESCs. The absence of ALDH2 reduced mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). Treatment of WT ESCs with AcH or 4-hydroxynonenal (4-HNE), another substrate of ALDH2, increased differentiation-associated transcripts compared to levels in untreated cells. mRNAs of genes involved in retinoic acid (RA) synthesis (Stra6 and Rdh10) were also increased by EtOH, AcH, and 4-HNE treatment. Retinoic acid receptor-γ (RARγ) is required for both EtOH- and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARγ signaling in AcH-induced ESC differentiation. ACSS2 knockouts showed no changes in differentiation phenotype, while pluripotency-related transcripts were decreased in ALDH2 knockout ESCs. We demonstrate that AcH increases differentiation-associated mRNAs in ESCs via RARγ.

Single-walled carbon nanohorn aggregates promotes mitochondrial dysfunction-induced apoptosis in hepatoblastoma cells by targeting SIRT3.

Single-walled carbon nanohorns (SWNHs) can accumulate in a variety of cell types or tissues and exert biological effects, which have been demonstrated to induce apoptosis in hepatoblastoma cells. However, the role and molecular mechanisms of SWNHs remain unclear. The mitochondrion is an important subcellular structure and may contribute to apoptosis that is induced by SWNHs in hepatoblastoma cells. To address this question, the mitochondrial function of HepG2 or L02 cells that were treated with SWNHs was examined. The results indicated that SWNHs were able to decrease the mitochondrial membrane potential and suppress the activity of the Na+/K+-ATPase. Secondly, HepG2 cells and L02 cells were treated with SWNHs in vivo and in vitro. The expression of mitochondrial-associated proteins [acyl-CoA synthetase short chain family member 1, Bax, cytochrome C (CYT-C), sodium channel epithelial 1α subunit, sirtuin 3 (SIRT3) and voltage-dependent anion channel 1] was analyzed by western blotting and immunohistochemical staining. The results revealed that SWNH treatment was able to alter the expression of multiple mitochondrial apoptotic pathway-associated proteins in HepG2 cells. SWNH treatment was able upregulate the expression of SIRT3, CYT-C and VDAC1 and downregulate the expression of AceCS2, but it had a more stable effect on SIRT3. However, similar findings were not observed in L02 cells. Therefore, the data from the present study indicated that SWNHs might be used as a safe anticancer agent, where it is able to trigger mitochondrial dysfunction-induced apoptosis by upregulating SIRT3 expression in HepG2 cells.

Responses of MAC-T Cells to Inhibited Stearoyl-CoA Desaturase 1 during cis-9, trans-11 Conjugated Linoleic Acid Synthesis.

Cis-9-conjugated, trans-11-conjugated linoleic acid (CLA) is known for its positive activities on human health. The synthesis of cis-9, trans-11 CLA in mammary glands is generally thought to be catalyzed by stearoyl-CoA desaturase 1 (SCD1), but this has not been rigorously established. In this study, we hypothesized that the inhibition of SCD1 (by CAY10566) would block the synthesis of cis-9, trans-11 CLA in bovine mammary alveolar cells (MAC-T) cells. Results showed that MAC-T cells incubated with 10 nM CAY10566 for 12 h (CAY) produced less cis-9, trans-11 CLA (p < 0.01), lower 14:1/(14:1 + 14:0)% (p < 0.01), more trans-11 18:1 (TVA) accumulation (p < 0.01), and reduced SCD1 mRNA levels (p < 0.01) compared with the control group (CON). Moreover, the mRNA abundances of sterol regulatory element-binding protein 1 [SREBPF1], acyl-CoA synthetase short-chain family member 2 [ACSS2], and lipin 1 [LPIN1] were significantly elevated when SCD1 was inhibited in the CAY group (p < 0.05). Taken together, CAY10566 inhibition of SCD1 resulted in lower cis-9, trans-11 CLA synthesis ability, and SREBF1, ACSSS2, and LPIN1 were negatively associated with SCD1. These findings not only provide the direct evidence that cis-9, trans-11 CLA synthesis is catalyzed by SCD1, but also help us understand the responses of MAC-T cells to SCD1 inhibition.