Expression Of ENO1 Research Articles

Targeted Drug Screening Leveraging Senescence-Induced T-Cell Exhaustion Signatures in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and a leading cause of cancer-related mortality globally, with most patients diagnosed at advanced stages and facing limited early treatment options. This study aimed to identify characteristic genes associated with T-cell exhaustion due to senescence in hepatocellular carcinoma patients, elucidating the interplay between senescence and T-cell exhaustion. We constructed prognostic models based on five signature genes (ENO1, STMN1, PRDX1, RAN, and RANBP1) linked to T-cell exhaustion, utilizing elastic net regression. The findings indicate that increased expression of ENO1 in T cells may contribute to T-cell exhaustion and Treg infiltration in hepatocellular carcinoma. Furthermore, molecular docking was employed to screen small molecule compounds that target the anti-tumor effects of these exhaustion-related genes. This study provides crucial insights into the diagnosis and treatment of hepatocellular carcinoma, establishing a strong foundation for the development of predictive biomarkers and therapeutic targets for affected patients.

Identification of ENO-1 positive extracellular vesicles as a circulating biomarker for monitoring of Ewing sarcoma.

The lack of circulating biomarkers for tumor monitoring is a major problem in Ewing sarcoma management. The development of methods for accurate tumor monitoring is required, considering the high recurrence rate of drug-resistant Ewing sarcoma. Here, we describe a sensitive analytical technique for tumor monitoring of Ewing sarcoma by detecting circulating extracellular vesicles secreted from Ewing sarcoma cells. Proteomic analysis of Ewing sarcoma cell-derived extracellular vesicles identified 564 proteins prominently observed in extracellular vesicles from three Ewing sarcoma cell lines. Among these, CD99, SLC1A5, and ENO-1 were identified on extracellular vesicles purified from sera of patients with Ewing sarcoma before treatment but not on extracellular vesicles from those after treatment and healthy individuals. Notably, not only Ewing sarcoma-derived extracellular vesicles but also Ewing sarcoma cells demonstrated proteomic expression of CD99 and ENO-1 on their surface membranes. ENO-1+CD63+ extracellular vesicle detection was reduced after tumor resection while both CD99+CD63+ and ENO-1+CD63+ extracellular vesicles were detected in serum from Ewing sarcoma-bearing mice. Finally, the accuracy of liquid biopsy targeting these candidates was assessed using extracellular vesicles from the sera of patients with Ewing sarcoma. Elevated ENO-1+CD81+ extracellular vesicles in the serum of patients before treatments distinguished patients with Ewing sarcoma from healthy individuals with an area under the curve value of 0.92 (P < 0.001) and reflected the tumor burden in patients with Ewing sarcoma during multidisciplinary treatments. Collectively, circulating ENO-1+CD81+ extracellular vesicle detection could represent a novel tool for tumor monitoring of Ewing sarcoma.

Effect of PLCE1-driven aerobic glycolysis through ENO1 modulation on immune evasion in esophageal squamous cell carcinoma (ESCC).

e16016 Background: Our prior investigations have demonstrated that PLCE1 actively promotes tumorigenesis, angiogenesis, and cell cycle progression in ESCC. However, the precise role of PLCE1 in glycolysis and its influence on the tumor immune microenvironment (TIME) remain elusive. Methods: To elucidate potential pathways regulated by PLCE1, we applied Affymetrix GeneChip, Affymetrix ClariomD Array, and IP-MS techniques to identify molecules interacting with PLCE1. Subsequently, we validated the involement of PLCE1 in glycolysis through assessments of glycolytic product production in both in vivo and in vitro assays. The underlying mechanisms by which PLCE1 influences glycolysis were further probed through a combination of IP assays, tumor growth experiments, and ubiquitination assays. Next, we conducted multi-color immunofluorescence staining to examine the infiltration of immune cells in human ESCC tissues. We utilized a transgenic PLCE1-/- C57BL/6 mice model to investigate the immune cell distribution in TIME. Results: PLCE1 emerges as a pivotal regulator in glycolytic processes within ESCC cell lines. Knockdown of PLCE1 instigates a notable attenuation of glycolysis, achieved through the modulation of ENO1 expression, a key enzyme in glycolysis. This effect was also observed in vivo. We have elucidated a novel pathway in which PLCE1 orchestrates a reduction in the expression of FBXW7, an E3 ubiquitin ligase, thereby attenuating FBXW7-mediated ubiquitination of ENO1 and fostering ENO1 stability. Based on mutations identified through sequencing of ESCC tissue and drawing from previous studies, we have pinpointed the specific interacting sites between FBXW7 and ENO1. Substrate phosphorylation is necessary for the recognition of FBXW7. We unveil a mechanism wherein PLCE1 interacts with CDK2 to augment ENO1 phosphorylation, potentiating FBXW7 recognition. Nonetheless, CDK2-mediated ENO1 phosphorylation fails to elicit FBXW7-mediated ubiquitination due to limited FBXW7 expression in ESCC. Elevated tumor glycolysis induced by PLCE1 via ENO1 engenders heightened lactate production, thereby dampening cytokine production and CD8+ T cell activation. In human ESCC tissues, a diminished CD8+ T cell population is observed proximal to PLCE1+ENO1+ tumor cells, concomitant with an augmented accumulation of exhausted CD8+ PD1+ T cells in their vicinity. In an ESCC murine model, a more robust infiltration and cytokine production, coupled with diminished PD-1 expression on both CD4+ and CD8+ T cells, is noted in the PLCE1-/- genotype. This effect was particularly prominent upon ENO1 inhibitor administration. Conclusions: Our investigation elucidates that PLCE1 orchestrates the glycolytic pathway in ESCC by upregulating the expression of ENO1, consequently hindering the infiltration and activation of CD8+ T cells, thus fostering an immunosuppressive TIME.

KAT2A changes the function of endometrial stromal cells via regulating the succinylation of ENO1.

Endometriosis is increasingly affecting women worldwide and research is focusing on identifying key targets in its pathogenesis. Changes in succinylation genes regulate the function of this protein and further influence the development of the disease. However, the role of succinylation genes in endometriosis is not clear from current studies. The expression of succinylation genes was determined in ectopic endometrium (EC) and ectopic patients with uterine fibroids (EN) by real-time quantitative PCR (qRT-PCR) and Western blot. Cell Counting Kit-8, transwell assays, and flow cytometry were used to assess endometrial stromal cells (ESCs) proliferation, apoptosis, migration, and invasion. KAT2A and ENO1 association was detected by qRT-PCR, immunofluorescence, and CoIP. We found that gene and protein levels of KAT2A were significantly increased in the EC group compared to EN group tissues. KAT2A silencing inhibited cell proliferation, migration, and invasion and promoted apoptosis. Western blot results showed that the expression of ENO1 and its succinylation was significantly upregulated in ECSc after KAT2A overexpression. CoIP results showed that KAT2A is positively bound to ENO1. Immunofluorescence also showed co-localized expression of KAT2A with ENO1. Furthermore, ENO1 overexpression reversed the effects of KAT2A silencing on the malignant behavior of ESCs. In summary, we found that succinylation of ENO1 mediated by KAT2A played a role in promoting the progression of endometriosis.

High Expression of ENO1 and Low Levels of Circulating Anti-ENO1 Autoantibodies in Patients with Myelodysplastic Neoplasms and Acute Myeloid Leukaemia.

In solid tumours, high expression of the glycolytic enzyme, α-enolase (ENO1), predicts for poor patient overall survival (OS), and circulating autoantibodies to ENO1 correlate positively with diagnosis and negatively with advanced disease. Although ENO1 is one of the most highly expressed genes in acute myeloid leukaemia (AML), its potential role as a biomarker in AML or its precursor, myelodysplastic neoplasms (MDS), has not been investigated. A meta-analysis of nine AML online datasets (n = 1419 patients) revealed that high ENO1 expression predicts for poor OS (HR = 1.22, 95% CI: 1.10-1.34, p < 0.001). Additionally, when compared to AML in remission (n = 5), ENO1 protein detected by immunohistochemistry was significantly higher at diagnosis in bone marrow from both AML (n = 5, p < 0.01) and MDS patients (n = 12, p < 0.05), and did not correlate with percentage of blasts (r = 0.28, p = 0.21). AML patients (n = 34) had lower circulating levels of ENO1 autoantibodies detected by ELISA compared to 26 MDS and 18 controls (p = 0.003). However, there was no difference in OS between AML patients with high vs. low levels of anti-ENO1 autoantibodies (p = 0.77). BM immunostaining for ENO1 and patient monitoring of anti-ENO1 autoantibody levels may be useful biomarkers for MDS and AML.

Valerenic acid attenuates pathological myocardial hypertrophy by promoting the utilization of multiple substrates in the mitochondrial energy metabolism

IntroductionValerenic acid (VA) is a unique and biologically active component in Valeriana officinalis L., which has been reported to have a regulatory effect on the cardiovascular system. However, its therapeutic effects on pathological myocardial hypertrophy (PMH) and the underlying mechanisms are undefined. ObjectivesOur study aims to elucidate how VA improves PMH, and preliminarily discuss its mechanism. MethodsThe efficacy of VA on PMH was confirmed by in vivo and in vitro experiments and the underlying mechanism was investigated by molecular dynamics (MD) simulations and specific siRNA interference. ResultsVA enhanced cardiomyocyte fatty acid oxidation (FAO), inhibited hyper-activated glycolysis, and improved the unbalanced pyruvate-lactate axis. VA could significantly improve impaired mitochondrial function and reduce the triglyceride (TG) in the hypertrophic myocardium while reducing the lactate (LD) content. Molecular mechanistic studies showed that VA up-regulated the expression of peroxisome proliferator-activated receptor-α (PPARα) and downstream FAO-related genes including CD36, CPT1A, EHHADH, and MCAD. VA reduced the expression of ENO1 and PDK4, the key enzymes in glycolysis. Meanwhile, VA improved the pyruvate-lactate axis and promoted the aerobic oxidation of pyruvate by inhibiting LDAH and MCT4. MD simulations confirmed that VA can bind with the F273 site of PPARα, which proposes VA as a potential activator of the PPARα. ConclusionOur results demonstrated that VA might be a potent activator for the PPARα-mediated pathway. VA directly targets the PPARα and subsequently promotes energy metabolism to attenuate PMH, which can be applied as a potentially effective drug for the treatment of HF.

SFXN1 as a potential diagnostic and prognostic biomarker of LUAD is associated with 18F-FDG metabolic parameters

BackgroundSideroflexin 1 (SFXN1) has been discovered as a novel tumor marker for lung adenocarcinoma, but data on its importance in the development of lung adenocarcinoma is still limited. This study evaluated the correlation between SFXN1 and parameters related to 18F-flurodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT), and further explored the role of SFXN1 in the value-added and glycolytic processes of LUAD. MethodThe expression and prognostic value of SFXN1 mRNA in LUAD were analyzed using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data base. Retrospective analysis of 18F-FDG PET imaging and metabolic parameters in 42 patients to explore the relationship between the expression of SFXN1 and glucose metabolism levels in lung adenocarcinoma and its clinical significance. H1975 cells were selected as the in vitro research object, and the biological effects of SFXN1 on LUAD were further elucidated through Edu proliferation assay, CCK8 activity assay, wound healing experiment, and cell flow cytometry. ResultSFXN1 is highly expressed in various tumors, including LUAD, and its high expression can serve as an independent predictor of overall survival in lung adenocarcinoma. In addition, the expression of SFXN1 in LUAD was significantly correlated with 18F-FDG PET/CT parameters: maximum and average standardized uptake values (SUVmax and SUVmean), as well as total lesion glycolysis (TLG) (rho = 0.574, 0.589, and 0.338, p < 0.05), which can predict the expression of SFXN1 with an accuracy of 0.934. In vitro functional experiments have shown that knocking down SFXN1 inhibits the proliferation and migration of LUAD cells, promotes cell apoptosis, and may inhibit tumor activity by regulating the expression of glycolytic related genes SLC2A1, HK2, GPI, ALDOA, GAPDH, ENO1, PKM, and LDHA. ConclusionThe overexpression of SFXN1 is closely related to FDG uptake, and SFXN1, as a promising prognostic biomarker, may mediate the development of LUAD through the glycolytic pathway.

Seven oxidative stress-related genes predict the prognosis of hepatocellular carcinoma.

Predicting the prognosis of hepatocellular carcinoma (HCC) is a major medical challenge and of guiding significance for treatment. This study explored the actual relevance of RNA expression in predicting HCC prognosis. Cox's multiple regression was used to establish a risk score staging classification and to predict the HCC patients' prognosis on the basis of data in the Cancer Genome Atlas (TCGA). We screened seven gene biomarkers related to the prognosis of HCC from the perspective of oxidative stress, including Alpha-Enolase 1(ENO1), N-myc downstream-regulated gene 1 (NDRG1), nucleophosmin (NPM1), metallothionein-3, H2A histone family member X, Thioredoxin reductase 1 (TXNRD1) and interleukin 33 (IL-33). Among them we measured the expression of ENO1, NGDP1, NPM1, TXNRD1 and IL-33 to investigate the reliability of the multi-index prediction. The first four markers' expressions increased successively in the paracellular tissues, the hepatocellular carcinoma samples (from patients with better prognosis) and the hepatocellular carcinoma samples (from patients with poor prognosis), while IL-33 showed the opposite trend. The seven genes increased the sensitivity and specificity of the predictive model, resulting in a significant increase in overall confidence. Compared with the patients with higher-risk scores, the survival rates with lower-risk scores are significantly increased. Risk score is more accurate in predicting the prognosis HCC patients than other clinical factors. In conclusion, we use the Cox regression model to identify seven oxidative stress-related genes, investigate the reliability of the multi-index prediction, and develop a risk staging model for predicting the prognosis of HCC patients and guiding precise treatment strategy.

Response of Propsilocerus akamusi (Diptera: Chironomidae) to the leachates from AMD-contaminated sediments: Implications for metal bioremediation of AMD-polluted areas

Acid mine water (AMD) is a global environmental problem caused by coal mining with the characteristics of low pH and high concentrations of metals and sulfates. It is a pertinent topic to seek both economical and environmentally friendly approaches to minimize the harmful effects of AMD on the environment. Insect larvae are considered a promising solution for pollution treatment. Chironomidae is the most tolerant family to contaminants in pools and its larvae have a strong capacity for metal accumulation from sediment. This paper aimed to evaluate the larvae of Propsilocerus akamusi, a dominant species in the chironomid community, as a new species for entomoremediation in AMD-polluted areas. We detected the toxic effects of AMD on P. akamusi larvae based on their survival and the trace metals bioaccumulation capabilities of P. akamusi larvae. Moreover, we analyzed the expression patterns of four stress-response genes, HSP70, Eno1, HbV, and Hb VII in P. akamusi larvae. Our results revealed that AMD exposure did not significantly affect the survival of the P. akamusi larvae and individuals exposed to some AMD gradients even exhibited higher survival. We also observed the significantly accumulated concentrations of Fe, Ni, and Zn as well as higher bioaccumulation factors (BAFs) for Ni and Zn in the P. akamusi larvae exposure to AMD. Induced expression of Eno1 and Hb VII may play important roles in the AMD tolerance of P. akamusi larvae. This study indicated the potential application of P. akamusi larvae in the metal bioremediation of AMD-polluted areas. Statement of environmental implicationAcid mine drainage (AMD) is a global environmental problem related to coal mining activities. AMD pollution has become a long-term, worldwide issue for its interactive and complex stress factors. Bioremediation is an effective method to remove the metals of AMD from wastewater to prevent downstream pollution. However, the disadvantages of the slow growth rate, susceptibility to seasonal changes, difficult post-harvest management, and small biomass of hyperaccumulating plants greatly limit the usefulness of phytoremediation. Insect larvae may be useful candidate organisms to overcome these shortcomings and have been considered a promising pollution solution. Propsilocerus akamusi is a dominant species in the chironomid community and is distributed widely in many lakes of eastern Asia. This species has extraordinary abilities to resist various stresses. This research is the first time to our knowledge to evaluate the application of P. akamusi as a new species in entomoremediation in AMD-contaminated areas.

Single-cell profiling reveals transcriptomic signatures of vascular endothelial cells in non-healing diabetic foot ulcers.

The treatment of diabetic foot ulcers (DFUs) poses a challenging medical problem that has long plagued individuals with diabetes. Clinically, wounds that fail to heal for more than 12 weeks after the formation of DFUs are referred to as non-healing/chronic wounds. Among various factors contributing to the non-healing of DFUs, the impairment of skin microvascular endothelial cell function caused by high glucose plays a crucial role. Our study aimed to reveal the transcriptomic signatures of non-healing DFUs endothelial cells, providing novel intervention targets for treatment strategies. Based on the GEO dataset (GSE165816), we selected DFU-Healer, DFU-Non-healer, and healthy non-diabetic controls as research subjects. Single-cell RNA transcriptomic sequencing technology was employed to analyze the heterogeneity of endothelial cells in different skin tissue samples and identify healing-related endothelial cell subpopulations. Immunofluorescence was applied to validate the sequencing results on clinical specimens. The number of endothelial cells and vascular density showed no significant differences among the three groups of skin specimens. However, endothelial cells from non-healing DFUs exhibited apparent inhibition of angiogenesis, inflammation, and immune-related signaling pathways. The expression of CCND1, ENO1, HIF1α, and SERPINE1 was significantly downregulated at the transcriptomic and histological levels. Further analysis demonstrated that healing-related endothelial cell subpopulations in non-healing DFUs has limited connection with other cell types and weaker differentiation ability. At the single-cell level, we uncovered the molecular and functional specificity of endothelial cells in non-healing DFUs and highlighted the importance of endothelial cell immune-mediated capability in angiogenesis and wound healing. This provides new insights for the treatment of DFUs.

Evaluating the Anti-Melanoma Effects and Toxicity of Cinnamaldehyde Analogues.

Cinnamaldehyde (CA) showed potent activity against melanoma in our previous study, and the structure of unsaturated aldehydes is envisaged to play a role. Nevertheless, its limited drug availability restricts its clinical application. Therefore, a series of CA analogues were synthesized to evaluate their anti-melanoma activities across various melanoma cell lines. These compounds were also tested for their toxicity against the different normal cell lines. The compound with the most potential, CAD-14, exhibited potent activity against the A375, A875 and SK-MEL-1 cells, with IC50 values of 0.58, 0.65, and 0.82 µM, respectively. A preliminary molecular mechanism study of CAD-14 indicated that it could inhibit the p38 pathway to induce apoptosis, and suppress tumor growth by inhibiting the expression of ENO1. Furthermore, an acute toxicity study depicted that CAD-14 has better safety and tolerability than CA in vivo. These findings indicate that CAD-14 might be a lead compound for exploring effective anti-melanoma drugs.

13C tracer analysis reveals the landscape of metabolic checkpoints in human CD8+ T cell differentiation and exhaustion

IntroductionNaïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells.MethodsHere we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions. ResultsThe quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate. DiscussionOverall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells.

High-Fat Mouse Model to Explore the Relationship between Abnormal Lipid Metabolism and Enolase in Pancreatic Cancer.

Malignant tumors have become a major social health problem that seriously threatens human health, among which pancreatic cancer has a high degree of malignancy, difficult diagnosis and treatment, short survival time, and high mortality. More and more attention has been paid to abnormal lipid metabolism as a momentous carcinogenesis mechanism. Here, we explored the relationship between abnormal lipid metabolism, enolase, and pancreatic cancer by clinical data analysis. A high-fat mouse model was constructed, and then, a subcutaneous tumorigenesis mouse model of carcinoma of pancreatic cells and a metastatic neoplasm mouse pattern of pancreatic carcinoma cells injected through the tail vein were constructed to explore whether abnormal lipid metabolism affects the progression of pancreatic cancer in mice. We constructed a high-lipid model of pancreatic carcinoma cell lines and knockdown and overexpressed enolase in pancreatic carcinoma cell lines and investigated whether high lipid regulates epithelial-mesenchymal transition (EMT) by upregulating enolase (ENO), thereby promoting the cells of pancreatic carcinoma to invade and migrate. Triglycerides, total cholesterol, free cholesterin, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and neuron-specific enolase (NSE) from pancreatic cancer patients and nonpancreatic cancer patients were tested. The differences in blood lipids between patients with and without pancreatic carcinoma were compared, and the correlation between blood lipids and neuron-specific enolase was analyzed. We confirmed that the serum triglyceride level of pancreatic cancer patients at initial diagnosis is overtopping nonpancreatic cancer patients, and the neuron-specific enolase level of patients with pancreatic carcinoma is better than nonpancreatic carcinoma sufferers. Triglyceride level is positively correlated with neuron-specific enolase level, and serum triglyceride level has predictive value for pancreatic cancer. Hyperlipidemia can promote tumor growth and increase the expression levels of ENO1, ENO2, and ENO3 in subcutaneous tumor formation of pancreatic cancer in mice. Additional hyperlipidemia promoted pancreatic carcinoma metastasis in the lung in mice injected through the tail vein, which confirmed that hyperlipidemia accelerated the process of EMT by increasing the expression of ENO1, ENO2, and ENO3, therefore promoting the pancreatic cancer cell metastasis.

Single-Cell Transcriptome Analysis Revealed Heterogeneity and Identified Novel Therapeutic Targets for Breast Cancer Subtypes.

Breast cancer (BC) is a heterogeneous disease, which is primarily classified according to hormone receptors and HER2 expression. Despite the many advances in BC diagnosis and management, the identification of novel actionable therapeutic targets expressed by cancerous cells has always been a daunting task due to the large heterogeneity of the disease and the presence of non-cancerous cells (i.e., immune cells and stromal cells) within the tumor microenvironment. In the current study, we employed computational algorithms to decipher the cellular composition of estrogen receptor-positive (ER+), HER2+, ER+HER2+, and triple-negative BC (TNBC) subtypes from a total of 49,899 single cells' publicly available transcriptomic data derived from 26 BC patients. Restricting the analysis to EPCAM+Lin- tumor epithelial cells, we identified the enriched gene sets in each BC molecular subtype. Integration of single-cell transcriptomic with CRISPR-Cas9 functional screen data identified 13 potential therapeutic targets for ER+, 44 potential therapeutic targets for HER2+, and 29 potential therapeutic targets for TNBC. Interestingly, several of the identified therapeutic targets outperformed the current standard of care for each BC subtype. Given the aggressive nature and lack of targeted therapies for TNBC, elevated expression of ENO1, FDPS, CCT6A, TUBB2A, and PGK1 predicted worse relapse-free survival (RFS) in basal BC (n = 442), while elevated expression of ENO1, FDPS, CCT6A, and PGK1 was observed in the most aggressive BLIS TNBC subtype. Mechanistically, targeted depletion of ENO1 and FDPS halted TNBC cell proliferation, colony formation, and organoid tumor growth under 3-dimensional conditions and increased cell death, suggesting their potential use as novel therapeutic targets for TNBC. Differential expression and gene set enrichment analysis in TNBC revealed enrichment in the cycle and mitosis functional categories in FDPShigh, while ENO1high was associated with numerous functional categories, including cell cycle, glycolysis, and ATP metabolic processes. Taken together, our data are the first to unravel the unique gene signatures and to identify novel dependencies and therapeutic vulnerabilities for each BC molecular subtype, thus setting the foundation for the future development of more effective targeted therapies for BC.

Allantopyrone A interferes with the degradation of hypoxia-inducible factor 1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Allantopyrone A is an α-pyrone metabolite that was originally isolated from the endophytic fungus Allantophomopsis lycopodina KS-97. We previously demonstrated that allantopyrone A exhibits anti-cancer, anti-inflammatory, and neuroprotective activities. In the present study, we showed that allantopyrone A up-regulated the protein expression of hypoxia-inducible factor (HIF)-1α in human fibrosarcoma HT-1080 cells. It also up-regulated the mRNA expression of BNIP3 and ENO1, but not other HIF target genes or HIF1A. Allantopyrone A did not inhibit the prolyl hydroxylation of HIF-1α, but enhanced the ubiquitination of cellular proteins. Consistent with this result, chymotrypsin-like and trypsin-like proteasome activities were reduced, but not completely inactivated by allantopyrone A. Allantopyrone A decreased the amount of proteasome catalytic subunits. Therefore, the present results showed that allantopyrone A interfered with the degradation of HIF-1α protein by reducing proteasome activity in human fibrosarcoma HT-1080 cells.

Sex-Specific Whole-Transcriptome Analysis in the Cerebral Cortex of FAE Offspring.

Fetal alcohol spectrum disorders (FASDs) are associated with systemic inflammation and neurodevelopmental abnormalities. Several candidate genes were found to be associated with fetal alcohol exposure (FAE)-associated behaviors, but a sex-specific complete transcriptomic analysis was not performed at the adult stage. Recent studies have shown that they are regulated at the developmental stage. However, the sex-specific role of RNA in FAE offspring brain development and function has not been studied yet. Here, we carried out the first systematic RNA profiling by utilizing a high-throughput transcriptomic (RNA-seq) approach in response to FAE in the brain cortex of male and female offspring at adulthood (P60). Our RNA-seq data analysis suggests that the changes in RNA expression in response to FAE are marked sex-specific. We show that the genes Muc3a, Pttg1, Rec8, Clcnka, Capn11, and pnp2 exhibit significantly higher expression in the male offspring than in the female offspring at P60. FAE female mouse brain sequencing data also show an increased expression of Eno1, Tpm3, and Pcdhb2 compared to male offspring. We performed a pathway analysis using a commercial software package (Ingenuity Pathway Analysis). We found that the sex-specific top regulator genes (Rictor, Gaba, Fmri, Mlxipl) are highly associated with eIF2 (translation initiation), synaptogenesis (the formation of synapses between neurons in the nervous system), sirtuin (metabolic regulation), and estrogen receptor (involved in obesity, aging, and cancer) signaling. Taken together, our transcriptomic results demonstrate that FAE differentially alters RNA expression in the adult brain in a sex-specific manner.

α-Enolase inhibits apoptosis and promotes cell invasion and proliferation of skin cutaneous melanoma

BackgroundThe glycolytic enzyme, α-Enolase (ENO1), catalyzes the production of phosphoenolpyruvate from 2-phosphoglycerate, thereby enhancing glycolysis and contributing to tumor progression. In the present study, we aimed to determine the role of ENO1 in skin cutaneous melanoma (SKCM) and the potential underlying mechanism.MethodsThe Sangerbox database was used to analyze the mRNA expression of ENO1 in SKCM. Western blotting was used to assess the levels of ENO1, c-Myc, β-catenin, MMP-9, PGAM1, and MMP-13 in SKCM-derived cell lines or tumor tissues from patients with SKCM. The pCMV-SPORT6-ENO1 and pET-28a-ENO1siRNA plasmids were used to overexpress and knockdown ENO1 in SKCM cells, respectively. To determine the function of ENO1 in the malignant behavior of SKCM cells, we performed a wound-healing assay, cell counting kit 8 assay, and transwell chamber analyses. The production of pyruvate and lactic acid in tumor cells was evaluated using their respective kits.ResultsCompared with non-tumor tissues, ENO1 was found to be overexpressed in SKCM tissues. In SKCM cells, ENO1 overexpression promoted invasion, migration, and proliferation of tumor cells; increased pyruvate and lactate production; and increased β-catenin, MMP-9, MMP-13, and c-Myc levels. The opposite effects were observed in SKCM cells silenced for ENO1.ConclusionsThese results indicate that ENO1 is involved in SKCM progression by enhancing the invasion and proliferation of tumor cells. In addition, ENO1 might have an important function in tumor cell glycolysis. Therefore, ENO1 represents a potential therapeutic target for treatment of SKCM.

Long non-coding RNA ANRIL promotes chemoresistance in triple-negative breast cancer via enhancing aerobic glycolysis

AimslncRNA ANRIL expression is dysregulated in many human cancers and is thus a useful prognostic marker for cancer patients. However, whether ANRIL is involved in drug resistance in triple-negative breast cancer (TNBC) has not yet been investigated. Main methodsA luciferase reporter assay was conducted to verify the binding between miR-125a and ANRIL. RT-PCR and western blotting were performed to detect the expression of miR-125a, ANRIL, and ENO1. Glycolysis stress was assessed using the Seahorse extracellular flux analyzer. Functional studies were performed using both in vitro and in vivo xenograft models. Key findingsANRIL was markedly upregulated in both patients with TNBC and TNBC cell lines. Knockdown of ANRIL increased the cytotoxic effect of ADR and repressed cellular glycolytic activity in TNBC cells. Mechanistic analysis showed that ANRIL may act as a competing endogenous RNA of miR-125a to relieve the repressive effect of miR-125a on its target glycolytic enzyme enolase (ENO1). Notably, 2-deoxy-glucose attenuated ANRIL-induced increase in drug resistance in TNBC cells. SignificanceThese results indicate that knockdown of ANRIL plays an active role in overcoming drug resistance in TNBC by inhibiting glycolysis through the miR-125a/ENO1 pathway, which may be useful for the development of novel therapeutic targets for treating patients with TNBC, especially those with drug resistance.

Identification of ENO1 as a prognostic biomarker and molecular target among ENOs in bladder cancer

BackgroundEnolase is an essential enzyme in the process of glycolysis and has been implicated in cancer progression. Though dysregulation of ENOs has been reported in multiple cancers, their prognostic value and specific role in bladder cancer (BLCA) remain unclear.MethodsMultiple databases were employed to examine the expression of ENOs in BLCA. The expression of ENO1 was also validated in BLCA cell lines and tissue samples by western blotting and immunohistochemistry. Kaplan–Meier analysis, ROC curve, univariate and multivariate Cox regression were performed to evaluate the predictive capability of the ENO1. Gene ontology (GO) and Gene Set Enrichment Analyses (GSEA) analysis were employed to perform the biological processes enrichment. Function experiments were performed to explore the biological role of ENO1 in BLCA. The correlation of ENO1 with immune cell infiltration was explored by CIBERSORT.ResultsBy analyzing three ENO isoforms in multiple databases, we identified that ENO1 was the only significantly upregulated gene in BLCA. High expression level of ENO1 was further confirmed in BLCA tissue samples. Aberrant ENO1 overexpression was associated with clinicopathological characteristics and unfavorable prognosis. Functional studies demonstrated that ENO1 depletion inhibited cancer cell aggressiveness. Furthermore, the expression level of ENO1 was correlated with the infiltration levels of immune cells and immune-related functions.ConclusionsTaken together, our results indicated that ENO1 might serve as a promising prognostic biomarker for prognosticating prognosis associated with the tumor immune microenvironment, suggesting that ENO1 could be a potential immune-related target against BLCA.

P870: DEFINING A NOVEL FUNCTION FOR THE POST-TRANSLATIONAL MODIFICATION UFMYLATION IN THE ADAPTIVE RESPONSE TO ARGININE DEPRIVATION IN MULTIPLE MYELOMA

Background: Our previous work showed that arginine deprivation is associated to multiple myeloma (MM) bortezomib refractoriness, but the molecular basis of this adaptive response is still largely unknown. Aims: We designed an integrative transcriptional and metabolic study to explore the contemporary transcriptional and metabolic changes occurring upon arginine deprivation in four human myeloma cell lines (HMCLs, U266, NCI-H929, OPM2 and MM1.s). Methods: HMCLs were individually cultured with customized complete with different Arg concentrations, correspondent respectively to 100%, 25% and 10% of the arginine concentration in MM bone marrow. After 24-48 hours of culture, the cells were collected for global metabolomic analysis (Metabolon Inc) and transcriptome profiling by Illumina platform. Apoptosis and mitochondrial depolarization were measured by FACS. Protein expression was evaluated by western blot analysis. Results: In all tested HMCLs the progressive arginine deprivation (range 1124-0 µM) induced delay in cell cycle until proliferation arrest (in total lack of arginine in culture media), with increase of G0-G1 length, at specific timepoints for each cell line tested. However global protein synthesis was not affected and surprisingly, in all tested cell lines, the monoclonal component secretion in extracellular medium was preserved after 96 hours of arginine starvation. At 48 hours of arginine deprivation (range 1124-100 µM) GCN2 pathway was induced in HMCLs with consequent increase of basal levels of ATF4, p62, GABARAP and autophagy flux. After 48 hours of arginine starvation, coupled metabolomics and RNA-seq, disclosed an impairment in the pool of nucleotides and activation of salvage purine pathway, that could explain changes in cell cycle and proliferation. Arginine-deprived MM cells had higher levels of choline and phospho-ethanolamine with lower levels of glycerophosphorylcholine (GPC) compared to controls suggesting an effect of arginine-deprivation on membrane dynamics, associated to increased production of fatty acids, palmitate (16:0) and stearate (18:0), required for the generation of new membranes. Culture in arginine deficient media resulted in complete depletion of reduced glutathione (GSH) and oxidized glutathione (GSSG), with depletion of gamma-glutamylcysteine, indicating increased levels of oxidative stress. Gene set enrichment analysis (GSEA) showed deep transcriptome rearrangements, involving upregulation of genes required for the unfolded protein response, NF-kB response to TNF, p53 pathway and networks and proteosome degradation, with a minimal effect on metabolism features, except the upregulation of genes involved in lactate generation and degradation. In all cell lines, transcripts involved in the UFMylation pathway were affected, with reduction of UFSP2 and increase of UFMYlation targets RPL26, PKM2, ENO-1, PGK-1 and SQSTM1/p62. In a large series of primary samples (9N - 20MGUS - 33SMM - 170MM - 24PPCL - 12SPCL) and 18 MM cell lines, through bioinformatic analysis of previously published datasets (GENE1.0_BAv20_Neri-Gutierrez), we found that myeloma progression was associated to increased expression of UFMYlation targets ENO-1, PGK1 and DLD. Summary/Conclusion: Taken together, our data clearly indicate how arginine starvation can induce an heterogeneous adaptation processes occur in individual tumour clones. The ER stress adaptive response triggered by arginine deprivation promotes UFMylation activation, due to downregulation of UFSP2 and accumulation of RPN1/RPL26 targets.