Significant Increase In Expression Research Articles

Vitamin D Receptor Regulates Liver Regeneration After Partial Hepatectomy in Male Mice.

Vitamin D signals through the vitamin D receptor (VDR) to induce its end-organ effects. Hepatic stellate cells control development of liver fibrosis in response to stressors and vitamin D signaling decreases fibrogenesis. VDR expression in hepatocytes, however, is low in healthy liver, and the role of VDR in hepatocyte proliferation is unclear. Hepatocyte-VDR null mice (hVDR) were used to assess the role of VDR and vitamin D signaling in hepatic regeneration. hVDR mice have impaired liver regeneration and impaired hepatocyte proliferation associated with significant differential changes in bile salts. Notably, mice lacking hepatocyte VDR had significant increases in expression of conjugated bile acids after partial hepatectomy, consistent with failure to normalize hepatic function by the 14-day time point tested. Real-time PCR of hVDR and control livers showed significant changes in expression of cell cycle genes including cyclins D1 and E1 and cyclin-dependent kinase 2. Gene expression profiling of hepatocytes treated with vitamin D or control showed regulation of groups of genes involved in liver proliferation, hepatitis, liver hyperplasia/hyperproliferation and liver necrosis/cell death. Together these studies demonstrate an important functional role for VDR in hepatocytes during liver regeneration. Combined with the known profibrotic effects of impaired VDR signaling in stellate cells, the studies provide a mechanism whereby vitamin D deficiency would both reduce hepatocyte proliferation and permit fibrosis, leading to significant liver compromise.

P-549 Differentially expressed genes in the immune cells obtained from preovulatory follicles undergoing progestin-primed ovarian stimulation and GnRH-antagonist: Insights from single-cell RNA sequencing analysis

Abstract Study question Could controlled ovarian stimulation protocol (progestin-primed ovarian stimulation [PPOS] vs GnRH-antagonist [GnRH-ant]) affect the gene expression of human immune cells (ICs) in preovulatory follicles? Summary answer Proinflammatory cytokine-related gene expression was significantly higher and proinflammatory pathways were also more prominently involved in ICs in PPOS than the GnRH-ant. What is known already PPOS has garnered attention, with several studies suggesting reproductive outcomes comparable to GnRH-ant. However, in our previous study, pregnancy outcomes with PPOS were significantly lower than GnRH-ant (38th ESHRE Annual Meeting [O-130]) and single-cell RNA sequencing (scRNA-seq) of granulosa cells revealed increased mitochondrial DNA gene expression in PPOS than GnRH-ant (39th ESHRE Annual Meeting [P-281]). Previous studies suggested that ICs play a pivotal role in follicular and embryonic developments, via proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. Notably, changes in gene expressions of ICs under different controlled ovarian stimulation (COS) protocols remain unresolved to date. Study design, size, duration From November 2021 to January 2022, all samples were obtained from the patients who provided written informed consent before the inclusion and underwent COS cycles at a single-institution. scRNA-seq was performed on 6,828 cells corresponding to ICs of the MII-oocyte follicles (56 from PPOS and 39 from GnRH-ant), involving 16 patients with normal ovarian reserves (aged < 40 years, AMH ≥1.1 ng/mL) undergoing PPOS with chlormadinone acetate (n = 8) or GnRH-ant with cetrorelix (n = 8). Participants/materials, setting, methods The ICs were collected via follicular aspiration from 16 patients. The follicular fluid (FF) from each follicle was collected and all FF were mixed together in MII-oocyte for sequencing. Sample sequencing was performed using Chromium Next GEM Single Cell V(D)J Reagent Kits (10X Genomics, Pleasanton, CA, USA). Libraries were sequenced using a DNBSEQ-G400, with reads subsequently analyzed using Cellxgene. The clustering was performed using fastcluster in scDblFinder. Functional enrichment analysis was carried out using Enrichr. Main results and the role of chance Two population of ICs were used in this investigation: PPOS and GnRH-ant of MII-oocyte. After the quality control, 4,447 cells for PPOS and 2,381 cells for GnRH-ant, respectively, were analyzed. Following single-cell clustering based on the Seurat package, cell proportion analysis revealed eight clusters, which consist of monocyte, myeloid dendritic cell, T cell, natural killer cell and naive B cell, in the PPOS and GnRH-ant groups. No significant difference was observed in gene expression and cell distribution in each cluster. Differentially expressed genes (DEGs) were identified in the two IC population. Interestingly, a significant increase in expression of proinflammatory cytokine including IL-1β and IL-6 was observed in PPOS compared to GnRH-ant, particularly in CD14-positive monocytes. Functional significance of DEGs was validated by identifying significantly regulated pathways based on Kyoto Encyclopedia of Genes and Genomes categories, revealing involvement in TNF signaling, IL-17 signaling, and NF-κB signaling pathways. Limitations, reasons for caution Our conclusions are limited due to the inclusion of Japanese patients at a single-institution. The results need to be validated across different centers and other ethnicities. Due to the small sample size, these results should prompt further study to confirm our findings. Wider implications of the findings This is the first report assessing the DEGs of ICs under different COS protocols at the single-cell level. This study suggests that progestin for PPOS induces elevation of the proinflammatory cytokine gene expression of ICs in preovulatory follicles, which may be associated with reproductive outcomes. Trial registration number Not applicable

Mitigative Effects of l-Arginine and N-Acetyl Cysteine against Cisplatin-Induced Testicular Dysfunction and Toxicity through the Regulation of Antioxidant, Anti-inflammatory, and Antiapoptotic Markers: Role of miR-155 and miR-34c Expression

Mitigative Effects of <scp>l</scp>-Arginine and N-Acetyl Cysteine against Cisplatin-Induced Testicular Dysfunction and Toxicity through the Regulation of Antioxidant, Anti-inflammatory, and Antiapoptotic Markers: Role of miR-155 and miR-34c Expression

Exercise-Induced Laminar Blood Flow Maintains Vascular Function by Enhanced Endothelial Homeostasis

PURPOSE: Laminar blood flow is known to play a critical role in maintaining endothelial homeostasis in blood vessels. It exerts a shear stress on the endothelial cells (ECs), which is essential for the regulation of various vascular functions. In this study, the effects of laminar blood flow-induced enhanced endothelial homeostasis on the maintenance of vascular function were investigated.METHODS: Human umbilical vein endothelial cells (HUVECs) were employed to study gene expression associated with vessel dilation and angiogenesis in response to varying shear stress levels. To assess angiogenesis, experimental mice participated in voluntary wheel running for 16 weeks. To evaluate the vascular autophagic function, the six weeks old C57BL/6 mice underwent an hour of forced treadmill running for 10 weeks. The resulting gene expression was evaluated through western blotting and quantitative real-time polymerase chain reaction (qRT-PCR).RESULTS: Disturbed flow resulting from partial ligation surgery in the mice’s carotid artery led to a decrease in angiogenic capacity compared to the laminar flow observed in ex vivo sprouting assays. Cultured HUVECs exhibited a significant increase in VEGF expression when exposed to increased durations of 20 dyne/cm² laminar shear stress (LSS). Following a 10-week treadmill exercise regimen, ECs in the carotid arteries of mice showed a significant increase in the expression of autophagic genes, such as LC3II, Atg3, and Atg7, which was not observed in smooth muscle cells (SMCs). LSS was found to boost the expression of both total and phosphorylated eNOS, genes involved in vascular dilation regulation, specifically in ECs.CONCLUSIONS: These findings suggest that laminar blood flow sustains vascular function, such as angiogenic capacity, through the enhancement of endothelial homeostasis.

Particulate matter exposure induces adverse effects on endometrium and fertility via aberrant inflammatory and apoptotic pathways in vitro and in vivo

This study aimed to evaluate the adverse effects of particulate matter (PM) exposure on endometrial cells and fertility and to identify possible underlying mechanisms. Thirteen women (aged 15–52 years) were included in this study. Enrolled patients underwent laparoscopic surgery at Gangnam Severance Hospital between 1 January and 31 December 2021. For in vivo experiments, 36 female and nine male C57BL/6 mice were randomly divided into control(vehicle), low-dose(10 mg/kg/d), and high-dose exposure groups(20 mg/kg/d). PM was inhaled nasally for four weeks and natural mating was performed. NIST® SRM® 1648a was used for PM exposure. qRT-PCR, western blotting and Masson's trichrome staining were performed. PM treatment in human endometrial stromal cells induced inflammation with significant upregulation of IL-1β, p–NF–kB, and p-c-Jun compared to those of controls. Additionally, PM treatment significantly increased apoptosis in human endometrial stromal cells by downregulating p-AKT and upregulating p-p53/p53, Cas-3, BAX/Bcl-2, p-AMPK, and p-ERK. After PM treatment, the relative expression of IL-1β, IL-6, TNF-α, p–NF–κB, p-c-Jun, and p-Nrf2/Nrf2 significantly increased in murine endometrium compared to those of the controls. Expression of apoptotic proteins p53, p27, and Cas-3, was also significantly elevated in murine endometrium of the PM exposure group compared to that of the controls. A significant increase in expression of procollagen Ⅰ, and Masson's trichrome staining scores in the murine endometrium was noted after PM treatment. PM treatment significantly decreased ERα expression. After natural mating, all 3 female mice in the control group gave birth to 25 offspring (mean 8.1), whereas in the low-dose PM treatment group, two of three female mice gave birth to nine offspring (mean 4.5). No pregnant mice or offspring was present in the high-dose PM treatment group. PM exposure induces adverse effects on the endometrium through aberrant activation of inflammatory and apoptotic pathways and is associated with detrimental effects on murine fertility.

Comprehensive genome-wide analysis of wheat xylanase inhibitor protein (XIP) genes: unveiling their role in Fusarium head blight resistance and plant immune mechanisms.

In this comprehensive genome-wide study, we identified and classified 83 Xylanase Inhibitor Protein (XIP) genes in wheat, grouped into five distinct categories, to enhance understanding of wheat's resistance to Fusarium head blight (FHB), a significant fungal threat to global wheat production. Our analysis reveals the unique distribution of XIP genes across wheat chromosomes, particularly at terminal regions, suggesting their role in the evolutionary expansion of the gene family. Several XIP genes lack signal peptides, indicating potential alternative secretion pathways that could be pivotal in plant defense against FHB. The study also uncovers the sequence homology between XIPs and chitinases, hinting at a functional diversification within the XIP gene family. Additionally, the research explores the association of XIP genes with plant immune mechanisms, particularly their linkage with plant hormone signaling pathways like abscisic acid and jasmonic acid. XIP-7A3, in particular, demonstrates a significant increase in expression upon FHB infection, highlighting its potential as a key candidate gene for enhancing wheat's resistance to this disease. This research not only enriches our understanding of the XIP gene family in wheat but also provides a foundation for future investigations into their role in developing FHB-resistant wheat cultivars. The findings offer significant implications for wheat genomics and breeding, contributing to the development of more resilient crops against fungal diseases.

#2229 miR-186, a potential noninvasive diagnostic biomarker of Membranous nephropathy in urinary microparticles

Abstract Background and Aims Membranous nephropathy (MN) is one of the most common causes of nephrotic syndrome, and is associated with podocyte damage and proteinuria. Invasive biopsy is the gold-standard diagnostic method for this disease. The use of specific biomarkers, such as microparticles (MPs) originating from damaged podocytes released in a patient's urine, has been proposed as an early, noninvasive, and repetitive diagnostic method. According to the previous studies, MPs derived from podocytes are present in the urine of patients with different glomerulonephritis, such as diabetic nephropathy, lupus nephritis, renal injury in preeclampsia, and renovascular hypertensive disorders. The analysis of urinary microparticle-derived miRNAs from patients with kidney diseases can help detect diagnostic biomarkers. This study aimed to analyze podocyte damage-related miRNAs in MN, miR-30c, miR-135, and miR-18 in the urinary MP of MN patients to identify appropriate candidate biomarkers for MN Method Urine samples from 30 patients with membranous nephropathy and 16 healthy individuals were collected to extract podocyte MPs using ultracentrifugation. Characterization of these particles was determined using the specific protein marker of MPs, TSG-101, and the podocyte-specific marker Nephrin, by western blotting, and SEM was used to check the size and morphology. The expression levels of microRNAs extracted from podocyte MPs, miR-30c, miR-135, and miR-186, were also evaluated by real-time PCR. Results The evaluation of the characteristics of MPs showed that there were many particles with a size of more than 100 nm in the urine sample, and there were higher levels of the specific markers TSG-101 and Nephrin in the urine samples of MGN patients than in the control samples (P &amp;lt; 0.001). The expression level of miR-186 was compared between the three studied groups and based on the results of the expression of this miRNA in the group of membranous nephropathy patients compared to healthy people group, there was a significant increase in expression (P = 0.000) Conclusion This study showed that although the expression levels of miR-186 in podocyte MPs samples extracted from the urine of patients with MN were significantly changed compared to the studied controls, more studies should be performed to determine the diagnostic values.

#883 P-cresyl sulphate and Indoxyl sulphate: two uremic toxins stimulating colon cancer progression in patients with chronic renal failure

Abstract Background and Aims Chronic kidney disease (CKD) leads to the accumulation and production of uremic toxins, such as Indoxyl Sulphate (IS) and P-cresyl sulphate (p-CS). These toxins have been found to activate various processes that promote tumor growth. The literature has reported a two-fold increase in the incidence of colon cancer (CRC) and a worse prognosis in CKD patients, although the specific mechanisms underlying this association have not been fully elucidated. Given the pro-oncogenic activities of p-CS and IS at both the systemic and intestinal levels, it is plausible that these toxins contribute to the heightened aggressiveness of CRC in CKD patients. Method In order to assess the impact of toxins detected in uremic patients, we conducted experiments using three different colorectal cancer (CRC) cell lines (HT29, Hct116, LoVo). We tested various concentrations of IS and p-CS and measured their effects on tumor cell proliferation through a colorimetric assay called WST-8. For further analysis of cell survival, we performed a clonogenic assay. To investigate the migratory abilities of tumor cells after toxins treatment, we conducted a wound healing assay. Additionally, we assessed the invasive capacities of the three CRC cell lines post treatment with IS and p-CS using an invasion assay. In order to identify the specific pathways affected by toxins exposure, we conducted Real Time PCR and Western blot analysis to evaluate the inflammatory pathway and the epithelial-mesenchymal transition (EMT), respectively. Results The results of this study are remarkable, as we observed a notable increase in cell growth after exposure of cultured cells to various concentrations of toxins, as compared to the control group. Furthermore, the concentrations of IS and p-CS induced a substantial enhancement in cell migration and invasion. In the case of HCT116 cells treated with uremic toxins, there was an up-regulation of Vimentin protein expression, while E-cadherin expression was down-regulated compared to the control group. These proteins are crucial in the process of Epithelial-Mesenchymal Transition (EMT). Additionally, we evaluated the expression of genes associated with the inflammatory process (TNF-alpha, INOS, and IL-6) in the HT-29 and LoVo cell lines. Interestingly, we observed an up-regulation of these genes. Of particular importance, in the LoVo cell line, IL-6 exhibited the most significant increase in expression. Conclusion Based on these preliminary results, it appears that IS and p-CS play a key role in the progression of CRC in patients with CKD.

P02 Accelerated ageing of hair follicles in androgenetic alopecia

Abstract Introduction and aims Androgenetic alopecia (AGA) is defined by hair loss from the frontal, parietal and vertex regions of the scalp; however, occipital follicles are spared. Hair follicles in AGA undergo miniaturization, which is the transition of terminal follicles into vellus follicles. Similarly with age, the diameter of the hair shaft decreases with loss of pigmentation, which is also seen in miniaturized follicles. To investigate whether frontal follicles have accelerated ageing compared with occipital follicles, we looked at methylation levels of genes associated with ageing. As the thickness and pigmentation of hair fibre are influenced by the dermal papilla (DP), we conducted microarray analysis of frontal and occipital DP to establish whether accelerated ageing of follicles is driven by DP. Methods The biological age of frontal and occipital follicles was calculated using methylation status of ELOVL2, ASPA, EDARADD and PDE4C (n = 3). For microarray analysis, DP was microdissected from the frontal (FDP) and occipital (ODP) follicles from patients undergoing hair transplantation surgeries to treat AGA (n = 4). Results Frontal follicles were found to have significantly higher biological age compared with patient-matched occipital follicles suggesting accelerated ageing of follicles in AGA. Transcriptomic analysis revealed significant increase in expression of several genes in FDP and notably EGR1, HSPB1, DNAJB1 and APOD, which have been shown to be upregulated in the DP of aged mice compared with young mice. We found downregulation of TRPS1 in FDP, which represses EGR1. Mutations in TRPS1 underlie trichorhinophalangeal syndrome which is associated with vellus-type hair. Conclusions Our transcriptional and molecular analysis revealed that FDP and frontal follicles have an advanced biological age relative to ODP. This increased age is also reflected in the transcriptional signature of FDP. We hypothesize that these changes are in part due to regulation of signalling by the transcriptional repressor TRPS1, whose expression is decreased in FDP.

SIRT3 protects endometrial receptivity in patients with polycystic ovary syndrome.

The sirtuin family is well recognized for its crucial involvement in various cellular processes. Nevertheless, studies on its role in the human endometrium are limited. This study aimed to explore the expression and localization of the sirtuin family in the human endometrium, focusing on sirtuin 3 (SIRT3) and its potential role in the oxidative imbalance of the endometrium in polycystic ovary syndrome (PCOS). Endometrial specimens were collected from both patients with PCOS and controls undergoing hysteroscopy at the Center for Reproductive Medicine, Peking University Third Hospital, from July to August 2015 and used for cell culture. The protective effects of SIRT3 were investigated, and the mechanism of SIRT3 in improving endometrial receptivity of patients with PCOS was determined using various techniques, including cellular bioenergetic analysis, small interfering ribonucleic acid (siRNA) silencing, real-time quantitative polymerase chain reaction, Western blot, immunofluorescence, immunohistochemistry, and flow cytometry analysis. The sirtuin family was widely expressed in the human endometrium, with SIRT3 showing a significant increase in expression in patients with PCOS compared with controls (P <0.05), as confirmed by protein and gene assays. Concurrently, endometrial antioxidant levels were elevated, while mitochondrial respiratory capacity was reduced, in patients with PCOS (P <0.05). An endometrial oxidative stress (OS) model revealed that the downregulation of SIRT3 impaired the growth and proliferation status of endometrial cells and reduced their receptivity to day 4 mouse embryos. The results suggested that SIRT3 might be crucial in maintaining normal cellular state by regulating antioxidants, cell proliferation, and apoptosis, thereby contributing to enhanced endometrial receptivity. Our findings proposed a significant role of SIRT3 in improving endometrial receptivity in patients with PCOS by alleviating OS and regulating the balance between cell proliferation and apoptosis. Therefore, SIRT3 could be a promising target for predicting and improving endometrial receptivity in this patient population.

Assessing the Ability to Reverse High Fat High Sugar Diet-Induced Hepatic Steatosis by Dietary Modification

Background: Metabolic syndrome includes a plethora of symptoms ultimately leading to obesity and type 2 diabetes. Increased consumption of ‘Western diets’ rich in fat and sugar increase the development of metabolic syndrome, diabetic obesity and furthermore increase the risk of non-alcoholic fatty liver disease. We aimed to better understand the influence of chronic consumption of a ‘western-type’ high fat-high sugar diet (HFS) on hepatic health and moreover we assessed the ability of dietary modifications (i.e., removal of fat and sugar from the diet) to reverse these detrimental hepatic effects. This would represent what is seen clinically, advocated by a provider in a clinical setting as a first line treatment for diabetic obese patients. Methods: Thirty male C57BL/6J mice were randomly assigned into the following groups: 12-weeks HFS then 12-weeks of standard diet and water (Std), control groups were fed standard chow and water for the entire 24-weeks (lean control) or fed HFS diet for the entire 24-weeks (HFS). At the end of the 24-week diet study, livers were removed and fixed (for sectioning and staining with Oil-Red-O or trichrome and quantified with Image J) or frozen and kept at -80°C (for PCR studies and assays). Serum was collected for assays. Results: At the end of the diet study, body weight was significantly increased 1.5-fold by HFS and reduced 34% (p&lt;0.05) in the Std group. Liver weight/body weight ratio followed the same trend as body weight. Percent fat area, total number of lipid droplets and average total lipid area were all significantly increased in the HFS group versus lean controls and were all significantly reduced by Std (p&lt;0.05). Serum ALT activity was increased 3-fold by HFS and then reduced to lean levels by Std. Serum TNF-α and MCP1 levels followed the same trend. Serum TBARS were significantly increased 1.6-fold, but unchanged with Std. Trichrome analysis demonstrated deposition of collagen in liver from HFS fed mice (15.53% area), which was significantly reversed by the switch to Std (4.58% area). This data will be complemented with mRNA levels currently being run for collagens 1, 3, 4. Preliminary findings indicate that HFS fed mice have a significant increase in expression of collagen-1 but not collagen-3. We aim to further explore the metabolic mechanisms involved in the reversal of hepatic steatosis and fibrosis between the three diet groups. Conclusions: We conclude that removal of the HFS diet and switching to standard chow and water (Std) is an effective treatment regimen and results in robust reversal of hepatic steatosis along with reversal of collagen deposition, with concomitant beneficial changes in key serum markers of hepatic health, systemic inflammatory profile, and body/liver weight. The mechanistic pathways triggering these effects are being explored. Midwestern-Arizona Alzheimer’s consortium and Midwestern University Intramural Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Growth performance, health status, gut microbiome, and expression of immune and growth-related genes of rainbow trout (Oncorhynchus mykiss) fed diets with pea protein replacement of fish meal

The need for fish meal constrains fish farming and significantly impacts sustainability of the aquaculture industry. Hence, it is important to investigate the use of plant-based protein sources in fish diets. The present study was conducted to determine the effects of different levels of fish meal (FM) replacement by pea protein (PP) in a 60-day feeding experiment in rainbow trout, Oncorhynchus mykiss. Effects on growth performance, body composition, hematology, serum biochemistry and immunology, and immune (TNF-α, IL1-ß and Il-8) and growth-related (GH and IGFI) gene expression were investigated. Five experimental diets (45% protein and 20% lipid) differed in replacement level of FM by PP at rates of 0% (control (PP0)), 25% (PP25), 50%(PP50), 75%(PP75) and 100%(PP100). Fish were fed with experimental diets in triplicate twice daily. The best growth performance was obtained in PP0 and PP25 groups. While fat ratios of fish fillets significantly differed (p < 0.05), there was no significant effects on protein ratios (p < 0.05). There was no significant change in the hematological values of fish, except those fed the PP100 diets, which displayed a reduction in eyrthocyte counts, hemoglobin content and hematocrit. As PP supplementation increased fish showed elevated serum glucose, total protein, cholesterol and myeloperoxidase activity and decreased glutamic pyruvic transaminase and alkaline phosphatase activity. Fish fed diets with between 25 and 75% replacement showed a decline in lactic acid bacteria in the gut. Significant increases in expression were observed in the liver of the PP25 fish relative to the 0% control for all immune and growth-related genes except for IL1-ß. These data suggest that up to 25% of FM can be replaced by PP without any adverse effects on rainbow trout.

Abstract 2855: ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3

Abstract Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor that affects children and young adults. Ewing sarcomas harbor few mutations beyond the chromosomal translocation that initiates disease, and the mechanistic basis for the metastasis of these tumors remains poorly understood. The epigenome of EWS cells reflects the regulatory state of genes associated with the DNA binding activity of the fusion oncoproteins EWSR1::FLI1 or EWSR1::ERG. In this study, we examined the EWSR1::FLI1/ERG’s repression of transcription factor genes, particularly those encoding transcriptional regulators of cell differentiation. To examine EWSR1::FLI1/ERG’s regulation of gene expression, we assayed DNA binding using ChIP-seq or CUT&amp;RUN, RNA using RNA-sequencing (RNA-seq) or qRT-PCR, and protein using immunoblotting or immunofluorescence. We depleted the expression of EWSR1::FLI1/ERG proteins using RNAi and overexpressed ETS1 using a full-length cDNA or CRISPR activation.RNA-seq analysis of control and EWSR1::FLI1-silenced TC-32, TC-71, and A673 EWS cells identified 67 genes encoding proteins with DNA binding activity that exhibited significant increases in expression following depletion of EWSR1::FLI1. Comparison of the expression of these 67 transcription factor genes in EWS cell lines (n=41) and tumors (n=79) showed 37 expressed at a wider range in tumors relative to cell lines (p &amp;lt;0.01). This more variable expression in EWS tumors suggests that one or more of these transcription factors could exert a phenotypic effect. Focusing on one of the EWSR1::FLI1-repressed target genes, ETS1, we detected EWSR1::FLI1 binding and an H3K27me3 repressive mark at this locus. Depletion of EWSR1::FLI1 results in ETS1’s binding of promoter regions. Analysis of EWS cells in which we profiled ETS1 binding and assessed the effects of ectopically expressed ETS1 defined 265 genes as positively regulated by ETS1 of which 103 also exhibited an increase in expression following depletion of EWSR1::FLI1. Of these 103 genes, we focused on ETS1’s regulation of TNS3, as assessment of multiple EWS tumor expression profiles, showed positive correlation of ETS1 and TNS3 expression. TNS3 encodes TENSIN3, a focal adhesion protein that regulates cytoskeletal reorganization and contributes to cell migration by connecting the cytoplasmic tail of integrins to the actin cytoskeleton. EWS cell lines (SK-N-MC (EWSR1::FLI1) and ES-5838 (EWSR1::ERG), in which we activated ETS1 expression (CRISPRa) exhibited a migratory phenotype and increased TNS3 expression. Critically, the activated ETS1 EWS cell lines show TNS3 accumulation at leading cell edges, with F-actin cytoskeletal reorganization, a phenotype associated with cell migration. Our study demonstrates that EWS cells expressing ETS1 exhibit a more migratory phenotype, which, has the potential to promote the dissemination of cells and, thus, metastasis. Citation Format: Vernon Justice Ebegboni, Tamara L. Jones, Tayvia Brownmiller, Erica Pehrsson, Patrick Zhao, Soumya Sundara Rajan, Natasha J. Caplen. ETS1, a target gene of the EWSR1::FLI1 fusion oncoprotein, regulates the expression of the focal adhesion protein TENSIN3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2855.

Abstract 3322: Sigma-2-Erastin inhibits PC tumor growth through lipid ROS-DUSP6-MAPK signaling

Abstract Introduction: Pancreatic cancer (PC) is a malignancy with poor prognosis and high mortality with a very low 5-year survival rate. Currently, apoptosis and autophagy are the central mechanisms of programmed cell death (PCD) that maintain cellular homeostasis and regulate cell fate. With the recent emergence of ‘ferroptosis’ as a new type of PCD and its close association with the pathophysiological processes of many diseases including tumors, it is viewed as a potential mechanism to improve systemic treatment of PC. Considering the opportunity to regulate both apoptosis and ferroptosis by a single agent, our group has developed a cancer-targeted small molecule consisting of a sigma-2 ligand (a known apoptotic inducer) delivery moiety linked to an inhibitor of the cystine importer xCT (dm-Erastin, a known ferroptotic inducer) and named it as Sigma-2-Erastin (S2E) or ACXT-3102. The study presented here is aimed at determining the efficacy of ACXT-3102 in PC and defining its underlying mechanism of action. Methods: Using in vitro and in vivo tumor xenograft models of human PC, we determined the effects of ACXT-3102 treatments in regulating PCD of PC cells and defined the signaling pathway involved in mediating the effects. Results: As assessed by TitreGlo assays, we observed dose-dependent inhibition of PC cell (BxPC-3, AsPC-1 &amp; PANC-1) viability. Following the WES-analysis of signaling proteins, we observed that ACXT-3102 induces PC cell death by combinatorial regulation of apoptotic and ferroptotic PCD via lipid ROS-DUSP6-MAPK/ERK-mediated signaling pathway. Interestingly, the results of this study demonstrated a hyperactivation of MAPK/ERK signaling by increased phosphorylation instead of inhibition involved in inducting apoptotic cell death as represented by significant increases in expression of cleaved Capase-3/7 and PARP. In addition, ACXT-3102 treatments deceased GPX4 expression along with an increase in lipid ROS production as key markers of inducing ferroptotic cell death. Results from the murine model of human tumor xenografts using AsPC-1 cells further demonstrated significant decreases in tumor volume and increases in survival proportions in ACXT-3102-treated mice. Conclusions: ACXT-3102, an orally administered conjugate of sigma-2 ligand and Erastin induces PCD of human PC by uniquely regulating apoptosis and ferroptosis together. Therefore, ACXT-3102 offers a novel and improved therapeutic strategy for treating patients of fatal human carcinomas, especially those, which are difficult to diagnose at an early stage. Citation Format: Kumar S. Bishnupuri, Kenneth F. Newcomer, Qingqing Gong, Li Ye, Suwanna Vangveravong, Rony Takchi, Bradley T. Keller, Dirk M. Spitzer, William G. Hawkins. Sigma-2-Erastin inhibits PC tumor growth through lipid ROS-DUSP6-MAPK signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3322.

Abstract 1799: Regulation of epidermal stem cell proliferation and adhesion by ACSS2

Abstract Acetyl-CoA Synthetase Short-Chain Family Member 2 (ACSS2) is an enzyme that converts acetate into acetyl-CoA, a crucial metabolite required for protein acetylation and lipogenesis. Acetyl-CoA is membrane-impermeable and must be synthesized in the compartment in which it will be used. Among ACSS enzymes, ACSS2 alone is found in the nucleocytoplasmic compartment rather than the mitochondria. This enzyme shows dynamic localization dependent on cell nutrient status, with nutrient stress promoting nuclear localization where ACSS2 activity promotes cell growth and survival. Emerging evidence suggests ACSS2 may also play roles in promoting metastasis, and has been proposed as a potential therapeutic target. However, the role of ACSS2 in controlling normal tissue homeostasis is understudied. Addressing this knowledge gap is critical to ensure safety and efficacy of cancer therapeutics. As 80% to 90% of human cancers originate from epithelial tissues, we have leveraged human epidermal stem cells to further interrogate the role of ACSS2. As progenitor keratinocytes differentiate, they detach from the basal lamina and migrate from the metabolically privileged basal layer to the more challenging environment at the surface of the epidermis. Using shRNA-mediated knockdown, we first found ACSS2 knockdown dramatically decreases progenitor keratinocyte clonogenicity, abolishing proliferative capacity. Analysis of RNA sequencing data following ACSS2 knockdown with five constructs showed significant increases in expression of genes related to cell adhesion, extracellular matrix organization, collagen catabolism, and cell migration, in addition to keratinocyte differentiation. Downregulated genes were associated with cell division, DNA repair, and DNA replication. Live cell imaging revealed knockdown cells show striking differences in cell adhesion compared to control. These results indicate a role for ACSS2 in regulating keratinocyte proliferation and adhesion during the maintenance of epidermal homeostasis. Citation Format: Nicholas K. Chamberlain, Xiaomin Bao. Regulation of epidermal stem cell proliferation and adhesion by ACSS2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1799.

Calotropis procera extract inhibits prostate cancer through regulation of autophagy.

Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen-independent PC-3 and androgen-sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound-healing assay and 2',7'-dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE-mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC-MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC-3 and 22Rv1, in a dose- and time-dependent manner. Also, CPE significantly inhibited the colony-forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF-κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE-treated PC-3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.

A200 EXPOSURE TO CANNABIS SMOKE IN UTERO AFFECTS THE DEVELOPMENT OF THE ENDOCANNABINOID SYSTEM IN THE GASTROINTESTINAL TRACT

Abstract Background Recreational use of cannabis has been legalized in Canada since 2018. Increasing numbers of women are using cannabis while pregnant, with reported rates ampersand:003E10%. There is a disconnect with societal perceptions, with up to one third of women believing that cannabis is safe for use in pregnancy, but with preclinical and clinical studies demonstrating negative neurodevelopmental outcomes. A key component of gut-brain axis regulation is found in the endocannabinoid system (ECS), of which receptors, ligands and enzymes are extensively distributed throughout the gastrointestinal (GI) tract and are actively involved in GI motility and sensation. Aims We tested the hypothesis that exposure to cannabis smoke in utero could influence the expression of ECS components in the developing GI tract. Methods We developed a “real world” cannabis smoke inhalation model in which CD-1 pregnant mouse dams were exposed daily to a delta-9-tetrahydrocannabinol (THC)-dominant strain of cannabis in a smoking chamber unit, from embryonic days 6 (E6) to E18. Control mice were placed in a restraining cage and exposed to room air for the equivalent time-period. Dams were sacrificed at E18, fetuses removed and the fetal GI tract (from stomach to distal colon) collected for RNA extraction. Key components of the ECS were investigated by developing a NanoString Custom CodeSet, including receptors (cannabinoid receptors 1 and 2; CB1 and CB2, transient receptor potential vanilloid type 1; TRVP1, GPR55) and enzymes (diacylglycerol lipase; DAGL, fatty acid amide hydrolase; FAAH, monoacylglycerol lipase; MAGL, N-acylphosphatidyletanolamine phospholipase D; NAPE-PLD). Samples (n=per litter; 7 cannabis-exposed and 8 control) were processed in the Farncombe Metagenomics Facility and NanoString data analyzed using nSolver Analysis Software. Results We found a significant increase in the expression of ECS receptors in the cannabis-exposed compared to control GI tract at E18, including CB2 (pampersand:003C0.05), TRPV1 (pampersand:003C0.05) and GPR55 (pampersand:003C0.0005). Interestingly, no significant change in mRNA expression was detected for CB1. We also found a significant increase in mRNA encoding the full panel of ECS enzymes, including DAGL (pampersand:003C0.005), FAAH (pampersand:003C0.05), MAGL (pampersand:003C0.05) and NAPE-PLD (pampersand:003C0.005) in cannabis-exposed compared to control GI tract. Conclusions In conclusion, we have found a significant increase in mRNA expression in a panel of key ECS receptors and enzymes in the GI tract at a point of maximal exposure to cannabis in utero. Future research will be needed to further characterize potential impacts on postnatal GI development and function. Funding Agencies CIHRThe Azrieli Foundation

ADORA3: A Key Player in the Pathogenesis of Intracranial Aneurysms and a Potential Diagnostic Biomarker.

The effects of genes on the development of intracranial aneurysms (IAs) remain to be elucidated, and reliable blood biomarkers for diagnosing IAs are yet to be established. This study aimed to identify genes associated with IAs pathogenesis and explore their diagnostic value by analyzing IAs datasets, conducting vascular smooth muscle cells (VSMC) experiments, and performing blood detection. IAs datasets were collected and the differentially expressed genes were analyzed. The selected genes were validated in external datasets. Autophagy was induced in VSMC and the effect of selected genes was determined. The diagnostic value of selected gene on the IAs were explored using area under curve (AUC) analysis using IAs plasma samples. Analysis of 61 samples (32 controls and 29 IAs tissues) revealed a significant increase in expression of ADORA3 compared with normal tissues using empirical Bayes methods of "limma" package; this was further validated by two external datasets. Additionally, induction of autophagy in VSMC lead to upregulation of ADORA3. Conversely, silencing ADORA3 suppressed VSMC proliferation and autophagy. Furthermore, analysis of an IAs blood sample dataset and clinical plasma samples demonstrated increased ADORA3 expression in patients with IA compared with normal subjects. The diagnostic value of blood ADORA3 expression in IAs was moderate when analyzing clinical samples (AUC: 0.756). Combining ADORA3 with IL2RB or CCR7 further enhanced the diagnostic ability for IAs, with the AUC value over 0.83. High expression of ADORA3 is associated with IAs pathogenesis, likely through its promotion of VSMC autophagy. Furthermore, blood ADORA3 levels have the potential to serve as an auxiliary diagnostic biomarker for IAs.

Jun/Fos promotes migration and invasion of hepatocellular carcinoma cells by enhancing BORIS promoter activity

The Brother of the Regulator of Imprinted Sites (BORIS), as a specific indicator of hepatocellular carcinoma, exhibits a significant increase in expression. However, its upstream regulatory network remains enigmatic. Previous research has indicated a strong correlation between the Hippo pathway and the progression of hepatocellular carcinoma. It is well established that the Activator Protein-1 (AP-1) frequently engages in interactions with the Hippo pathway. Thus, we attempt to prove whether Jun and Fos, a major member of the AP-1 family, are involved in the regulation of BORIS expression. Bioinformatics analysis revealed the existence of binding sites for Jun and Fos within the BORIS promoter. Through a series of overexpression and knockdown experiments, we corroborated that Jun and Fos have the capacity to augment BORIS expression, thereby fostering the migration and invasion of hepatocellular carcinoma cells. Moreover, Methylation-Specific PCR and Bisulfite Sequencing PCR assays revealed that Jun and Fos do not have a significant impact on the demethylation of the BORIS promoter. However, luciferase reporter and chromatin immunoprecipitation experiments substantiated that Jun and Fos could directly bind to the BORIS promoter, thereby enhancing its transcription. In conclusion, these results suggest that Jun and Fos can promote the development of hepatocellular carcinoma by directly regulating the expression of BORIS. These findings may provide experimental evidence positioning BORIS as a novel target for the clinical intervention of hepatocellular carcinoma.

IFN-λ3 is induced by Leishmania donovani and can inhibit parasite growth in cell line models but not in the mouse model, while it shows a significant association with leishmaniasis in humans.

The intracellular protozoan parasite Leishmania donovani causes debilitating human diseases that involve visceral and dermal manifestations. Type 3 interferons (IFNs), also referred to as lambda IFNs (IFNL, IFN-L, or IFN-λ), are known to play protective roles against intracellular pathogens at the epithelial surfaces. Herein, we show that L. donovani induces IFN-λ3 in human as well as mouse cell line-derived macrophages. Interestingly, IFN-λ3 treatment significantly decreased parasite load in infected cells, mainly by increasing reactive oxygen species production. Microscopic examination showed that IFN-λ3 inhibited uptake but not replication, while the phagocytic ability of the cells was not affected. This was confirmed by experiments that showed that IFN-λ3 could decrease parasite load only when added to the medium at earlier time points, either during or soon after parasite uptake, but had no effect on parasite load when added at 24 h post-infection, suggesting that an early event during parasite uptake was targeted. Furthermore, the parasites could overcome the inhibitory effect of IFN-λ3, which was added at earlier time points, within 2-3 days post-infection. BALB/c mice treated with IFN-λ3 before infection led to a significant increase in expression of IL-4 and ARG1 post-infection in the spleen and liver, respectively, and to different pathological changes, especially in the liver, but not to changes in parasite load. Treatment with IFN-λ3 during infection did not decrease the parasite load in the spleen either. However, IFN-λ3 was significantly increased in the sera of visceral leishmaniasis patients, and the IFNL genetic variant rs12979860 was significantly associated with susceptibility to leishmaniasis.