Biology subjects

Vasiliou, V.

Publications and source records attributed to Vasiliou, V..

10 recordsLinked to original sources

bioRxiv2025

ALDH2 Deficiency Drives Proliferative Mammary Morphogenesis and Epithelial Cell Stemness via Oxidative Stress and Estrogen Receptor Activation

Alcohol consumption has been linked to breast cancer, partly due to the accumulation of toxic aldehydes like acetaldehyde, a carcinogenic byproduct of ethanol metabolism. ALDH2, a key mitochondrial enzyme, detoxifies acetaldehyde and other harmful aldehydes that drive oxidative stress, DNA damage, and hormonal dysregulation, key factors in carcinogenesis. Despite the possible link between alcohol consumption and breast cancer risk, little is known about how ALDH2 deficiency itself, independent of alcohol exposure, affects mammary gland biology and cancer susceptibility. Genetic variants leading to ALDH2 deficiency are highly prevalent in East Asian populations, where individuals carrying inactive ALDH2 alleles experience impaired aldehyde detoxification. While these individuals are at increased risk for alcohol-related cancers, the impact of ALDH2 deficiency on mammary gland development and homeostasis in the absence of alcohol exposure remains unexplored. To address this, we utilized a C57BL/6-based ALDH2 knockout (Aldh2-/-) mouse model to investigate its effects on mammary proliferation and development. Our findings revealed that Aldh2-/- mice exhibited hyperproliferative mammary glands, characterized by increased epithelial cell density, ductal expansion, and elevated Ki67+ cells. Flow cytometry analysis demonstrated a rise in luminal and basal epithelial subpopulations, alongside enhanced mammary epithelial stemness, as evidenced by increased mammosphere formation and colony-forming efficiency. At the molecular level, ALDH2 deficiency activated oxidative stress pathways, marked by elevated 8-OHdG, p38 MAPK, NF-{kappa}B, and Nrf2 signaling, alongside DNA damage responses involving p53 and H2A.X. Importantly, we also identified a previously unrecognized upregulation of RANKL in Aldh2-/- mammary glands, implicating the RANK/RANKL axis as a critical mediator linking aldehyde stress to NF-{kappa}B/p38 MAPK activation and enhanced mammary stemness. Furthermore, hormonal dysregulation was observed, with a significant increase in ER and PR expression and phosphorylation. Dysregulated ER signaling was further linked to enhanced erbB3 activation and downstream signaling, including the cyclin D1-pRb-E2F1 axis. Our results suggest that the accumulation of endogenous aldehydes, independent of alcohol exposure, profoundly alters mammary morphogenesis, epithelial repopulation, and stemness. Mechanistically, this occurs through oxidative stress activation and DNA damage pathways, leading to metabolic changes and upregulation of estrogen receptor and receptor tyrosine kinase signaling. This study highlights for the first time the potential role of ALDH2 deficiency in increasing mammary tissue susceptibility to oncogenic factors and breast cancer risk.

cancer biology↗

bioRxiv2024

Catabolism of extracellular glutathione supplies amino acids to support tumor growth

Restricting amino acids from tumors is an emerging therapeutic strategy with significant promise. While typically considered an intracellular antioxidant with tumor-promoting capabilities, glutathione (GSH) is a tripeptide of cysteine, glutamate, and glycine that can be catabolized, yielding amino acids. The extent to which GSH-derived amino acids are essential to cancers is unclear. Here, we find that GSH catabolism promotes tumor growth. We show that depletion of intracellular GSH does not perturb tumor growth, and extracellular GSH is highly abundant in the tumor microenvironment, highlighting the potential importance of GSH outside of tumors. We find supplementation with GSH can rescue cancer cell survival and growth in cystine-deficient conditions, and this rescue is dependent on the catabolic activity of {gamma}-glutamyltransferases (GGTs). Finally, pharmacologic targeting of GGTs activity prevents the breakdown of circulating GSH, lowers tumor cysteine levels, and slows tumor growth. Our findings indicate a non-canonical role for GSH in supporting tumors by acting as a reservoir of amino acids. Depriving tumors of extracellular GSH or inhibiting its breakdown is potentially a therapeutically tractable approach for patients with cancer. Further, these findings change our view of GSH and how amino acids, including cysteine, are supplied to cells.

cancer biology↗

bioRxiv2024

Glycosite Mapping and in situ Mass Spectrometry Imaging of MUC2 Glycopeptides via On-slide Digestion with Mucinase StcE

Many cancers are characterized by altered mucin expression and glycosylation, although the mechanistic relationship between tumor glycosylation and disease progression is not well-defined. Herein, our goal was to map specific mucin glycoforms in diseased tissue, enabling correlation of the tumor glycan profile with malignant features. To this end, we developed a workflow implementing on-tissue digestion with mucinase StcE, followed by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography coupled to mass spectrometry (LC-MS). To optimize our workflow, we analyzed four different mucinous carcinomas derived from colon, esophageal, and salivary gland tissue. Using this technique, we deduced the spatial distribution of StcE-generated O-glycopeptides within mucinous tumors using MALDI-IMS. Subsequent LC-MS analyses revealed the identity of different species detected in imaging experiments, in addition to comprehensively characterizing the mucinome and proteome of each tissue. Our coupled MS approach unveiled a striking mucin 2 (MUC2) expression pattern in two colorectal mucinous adenocarcinomas, in which different glycoforms clearly stratified regions within the tumor. Notably, our LC-MS experiments obtained near-complete sequence coverage over the mucin domains of MUC2, enabling glycoproteomic mapping of this canonical mucin in unprecedented depth. MUC2 glycosylation was dominated by the T and Tn antigens, with surprisingly little sialylation detected. However, O-glycans containing mono- and di-O-acetylated sialic acid were detected in low abundance. Finally, we obtained spectral evidence for an endogenous O-acetylated GalNAc, an O-glycan structure not previously reported in the literature. Overall, this proof-of-concept work underscores the potential of this technique to generate new research avenues in oncology and beyond. Significance StatementAberrant mucin expression and glycosylation are hallmarks of cancer, but how these changes promote malignant processes are not well understood. Solid tumors are highly heterogeneous in their cellular and molecular composition, and many advanced spatial techniques have emerged in recent years to study the tumor microenvironment (TME) for better understanding disease progression. Spatially resolved glycoprotein analyses typically detect either the protein or glycan components, but not both. We developed a workflow using a dual mass spectrometry approach to map the location of intact glycopeptides in mucinous tumors, enabled by on-tissue digestion with the mucin-specific protease StcE. Future applications of this method on larger patient cohorts will enhance our understanding of glycans in malignancy, identify disease biomarkers, and define therapeutic targets.

cancer biology↗

bioRxiv2023

A Th17 cell-intrinsic glutathione/mitochondrial-IL-22 axis protects against intestinal inflammation

Although the intestinal tract is a major site of reactive oxygen species (ROS) generation, the mechanisms by which antioxidant defense in gut T cells contribute to intestinal homeostasis are currently unknown. Here we show, using T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that the ensuing loss of glutathione (GSH) impairs the production of gut-protective IL-22 by Th17 cells within the lamina propria. Although Gclc ablation does not affect T cell cytokine secretion in the gut of mice at steady-state, infection with C. rodentium increases ROS, inhibits mitochondrial gene expression and mitochondrial function in Gclc-deficient Th17 cells. These mitochondrial deficits affect the PI3K/AKT/mTOR pathway, leading to reduced phosphorylation of the translation repressor 4E-BP1. As a consequence, the initiation of translation is restricted, resulting in decreased protein synthesis of IL-22. Loss of IL-22 results in poor bacterial clearance, enhanced intestinal damage, and high mortality. ROS-scavenging, reconstitution of IL-22 expression or IL-22 supplementation in vivo prevent the appearance of these pathologies. Our results demonstrate the existence of a previously unappreciated role for Th17 cell-intrinsic GSH coupling to promote mitochondrial function, IL-22 translation and signaling. These data reveal an axis that is essential for maintaining the integrity of the intestinal barrier and protecting it from damage caused by gastrointestinal infection. Executive summary- GSH-regulated Th17 cell-derived IL-22, but not IL-17 is required to maintain intestinal barrier integrity and to revent lethality following C. rodentium infection. - GCLC expression in IBD patients correlates positively with expression of genes related to gut integrity. - Gclc-deficient Th17 cells accumulate mitochondrial ROS, which is linked to impaired mitochondrial function, ysregulated PI3K/AKT/mTOR signaling and impaired translation of IL-22. - ROS-scavenging, IL-22 reconstitution or T cell-specific expression of IL-22 in Gclc-deficient T cells rescues utant mice from the lethal infection outcome in vivo.

immunology↗

bioRxiv2023

Endocrine pancreas-specific Gclc gene deletion causes a severe diabetes phenotype

Reduced glutathione (GSH) is an abundant antioxidant that regulates intracellular redox homeostasis by scavenging reactive oxygen species (ROS). Glutamate-cysteine ligase catalytic (GCLC) subunit is the rate-limiting step in GSH biosynthesis. Using the Pax6-Cre driver mouse line, we deleted expression of the Gclc gene in all pancreatic endocrine progenitor cells. Intriguingly, Gclc knockout (KO) mice, following weaning, exhibited an age-related, progressive diabetes phenotype, manifested as strikingly increased blood glucose and decreased plasma insulin levels. This severe diabetes trait is preceded by pathologic changes in islet of weanling mice. Gclc KO weanlings showed progressive abnormalities in pancreatic morphology including: islet-specific cellular vacuolization, decreased islet-cell mass, and alterations in islet hormone expression. Islets from newly-weaned mice displayed impaired glucose-stimulated insulin secretion, decreased insulin hormone gene expression, oxidative stress, and increased markers of cellular senescence. Our results suggest that GSH biosynthesis is essential for normal development of the mouse pancreatic islet, and that protection from oxidative stress-induced cellular senescence might prevent abnormal islet-cell damage during embryogenesis.

physiology↗

bioRxiv2023

Glutathione supports lipid abundance in vivo

Cells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series of in vivo models to induce Gclc deletion in adult animals. We find that GSH is essential to lipid abundance in vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the livers balance of redox buffering and triglyceride production.

biochemistry↗

bioRxiv2021

Oxidative stress induces inflammation of lens cells and triggers immune surveillance of ocular tissues

Recent reports have challenged the notion that the lens is immune-privileged. However, these studies have not fully identified the molecular mechanism(s) that promote immune surveillance of the lens. Using a mouse model of targeted glutathione (GSH) deficiency in ocular surface tissues, we have investigated the role of oxidative stress in upregulating cytokine expression and promoting immune surveillance of the eye. RNA-sequencing of lenses from postnatal day (P) 1- aged Gclcf/f;Le-CreTg/- (KO) and Gclcf/f;Le-Cre-/- control (CON) mice revealed upregulation of many cytokines (e.g., CCL4, GDF15, CSF1) and immune response genes in the lenses of KO mice. The eyes of KO mice had a greater number of cells in the aqueous and vitreous humors at P1, P20 and P50 than age-matched CON and Gclcw/w;Le-CreTg/- (CRE) mice. Histological analyses revealed the presence of innate immune cells (i.e., macrophages, leukocytes) in ocular structures of the KO mice. At P20, the expression of cytokines and ROS content was higher in the lenses of KO mice than in those from age-matched CRE and CON mice, suggesting that oxidative stress may induce cytokine expression. In vitro administration of the oxidant, hydrogen peroxide, and the depletion of GSH (using buthionine sulfoximine (BSO)) in 21EM15 lens epithelial cells induced cytokine expression, an effect that was prevented by co-treatment of the cells with N-acetyl-L-cysteine (NAC), a antioxidant. The in vivo and ex vivo induction of cytokine expression by oxidative stress was associated with the expression of markers of epithelial-to-mesenchymal transition (EMT), -SMA, in lens cells. Given that EMT of lens epithelial cells causes posterior capsule opacification (PCO), we propose that oxidative stress induces cytokine expression, EMT and the development of PCO in a positive feedback loop. Collectively these data indicate that oxidative stress induces inflammation of lens cells which promotes immune surveillance of ocular structures. HighlightsO_LIImmune surveillance of ocular structures occurs in mouse eyes deficient in glutathione. C_LIO_LIOxidative stress upregulates the expression of pro-inflammatory cytokines (e.g., GDF15, CSF1) in lens cells in vitro and in vivo. C_LIO_LIThe upregulation of cytokines in lens cells is associated with markers of an epithelial-to-mesenchymal transition phenotype. C_LIO_LIOxidative stress-induced inflammation and associated epithelial-to-mesenchymal transition may play a role in the development of posterior capsule opacification. C_LI

developmental biology↗

bioRxiv2020

Novel insights into the mode of action of 1,4-dioxane using a systems screening approach

1,4-Dioxane (1,4-DX) is an environmental contaminant found in drinking water throughout the United States (US). While it is a suspected liver carcinogen, there is no federal or state maximum contaminant level for 1,4-DX in drinking water. Very little is known about the mechanisms by which this chemical elicits liver carcinogenicity. In the present study, female BDF-1 mice were exposed to 1,4-DX (0, 50, 500 and 5,000 mg/L) in their drinking water for one or four weeks, to explore the toxic effects. Histopathological studies and a multi-omics approach (transcriptomics and metabolomics) were performed to investigate potential mechanisms of toxicity. Immunohistochemical analysis of the liver revealed increased H2AX{gamma}-positive hepatocytes (a marker of DNA double strand breaks), and an expansion of precholangiocytes (reflecting both DNA damage and repair mechanisms) after exposure. Liver transcriptomics revealed 1,4-DX-induced perturbations in signaling pathways predicted to impact the oxidative stress response, detoxification, and DNA damage. Liver, kidney, feces and urine metabolomic profiling revealed no effect of 1,4-DX exposure, and bile acid quantification in liver and feces similarly showed no effect of exposure. We speculate that the results may be reflective of DNA damage being counterbalanced by the repair response, with the net result being a null overall effect on the systemic biochemistry of the exposed mice. Our results show a novel approach for the investigation of environmental chemicals that do not elicit cell death but have activated the repair systems in response to 1,4-DX exposure.

pharmacology and toxicology↗

bioRxiv2019

Gclc deletion in surface-ectoderm tissues induces microphthalmia

Glutamate cysteine ligase catalytic subunit (Gclc) is the catalytic subunit for the glutamate-cysteine ligase (Gcl) enzyme. Gcl catalyzes the rate limiting step in glutathione (GSH) synthesis. Gclc is highly expressed in the developing eye. To define the regulatory role of Gclc in eye development, we developed a novel, Le-Cre transgene-driven, Gclc knockout mouse model. Gclcf/f/Le-CreTg/- mice present with deformation of the retina, cornea, iris, and lens, consistent with a microphthalmia phenotype. Controlling for the microphthalmia phenotype of Gclcwt/wt/Le-CreTg/- mice revealed that Gclcf/f/Le-CreTg/- mice have a more severe microphthalmia phenotype. Thus, the loss of Gclc expression exacerbates the microphthalmia phenotype in Le-Cre mice. Gclcf/f/Le-CreTg/- eyes present with reduced retinal and lens epithelium proliferation and increased lens cell death. Imaging mass spectrometry of ocular tissues revealed changes in the intensity and distribution of several lipid species and proteins in the retina and corneas of Gclcf/f/Le-CreTg/- eyes. Lastly, using splice-blocking morpholinos and CRISPR/Cas9, we created two gclc knockdown zebrafish models, both of which display a microphthalmia phenotype. Combined, the mouse and zebrafish results indicate that, in chordates, Gclc has a conserved role in regulating eye development. In summary, these novel animal models are useful tools for elucidating the mechanisms involved in microphthalmia development.

developmental biology↗

bioRxiv2019

Molecular pathway analysis indicates a distinct metabolic and immune phenotype in women with right-sided colon cancer

Colon cancer is the third most commonly diagnosed cancer in the United States. Recent reports have shown that the location of the primary tumor is of clinical importance. Patients with right-sided cancers (RCCs) (tumors arising between the cecum and proximal transverse colon) have poorer clinical outcomes than those with left-sided colon cancers (LCCs) (tumors arising between the distal transverse colon and sigmoid colon, excluding the rectum). Interestingly, women have a lower incidence of colon cancer than men do. However, women have a higher propensity for RCC than men. Identification of gene expression differences between RCC and LCC is considered a potential means of prognostication. Furthermore, studying colon cancer sidedness could reveal important predictive markers for response to various treatments. This study provides a comprehensive bioinformatic analysis of various genes and molecular pathways that correlated with sex and anatomical location of colon cancer using four publicly available annotated datasets housed in the National Center for Biotechnology Informations Gene Expression Omnibus (GEO). We identified differentially expressed genes in tumor tissues from women with RCC, which showed attenuated energy and nutrient metabolism when compared to women with LCC. Specifically, we showed that the downregulation of 5 AMP-activated protein kinase alpha subunit (AMPK) and downregulated anti-tumor immune response in women with RCC. This difference was not seen when comparing tumor tissues from men with RCC to men with LCC. Therefore, women with RCC may have a specific metabolic and immune phenotype which accounts for differences in prognosis and treatment response.

cell biology↗