Extracellular Uptake Research Articles

Highly expressed VGLL3 in keloid fibroblasts promotes glycolysis and collagen production via the activation of Wnt/β-catenin signaling.

This study investigated the effects and related mechanisms of Vestigial-like family member 3 (VGLL3) on keloid fibroblast (KF) proliferation, apoptosis, collagen production, and glycolysis. Western blot, qRT-PCR, and immunohistochemistry were used for determining VGLL3 expression. KF viability, proliferation, and apoptosis were assessed using CCK-8 assay, EdU assay, and flow cytometry. Changes in the protein expression levels of α-SMA, fibronectin, collagen I, and collagen III were examined utilizing western blotting. The pathways related to VGLL3 were analyzed using Gene Set Enrichment Analysis. Changes in glycolysis were assessed by measuring oxygen consumption rate (OCR), extracellular acidification rate (ECAR), glucose uptake, and lactate production. WNT2 and β-catenin protein levels were measured using western blotting. VGLL3 was upregulated in human keloid tissues. In KFs, overexpression of VGLL3 inhibited cell apoptosis, promoted cell proliferation and protein expression of α-SMA, fibronectin, collagen I, and collagen III. Moreover, it reduced OCR level, and increased the levels of ECAR, glucose uptake, and lactate production. On the other hand, the knockdown of VGLL3 had the opposite effect. WNT2 and β-catenin protein levels were enhanced by overexpression of VGLL3 and reduced by VGLL3 knockdown. Silencing of WNT2 reversed the effects of VGLL3 on apoptosis, proliferation, collagen production, and glycolysis in KFs. VGLL3 promoted glycolysis in KFs and keloid progression, which was achieved through the activation of Wnt signaling pathway. Therefore, targeting VGLL3 may be a promising therapeutic strategy for the treatment of keloids.

SUMOylated GLUT1 inhibited the glycometabolism disorder in chondroctyes during osteoarthritis.

Reduction of glucose transporter 1 (GLUT1), even deletion, may results in cartilage fibrosis and osteoarthritis. This study aims to investigate the SUMOylation of GLUT1 in osteoarthritis through small ubiquitin-like modifier 1(SUMO1), and explore the role of SUMOylated GLUT1 in glycometabolism, proliferation and apoptosis in chondrocytes. Human chondrocytes were incubated with 10 ng/mL of IL-1β to mimic osteoarthritis in vitro. GLUT1, SUMO1 and Chondrocyte-related genes including COL2A1, MMP13 and ADAMTS4 were evaluated using western blot. Cell viability and cell apoptosis of chondrocytes were measured by cell counting kit-8 assay and flow cytometry, respectively. The changes in glycometabolism were evaluated using extracellular acidification rate (ECAR) and glucose uptake assay. Co-immunoprecipitation (Co-IP) was used to verify the interaction between GLUT1 and SUMO1. The stabilization role of SUMO1 in GLUT1 was determined by cycloheximide assay. IL-1β induced the decrease of GLUT1, cell viability, ECAR, glucose uptake and COL2A1 and the increase of cell apoptosis, MMP13 and ADAMTS4 in chondrocytes. However, overexpression of SUMO1 led to the reduction of cell apoptosis, MMP13 and ADAMTS4 and the elevation of GLUT1, cell viability, ECAR, glucose uptake and COL2A1 in IL-1β-stimulated chondrocytes. There was SUMOylation sites on GLUT1. Intriguingly, SUMO1 was significantly enriched in GLUT1 using Co-IP assay, and stabilized GLUT1 in chondrocytes. SUMO1-mediated SUMOylation is capable of stabilizing GLUT1 to inhibit glycometabilsm disorder and cell apoptosis in IL-1β-stimulated chondrocytes.

Comparative assessment of chondral defect repair using human bone marrow- and adipose tissue-derived mesenchymal stem cells, adult and foetal articular cartilage-derived chondrocytes, and chondroprogenitors: an ex-vivo model.

Cartilage repair necessitates adjunct therapies such as cell-based approaches, which commonly use MSCs and chondrocytes but is limited by the formation of fibro-hyaline cartilage. Articular cartilage-derived chondroprogenitors(CPs) offer promise in overcoming this, as they exhibit higher chondrogenic and lower hypertrophic phenotypes. The study aimed to compare the efficacy of various cell types derived from adult and foetal cartilage suspended in platelet-rich plasma(PRP) in repairing chondral defects in an Ex-vivo Osteochondral Unit(OCU) model. In-vitro characterization of the cells included growth kinetics, FACS, qRT-PCR, and multilineage differentiation potential using histology and GAG analysis. Ex-vivo human OCUs with chondral defects containing the different cells in PRP were cultured and subjected to analysis for matrix and collagen staining. The ex-vivo OCU analysis, in terms of defect repair, showed that adult chondrocytes, sorted-CPs, and foetal MCPs displayed better host integration and filling. The In-vitro analysis of adult chondrocytes displayed greater chondrogenic genes ACAN and COL2A1 expression, with sorted-CPs also showing higher levels of ACAN. In terms of accumulation of extracellular matrix uptake evident by Safranin O staining and collagen type II fibrillar uptake, the AD-MSCs, BM-MSCs, and sorted CPs outperformed the other groups. BM-MSCs also showed corroborative higher CD146 levels, however, the gene analysis of the AD-MSCs showed a high hypertrophic tendency in terms of its COL1A1 and RUNX2 expression. Sorted chondroprogenitors outperformed both in terms of filling and hyaline-like repair, with AD-MSC and BM-MSC groups also achieving functional cartilage of a hyaline nature, warranting further evaluation using in-vivo and clinical studies.

Transcription Factor MAZ Potentiates the Upregulated NEIL3-mediated Aerobic Glycolysis, thereby Promoting Angiogenesis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is characterized by high vascularity and notable abnormality of blood vessels, where angiogenesis is a key process in tumorigenesis and metastasis. The main functions of Nei Like DNA Glycosylase 3 (NEIL3) include DNA alcoholization repair, immune response regulation, nervous system development and function, and DNA damage signal transduction. However, the underlying mechanism of high expression NEIL3 in the development and progression of HCC and whether the absence or silencing of NEIL3 inhibits the development of cancer remain unclear. Therefore, a deeper understanding of the mechanisms by which increased NEIL3 expression promotes cancer development is needed. Expression of NEIL3 and its upstream transcription factor MAZ in HCC tumor tissues was analyzed in bioinformatics efforts, while validation was done by qRT-PCR and western blot in HCC cell lines. The migration and tube formation capacity of HUVEC cells were analyzed by Transwell and tube formation assays. Glycolytic capacity was analyzed by extracellular acidification rate, glucose uptake, and lactate production levels. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter gene assays were utilized to investigate specific interactions between MAZ and NEIL3. NEIL3 and MAZ were substantially upregulated in HCC tissues and cells. NEIL3 was involved in modulating the glycolysis pathway, suppression of which reversed the stimulative impact of NEIL3 overexpression on migration and angiogenesis in HUVEC cells. MAZ bound to the promoter of NEIL3 to facilitate NEIL3 transcription. Silencing MAZ reduced NEIL3 expression and suppressed the glycolysis pathway, HUVEC cell migration, and angiogenesis. MAZ potentiated the upregulated NEIL3-mediated glycolysis pathway and HCC angiogenesis. This study provided a rationale for the MAZ/NEIL3/glycolysis pathway as a possible option for anti-angiogenesis therapy in HCC.

SPC25 Activates the Warburg Effect to Inhibit Ferroptosis in Prostate Cancer Cells.

SPC25 is associated with unfavorable outcomes in various cancers, but its role in prostate cancer (PRAD) is unclear. More research is needed on glycolysis and ferroptosis targets in PRAD. Bioinformatics tools were used to analyze SPC25 expression disparities. Gene set enrichment analysis (GSEA) identified pathways enriched by SPC25 and its correlation with glycolytic proteins. SPC25 mRNA transcriptional activity was analyzed by quantitative polymerase chain reaction (qPCR), while protein levels of SPC25, glycolytic markers, and ferroptosis markers were assessed using Western blot. CCK-8 was used to evaluate the effects of SPC25 on cell survival. Ferroptosis levels were measured by flow cytometry and assays for Fe2+ and malondialdehyde (MDA) content. Glycolytic capacity was assessed using glucose uptake assays, lactate tests, and a Seahorse XF analyzer. In PRAD tissues and cells, SPC25 was notably upregulated and correlated with adverse outcomes. It enhanced cancer cell vitality. GSEA showed SPC25's strong association with ferroptosis and glycolytic pathways, while Pearson correlation analysis indicated a positive relationship between SPC25 and glycolytic proteins. Overexpression of SPC25 in cell lines noticeably curbed the accumulation of lipid reactive oxygen species, MDA formation, and Fe2+ content, while it augmented the protein expression of ferroptosis markers. SPC25 stimulated an increase in cellular extracellular acidification rate, glucose uptake, and lactate secretion, while it dampened oxygen consumption rate, and this effect could be counteracted by 2-deoxy-d-glucose (2-DG). Conversely, 2-DG mitigated the ferroptosis indicators that were diminished by SPC25 downregulation, including the reduction of ferroptosis marker protein expression. By upregulating glycolysis in PRAD cells, SPC25 suppresses the occurrence of ferroptosis.

ST3GAL4 promotes tumorigenesis in breast cancer by enhancing aerobic glycolysis.

Sialyltransferases are enzymes that play a crucial role in regulating cancer progression by modifying glycoproteins through sialylation. In particular, the ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) enzyme is known to be upregulated in breast cancer, but its specific biological functions have not been fully understood. This study aimed to investigate the impact and mechanisms of ST3GAL4 on aerobic glycolysis in breast cancer. We examined ST3GAL4 expression in tumor tissue samples and breast cancer cell lines and also manipulated ST3GAL4 expression in breast cancer cells using lentivirus transduction. The study evaluated cellular processes such as cell viability, cell cycle progression, and aerobic glycolysis by measuring parameters like extracellular acidification rate, glucose uptake, lactate production, and lactate dehydrogenase A (LDHA) expression. We found that ST3GAL4 expression was consistently increased in tumor tissues and breast cancer cell lines. High ST3GAL4 expression was associated with a poor prognosis for patients with breast cancer. Inhibiting ST3GAL4 expression decreased cell viability, disrupted cell cycle progression, and reduced aerobic glycolysis and LDHA expression. Furthermore, suppressing ST3GAL4 expression in animal models reduced tumor growth and cell proliferation. Conversely, overexpressing ST3GAL4 promoted cell viability and cell cycle progression, but these effects were reversed when an inhibitor of aerobic glycolysis was used. The study provided evidence in cells and animal models that ST3GAL4 promotes tumorigenesis in breast cancer by enhancing aerobic glycolysis. These findings suggest that targeting ST3GAL4 may be a potential strategy for the treatment of breast cancer.

RON receptor tyrosine kinase regulates glycolysis through MAPK/CREB signaling to affect ferroptosis and chemotherapy sensitivity of thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is one of the deadliest and most aggressive human malignancies for which there is currently no effective treatment. Tyrosine kinase receptor RON is highly expressed in various cancer types, including colon, pancreatic and thyroid cancer. However, its underlying role in ATC is not fully understood. The present study investigated the therapeutic potential and molecular mechanism of RON in ATC. RON expression in thyroid cancer cells was detected by western blotting. Glycolysis was assessed by measuring the extracellular acidification rate, glucose uptake, lactate concentration, and expression levels of glucose transporter 1, hexokinase 2 and pyruvate kinase M1/2. In addition, ferroptosis was assessed by detecting the levels of total iron, lipid peroxide and reactive oxygen species, and the expression levels of ferroptosis‑related proteins. Furthermore, mitochondrial function were assessed by JC‑1 staining and detection kits, respectively. The results demonstrated that RON was highly expressed in thyroid cancer cell lines. Furthermore, RON interference inhibited glycolysis, promoted ferroptosis, elevated cell sensitivity to chemotherapy and affected mitochondrial function in thyroid cancer cells. Further experiments demonstrated that RON interference affected the ferroptosis levels in thyroid cancer cells by inhibiting the glycolysis process. Mechanistically, the present results indicated that RON may affect ferroptosis, glycolysis and chemotherapy sensitivity by regulating MAPK/cAMP‑response element binding protein (CREB) signaling in thyroid cancer cells. In conclusion, the present study demonstrated that RON affected ferroptosis, glycolysis and chemotherapy sensitivity in thyroid cancer cells by regulating MAPK/CREB signaling, demonstrating its potential as a therapeutic target in thyroid cancer cells.

Natural Organic Matter Enhances Natural Transformation of Extracellular Antibiotic Resistance Genes in Sunlit Water.

Antibiotic resistance genes (ARGs) as emerging environmental contaminants exacerbate the risk of spreading antibiotic resistance. Natural organic matter (NOM) is ubiquitous in aquatic environments and plays a crucial role in biogeochemical cycles. However, its impact on the dissemination of extracellular antibiotic resistance genes (eARGs) under sunlight exposure remains elusive. This study reveals that environmentally relevant levels of NOM (0.1-20 mg/L) can significantly enhance the natural transformation frequency of the model bacterium Acinetobacter baylyi ADP1 by up to 7.6-fold under simulated sunlight. Similarly, this enhancement was consistently observed in natural water and wastewater systems. Further mechanism analysis revealed that reactive oxygen species (ROS) generated by NOM under sunlight irradiation, primarily singlet oxygen and hydroxyl radicals, play a crucial role in this process. These ROS induce intracellular oxidative stress and elevated cellular membrane permeability, thereby indirectly boosting ATP production and enhancing cell competence of extracellular DNA uptake and integration. Our findings highlight a previously underestimated role of natural factors in the dissemination of eARGs within aquatic ecosystems and deepen our understanding of the complex interplay between NOM, sunlight, and microbes in environmental water bodies. This underscores the importance of developing comprehensive strategies to mitigate the spread of antibiotic resistance in aquatic environments.

Polyamine Derived Photosensitizer: A Novel Approach for Photodynamic Therapy of Cancer.

Polyamines play a pivotal role in cancer cell proliferation. The excessive polyamine requirement of these malignancies is satisfied through heightened biosynthesis and augmented extracellular uptake via the polyamine transport system (PTS) present on the cell membrane. Meanwhile, photodynamic therapy (PDT) emerges as an effective anti-cancer treatment devoid of drug resistance. Recognizing these intricacies, our study devised a novel polyamine-derived photosensitizer (PS) for targeted photodynamic treatment, focusing predominantly on pancreatic cancer cells. We synthesized and evaluated novel spermine-derived fluorescent probes (N2) and PS (N3), exhibiting selectivity towards pancreatic cancer cells via PTS. N3 showed minimal dark toxicity but significant phototoxicity upon irradiation, effectively causing cell death in vitro. A significant reduction in tumor volume was observed post-treatment with no pronounced dark toxicity using the pancreatic cancer CDX mouse model, affirming the therapeutic potential of N3. Overall, our findings introduce a promising new strategy for cancer treatment, highlighting the potential of polyamine-derived PSs in PDT.

PhysioFit: a software to quantify cell growth parameters and extracellular fluxes.

Quantification of growth parameters and extracellular uptake and production fluxes is central in systems and synthetic biology. Fluxes can be estimated using various mathematical models by fitting time-course measurements of the concentration of cells and extracellular substrates and products. A single tool is available to non-computational biologists to calculate extracellular fluxes, but it is hardly interoperable and is limited to a single hard-coded growth model. We present our open-source flux calculation software, PhysioFit, which can be used with any growth model and is interoperable by design. PhysioFit includes some of the most common growth models, and advanced users can implement additional models to calculate extracellular fluxes and other growth parameters for metabolic systems or experimental setups that follow alternative kinetics. PhysioFit can be used as a Python library and offers a graphical user interface for intuitive use by end-users and a command-line interface to streamline integration into existing pipelines. PhysioFit v3 is implemented in Python 3 and was tested on Windows, Unix, and MacOS platforms. The source code and the documentation are freely distributed under GPL3 license at https://github.com/MetaSys-LISBP/PhysioFit/ and https://physiofit.readthedocs.io/.

HOXC4 promotes proliferation of pancreatic cancer cells by increasing LDHA-mediated glycolysis.

Homeobox C4 (HOXC4) is a member of homeobox family and acts as a transcription factor in regulating morphological development. The current study aimed to determine its role in pancreatic cancer (PC). Bioinformatics analysis was employed to assess the expression and clinical significance of HOXC4 in PC, while the expression of HOXC4 was further confirmed in PC tissues through quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The impact of HOXC4 on PC cell proliferation was evaluated using various assays including Cell Counting Kit-8, colony formation, apoptosis detection, cell cycle analysis, and subcutaneous tumorigenesis. Extracellular acidification rate, glucose uptake, and lactate production measurements were detected to examine the impact of HOXC4 on glycolysis. The relationship between HOXC4 and lactate dehydrogenase A (LDHA) was investigated using CHIP assay, luciferase reporter assay, and western blot. Notably, there was a substantial increase in HOXC4 expression in PC, and patients with elevated HOXC4 levels exhibited shorter survival durations. HOXC4 knockdown resulted in significantly reduced proliferation and colony formation in PC cells, accompanied by increased apoptosis and G1 phase arrest. The overexpression of HOXC4 resulted in contrasting effects. In vivo, the proliferation of PC cells was diminished upon the knockdown of HOXC4. HOXC4 exhibited an increase in LDHA expression by binding to its promoter. The suppressive effects of HOXC4 knockdown on PC cells were counteracted upon the restoration of LDHA. In conclusion, HOXC4 promoted the proliferation of PC cells by increasing LDHA-mediated glycolysis. HOXC4 can act as a target for PC therapy.

Cryo-EM analysis reveals human SID-1 transmembrane family member 1 dynamics underlying lipid hydrolytic activity

Two mammalian homologs of systemic RNA interference defective protein 1 (SID-1) (SIDT1/2) are suggested to function as double-stranded RNA (dsRNA) transporters for extracellular dsRNA uptake or for release of incorporated dsRNA from lysosome to cytoplasm. SIDT1/2 is also suggested to be involved in cholesterol transport and lipid metabolism. Here, we determine the cryo-electron microscopy structures of human SIDT1, homodimer in a side-by-side arrangement, with two distinct conformations, the cholesterol-bound form and the unbound form. Our structures reveal that the membrane-spanning region of SIDT1 harbors conserved histidine and aspartate residues coordinating to putative zinc ion, in a structurally similar manner to alkaline ceramidases or adiponectin receptors that require zinc for ceramidase activity. We identify that SIDT1 has a ceramidase activity that is attenuated by cholesterol binding. Observations from two structures suggest that cholesterol molecules serve as allosteric regulator that binds the transmembrane region of SIDT1 and induces the conformation change and the reorientation of the catalytic residues. This study represents a contribution to the elucidation of the cholesterol-mediated mechanisms of lipid hydrolytic activity and RNA transport in the SID-1 family proteins.

Abstract 1792: Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer

Abstract Reprogramming of metabolic processes by tumor cells is essential to cope with their increased proliferative activity, and with the challenges of the unique microenvironment they inhabit. These metabolic alterations are attractive targets to design novel therapeutic approaches.The most aggressive subtype of thyroid cancer, Anaplastic Thyroid Cancer (ATC), is often unresectable at presentation, highly resistant to therapy and usually lethal. We have recently discovered that ATCs overexpress several genes encoding enzymes involved in one-carbon metabolism (1C-Met), which utilizes serine and dietary folates to produce glycine and tetrahydrofolate-bound one-carbon units, required for nucleotide biosynthesis and for NADPH and glutathione (GSH) production. We have shown that i) the increased activity of the 1C-Met pathway supports the high purine demand of ATC; ii) inhibition of 1C-Met impairs tumor growth in vitro and in vivo, leading to growth arrest; iii) inhibition of 1C-Met renders thyroid cancer cells glycine-auxotroph.We now demonstrate that the profound addiction of ATC cells to 1C-Met creates a series of targetable vulnerabilities that can be harnessed to induce massive ATC cell death.Our new data demonstrate that:1- The purine depletion consequent impaired 1C-Met induces replicative stress and triggers the DNA Damage Response (DDR). Activation of DDR, coupled with the absence of functional TP53 in the vast majority of ATCs, leads to synthetic lethality between inhibition of 1C-Met and G2/M checkpoint kinases.2- Inhibition of 1C-Met in ATC cells generates an absolute dependence on extracellular glycine uptake. We have identified and validated the main glycine transporter(s) in ATC cells and show that inhibition of 1C-Met is synthetic lethal with inhibition of glycine uptake.3- Impaired 1C-Met leads to increased oxidative stress and decrease of NADPH and glutathione levels. These alterations create a synthetic lethality relationship with the glutathione-depleting activity of PRIMA-1 (APR246), a small molecule originally identified as a compound restoring mutant p53 conformation and function, but later shown to be converted within cells into the reactive electrophile methylene quinuclidinone, which acts in a p53-independent manner through a variety of mechanisms, including reduction of cellular GSH levels.Our data shed light on the molecular consequences of 1C-Met upregulation in ATC and support the efficacy of novel rationally designed therapeutic approaches with curative intent not only for ATC, but also for other aggressive, dedifferentiated solid tumors. Citation Format: Alexander Forrest, Anil Ahsan, Antonio Di Cristofano. Synthetic lethal vulnerabilities stemming from inhibition of one carbon metabolism in anaplastic thyroid cancer [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 1792.

Biallelic NAA60 variants with impaired N-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Abstract A110: Targeting methionine metabolism inhibits ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the leading cause of gynecologic cancer death. Platinum (Pt)- based chemotherapy can reprogram cancer cells to ovarian cancer stem cells (OCSCs) and contribute to disease recurrence. In the current study, we demonstrated that platinum drugs can enrich for OCSCs. In high grade serous ovarian cancer (HGSOC) cell lines, cisplatin treatment (IC50 for 16hrs) increased the percentage of cells with high aldehyde dehydrogenase (ALDH+) activity, a known OCSCs marker, and altered methionine (Met) metabolism. Extracellular Met uptake is converted to S-adenosyl-methionine (SAM) by methionine adenosyltransferase 2A (MAT2A), the rate-limiting enzyme of Met utilization. Short-term cisplatin treatment decreased SAM level and increased expression of MAT2A. It has been reported that CSCs have a high-flux metabolic state and are dependent on Met metabolism. We observed higher MAT2A expression in ALDH+ cells compared to the whole cell population, and Met depletion blocked the cisplatin-induced increase in %ALDH+ cells and spheroid formation ability. Furthermore, both MAT2A siRNA knockdown and the MAT2A inhibitor FIDAS-5 prevented cisplatin-induced increase in %ALDH+ cells, suggesting that blocking Met metabolism phenotypically inhibited the platinum enrichment of OCSCs. MAT2A inhibition also downregulated Cyclin E and arrested HGSOC cells in G1/S phase, indicating that a lower level of Met utilization re-established G1/S checkpoint in p53-mutant OC. As SAM is the universal methyl-group donor, it was of interest to examine whether the observed cisplatin-induced alterations in SAM level resulted in changes in DNA methylation. Methylation specific-PCR analysis demonstrated that siMAT2A or FIDAS-5 treatment of HGSOC cells inhibited cisplatin-induced hypermethylation of CpG islands of BRCA1 and ALDH1L1, a folate metabolic enzyme of which promoter hypermethylation is associated to tumor progression. Collectively, these results demonstrated a crucial role of Met metabolism in cisplatin-induced OCSCs enrichment. MAT2A has been recognized as a therapeutic target for the treatment of solid tumors and Met inhibition by targeting MAT2A could represent a therapeutic approach for inhibiting CSC in ovarian cancer. Citation Format: Shu Zhang, Tara X. Metcalfe, Christiane A. Hassel, Heather M. O'Hagan, Kenneth P. Nephew. Targeting methionine metabolism inhibits ovarian cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A110.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

AKR1C3 silencing inhibits autophagy-dependent glycolysis in thyroid cancer cells by inactivating ERK signaling.

Thyroid cancer is a highly differentiated and poorly malignant tumor. Interfering with glycolysis has become an effective means of controlling cancer progression and autophagy is negatively correlated with glycolysis. Aldo-keto reductase family 1 member C3 (AKR1C3) has been demonstrated to be highly expressed in thyroid cancer tissue and the higher AKR1C3 expression predicted the worse prognosis. We aimed to explore whether AKR1C3 could affect thyroid cancer progression by regulating autophagy-dependent glycolysis. AKR1C3 expression in thyroid cancer cells was detected by western blot. Then, AKR1C3 was knocked down by transfection with short hairpin RNA specific to AKR1C3 in the absence or presence of 3-methyladenine (3-MA) or PMA treatment. Cell cycle and apoptosis was detected by flow cytometry. Immunofluorescence staining was used to analyze LC3B expression. Extracellular acidification, glucose uptake and lactic acid secretion were detected. To evaluate the tumorigenicity of AKR1C3 insufficiency on thyroid cancer in vivo, TPC-1 cells with AKR1C3 knockdown were injected subcutaneously into nude mice. Then, cyclinD1 and Ki67 expression in tumorous tissues was measured by immunohistochemical analysis. Apoptosis was assessed by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining. Additionally, the expression of proteins related to cell cycle, apoptosis, glycolysis, autophagy, and extracellular signal-regulated kinase (ERK) signaling in cells and tumor tissues was assessed by western blot. Highly expressed AKR1C3 was observed in thyroid cancer cells. AKR1C3 knockdown induced cell cycle arrest and apoptosis of TPC-1 cells. Besides, autophagy was activated and glycolysis was inhibited following AKR1C3 silencing, and 3-MA treatment restored the impacts of AKR1C3 silencing on glycolysis. The further experiments revealed that AKR1C3 insufficiency inhibited ERK signaling and PMA application reversed AKR1C3 silencing-induced autophagy in TPC-1 cells. The in vivo results suggested that AKR1C3 knockdown inhibited the development of subcutaneous TPC-1 tumors in nude mice and inactivated the ERK signaling. Collectively, AKR1C3 silencing inhibited autophagy-dependent glycolysis in thyroid cancer by inactivating ERK signaling.

The futile creatine cycle and the synthesis of fatty acids in inguinal white adipose tissue from growing rats, submitted to a hypoprotein-hyperglycidic diet for 15 days.

The low-protein, high-carbohydrate (LPHC) diet administered to growing rats soon after weaning, for 15 days, promoted an increase in energy expenditure by uncoupling protein 1 (UCP1) in interscapular brown adipose tissue, and also due to the occurrence of the browning process in the perirenal white adipose tissue (periWAT). However, we believe that inguinal white adipose tissue (ingWAT) may also contribute to energy expenditure through other mechanisms. Therefore, the aim of this work is to investigate the presence of the futile creatine cycle, and the origin of lipids in ingWAT, since that tissue showed an increase in the lipids content in rats submitted to the LPHC diet for 15 days. We observed increases in creatine kinase and alkaline phosphatase activity in ingWAT, of the LPHC animals. The mitochondrial Nicotinamide adenine dinucleotide reduced/nicotinamide adenine dinucleotide oxidized ratio is lower in ingWAT of LPHC animals. In the LPHC animals treated with β-guanidinopropionic acid, the extracellular uptake of creatine in ingWAT was lower, as was the rectal temperature. Regarding lipid metabolism, we observed that in ingWAT, lipolysis in vitro when stimulated with noradrenaline is lower, and there were no changes in baseline levels. In addition, increases in the activity of enzymes were also observed: malic, glucose-6-phosphate dehydrogenase, and ATP-citrate lyase, in addition to an increase in the PPARγ content. The results show the occurrence of the futile creatine cycle in ingWAT, and that the increase in the relative mass may be due to an increase in de novo fatty acid synthesis.

Discovery of (2-(4-Substituted phenyl)quinolin-4-yl)(4-isopropylpiperazin-1-yl)methanone Derivatives as Potent Proprotein Convertase Subtilisin/Kexin Type 9 Inhibitors.

Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an increasingly important role in the treatment of hyperlipidemia. In pursuit of potent small molecules that block the PCSK9/low-density lipoprotein receptor (LDLR) protein-protein interaction (PPI), a series of 2-phenylquinoline-4-carboxylic acid derivatives were designed and synthesized based on previously derived molecules. In the in vitro PPI inhibition test, compounds M1, M12, M14, M18 and M27 exhibited potent activities with IC50 values of 6.25 μM, 0.91 μM, 2.81 μM, 4.26 μM and 0.76 μM, respectively, compared with SBC-115337 (IC50 value of 9.24 μM). Molecular docking and molecular dynamics simulations revealed the importance of hydrophobic interactions in the binding of inhibitors to the PPI interface of PCSK9. In LDLR expression and LDL uptake assays, the tested compounds M1, M12 and M14 were found to restore LDLR expression levels and to increase the extracellular LDL uptake capacity of HepG2 cells in the presence of exogenous PCSK9. Collectively, novel small-molecule PCSK9/LDLR PPI inhibitors (especially M12) with in vitro lipid lowering ability, were discovered as lead compounds for further development of hypolipidemic drugs.

Phospholipase D3 drives microglial response to amyloid pathology in Alzheimer’s disease

AbstractBackgroundAmyloid‐beta (Aβ) plaque formation is a well‐established hallmark for Alzheimer’s disease (AD). However, the processes behind plaque formation are not understood. Previous work from our group identified rare coding variants for PLD3, with one variant in particular, p.A442A, disrupting a splicing enhancer binding site within the mRNA transcript. In this study, we present findings that PLD3 regulates Aβ clearance in the brain through both cell autonomous and non‐autonomous means.MethodTo replicate the splicing effect observed in patient brain samples in vitro, PLD3 p.A442A expressing induced pluripotent stem cells (iPSCs) were differentiated into neurons or microglia and evaluated for extracellular Aβ and Aβ uptake, respectively. In an APP/PSEN1xPld3KO mouse model, Aβ dynamics were assessed with in vivo microdialysis and plaque pathology was assessed with immunohistochemistry. Frozen cortical tissue from these mice was analyzed through bulk RNAseq to identify transcriptomic and molecular changes that are attributed to Pld3 deficiency. PLD3 expression was assessed in an independent single‐cell RNAseq (scRNAseq) dataset from iPSC‐derived microglia to assess a potential cell‐autonomous role for PLD3 in microglia.ResultTranscriptomics from human samples reveal a significant decrease of PLD3 expression in AD brains versus controls, particularly in the brain regions most susceptible to amyloid pathology. Similar to PLD3‐variant brains, iPSC‐derived neurons carrying PLD3 p.A442A exhibited alternative splicing and displayed elevated Aβ levels in the conditioned media. Loss of Pld3 in APP/PSEN1 mice led to increased interstitial levels of Aβ and reduced Aβ clearance. Immunohistochemistry revealed an increase in diffuse, nonfibrillar plaque area and reduced microglial recruitment in APP/PSEN1xPld3KO. Transcriptomics data from brain tissue in these mice revealed altered expression of genes that define the disease associated microglia signatures. To begin to understand the role of PLD3 in microglia, we analyzed scRNAseq from CRISPRi screen in iPSC‐derived microglia. We found that PLD3 expression is enriched in interferon‐associated microglia. Finally, we demonstrate that iPSC‐derived microglia carrying PLD3 p.A442A exhibit reduced internalization of Aβ and myelin, suggesting a defect in phagocytic capacity.ConclusionTogether, we show that PLD3 plays a critical role in the brain by facilitating the microglial response to Aβ plaque pathology, modulating plaque composition.