Extracellular Sources Research Articles

Dynamic composition of stress granules in Trypanosoma brucei.

Stress granules (SGs) are stress-induced RNA condensates consisting of stalled initiation complexes resulting from translational inhibition. The biochemical composition and function of SGs are highly diverse, and this diversity has been attributed to different stress conditions, signalling pathways involved and specific cell types. Interestingly, mRNA decay components, which are found in ubiquitous cytoplasmic foci known as processing bodies (PB), have also been identified in SG proteomes. A major challenge in current SG studies is to understand the cause of SG diversity, as well as the function of SG under different stress conditions. Trypanosoma brucei is a single-cellular parasite that causes Human African Trypanosomiasis (sleping sickness). In this study, we showed that by varying the supply of extracellular carbon sources during starvation, cellular ATP levels changed rapidly, resulting in SGs of different compositions and dynamics. We identified a subset of SG components, which dissociated from the SGs in response to cellular ATP depletion. Using expansion microscopy, we observed sub-granular compartmentalization of PB- and SG-components within the stress granules. Our results highlight the importance of cellular ATP in SG composition and dynamics, providing functional insight to SGs formed under different stress conditions.

New insight into enhanced carbon recovery from anaerobic fermentation of waste activated sludge with cation exchange resin coupled with NaCl pretreatment

Carbon recovery from waste activated sludge has been attracting considerable attention. However, the migration and transformation patterns of carbon sources between the phases have rarely been reported. In this study, a novel strategy using cation exchange resin (CER) coupled with sodium chloride (NaCl) to enhance carbon recovery through anaerobic fermentation (AF) was proposed. The results demonstrated that CER coupled with NaCl destroyed OH and CO stretching in amide I while promoting the formation of β-sheet and random coil structures, leading to sludge disintegration. This significantly improved the kinetics of endogenous carbon release, resulting in the release of 1146.33 mg/L of carbon from the solid sludge into the liquid phase. Approximately 75.61 % of the initial carbon source was bio-transformed into short-chain fatty acids. Correspondingly, carbon recovery was significantly increased up to 852.23 mg C/L, 4.57 times that of the control. Mechanism exploration revealed that carbon source recovery was significantly elevated by the synergistic effect of CER and NaCl. CER effectively removed high-valence cations from extracellular polymeric substance (EPS), weakening its bridging and adsorption-electro neutralization capabilities, promoting protein deflocculation, and triggering EPS disruption to release extracellular carbon sources. NaCl disrupted the ionic strength and distribution inside and outside microbial cells, creating an osmotic pressure difference that resulted in cell plasmolysis and lysis, ultimately inducing the release of intracellular carbon sources. Economic and carbon emission reduction benefit analyses verified that the CER coupled with NaCl pretreatment is a cost-effective sludge treatment strategy. This study illustrates the carbon source migration and transformation pathways in the CER coupled with NaCl-assisted AF process, providing guidance for sustainable sludge management.

Asparagine Dependency Is a Targetable Metabolic Vulnerability in TP53-Altered Castration-Resistant Prostate Cancer.

TP53 tumor suppressor is frequently altered in lethal, castration-resistant prostate cancer (CRPC). However, to date there are no effective treatments that specifically target TP53 alterations. Using transcriptomic and metabolomic analyses, we have shown here that TP53-altered prostate cancer exhibits an increased dependency on asparagine (Asn) and overexpresses Asn synthetase (ASNS), the enzyme catalyzing the synthesis of Asn. Mechanistically, the loss or mutation of TP53 transcriptionally activated ASNS expression, directly and via mTORC1-mediated ATF4 induction, driving de novo Asn biosynthesis to support CRPC growth. TP53-altered CRPC cells were sensitive to Asn restriction by knockdown of ASNS or L-asparaginase treatment to deplete the intracellular and extracellular sources of Asn, respectively, and cell viability was rescued by Asn addition. Notably, pharmacological inhibition of intracellular Asn biosynthesis using a glutaminase inhibitor and depletion of extracellular Asn with L-asparaginase significantly reduced Asn production and effectively impaired CRPC growth. This study highlights the significance of ASNS-mediated metabolic adaptation as a synthetic vulnerability in CRPC with TP53 alterations, providing a rationale for targeting Asn production to treat these lethal prostate cancers. Significance: TP53-mutated castration-resistant prostate cancer is dependent on asparagine biosynthesis due to upregulation of ASNS and can be therapeutically targeted by approaches that deplete intracellular and extracellular asparagine.

Discrimination of extracellular miRNA sources for the identification of tumor-related functions based on nanowire thermofluidics

Discrimination of extracellular miRNA sources for the identification of tumor-related functions based on nanowire thermofluidics

Abstract 2120: Asparaginases-Treatment potential for the biomarker selected hepatocellular carcinoma (HCC) population

Abstract Asparaginase is a key component of the therapeutic regimen used to treat patients with acute lymphoblastic leukemia (ALL). Calaspargase pegol (Asparlas) is composed of E. coli L-asparaginase and is PEGylated with approximately 5000 Da polyethylene glycol (PEG). Asparlas depletes asparagine (Asn) by deamination into aspartic acid. Asn synthetase (ASNS) is the key enzyme responsible for de novo Asn synthesis. In the absence of Asn in their direct environment, normal cells having adequate ASNS activity can survive with their own Asn supply while cancer cells lacking ASNS activity undergo apoptosis as they depend on extracellular sources of Asn. The ASNS gene has a typical CpG island in its promoter and its methylation status in tumor cells has been reported to be associated with asparaginase sensitivity clinically in T-ALL and in solid tumors preclinically. In this study we have investigated ASNS promoter hypermethylation as a biomarker for Asparlas activity in solid tumors and explored the feasibility of translating these results into the clinic. Analyses of the TCGA datasets indicated that a relatively high fraction of hepatocellular carcinoma (HCC) and gastric cancers are hypermethylated at the ASNS promoter. Testing Asparlas across a panel of HCC cell lines demonstrated that ASNS promoter-hypermethylated cell lines with lowest ASNS mRNA expression are the most responsive to Asparlas treatment in vitro. In vivo efficacy studies in mice bearing human ASNS promoter-hypermethylated xenograft models of HCC and gastric cancer showed that Asparlas treatment inhibited tumor growth in a dose-dependent manner and resulted in tumor rejections at the highest doses. Data from the HCC model was used to build a PK/PD efficacy model to project efficacious concentrations in patients. A panel of HCC PDX models with different levels of ASNS promoter methylation was evaluated to better understand the relationship between this biomarker and response to Asparlas. Treatment with Asparlas resulted in complete depletion of Asn in plasma and a partial depletion of Asn in the tumor for all models tested and strong anti-tumor activity against a model that had high methylation of the ASNS promoter. This activity was lost in models with lesser degrees of methylation indicating a strict threshold of promoter methylation for efficacy. These data confirm and extend previous findings that hypermethylation of the ASNS promoter has a synthetic lethal relationship with Asn-depleting therapies including Asparlas and highlight the need to carefully define the minimum threshold of hypermethylation to select HCC patients who could potentially benefit from Asparlas treatment. Citation Format: Ethika Tyagi, Magdalena Pohorecka, Valérie Duvivier, Aurelie Studeny, Damien Valour, Natacha Moulharat, Hélène Darville, Nicolas Provost, Pierre Barbier Saint Hilaire, Eef Hoeben, Donald Simons. Asparaginases-Treatment potential for the biomarker selected hepatocellular carcinoma (HCC) population [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 2120.

Bacterial hemophilin homologs and their specific type eleven secretor proteins have conserved roles in heme capture and are diversifying as a family.

Cellular life relies on enzymes that require metals, which must be acquired from extracellular sources. Bacteria utilize surface and secreted proteins to acquire such valuable nutrients from their environment. These include the cargo proteins of the type eleven secretion system (T11SS), which have been connected to host specificity, metal homeostasis, and nutritional immunity evasion. This Sec-dependent, Gram-negative secretion system is encoded by organisms throughout the phylum Proteobacteria, including human pathogens Neisseria meningitidis, Proteus mirabilis, Acinetobacter baumannii, and Haemophilus influenzae. Experimentally verified T11SS-dependent cargo include transferrin-binding protein B (TbpB), the hemophilin homologs heme receptor protein C (HrpC), hemophilin A (HphA), the immune evasion protein factor-H binding protein (fHbp), and the host symbiosis factor nematode intestinal localization protein C (NilC). Here, we examined the specificity of T11SS systems for their cognate cargo proteins using taxonomically distributed homolog pairs of T11SS and hemophilin cargo and explored the ligand binding ability of those hemophilin cargo homologs. In vivo expression in Escherichia coli of hemophilin homologs revealed that each is secreted in a specific manner by its cognate T11SS protein. Sequence analysis and structural modeling suggest that all hemophilin homologs share an N-terminal ligand-binding domain with the same topology as the ligand-binding domains of the Haemophilus haemolyticus heme binding protein (Hpl) and HphA. We term this signature feature of this group of proteins the hemophilin ligand-binding domain. Network analysis of hemophilin homologs revealed five subclusters and representatives from four of these showed variable heme-binding activities, which, combined with sequence-structure variation, suggests that hemophilins are diversifying in function.IMPORTANCEThe secreted protein hemophilin and its homologs contribute to the survival of several bacterial symbionts within their respective host environments. Here, we compared taxonomically diverse hemophilin homologs and their paired Type 11 secretion systems (T11SS) to determine if heme binding and T11SS secretion are conserved characteristics of this family. We establish the existence of divergent hemophilin sub-families and describe structural features that contribute to distinct ligand-binding behaviors. Furthermore, we demonstrate that T11SS are specific for their cognate hemophilin family cargo proteins. Our work establishes that hemophilin homolog-T11SS pairs are diverging from each other, potentially evolving into novel ligand acquisition systems that provide competitive benefits in host niches.

Abstract B050: Metabolic rewiring in human brain cancer

Abstract Metabolic adaptation can promote oncogenic phenotypes in glioblastoma (GBM), but the metabolic pathways utilized by GBM and how they differ from the pathways utilized in normal cortex are poorly understood. Here, we utilize in vivo stable isotope tracing in mice and patients with glioblastoma to define carbon fate and metabolic rewiring in cancer. We infused uniformly labeled 13C glucose into mice bearing orthotopic GBM patient-derived xenografts or into patients undergoing surgical resection for likely GBM. Tumor and cortex were physically separated in mice by fluorescence-guided microdissection. In humans, samples of enhancing tumor, non-enhancing tumor and normal cortex were separated intraoperatively using MRI guidance. Samples were subsequently analyzed by liquid chromatography mass spectrometry and MALDI imaging mass spectrometry to determine the downstream metabolic fates of infused 13C glucose. Of eight patients infused with 13C6-glucose, six were confirmed to have GBM while the others had non-GBM high grade gliomas. In mice and in patients, glucose-derived carbon effectively entered glycolysis and labeled glycolytic intermediates equivalently in tumor and cortex. Compared to GBM tissue, cortical tissue preferentially oxidized glucose-derived carbon in the TCA cycle and used glucose-derived carbon to synthesize the neurotransmitter GABA. Cortical tissue utilized glucose-derived carbon to synthesize the amino acid and neurotransmitter precursor serine, while GBM tissue preferentially derived serine from extracellular sources. By contrast, GBM preferentially utilized glucose-derived carbon for the synthesis of metabolites required for growth: purines, pyrimidines and NAD+/NADH. These studies are the first measurements of numerous metabolic pathways in human brain cancer and cortex and suggest that GBMs suppress the physiologic utilization of glucose carbons for ATP and neurotransmitter generation and instead use these carbons to synthesize the biomolecules they need to proliferate. Consistent with this hypothesis, eliminating dietary serine in mice slowed GBM PDX growth and forced tumors to synthesize serine from glucose, which in turn lowered tumor nucleotide and NAD+/NADH levels. Dietary serine depletion had minimal effects on cortical metabolism, consistent with glucose being the primary serine source in the normal brain. These direct measurements of metabolic pathway activity in human and mouse brain cancer reveal adaptive metabolic rewiring and new therapeutic targets. Citation Format: Andrew Scott, Anjali Mittal, Sravya Palavalasa, Baharan Meghdadi, Nathan Qi, Alexandra O'Brien, Ayesha Kothari, Nathalie Agar, Ethan Yang, Joshua Fischer, Vijay Tarnal, Wajd Al-Holou, Costas Lyssiotis, Deepak Nagrath, Daniel Wahl. Metabolic rewiring in human brain cancer [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B050.

Estimations of Charge Deposition Onto Convoluted Axon Surfaces Within Extracellular Electric Fields.

Biophysical models of neural stimulation are a valuable approach to explaining the mechanisms of neuronal recruitment via applied extracellular electric fields. Typically, the applied electric field is estimated via a macroscopic finite element method solution and then applied to cable models as an extracellular voltage source. However, the field resolution is limited by the finite element size (typically 10's-100's of times greater than average neuronal cross-section). As a result, induced charges deposited onto anatomically realistic curved membrane interfaces are not taken into consideration. However, these details may alter estimates of the applied electric field and predictions of neural tissue activation. To estimate microscopic variations of the electric field, data for intra-axonal space segmented from 3D scanning electron microscopy of the mouse brain genu of corpus callosum were used. The boundary element fast multipole method was applied to accurately compute the extracellular solution. Neuronal recruitment was then estimated via an activating function. Taking the physical structure of the arbor into account generally predicts higher values of the activating function. The relative integral 2-norm difference is 90% on average when the entire axonal arbor is present. A large fraction of this difference might be due to the axonal body itself. When an isolated physical axon is considered with all other axons removed, the relative integral 2-norm difference between the single-axon solution and the complete solution is 25% on average. Our result may provide an explanation as to why Deep Brain Stimulation experiments typically predict lower activation thresholds than commonly used FEM/Cable model approaches to predicting neuronal responses to extracellular electrical stimulation. These results may change methods for bi-domain neural modeling and neural excitation.

Unresolved questions regarding cellular cysteine sources and their possible relationships to ferroptosis.

Cysteine is required for synthesis of glutathione (GSH), coenzyme A, other sulfur-containing metabolites, and most proteins. In most cells, cysteine comes from extracellular disulfide sources including cystine, glutathione-disulfide, and peptides. The thioredoxin reductase-1 (TrxR1)- or glutathione-disulfide reductase (GSR)-driven enzymatic systems can fuel cystine reduction via thioredoxins, glutaredoxins, or other thioredoxin-fold proteins.Free cystine enters cells thorough the cystine-glutamate antiporter, xCT, but systemically, plasma glutathione-disulfide might predominate as a cystine source. Erastin, inhibiting both xCT and voltage-dependent anion channels, induces ferroptotic cell death, so named because this type of cell death is antagonized by iron-chelators. Many cancer cells seem to be predisposed to ferroptosis, which has been proposed as a targetable cancer liability. Ferroptosis is associated with lipid peroxidation and loss of either glutathione peroxidase-4 (GPX4) or ferroptosis suppressor protein-1 (FSP1), which each prevent accumulation of lipid peroxides. It has been suggested that an xCT inhibition-induced cellular cysteine-deficiency lowers GSH levels, starving GPX4 for reducing power and allowing membrane lipid peroxides to accumulate, thereby causing ferroptosis. Aspects of ferroptosis are however not fully understood and need to be further scrutinized, for example that neither disruption of GSH synthesis, loss of GSH, nor disruption of glutathione disulfide reductase (GSR), triggers ferroptosis in animal models. Here we reevaluate the relationships between Erastin, xCT, GPX4, cellular cysteine and GSH, RSL3 or ML162, and ferroptosis. We conclude that, whereas both Cys and ferroptosis are potential liabilities in cancer, their relationship to each other remains insufficiently understood.

TMET-03. REPROGRAMMING OF GLIOBLASTOMA GLUCOSE METABOLISM FUELS TUMOR GROWTH

Abstract Metabolic adaptation can promote oncogenic phenotypes in glioblastoma (GBM), but the metabolic pathways utilized by GBM and how they differ from the pathways utilized in normal cortex are poorly understood. Here, we utilize in vivo stable isotope tracing in mice and patients with GBM to define carbon fate and metabolic rewiring in cancer. We infused uniformly-labeled 13C glucose into mice bearing orthotopic GBM patient-derived xenografts or into patients undergoing surgical resection for likely GBM. Tumor and cortex were physically separated in mice by fluorescence-guided microdissection. In humans, samples of enhancing tumor, non-enhancing tumor and normal cortex were separated intraoperatively using MRI guidance. Samples were subsequently analyzed by liquid chromatography mass spectrometry to determine the downstream metabolic fates of infused 13C glucose. Of eight patients infused with 13C6-glucose, six were confirmed to have GBM while the others had non-GBM high grade gliomas. In mice and in patients, glucose-derived carbon effectively entered glycolysis and labeled glycolytic intermediates equivalently in tumor and cortex. Compared to GBM tissue, cortical tissue preferentially oxidized glucose-derived carbon in the TCA cycle and used glucose-derived carbon to synthesize neurotransmitters. Cortical tissue utilized glucose-derived carbon to synthesize the amino acid and neurotransmitter precursor serine, while GBM tissue derived serine from extracellular sources. By contrast, GBM tissue preferentially utilized glucose-derived carbon for the synthesis of purines, pyrimidines and NAD+/NADH. Using time-course isotope tracing studies in mice with GBM and a novel metabolic flux analysis framework, we quantified absolute metabolic fluxes in vivo and confirmed elevated nucleotide synthesis and serine salvage in GBM. Consistent with these results, eliminating dietary serine in mice slowed GBM PDX growth. These studies are the first measurements of numerous metabolic pathways in humans and indicate that GBMs suppress the physiologic utilization of glucose carbons to fuel biosynthesis, which represents a targetable metabolic liability.

Targeted synergy of cation exchange-driven extracellular carbon release and lysozyme-catalyzed intracellular carbon release for maximizing carbon source recovery from waste activated sludge

Short-chain fatty acids as superior carbon recovery from waste activated sludge have received rising attentions. This study quantified contributions of cation exchange (CE) and lysozyme on the targeted carbon source migration. The innovative targeted pattern for extracellular and intracellular carbon source release was proposed. The 1-day synergetic treatment at optimal CE resin dosage of 1.75 g/g SS and lysozyme dosage of 10 % drove considerable carbon source release and particulate carbon source solubilization, which occupied 35.31 % of total sludge solid carbon sources. The CE dominantly contributed to extracellular polymeric substances disruption and extracellular carbon source release, with the percentage of 39.47–48.13 %. In this case, The Lyso was the strongest contributor to microbial cell lysis and intracellular carbon source release, with the proportions of 49.53–75.76 %. Subsequently, approximately 56.54–58.49 % of the originally dissolved carbon sources (mostly proteins and carbohydrates) were bio-degraded into micromolecular carbon sources with high bioavailability in a short-term anaerobic fermentation (2 days). Correspondingly, satisfactory carbon recovery rate of 23.06 % was obtained, which was 7.30 times higher than the control. The synergy of CE and lysozyme greatly improved kinetic speeds and performances of endogenous carbon source migration and recovery. Such synergetic treatment of CE and lysozyme not only provided novel insights into carbon fate in anaerobic fermentation, but also achieved remarkable carbon source release and recovery benefits with economic incomes of 190.8–283.4 CNY/ton sludge.

Enhanced nutrients removal and microbial mechanisms in a pilot-scale anaerobic-oxic-anoxic (A/O/A) system: Synergistic roles of denitrifying polyphosphate accumulating organisms and endogenous denitrifiers

The realization of low-cost and high-standard nutrients removal are not acquired effectively in conventional pre-denitrification biological nitrogen removal processes (e.g., anaerobic/anoxic/oxic (A/A/O) process) for the treatment of domestic sewage. In this study, a pilot-scale bioreactor system based on the synergism of denitrifying phosphorus removal (DPR) and endogenous denitrification (ED) was proposed for advanced nutrients removal, which was operated under alternating anaerobic/oxic/anoxic (A/O/A) mode with no extracellular carbon source in the anoxic stage. The results showed that A/O/A and A/A/O systems were successfully established and operated in four stages with different mixed liquor suspended solid (MLSS, I: 3.32 ± 0.25 g/L, II: 5.89 ± 0.94 g/L, III: 8.66 ± 0.51 g/L, IV: 9.11 ± 5.72 g/L). Compared with the A/A/O system, the A/O/A system showed significantly higher nitrogen and phosphorus removal effectiveness within hydraulic retention time of 10 h. At MLSS of 8.66 ± 0.51 g/L, the A/O/A system could achieve simultaneously efficient removal of COD (91.47% ± 2.06%, 2.97 ± 0.26 mg COD/(g MLSS·h)), NH4+-N (97.27% ± 1.23%, 0.37 ± 0.06 mg NH4+-N/(g MLSS·h)), TN (89.94% ± 1.47%, 0.45 ± 0.03 mg TN/(g MLSS·h)), and PO43--P (96.67% ± 0.88%, 0.04 ± 0.01 mg PO43--P/(g MLSS·h)), and the effluent concentrations of COD, NH4+-N, TN, and PO43--P were 24.0 ± 3.1, 0.9 ± 0.4, 4.4 ± 0.6, and 0.13 ± 0.03 mg/L, respectively. In addition, microbial community results indicated that denitrifying polyphosphate accumulating organisms and endogenous denitrifiers synergistically enhanced the nutrients removal capacity in the A/O/A system. Overall, this work provides new insight for further scaling and optimization of the A/O/A system.

Extracellular Vesicles-A Source of RNA Biomarkers for the Detection of Breast Cancer in Liquid Biopsies.

Over the past decade, extracellular vesicles (EVs) have emerged as a promising source of cancer-derived RNAs for liquid biopsies. However, blood contains a pool of heterogeneous EVs released by a variety of cell types, making the identification of cancer RNA biomarkers challenging. Here, we performed deep sequencing of plasma EV RNA cargo in 32 patients with locally advanced breast cancer (BC) at diagnosis and 7 days after breast surgery and in 30 cancer-free healthy controls (HCs). To identify BC-derived RNA biomarkers, we searched for RNAs that had higher levels in BC EVs at the time of diagnosis compared with HCs and decreased after surgery. Data analysis showed that the fractions of miRNAs, snRNAs, snoRNAs, and tRFs were increased, but the fraction of lncRNAs was decreased in BC EVs as compared to HCs. BC-derived biomarker candidates were identified across various RNA biotypes. Considered individually, they had very high specificity but moderate sensitivity for the detection of BC, whereas a biomarker model composed of eight RNAs: SNORD3H, SNORD1C, SNORA74D, miR-224-5p, piR-32949, lnc-IFT-122-2, lnc-C9orf50-4, and lnc-FAM122C-3 was able to distinguish BC from HC EVs with an AUC of 0.902 (95% CI = 0.872-0.931, p = 3.4 × 10-9) in leave-one-out cross-validation. Furthermore, a number of RNA biomarkers were correlated with the ER and HER2 expression and additional biomarker models were created to predict hormone receptor and HER2 status. Overall, this study demonstrated that the RNA composition of plasma EVs is altered in BC patients and that they contain cancer-derived RNA biomarkers that can be used for BC detection and monitoring using liquid biopsies.

Preparation and characterization of ATP-loaded chitosan/alginate nanoparticles for therapeutic applications

Adenosine triphosphate (ATP) is known as an energy source and is generated by mitochondria which is the powerhouse of the cell. The ATP supplies energy through the disassociation of the phosphate group by the cellular enzymes. The extracellular ATP in the extracellular environment acts as a signaling molecule and can act as an anti-inflammatory molecule, can promote cancer progression, and also can act as a pro-inflammatory molecule. The degradation of ATP is a major disadvantage by ectonucleotidases that attenuates the therapeutic property of ATP in the extracellular environment. We have formulated chitosan/alginate nanoparticles loaded with ATP for increasing their encapsulation efficiency and for sustained drug release. This encapsulation can avoid ATP degradation from ectonucleotidases. Nanoparticle characterization by DLS, FTIR, SEM, encapsulation efficiency, and cytotoxicity assay by XTT assay and Live-dead assay was monitored for the synthesized nano formulated ATP. Our results showed that the formulated ATP-loaded chitosan/alginate nanoparticle sized about 342 nm with the optimum encapsulation efficiency of about 92.03% with a sustained drug release profile. These nano formulated ATP could be used for calorie restriction conditions where ATP can be supplied as an extracellular source for bypassing oxidative phosphorylation, and we can circumvent the oxidant production during oxidative phosphorylation. The concept of avoiding oxidative stress by bypassing oxidative phosphorylation can open an avenue for healthy aging.

Abstract 678: Use Of Extracellular Substrates By Alveolar Type 2 Pneumocytes For Surfactant Lipid Synthesis.

Introduction: Pulmonary surfactant is a lipoprotein complex essential for lung function. It reduces surface tension during inspiration and avoids alveolar collapse during expiration. It is synthetized by alveolar type 2 pneumocytes (T2C) and it is composed of phospholipids (~90%), mainly phosphatidylcholine (PC), and specific proteins. Decreased surfactant and altered composition are associated with multiple adult lung diseases. However, T2C lipid metabolism and their use of extracellular glucose and lipid for surfactant synthesis has not been studied. Our prior results showed that the low density lipoprotein receptor-related 1 (LRP1) is needed for surfactant synthesis. We hypothesized that nutritional conditions and LRP1 function impact the pathways used by T2C to synthetize surfactant. Methods: Control and LRP1 knockdown T2C-derived A549 cells were cultured in DMEM (4.5 g/L glucose) with 10% FBS and maintained at the air-liquid interface for 24 hours to promote their differentiation and polarization. Then, cells were cultured in DMEM with delipidated FBS and BSA-complexed palmitic acid (PA) at different concentrations (0-0.25 mM) for 2 hours and mRNA of surfactant synthetic enzymes was analyzed. Results: mRNA expression of Fatty acid synthase (FASN) and Choline kinase (CK) decreased 80% and 93% (p<0.001) respectively when at least 0.05 mM PA was present. Phospholipase A2 ( PLA2) followed the opposite pattern, with 21.5-fold increase (p<0.001) when PA was present. FASN and CK expression was downregulated in the absence of LRP1. mRNA expression of genes involved in cholesterol metabolism was assessed too. HMG-CoA reductase decreased only at the highest concentration of PA (41% decrease, p<0.01), while expression of HMG-CoA synthase and lipid exporters ABCA1 and ABCG1 was not affected. ABCG1 expression was compromised in LRP1 knockdown cells. Conclusions: Extracellular availability of glucose and fatty acids affects mRNA expression of genes involved in surfactant lipid synthesis in T2C, suggesting metabolic pathway flexibility during different nutritional conditions. In the absence of extracellular lipid sources, glucose can be used by T2C for fatty acid synthesis. Furthermore, LRP1 expression affects the metabolic pathways used by T2C.

Abstract 1589: Characterization of polyaminohydroxamic acids and polyaminobenzamides as inhibitors of HDAC10

Abstract Elevated polyamine levels are essential for continual proliferation of cancer cells. Cytosolic histone deacetylase-10 (HDAC10) specifically deacetylates the modified polyamine N8-acetylspermidine (N8-AcSpd), of which extracellular sources can be abundant, particularly in the colonic lumen. We previously demonstrated that exogenously supplied N8-AcSpd can support tumor cell growth through HDAC10-mediated deacetylation into spermidine, which is then redistributed within the polyamine interconversion pathway to maintain homeostasis.In the current study, we investigate the utility of polyamine-based hydroxamic acids (PAHAs) and benzamides (PABAs) as pharmacological inhibitors of HDAC10-mediated polyamine deacetylation. Incorporation of the polyamine moiety enables these compounds to enter the cell selectively through the polyamine transport system, which is upregulated in cancer cells. A total of 42 compounds, including 21 PAHAs and 21 PABAs, were screened for HDAC10 inhibition using a cell-based growth assay in which polyamine-depleted, growth-arrested cells are exposed to N8-AcSpd: in the presence of HDAC10 activity, this polyamine is deacetylated and proliferation is restored. Several compounds in our screen inhibited HDAC10 activity in this assay, as indicated by a lack of growth rescue, and were selected for further analysis in which treatment was expanded over a range of doses, thereby allowing a dose-dependent evaluation and comparison of sensitivity. HPLC-based analysis of cell lysates functionally confirmed that deacetylation of N8-AcSpd into spermidine was inhibited by the test compounds. Finally, specificity of the selected PAHAs and PABAs for HDAC10 was evaluated by immunoblotting for increased acetylation of the substrates of related HDACs, including histones H3 and H4, and alpha-tubulin. These studies provide a basis for future drug design strategies targeting the polyamine deacetylase activity of HDAC10 as an antitumor strategy, which may be particularly important in the context of polyamine-blocking therapies. Citation Format: Tracy Murray Stewart, Jackson R. Foley, Patrick M. Woster, Robert A. Casero. Characterization of polyaminohydroxamic acids and polyaminobenzamides as inhibitors of HDAC10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1589.

Abstract 1712: Targeting asparagine dependency as a therapy for TP53-mutated castration-resistant prostate cancer

Abstract Background: The TP53 tumor suppressor is frequently altered in lethal, castration-resistant prostate cancer (CRPC). However, to date there are no effective treatments that specifically target TP53 gain-of-function (GOF) mutants. Thus, there is an urgent need to identify and target vulnerabilities that perpetuate disease progression and treatment resistance resulting from TP53 GOF mutants. Methods: We performed RNA sequencing (RNA-seq) and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics on GEM and PDX CRPC models with or without TP53 GOF mutations. Our findings were validated using genetic and pharmacologic approaches in relevant in vitro and in vivo models. We assessed the effect of L-asparaginase (ASNase) or/and glutaminase (GLS) inhibitor CB-839 in cell lines, organoids and PDX models. Tumors were characterized by histology and immunohistochemistry for relevant markers. Results: Using transcriptomic and metabolomic analyses, we show here that TP53-mutated prostate cancer (PCa) exhibits an increased dependency on asparagine and overexpresses asparagine synthetase (ASNS, the enzyme catalyzing the synthesis of asparagine). Mechanistically, TP53 GOF mutations transcriptionally activate ASNS expression, directly as well as via ATF4, driving de novo asparagine biosynthesis to support CRPC growth. TP53-mutated CRPC cells are sensitive to asparagine restriction by knockdown of ASNS and L-asparaginase treatment to deplete the intracellular and extracellular sources of asparagine respectively. This effect was rescued by asparagine addition. Notably, pharmacological inhibition of intracellular asparagine biosynthesis using a glutaminase (GLS) inhibitor and depletion of extracellular asparagine with L-asparaginase significantly reduced asparagine production and effectively impaired CRPC growth. Conclusions: This study highlights the significance of ASNS-mediated metabolic adaptation as a synthetic vulnerability in CRPC with TP53 GOF mutations, providing a rationale for co-targeting intracellular and extracellular asparagine production to treat these lethal prostate cancers. Citation Format: Young A Yoo. Targeting asparagine dependency as a therapy for TP53-mutated castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1712.

Aging reduces calreticulin expression and alters spontaneous calcium signals in astrocytic endfeet of the mouse dorsolateral striatum

Aging-related impairment of the blood brain barrier (BBB) and neurovascular unit (NVU) increases the risk for neurodegeneration. Among various cells that participate in BBB and NVU function, calcium signals in astrocytic endfeet are crucial for maintaining BBB and NVU integrity. To assess if aging is associated with altered calcium signals within astrocytic endfeet of the dorsolateral striatum (DLS), we expressed GCaMP6f in DLS astrocytes of young (3–4 months), middle-aged (12–15 months) and aging (20–30 months) mice. Compared to endfeet in young mice, DLS endfeet in aging mice demonstrated decreased calreticulin expression, and alterations to both spontaneous membrane-associated and mitochondrial calcium signals. While young mice required both extracellular and endoplasmic reticulum calcium sources for endfoot signals, middle-aged and aging mice showed heavy dependence on endoplasmic reticulum calcium. Thus, astrocytic endfeet show significant changes in calcium buffering and sources throughout the lifespan, which is important for understanding mechanisms by which aging impairs the BBB and NVU.

Antioxidant Activity of Panax ginseng to Regulate ROS in Various Chronic Diseases

Reactive oxygen species (ROS)-the byproduct of regular cell activity formed by various cellular components—play a significant role in pathological and physiological conditions. Alternatively, antioxidants are compounds that reduce or scavenge reactive species in cells. An asymmetry between the antioxidant defense system and ROS from intracellular and extracellular sources cause chronic diseases such as cancer, inflammation, tumorigenesis, cardiovascular and neurogenerative diseases. However, Panax ginseng and its secondary metabolites (known as ginsenosides, phenolic compounds, peptides, acid polysaccharides, polyacetylene, and alkaloids) are well-recognized as antioxidants in many in vitro and in vivo experiments which show beneficial activity in regulating ROS in these diseases. There are extensive evidences that P. ginseng can destroy cancer cells specifically by increasing oxidative stress through ROS generation without significantly harming normal cells. Additionally, numerous studies have examined the antioxidant activity of ginseng and its derivatives on ROS-mediated signaling pathways which are discussed herein. This review summarizes the potential antioxidant activity of P. ginseng in several chronic diseases, and gives updated research evidence with related mechanisms and the future possibilities of nano-formulated compounds of P. ginseng and other polyphenols.

Spatial and temporal conversion of nitrogen using Arthrobacter sp. 24S4-2, a strain obtained from Antarctica.

According to average nucleotide identity (ANI) analysis of the complete genomes, strain 24S4-2 isolated from Antarctica is considered as a potential novel Arthrobacter species. Arthrobacter sp. 24S4-2 could grow and produce ammonium in nitrate or nitrite or even nitrogen free medium. Strain 24S4-2 was discovered to accumulate nitrate/nitrite and subsequently convert nitrate to nitrite intracellularly when incubated in a nitrate/nitrite medium. In nitrogen-free medium, strain 24S4-2 not only reduced the accumulated nitrite for growth, but also secreted ammonia to the extracellular under aerobic condition, which was thought to be linked to nitrite reductase genes nirB, nirD, and nasA by the transcriptome and RT-qPCR analysis. A membrane-like vesicle structure was detected in the cell of strain 24S4-2 by transmission electron microscopy, which was thought to be the site of intracellular nitrogen supply accumulation and conversion. This spatial and temporal conversion process of nitrogen source helps the strain maintain development in the absence of nitrogen supply or a harsh environment, which is part of its adaption strategy to the Antarctic environment. This process may also play an important ecological role, that other bacteria in the environment would benefit from its extracellular nitrogen source secretion and nitrite consumption characteristics.