Decrease In Glycolysis Research Articles

Abstract Tu103: SIRT2 Inhibition Decreases Glycolysis and Attenuates Hypertrophic Response in H9c2 Cardiomyocytes

Introduction: Myocardial glycolysis increases in hypertrophic and failing hearts. Hyperacetylation also occurs in the failing heart, and many glycolytic enzymes are known to be subject to acetylation. However, it is generally considered that acetylation has inhibitory effects on glycolysis. As a result, it is not clear whether acetylation changes directly contribute to glycolysis changes in cardiac hypertrophy. We therefore determined whether changes in the acetylation of glycolytic enzymes and the activity of the cytosolic deacetylase SIRT2 regulate cardiac glycolysis. Methods: Glycolysis rates were directly measured in rat heart-derived H9c2 cardiomyocytes perfused with 5 mM [5- 3 H] glucose, 0.8 mM palmitate, and 4% bovine serum albumin. Before these metabolic measurements, H9c2 cells were treated with either a SIRT2 inhibitor (10 µM AGK2) or a vehicle for 24 hours. In separate experiments, SIRT2 was also knocked down in H9c2 cells using siRNA, followed by glycolysis rate determinations. The impact of SIRT2 inhibition or SIRT2 knockdown on the acetylation status of glycolytic enzyme was also assessed. Furthermore, the effects of SIRT2 inhibition on hypertrophic signalling were assessed by treating H9c2 cells with phenylephrine. Results: SIRT2 inhibition markedly decreased glycolysis rates in H9c2 cells compared to vehicle-treated cells (524±108 vs 2631±372 nmol . g dry wt -1. min -1 , p<0.05). Similarly, SIRT2 knockdown resulted in a significant reduction in glycolysis rates compared to scrambled siRNA-treated H9c2 cells (745±31 vs 1659±168 nmol . g dry wt -1. min -1 , p<0.05). The decrease in SIRT2 was accompanied by an increase in the acetylation status of the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GPDH). Moreover, a trend towards increased phosphofructokinase (PFK) acetylation was also observed in SIRT2 knockdown H9c2 cells compared to scrambled siRNA-treated cells. Lastly, AGK2 treatment also attenuated phenylephrine-mediated hypertrophic responses in H9c2 cells. Conclusions: Increased acetylation of glycolytic enzymes is associated with a decrease in glycolysis, and SIRT2 inhibition or deletion in H9c2 cells significantly decreases glycolysis rates and attenuates hypertrophy. SIRT2 may therefore contribute to the increased glycolysis seen in hypertrophy and heart failure.

Systemically Administered D-allose Inhibits the Tumor Energy Pathway and Exerts Synergistic Effects With Radiation.

The present study investigated the anticancer effects of intraperitoneally administered D-allose in in vivo models of head and neck cancer cell lines. To assess the direct effects of D-allose, its dynamics in blood and tumor tissues were examined. D-allose was detected in blood and tumor tissues 10 min after its intraperitoneal administration and then gradually decreased. In vivo experiments revealed that radiation plus D-allose was more effective than either treatment alone. Thioredoxin-interacting protein (TXNIP) mRNA over-expression was detected after the addition of D-allose in in vitro and in vivo experiments. D-allose inhibited cell growth, which was associated with decreases in glycolysis and intracellular ATP levels and the prolonged activation of AMPK. The phosphorylation of p38-MAPK was also observed early after the administration of D-allose and was followed by the activation of AMPK and up-regulated expression of TXNIP in both in vitro and in vivo experiments. Systemically administered D-allose appears to exert antitumor effects. Further studies are needed to clarify the appropriate dosage and timing of the administration of D-allose and its combination with other metabolic agents.

The E-liquid flavoring vanillin alters energy and autophagic pathways in human proximal tubule (HK-2) epithelial cells

The use of flavored e-liquids in electronic nicotine delivery systems (ENDS) has become very popular in recent years, but effects of these products have not been well characterized outside the lung. In this study, acute exposure to the popular flavoring vanillin (VAN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0–1000 μM VAN for 24 or 48 h and cellular stress responses were determined. Mitochondrial viability using MTT assay showed a significant decrease between the control and 1000 μM group by 48 h. Seahorse XFp analysis showed significantly increased basal respiration, ATP production, and proton leak after 24 h exposure. By 48 h exposure, these parameters remained significantly increased in addition to non-mitochondrial respiration and maximal respiration. Glycolytic activity after 24 h exposure showed significant decreases in glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. The autophagy markers microtubule-associated protein 1A/1B light chain 3 (LC3B–I and LC3B-II) were probed via western blotting. The ratio of LC3B-II/LC3B–I was significantly increased after 24 h exposure to VAN, but by 48 h this ratio significantly decreased. The mitophagy marker PINK1 showed an increasing trend at 24 h, and its downstream target Parkin was significantly increased between the control and 750 μM group only. Finally, the oxidative stress marker 4-HNE was significantly decreased after 48 h exposure to VAN. These results indicate that acute exposure to VAN in the kidney HK-2 model can induce energy and autophagic changes within the cell.

Tumor microenvironment induced switch to mitochondrial metabolism promotes suppressive functions in immune cells.

Understanding the intricacies of the metabolic phenotype in immune cells and its plasticity within the tumor microenvironment is pivotal in understanding the pathology and prognosis of cancer. Unfavorable conditions and cellular stress in the tumor microenvironment (TME) exert a profound impact on cellular functions in immune cells, thereby influencing both tumor progression and immune responses. Elevated AMP:ATP ratio, a consequence of limited glucose levels, activate AMP-activated protein kinase (AMPK) while concurrently repressing the activity of mechanistic target of rapamycin (mTOR) and hypoxia-inducible factor 1-alpha (HIF-1α). The intricate balance between AMPK, mTOR, and HIF-1α activities defines the metabolic phenotype of immune cells in the TME. These Changes in metabolic phenotype are strongly associated with immune cell functions and play a crucial role in creating a milieu conducive to tumor progression. Insufficiency of nutrient and oxygen supply leads to a metabolic shift in immune cells characterized by a decrease in glycolysis and an increase in oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) rates. In most cases, this shift in metabolism is accompanied by a compromise in the effector functions of these immune cells. This metabolic adaptation prompts immune cells to turn down their effector functions, entering a quiescent or immunosuppressive state that may support tumor growth. This article discusses how tumor microenvironment alters the metabolism in immune cells leading to their tolerance and tumor progression, with emphasis on mitochondrial metabolism (OXPHOS and FAO).

Multi‐omic profiling of isogenic ApoE3/3 and ApoE4/4 iPSC‐derived astrocytes uncovers altered lipid metabolism and mitochondrial function of ApoE4 astrocytes

AbstractBackgroundAs the main genetic risk factor for AD, glial ApoE4 has been implicated in a number of early disease relevant processes, such as lipid dyshomeostasis, mitochondrial dysfunction and altered immune function. This ApoE4 glial phenotype is not yet well characterized and this hampers the rationale design of ApoE4‐targeting interventions.MethodWe characterize the phenotypes of ApoE3/3 and ApoE4/4 iPSC‐derived astrocytes by multi‐omic profiling. In addition, we use lipidomic and metabolic profiling of interventions in ApoE4 astrocytes to define compounds that normalize lipid counts and metabolic function.ResultIn the ApoE4 astrocytes we observe strong accumulation of cholesterol esters (storage product of cholesterol) and triglycerides (storage products of fatty acids), in comparison to the ApoE3 astrocytes. Interestingly, our proteomic and transcriptomic profiling of these cells reveal an increase in cholesterol synthesis, despite the already high levels of cholesterol esters in the cell. Moreover, our proteomic and transcriptomic results display an increase in mitochondrial pathways, such as beta‐oxidation, TCA cycle and oxidative phosphorylation and a decrease in glycolysis. Not only mitochondrial function is altered but also mitochondrial organization, as we see an upregulation of the most relevant proteins for mitochondrial fusion, MFN1, MFN2 and OPA1. Our lipidomic and metabolic profiling of ApoE4 astrocytes after treatment with a number of different interventions reveals new insights into these altered lipid metabolic pathways.ConclusionOur multi‐omic profiling of iPSC‐derived ApoE3/3 and ApoE4/4 astrocytes uncovers that ApoE4 astrocytes have altered lipid metabolism, mitochondrial function and mitochondrial organization. Treatment with interventions reveals new insights into these altered pathways.

TMET-01. THERAPEUTIC TARGETING OF CROSSTALK BETWEEN MITOCHONDRIAL FITNESS AND CELL ADHESION/MIGRATION IN DIFFUSE MIDLINE GLIOMA (DMG)

Abstract Diffuse midline gliomas (DMGs) are aggressive tumors characterized by infiltration into normal midline brain tissue, hindering surgical resection and contributing to overall morbidity and mortality. To study which genes are driving this invasive phenotype, we established a novel two-step pooled whole genome CRISPR-Migration screen for DMG primary cell cultures (n = 3) derived from patients. After selecting for genes critical for cell survival, each cell line was seeded in the upper chamber of a transwell assay optimized for DMG cells to promote complete serum-free cell attachment and migration through laminin, one of the major components of the blood vessels basement membrane. Following sgRNA library sequencing of low and high-migratory populations, we found a strong positive correlation with genes directly involved in the focal adhesion machinery (ITGB1, CRKL, RAPGEF1, PARVA, PTK2, FERMT2). Stable CRISPR knockout of these genes validated significant reduction in adhesion/migration, without affecting proliferation. Interestingly, we also identified in the screen genes controlling different aspects of mitochondrial function (NDUFAF6, PITRM1, MINOS1, MICU3, LIPT1, MRPL38), especially affecting outer-inner mitochondrial membrane integrity. This finding is in line with the higher sensitivity of multiple DMG cell cultures to Bcl-2 family inhibitors, readily prompting apoptosis in combination with sub-lethal doses of various drugs. Agents that specifically modulate mitochondrial oxidative phosphorylation (OXPHOS) (Rotenone, Oligomycin A, Antimycin A) reduced DMG cell migration without affecting proliferation. Surprisingly, silencing of ITGB1 and CRKL led to a marked decrease in glycolysis and OXPHOS levels, again without any changes in proliferation. Additionally, silencing of ITGB1 (primarily responsible for cell adhesion to laminin) led to the decreased expression (RNA) of multiple mitochondrial NADH dehydrogenases, pointing to an intricate bi-directional mechanism connecting mitochondrial fitness to DMG cell migration. Studies are underway to validate these findings in vivo and to determine an optimal therapeutic disruption of this critical DMG malignant phenotype.

In vitro-generated human muscle reserve cells are heterogeneous for Pax7 with distinct molecular states and metabolic profiles

BackgroundThe capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies.MethodsIn the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile.ResultsWe observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake.ConclusionsOverall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.

Improving anti-tumor CD8+ T cell function through manipulating glutamine metabolism

Abstract Immunotherapies that bolster the anti-tumor effects of cytotoxic CD8 T lymphocytes (CTLs) have improved outcomes for many, yet most patients fail to achieve durable responses. Investigations into strategies to further enhance the cancer killing capacity of these cells are crucial in the effort to improve immunotherapies. T cells radically alter their metabolism upon activation, and prior studies have shown pan-inhibition of glutamine metabolism (glutaminolysis) using the inhibitor 6-Diazo-5-oxo-norleucine (DON) enhances the function of CTLs in the tumor microenvironment. However, DON’s toxicity combined with this drug’s lack of specificity demonstrate a need for a more directed glutaminolysis targeting approach. I utilized a targeted glutaminolysis CRISPR library to investigate the role of individual enzymes targeted by DON. To interrogate the importance of these enzymes specifically in anti-tumor CTLs, I developed a model in which CRISPR-library edited antigen-specific CTLs were adoptively transferred into tumor-bearing mice. After 7 days of in vivo selection, next generation sequencing was performed on isolated CTLs to evaluate the survival advantage or disadvantage of these gene knockouts. While multiple DON targets resulted in decreased CTL fitness, deletion of the gene encoding for glutamine synthetase (GS) – which converts glutamate to glutamine – conferred an advantage to CTLs. Upon inhibition of GS, cells may increase cellular levels of glutamate, and preliminary data suggest this results in a decrease in glycolysis and increase in mitochondrial respiration capacity. Lastly, in vitro data demonstrate increased inflammatory cytokine production of CTLs upon genetic knockout or pharmacologic inhibition of GS. Supported by grants from NIH (T32 GM007347, R01 CA217987)

Abstract 3860: ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism

Abstract Background: Primary or secondary resistance to venetoclax is pervasively associated with mutational/non-mutational activation of RAS/RAF/ERK pathway which confers metabolic alterations as a compensatory adaptation. Preclinical models of venetoclax resistance and primary patient samples (n=8) were used to assess the synergy of concomitant Bcl2 and ERK1/2 inhibition followed by RNAseq analysis to identify molecular mechanisms responsible for overcoming resistance to venetoclax. Results: ERK1/2 inhibition using Compound 27 (ERKi, Heightman et al., J Med Chem. 2018), an analog of ASTX029, was highly synergistic with venetoclax at inducing apoptosis in AML. NRAS-mutant OCI-AML3 and THP1 cells, that are inherently resistant to venetoclax, were sensitized to venetoclax when combined with ERKi with ~90% apoptosis compared to 20% by venetoclax alone (Combination Index = 0.008). Synergy was also confirmed using isogenic OCI-AML2 cells with lab-generated acquired resistance to venetoclax, resulting in 75% apoptosis versus 22% by venetoclax (CI=0.6). Leukemia progenitor cells in primary AML samples were also depleted by the ven+ERKi combination (CI:0.03-0.23). In line with our in-vitro findings, treatment with ASTX029 as a single agent or in combination with venetoclax significantly inhibited leukemia burden and increased survival in an OCIAML3 xenograft model (p<0.05). Single agent ERKi and combination treatment using an NRAS-mutant PDX resulted in significantly impaired clonogenic potential and decreased expression of cMyc and CD44 in CD34+CD38- population as demonstrated by CyTOF, indicating depletion of progenitors. Transcriptomic profiling of OCIAML3 cells revealed upregulation of 6 pathways in response to venetoclax including oxidative phosphorylation (OXPHOS), electron transport chain, Myc targets, E2F targets, epithelial mesenchymal transition and KRAS signaling. Interestingly, these pathways were downregulated after ERKi+venetoclax treatment along with decrease in glycolysis and fatty acid metabolism (FDR<0.05). Since, venetoclax treatment significantly enriched for OXPHOS, we examined the metabolic effect of ERKi to overcome venetoclax resistance. Seahorse analysis showed increased basal and maximal respiration, and ATP production in OCIAML2 venetoclax resistant versus parental cells. Combining ERKi with venetoclax reduced OCR and ATP production in OCIAML3 and OCIAML2-venetoclax resistant cells. This was accompanied by an increase in membrane depolarization (p=0.001) as well as enhanced mitochondrial ROS (p<0.001). Conclusion: The inhibition of ERK1/2 alters mitochondrial metabolism and functional integrity to overcome resistance to venetoclax by causing apoptosis in venetoclax resistant AML cells. These data provide a strong rationale for the combination of ERK1/2 and Bcl-2 inhibitors in treatment of AML and warrant further investigation in the clinic. Citation Format: Priyanka Sharma, Lauren Ostermann, Sujan Piya, Natalia Baran, Anudishi Tyagi, Christopher Hindley, Kim-Hien Dao, Martin Sims, V. Lokesh Battula, Michael Andreeff, Gautam Borthakur. ERK1/2 inhibition overcomes resistance to venetoclax in AML by altering mitochondrial metabolism. [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 3860.

Abstract P5-08-14: Blockade of CD47/Thrombospondin-1 signaling increases glycolytic metabolism as a protective mechanism against chemotherapy-associated cardiac injury in a model of Triple-Negative Breast Cancer

Abstract Due to advances in diagnosis and treatment, cancer–related mortality has decreased, and by the year 2030, there will be 22 million cancer survivors in the United States. This success comes with an increased incidence of serious adverse effects, mainly in the cardiovascular system. While new treatment modalities are emerging for Triple-negative breast cancer (TNBC), most current strategies include anthracycline-based regimens to manage disease. Therefore, novel strategies are needed to overcome anthracycline-induced cardiotoxicities in this patient population. Activation of the TSP1/CD47 signaling axis is implicated in the progression of heart failure, with reported increases in TSP1 levels following myocardial infarction. Therefore, we examined the potential of CD47 blockade as a strategy to prevent cardiac injury as a consequence of cancer chemotherapy. Our data in a syngeneic orthotopic breast cancer model shows that blockade of CD47 using an in vivo anti-sense phosphodiesterase morpholino (PMO) preserved ejection fraction, fractional shortening, and cardiac output when compared to DOX treatment while preserving oncologic efficacy of chemotherapy. To determine a potential mechanism of cardioprotection, hearts of control and CD47 PMO-treated mice were subjected to RNA sequencing. Gene set enrichment analysis (GSEA) showed significant positive enrichment for metabolic pathways including pyruvate metabolism (NES= 2.3 , p< 0.002), and oxidative phosphorylation (NES=2.0, p< 0.01). During cardiac insult, metabolic flexibility of cardiomyocytes results in metabolic reprogramming from fatty acid oxidation to a glycolytic mechanism to overcome injury. Thus, DOX-associated cardiotoxicity may be mediated by an increase in TSP1 and a decrease in glycolysis, leading to the inability to overcome acute cellular stress. In vitro cellular bioenergetics, analysis revealed that TSP1 caused a dose-dependent reduction in glycolytic flux and glycolytic capacity in cardiac myoblast. This, coupled with preserved cardiac viability of cardiac cells treated with CD47 PMO in the presence of DOX, suggests that TSP1 may act through CD47 to prevent cardiac cell metabolic reprogramming needed to overcome injury. Furthermore, anti-sense experiments with siRNAs to Glut-4 and Hexokinase-II showed that the protection conferred by CD47 is mediated by activating these proteins. Therefore our studies suggest that the TSP1/CD47 axis may be central to the interplay of metabolism to preserve cardiac tissue integrity; thus, targeting this pathway may prevent the onset of chronic cardiac disease due to chemotherapy in cancer patients. Citation Format: Steven M. Bronson, Jessica D. Mackert, Mitra Kooshki, Adam S. Wilson, Nildris Cruz-Diaz, Pierre L Triozzi, Alexandra Thomas, Katherine L. Cook, David R. Soto-Pantoja. Blockade of CD47/Thrombospondin-1 signaling increases glycolytic metabolism as a protective mechanism against chemotherapy-associated cardiac injury in a model of Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-08-14.

Pyruvate Dehydrogenase Inhibition Leads to Decreased Glycolysis, Increased Reliance on Gluconeogenesis and Alternative Sources of Acetyl-CoA in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease characterized by poor outcomes and therapy resistance. Devimistat is a novel agent that inhibits pyruvate dehydrogenase complex (PDH). A phase III clinical trial in AML patients combining devimistat and chemotherapy was terminated for futility, suggesting AML cells were able to circumvent the metabolic inhibition of devimistat. The means by which AML cells resist PDH inhibition is unknown. AML cell lines treated with devimistat or deleted for the essential PDH subunit, PDHA, showed a decrease in glycolysis and decreased glucose uptake due to a reduction of the glucose transporter GLUT1 and hexokinase II. Both devimistat-treated and PDHA knockout cells displayed increased sensitivity to 2-deoxyglucose, demonstrating reliance on residual glycolysis. The rate limiting gluconeogenic enzyme phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly upregulated in devimistat-treated cells, and its inhibition increased sensitivity to devimistat. The gluconeogenic amino acids glutamine and asparagine protected AML cells from devimistat. Non-glycolytic sources of acetyl-CoA were also important with fatty acid oxidation, ATP citrate lyase (ACLY) and acyl-CoA synthetase short chain family member 2 (ACSS2) contributing to resistance. Finally, devimistat reduced fatty acid synthase (FASN) activity. Taken together, this suggests that AML cells compensate for PDH and glycolysis inhibition by gluconeogenesis for maintenance of essential glycolytic intermediates and fatty acid oxidation, ACLY and ACSS2 for non-glycolytic production of acetyl-CoA. Strategies to target these escape pathways should be explored in AML.

Lactate metabolism is essential in early-onset mitochondrial myopathy.

Myopathies secondary to mitochondrial electron transport chain (ETC) dysfunction can result in devastating disease. While the consequences of ETC defects have been extensively studied in culture, little in vivo data are available. Using a mouse model of severe, early-onset mitochondrial myopathy, we characterized the proteomic, transcriptomic, and metabolic characteristics of disease progression. Unexpectedly, ETC dysfunction in muscle results in reduced expression of glycolytic enzymes in our animal model and patient muscle biopsies. The decrease in glycolysis was mediated by loss of constitutive Hif1α signaling, down-regulation of the purine nucleotide cycle enzyme AMPD1, and activation of AMPK. In vivo isotope tracing experiments indicated that myopathic muscle relies on lactate import to supply central carbon metabolites. Inhibition of lactate import reduced steady-state levels of tricarboxylic acid cycle intermediates and compromised the life span of myopathic mice. These data indicate an unexpected mode of metabolic reprogramming in severe mitochondrial myopathy that regulates disease progression.

Homeostasis of carbohydrates and reactive oxygen species is critically changed in the brain of middle-aged mice: Molecular mechanisms and functional reasons

The brain is an organ that consumes a lot of energy. In the brain, energy is required for synaptic transmission, numerous biosynthetic processes and axonal transport in neurons, and for many supportive functions of glial cells. The main source of energy in the brain is glucose and to a lesser extent lactate and ketone bodies. ATP is formed at glucose catabolism via glycolysis and oxidative phosphorylation in mitochondrial electron transport chain (ETC) within mitochondria being the main source of ATP. With age, brain's energy metabolism is disturbed, involving a decrease in glycolysis and mitochondrial dysfunction. The latter is accompanied by intensified generation of reactive oxygen species (ROS) in ETC leading to oxidative stress. Recently, we have found that crucial changes in energy metabolism and intensity of oxidative stress in the mouse brain occur in middle age with minor progression in old age. In this review, we analyze the metabolic changes and functional causes that lead to these changes in the aging brain.

LPLUNC1 reduces glycolysis in nasopharyngeal carcinoma cells through the PHB1-p53/c-Myc axis.

Cancer cells prefer glycolysis to support their proliferation. Our previous studies have shown that the long palate, lung, and nasal epithelial cell clone 1 (LPLUNC1) can upregulate prohibitin 1 (PHB1) expression to inhibit the proliferation of nasopharyngeal carcinoma (NPC) cells. Given that PHB1 is an important regulator of cell energy metabolism, we explored whether and how LPLUNC1 regulated glucose glycolysis in NPC cells. LPLUNC1 or PHB1 overexpression decreased glycolysis and increased oxidative phosphorylation (OXPHOS)-related protein expression in NPC cells, promoting phosphorylated PHB1 nuclear translocation through 14-3-3σ. LPLUNC1 overexpression also increased p53 but decreased c-Myc expression in NPC cells, which were crucial for the decrease in glycolysis and increase in OXPHOS-related protein expression induced by LPLUNC1 overexpression. Finally, we found that treatment with all-trans retinoic acid (ATRA) reduced the viability and clonogenicity of NPC cells, decreased glycolysis, and increased OXPHOS-related protein expression by enhancing LPLUNC1 expression in NPC cells. Therefore, the LPLUNC1-PHB1-p53/c-Myc axis decreased glycolysis in NPC cells, and ATRA upregulated LPLUNC1 expression, ATRA maybe a promising drug for the treatment of NPC.

Hypoxemia in the presence or absence of systemic inflammation does not increase blood lactate levels in healthy volunteers.

Elevated lactate levels are a sign of critical illness and may result from insufficient oxygen delivery. We investigated whether hypoxemia and/or systemic inflammation, results in increased lactate levels in healthy volunteers. 30 healthy volunteers were exposed to either 3.5 h of hypoxemia (FiO2 ± 11.5%), normoxemic endotoxemia (FiO2 21%, administration of 2 ng/kg endotoxin), or hypoxemic endotoxemia (n = 10 per group). Blood lactate, hemoglobin, SpO2, PaO2, PaCO2, pH, and hemodynamic parameters were serially measured. Hypoxemic treatment resulted in lower SpO2 (81.7 ± 2.6 and 81.4 ± 2.4% in the hypoxemia and hypoxemic endotoxemia groups, respectively) and hyperventilation with a PaCO2 decrease of 0.8 ± 0.5 and 1.5 ± 0.6 kPa and an increase in pH. Arterial oxygen content (CaO2) decreased by 20.5 ± 2.9 and 23.5 ± 4.4%, respectively. Lactate levels were slightly, but significantly higher in both hypoxemic groups compared with the normoxemic endotoxemia group over time (p < 0.0001 for both groups), but remained below 2.3 mmol/L in all subjects. Whereas PaO2 and SpO2 did not correlate with lactate levels, PaCO2, pH and CaO2 did. Hypoxemia, in the absence or presence of inflammation does not result in relevant increases of lactate. The small increases in lactate observed are likely to be due to hyperventilation-related decreases in glycolysis.

Abstract 2344: BRAF inhibitor resistant thyroid cancer cells exhibit altered cellular energetics

Abstract Thyroid cancer is the most commonly diagnosed endocrine malignancy. Although prognosis for patients is positive overall, dedifferentiated thyroid tumors and invasive thyroid tumors are difficult to treat. There is one FDA approved targeted therapy in use for in BRAF mutant, highly dedifferentiated anaplastic thyroid cancer, a combination of the BRAF inhibitor dabrafenib and the ERK inhibitor trametinib. While this treatment improves outcomes, resistance is of great concern. In other cancers driven by BRAF mutations, particularly melanoma, a known driver of BRAF inhibitor resistance is enhanced oxidative phosphorylation. We therefore investigated the energetics of thyroid cancer cells to determine whether metabolic changes can drive resistance in thyroid cancer. We have previously determined sensitivity to dabrafenib by area under the curve to classify cell lines as sensitive or resistant (Hicks HM et al, 2021). We used CUTC5 cells as our primary model of intrinsically sensitive cells and CUTC60 cells to model intrinsic resistance. We observed both cell lines had similar basal oxygen consumption rates (OCR), however dabrafenib treatment reduced this in the sensitive CUTC5 cells and did not in the resistant CUTC60 cells. Additionally, maximum OCR was reduced in the CUTC5 cells with as little as 10 nM dabrafenib. In a preliminary experiment, CUTC60 cells were co-treated with dabrafenib and rotenone, an inhibitor of the electron transport chain, and synergistic inhibition of proliferation was observed. We further investigated energetics in the dabrafenib sensitive cell line KTC1 and KTC1-VA7, which are derived from KTC1 and BRAF-inhibitor resistant (Danysh BP et al, 2016). Unlike the other cells evaluated these cells did not have spare respiratory capacity. Additionally, dabrafenib treatment did not alter basal OCR except at the highest doses. Although, we did observe a reduction in glycolysis in the parental KTC1 upon dabrafenib treatment, which was not observed in the KTC1-VA7 cells. Sensitive CUTC5 cells also exhibited a greater decrease in glycolysis than resistant CUTC60 cells. From this, we conclude that glycolysis may contribute to dabrafenib resistance and merits further investigation. With regards to oxidative metabolism, we only observed a reduction in OCR in a dabrafenib sensitive cell line and there was synergistic inhibition in the resistant CUTC60 cells treated with both dabrafenib and rotenone, suggesting that oxidative metabolism aids the cells in tolerating BRAF inhibition. Further screening of dabrafenib sensitive and resistant cells is necessary to determine if this is a function of dabrafenib sensitivity. Citation Format: Eric Bolf, Rebecca Schweppe. BRAF inhibitor resistant thyroid cancer cells exhibit altered cellular energetics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2344.

An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells.

Corneal epithelial cells derived from human pluripotent stem cells (hPSCs) are an important cell source for preclinical models to test ophthalmic drugs. However, current differentiation protocols lack instructions regarding optimal culturing conditions, which hinders the quality of cells and limits scale-up. Here, we introduce a simplified small molecule-based corneal induction method (SSM-CI) to generate corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimizing cell-culturing time using two defined culturing media containing TGF-β, and Wnt/β-catenin pathway inhibitors, and bFGF growth factor over 25days. Compared to the conventional human corneal epithelial cell line (HCE-T) and human primary corneal epithelial cells (hPCEpCs), corneal epithelial cells generated by SSM-CI are well differentiated and express relevant maturation markers, including PAX6 and CK12. RNA-seq analysis indicated the faithful differentiation of hPSCs into corneal epithelia, with significant upregulation of corneal progenitor and adult corneal epithelial phenotypes. Furthermore, despite the initial inhibition of TGF-β and Wnt/β-catenin, upregulation of these pathway-related transcripts was observed in the later stages, indicating their necessity in the generation of mature corneal epithelial cells. Moreover, we observed a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells, involving a decrease in glycolysis and an increase in fatty acid oxidation. This was also attributed to the overexpression of metabolic enzymes and transporter-related transcripts responsible for fatty acid metabolism. Thus, SSM-CI provides a comprehensive method for the generation of functional corneal epithelial cells for use in preclinical models.

Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome

ObjectivePolycystic ovary syndrome (PCOS) is characterized by follicular dysplasia. An insufficient glycolysis-derived energy supply of granulosa cells (GCs) is an important cause of follicular dysplasia in PCOS. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been proven to regulate the function of GCs. In this study, exosomes extracted from clinical FF samples were used for transcriptome sequencing (RNA-seq) analysis, and a human ovarian granulocyte tumour cell line (KGN cells) was used for in vitro mechanistic studies.Methods and resultsIn FF exosomal RNA-seq analysis, a decrease in glycolysis-related pathways was identified as an important feature of the PCOS group, and the differentially expressed miR-143-3p and miR-155-5p may be regulatory factors of glycolysis. By determining the effects of miR-143-3p and miR-155-5p on hexokinase (HK) 2, pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate, lactate and apoptosis in KGN cells, we found that upregulated miR-143-3p expression in exosomes from the PCOS group inhibited glycolysis in KGN cells; knockdown of miR-143-3p significantly alleviated the decrease in glycolysis in KGN cells in PCOS. MiR-155-5p silencing attenuated glycolytic activation in KGN cells; overexpression of miR-155-5p significantly promoted glycolysis in KGN cells in PCOS. In this study, HK2 was found to be the mediator of miR-143-3p and miR-155-5p in FF-derived exosome-mediated regulation of glycolysis in KGN cells. Reduced glycolysis accelerated apoptosis of KGN cells, which mediated follicular dysplasia through ATP, lactate and apoptotic pathways.ConclusionsIn conclusion, these results indicate that miR-143-3p and miR-155-5p in FF-derived exosomes antagonistically regulate glycolytic-mediated follicular dysplasia of GCs in PCOS.9LsDb7oAvmV44Q9GUxhugtVideo

Curcumae Radix Decreases Neurodegenerative Markers through Glycolysis Decrease and TCA Cycle Activation.

Neurodegenerative diseases (ND) are being increasingly studied owing to the increasing proportion of the aging population. Several potential compounds are examined to prevent neurodegenerative diseases, including Curcumae radix, which is known to be beneficial for inflammatory conditions, metabolic syndrome, and various types of pain. However, it is not well studied, and its influence on energy metabolism in ND is unclear. We focused on the relationship between ND and energy metabolism using Curcumae radix extract (CRE) in cells and animal models. We monitored neurodegenerative markers and metabolic indicators using Western blotting and qRT-PCR and then assessed cellular glycolysis and metabolic flux assays. The levels of Alzheimer’s disease-related markers in mouse brains were reduced after treatment with the CRE. We confirmed that neurodegenerative markers decreased in the cerebrum and brain tumor cells following low endoplasmic reticulum (ER) stress markers. Furthermore, glycolysis related genes and the extracellular acidification rate decreased after treatment with the CRE. Interestingly, we found that the CRE exposed mouse brain and cells had increased mitochondrial Tricarboxylic acid (TCA) cycle and Oxidative phosphorylation (OXPHOS) related genes in the CRE group. Curcumae radix may act as a metabolic modulator of brain health and help treat and prevent ND involving mitochondrial dysfunction.

PFKFB3 Regulates Chemoresistance, Metastasis and Stemness via IAP Proteins and the NF-κB Signaling Pathway in Ovarian Cancer.

Glycolysis has been reported to be critical for cancer stem cells (CSCs), which are associated with tumor chemoresistance, metastasis and recurrence. Thus, selectively targeting glycolytic enzymes may be a potential therapy for ovarian cancer. 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 3 (PFKFB3), the main source of fructose-2,6-bisphosphate, controls the first committed step in glycolysis. We investigate the clinical significance and roles of PFKFB3 in ovarian cancer using in vitro and in vivo experiments. We demonstrate that PFKFB3 is widely overexpressed in ovarian cancer and correlates with advanced stage/grade and poor outcomes. Significant up-regulation of PFKFB3 was found in ascites and metastatic foci, as well as CSC-enriched tumorspheres and ALDH+CD44+ cells. 3PO, a PFKFB3 inhibitor, reduced lactate level and sensitized A2780CP cells to cisplatin treatment, along with the modulation of inhibitors of apoptosis proteins (c-IAP1, c-IAP2 and survivin) and an immune modulator CD70. Blockade of PFKFB3 by siRNA approach in the CSC-enriched subset led to decreases in glycolysis and CSC properties, and activation of the NF-κB cascade. PFK158, another potent inhibitor of PFKFB3, impaired the stemness of ALDH+CD44+ cells in vitro and in vivo, whereas ectopic expression of PFKFB3 had the opposite results. Overall, PFKFB3 was found to mediate metabolic reprogramming, chemoresistance, metastasis and stemness in ovarian cancer, possibly via the modulation of inhibitors of apoptosis proteins and the NF-κB signaling pathway; thus, suggesting that PFKFB3 may be a potential therapeutic target for ovarian cancer.