Glycolytic Phenotype Research Articles

Temperature Dependence of Platelet Metabolism.

Temperature plays a fundamental role in biology, influencing cellular function, chemical reaction rates, molecular structures, and interactions. While the temperature dependence of many biochemical reactions is well defined in vitro, the effect of temperature on metabolic function at the network level is poorly understood, and it remains an important challenge in optimizing the storage of cells and tissues at lower temperatures. Here, we used time-course metabolomic data and systems biology approaches to characterize the effects of storage temperature on human platelets (PLTs) in a platelet additive solution. We observed that changes to the metabolome with storage time do not simply scale with temperature but instead display complex temperature dependence, with only a small subset of metabolites following an Arrhenius-type relationship. Investigation of PLT energy metabolism through integration with computational modeling revealed that oxidative metabolism is more sensitive to temperature changes than glycolysis. The increased contribution of glycolysis to ATP turnover at lower temperatures indicates a stronger glycolytic phenotype with decreasing storage temperature. More broadly, these results demonstrate that the temperature dependence of the PLT metabolic network is not uniform, suggesting that efforts to improve the health of stored PLTs could be targeted at specific pathways.

AKT1 phosphorylation of cytoplasmic ME2 induces a metabolic switch to glycolysis for tumorigenesis

Many types of tumors feature aerobic glycolysis for meeting their increased energetic and biosynthetic demands. However, it remains still unclear how this glycolytic phenomenon is achieved and coordinated with other metabolic pathways in tumor cells in response to growth stimuli. Here we report that activation of AKT1 induces a metabolic switch to glycolysis from the mitochondrial metabolism via phosphorylation of cytoplasmic malic enzyme 2 (ME2), named ME2fl (fl means full length), favoring an enhanced glycolytic phenotype. Mechanistically, in the cytoplasm, AKT1 phosphorylates ME2fl at serine 9 in the mitochondrial localization signal peptide at the N-terminus, preventing its mitochondrial translocation. Unlike mitochondrial ME2, which accounts for adjusting the tricarboxylic acid (TCA) cycle, ME2fl functions as a scaffold that brings together the key glycolytic enzymes phosphofructokinase (PFKL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as Lactate dehydrogenase A (LDHA), to promote glycolysis in the cytosol. Thus, through phosphorylation of ME2fl, AKT1 enhances the glycolytic capacity of tumor cells in vitro and in vivo, revealing an unexpected role for subcellular translocation switching of ME2 mediated by AKT1 in the metabolic adaptation of tumor cells to growth stimuli.

MEK1/2-PKM2 Pathway Modulates the Immunometabolic Reprogramming of Proinflammatory Allograft-infiltrating Macrophages During Heart Transplant Rejection.

Emerging evidence has highlighted the role of macrophages in heart transplant rejection (HTR). However, the molecular signals modulating the immunometabolic phenotype of allograft-infiltrating macrophages (AIMs) during HTR remain unknown. We analyzed single-cell RNA sequencing data from cardiac graft-infiltrating immunocytes to characterize the activation patterns and metabolic features of AIMs. We used flow cytometry to determine iNOS and PKM2 expression and MEK/ERK signaling activation levels in AIMs. We then generated macrophage-specific Mek1/2 knockout mice to determine the role of the MEK1/2-PKM2 pathway in the proinflammatory phenotype and glycolytic capacity of AIMs during HTR. Single-cell RNA sequencing analysis showed that AIMs had a significantly elevated proinflammatory and glycolytic phenotype. Flow cytometry analysis verified that iNOS and PKM2 expressions were significantly upregulated in AIMs. Moreover, MEK/ERK signaling was activated in AIMs and positively correlated with proinflammatory and glycolytic signatures. Macrophage-specific Mek1/2 deletion significantly protected chronic cardiac allograft rejection and inhibited the proinflammatory phenotype and glycolytic capacity of AIMs. Mek1/2 ablation also reduced the proinflammatory phenotype and glycolytic capacity of lipopolysaccharides + interferon-γ-stimulated macrophages. Mek1/2 ablation impaired nuclear translocation and PKM2 expression in macrophages. PKM2 overexpression partially restored the proinflammatory phenotype and glycolytic capacity of Mek1/2 -deficient macrophages. Moreover, trametinib, an Food and Drug Administration-approved MEK1/2 inhibitor, ameliorated chronic cardiac allograft rejection. These findings suggest that the MEK1/2-PKM2 pathway is essential for immunometabolic reprogramming of proinflammatory AIMs, implying that it may be a promising therapeutic target in clinical heart transplantation.

Exploring the Role of Desmoplastic Physical Stroma in Pancreatic Cancer Progression Using a Three-Dimensional Collagen Matrix Model.

Pancreatic ductal adenocarcinoma (PDAC) is a refractory tumor with a poor prognosis, and its complex microenvironment is characterized by a fibrous interstitial matrix surrounding PDAC cells. Type I collagen is a major component of this interstitial matrix. Abundant type I collagen promotes its deposition and cross-linking to form a rigid and dense physical barrier, which limits drug penetration and immune cell infiltration and provides drug resistance and metabolic adaptations. In this study, to identify the physical effect of the stroma, type I collagen was used as a 3D matrix to culture Capan-1 cells and generate a 3D PDAC model. Using transcriptome analysis, a link between type I collagen-induced physical effects and the promotion of Capan-1 cell proliferation and migration was determined. Moreover, metabolomic analysis revealed that the physical effect caused a shift in metabolism toward a glycolytic phenotype. In particular, the high expression of proline in the metabolites suggests the ability to maintain Capan-1 cell proliferation under hypoxic and nutrient-depleted conditions. In conclusion, we identified type I collagen-induced physical effects in promoting Capan-1 cells, which cause PDAC progression, providing support for the role of dense stroma in the PDAC microenvironment and identifying a fundamental method for modeling the complex PDAC microenvironment.

Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing 13C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

Epigenetic and Metabolic Reprogramming of Fibroblasts in Crohn's Disease Strictures Reveals Histone Deacetylases as Therapeutic Targets.

No effective therapeutic intervention exists for intestinal fibrosis in Crohn's disease [CD]. We characterized fibroblast subtypes, epigenetic and metabolic changes, and signalling pathways in CD fibrosis to inform future therapeutic strategies. We undertook immunohistochemistry, metabolic, signalling pathway and epigenetic [Transposase-Accessible Chromatin using sequencing] analyses associated with collagen production in CCD-18Co intestinal fibroblasts and primary fibroblasts isolated from stricturing [SCD] and non-stricturing [NSCD] CD small intestine. SCD/NSCD fibroblasts were cultured with TGFβ and valproic acid [VPA]. Stricturing CD was characterized by distinct histone deacetylase [HDAC] expression profiles, particularly HDAC1, HDAC2, and HDAC7. As a proxy for HDAC activity, reduced numbers of H3K27ac+ cells were found in SCD compared to NSCD sections. Primary fibroblasts had increased extracellular lactate [increased glycolytic activity] and intracellular hydroxyproline [increased collagen production] in SCD compared to NSCD cultures. The metabolic effect of TGFβ stimulation was reversed by the HDAC inhibitor VPA. SCD fibroblasts appeared 'metabolically primed' and responded more strongly to both TGFβ and VPA. Treatment with VPA revealed TGFβ-dependent and TGFβ-independent Collagen-I production in CCD-18Co cells and primary fibroblasts. VPA altered the epigenetic landscape with reduced chromatin accessibility at the COL1A1 and COL1A2 promoters. Increased HDAC expression profiles, H3K27ac hypoacetylation, a significant glycolytic phenotype and metabolic priming characterize SCD-derived as compared to NSCD fibroblasts. Our results reveal a novel epigenetic component to Collagen-I regulation and TGFβ-mediated CD fibrosis. HDAC inhibitor therapy may 'reset' the epigenetic changes associated with fibrosis.

LIPH contributes to glycolytic phenotype in pancreatic ductal adenocarcinoma by activating LPA/LPAR axis and maintaining ALDOA stability

BackgroundLIPH, a membrane-associated phosphatidic acid-selective phospholipase A1a, can produce LPA (Lysophosphatidic acid) from PA (Phosphatidic acid) on the outer leaflet of the plasma membrane. It is well known that LIPH dysfunction contributes to lipid metabolism disorder. Previous study shows that LIPH was found to be a potential gene related to poor prognosis with pancreatic ductal adenocarcinoma (PDAC). However, the biological functions of LIPH in PDAC remain unclear.MethodsCell viability assays were used to evaluate whether LIPH affected cell proliferation. RNA sequencing and immunoprecipitation showed that LIPH participates in tumor glycolysis by stimulating LPA/LPAR axis and maintaining aldolase A (ALDOA) stability in the cytosol. Subcutaneous, orthotopic xenograft models and patient-derived xenograft PDAC model were used to evaluate a newly developed Gemcitabine-based therapy.ResultsLIPH was significantly upregulated in PDAC and was related to later pathological stage and poor prognosis. LIPH downregulation in PDAC cells inhibited colony formation and proliferation. Mechanistically, LIPH triggered PI3K/AKT/HIF1A signaling via LPA/LPAR axis. LIPH also promoted glycolysis and de novo synthesis of glycerolipids by maintaining ALDOA stability in the cytosol. Xenograft models show that PDAC with high LIPH expression levels was sensitive to gemcitabine/ki16425/aldometanib therapy without causing discernible side effects.ConclusionLIPH directly bridges PDAC cells and tumor microenvironment to facilitate aberrant aerobic glycolysis via activating LPA/LPAR axis and maintaining ALDOA stability, which provides an actionable gemcitabine-based combination therapy with limited side effects.Graphical

Enhancing Immune-Mediated Radiotherapy Efficacy in Murine Models of Head and Neck Cancer through Peroxisome Proliferator-Activated Receptor Modulation

The tumor microenvironment (TME) in HPV-unrelated head and neck cancer (HNC) is considered to be immunologically "cold," and immunometabolic adaptations to radiotherapy (RT) can exacerbate these conditions and lead to radioresistance. Peroxisome proliferator-activated receptor-α (PPARα) agonism may improve the immune response to RT by reducing glucose scarcity through upregulation of fatty acid utilization in cancer cells. Our clinical trial data shows that high serum oleic acid (OA), a PPAR ligand, correlates with response to radioimmunotherapy. Orthotopic buccal tumor implantations of MOC2 and LY2 HNC cell lines were performed in C57BL/6 and BALB/c mice, respectively. Mice were placed on a high OA diet (38% kcal from OA) 3 weeks prior to tumor implantation. Control diet consisted of 1% kcal from OA. In vitro RT was performed to characterize the metabolic effects on the cancer cell lines. For in vivo studies, tumors were irradiated upon reaching 100 mm^3 with three 8 Gy fractions, given every 4-5 days. The PPARα agonist fenofibrate (FF, 100mg/kg/day) was given via oral gavage or intraperitoneal injection starting 5 days after RT. Tumor volume and overall survival were measured as objective outcomes, and flow cytometry was used to characterize the immune landscape of the tumor, blood, and lymph nodes. Mass spectrometry was employed for bulk proteomics and metabolomics on serum samples, and stable isotope labeling was performed on CD8 T cells from treated mice ex vivo. Extracellular flux analyses were performed on tumor cells and CD8 T cells to determine metabolic phenotypic changes. In vitro cytotoxicity was met for MOC2 and LY2 cancer cells in 100 uM FF and OA in comparison to solvent (p<0.01). Extracellular flux analyses show that RT upregulates the glycolytic phenotype of MOC2 and LY2 cancer cells; and that FF and/or OA decrease the glycolytic capacity of the cancer cells. Ex vivo extracellular flux analyses showed increased glycolytic capacity of CD8 T cells in mice treated with FF in comparison to RT alone. RT in combination with FF significantly improved tumor volume response in comparison to RT alone (mean 99.4 vs 182.7 mm^3, p = 0.001) and was comparable to combination radioimmunotherapy (with anti-PDL1 therapy; mean 99.4 vs. 132.3 mm^3). Unexpectedly, OA negated this improvement in efficacy in both MOC2 and LY2 tumor models; mice on the high OA diet had significantly higher average body weight (p<0.01). Significant changes in adaptive immune response were observed within the TME with the addition of FF. This was mirrored by changes in serum metabolomics. PPARα agonism improved the efficacy of RT in murine HNC models and could be considered in future translational clinical trial design.

The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: implications for retinal ischemic injury

The enzyme arginase 1 (A1) hydrolyzes the amino acid arginine to form L-ornithine and urea. Ornithine is further converted to polyamines by the ornithine decarboxylase (ODC) enzyme. We previously reported that deletion of myeloid A1 in mice exacerbates retinal damage after ischemia/reperfusion (IR) injury. Furthermore, treatment with A1 protects against retinal IR injury in wild-type mice. PEG-A1 also mitigates the exaggerated inflammatory response of A1 knockout (KO) macrophages in vitro. Here, we sought to identify the anti-inflammatory pathway that confers macrophage A1-mediated protection against retinal IR injury. Acute elevation of intraocular pressure was used to induce retinal IR injury in mice. A multiplex cytokine assay revealed a marked increase in the inflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in the retina at day 5 after IR injury. In vitro, blocking the A1/ODC pathway augmented IL-1β and TNF-α production in stimulated macrophages. Furthermore, A1 treatment attenuated the stimulated macrophage metabolic switch to a pro-inflammatory glycolytic phenotype, whereas A1 deletion had the opposite effect. Screening for histone deacetylases (HDACs) which play a role in macrophage inflammatory response showed that A1 deletion or ODC inhibition increased the expression of HDAC3. We further showed the involvement of HDAC3 in the upregulation of TNF-α but not IL-1β in stimulated macrophages deficient in the A1/ODC pathway. Investigating HDAC3 KO macrophages showed a reduced inflammatory response and a less glycolytic phenotype upon stimulation. In vivo, HDAC3 co-localized with microglia/macrophages at day 2 after IR in WT retinas and was further increased in A1-deficient retinas. Collectively, our data provide initial evidence that A1 exerts its anti-inflammatory effect in macrophages via ODC-mediated suppression of HDAC3 and IL-1β. Collectively we propose that interventions that augment the A1/ODC pathway and inhibit HDAC3 may confer therapeutic benefits for the treatment of retinal ischemic diseases.

Cholesterol reprograms glucose and lipid metabolism to promote proliferation in colon cancer cells

Hypercholesterolemia is often correlated with obesity which is considered a risk factor for various cancers. With the growing population of hypercholesterolemic individuals, there is a need to understand the role of increased circulatory cholesterol or dietary cholesterol intake towards cancer etiology and pathology. Recently, abnormality in the blood cholesterol level of colon cancer patients has been reported. In the present study, we demonstrate that alteration in cholesterol levels (through a high-cholesterol or high-fat diet) increases the incidence of chemical carcinogen-induced colon polyp occurrence and tumor progression in mice. At the cellular level, low-density lipoprotein cholesterol (LDLc) and high-density lipoprotein cholesterol (HDLc) promote colon cancer cell proliferation by tuning the cellular glucose and lipid metabolism. Mechanistically, supplementation of LDLc or HDLc promotes cellular glucose uptake, and utilization, thereby, causing an increase in lactate production by colon cancer cells. Moreover, LDLc or HDLc upregulates aerobic glycolysis, causing an increase in total ATP production through glycolysis, and a decrease in ATP generation by OXPHOS. Interestingly, the shift in the metabolic status towards a more glycolytic phenotype upon the availability of cholesterol supports rapid cell proliferation. Additionally, an alteration in the expression of the molecules involved in cholesterol uptake along with the increase in lipid and cholesterol accumulation was observed in cells supplemented with LDLc or HDLc. These results indicate that colon cancer cells directly utilize the cholesterol associated with LDLc or HDLc. Moreover, targeting glucose metabolism through LDH inhibitor (oxamate) drastically abrogates the cellular proliferation induced by LDLc or HDLc. Collectively, we illustrate the vital role of cholesterol in regulating the cellular glucose and lipid metabolism of cancer cells and its direct effect on the colon tumorigenesis.Graphical

P02.04.B TARGETING METABOLIC VULNERABILITIES IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma is the most common and aggressive primary brain cancer, with a median survival of 16 months. A subset of cells within these tumours, termed glioblastoma stem cells (GSCs), displays high tumourigenic capacity and resistance to conventional therapies, and are responsible for tumour recurrence. Glioblastomas have been initially shown to mainly rely on glycolysis as a source of energy. However, a growing body of literature reports that GSCs are less glycolytic than their differentiated progeny and can thrive on oxidative phosphorylation (OXPHOS) to survive, sustain stemness and fuel tumourigenic potential. Thus, targeting OXPHOS could be a promising strategy to halt energy production and starve GSCs to death. However, under metabolic stress, GSCs can also switch to a more glycolytic phenotype, suggesting a metabolic plasticity in GSCs, and thus adding a layer of complexity to target GSC metabolism. In the current study, we have investigated the impact of compound X (CX), recently shown to impair mitochondrial respiration, on the fate and tumourigenesis of GSCs. MATERIAL AND METHODS Multiple patient-derived and murine GSC models were used to test the impact of CX on GSCs in vitro. The effect of CX on glioblastoma tumors was evaluated using patient-derived xenografts and syngeneic mouse models. RESULTS We have established that CX inhibits GSC proliferation and impairs stemness-related pathways in vitro. CX delays glioblastoma tumourigenesis in vivo. While a drastic decrease in cell proliferation was observed, cell death assays revealed that CX does not induce cell death. This suggests that GSCs gain a slow-cycling phenotype to resist energy depletion. Mechanistically, we have established that CX targets complex I of the mitochondrial respiratory chain, leading to an impairment of OXPHOS. Interestingly, inhibition of OXPHOS by CX was associated with an extensive production of extracellular lactate, indicating that at least, a subset of GSCs gain metabolic adaptability by co-opting glycolysis to survive. Thus, to tackle this metabolic plasticity in GSCs, we adopted a new strategy that involves sequestration of the lactic acid intracellularly via disruption of lactate transporters. Treatment of GSCs with monocarboxylate transporter inhibitors, AZD3965 and syrosingopine, alone did not significantly impact GSC growth and viability. Strikingly, combined CX treatment with either AZD3965 or syrosingopine resulted in high intracellular lactate buildup, increased reactive oxygen species, and led to drastic cell lethality in multiple GSC models. CONCLUSION This study highlights that re-routing the OXPHOS-dependent GSCs to glycolysis offers a vulnerability to sensitize GSCs to cell death. Dual inhibition of OXPHOS and lactate export could be a promising therapeutic approach to deplete malignant GSCs and suppress glioblastoma tumourigenesis.

Metabolic reprogramming and polarization of microglia in Parkinson's disease: Role of inflammasome and iron.

Parkinson's disease (PD) is a slowly progressive neurodegenerative disease characterized by α-synuclein aggregation and dopaminergic neuronal death. Recent evidence suggests that neuroinflammation is an early event in the pathogenesis of PD. Microglia are resident immune cells in the central nervous system that can be activated into either pro-inflammatory M1 or anti-inflammatory M2 phenotypes as found in peripheral macrophages. To exert their immune functions, microglia respond to various stimuli, resulting in the flexible regulation of their metabolic pathways. Inflammasomes activation in microglia induces metabolic shift from oxidative phosphorylation to glycolysis, and leads to the polarization of microglia to pro-inflammatory M1 phenotype, finally causing neuroinflammation and neurodegeneration. In addition, iron accumulation induces microglia take an inflammatory and glycolytic phenotype. M2 phenotype microglia is more sensitive to ferroptosis, inhibition of which can attenuate neuroinflammation. Therefore, this review highlights the interplay between microglial polarization and metabolic reprogramming of microglia. Moreover, it will interpret how inflammasomes and iron regulate microglial metabolism and phenotypic shifts, which provides a promising therapeutic target to modulate neuroinflammation and neurodegeneration in PD and other neurodegenerative diseases.

The fate of mitochondria during platelet activation

AbstractBlood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam–scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

Hypoxia induces a glycolytic complex in intestinal epithelial cells independent of HIF-1-driven glycolytic gene expression

The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic adenosine triphosphate (ATP) production, an event with consequences for cellular bioenergetics and cell fate. This response is regulated at the transcriptional level by the hypoxia-inducible factor-1(HIF-1)-dependent transcriptional upregulation of glycolytic enzymes (GEs) and glucose transporters. However, this transcriptional upregulation alone is unlikely to account fully for the levels of glycolytic ATP produced during hypoxia. Here, we investigated additional mechanisms regulating glycolysis in hypoxia. We observed that intestinal epithelial cells treated with inhibitors of transcription or translation and human platelets (which lack nuclei and the capacity for canonical transcriptional activity) maintained the capacity for hypoxia-induced glycolysis, a finding which suggests the involvement of a nontranscriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. In our investigations into potential nontranscriptional mechanisms for glycolytic induction, we identified a hypoxia-sensitive formation of complexes comprising GEs and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of such glycolytic complexes occurs independent of HIF-1-driven transcription. Finally, we provide evidence for the presence of HIF-1α in cytosolic fractions of hypoxic cells which physically interacts with the glucose transporter GLUT1 and the GEs in a hypoxia-sensitive manner. In conclusion, we provide insights into the nontranscriptional regulation of hypoxia-induced glycolysis in intestinal epithelial cells.

Metabolic Oscillations and Glycolytic Phenotypes of Cancer Cells.

Cancer cells show several metabolic phenotypes depending on the cancer types and the microenvironments in tumor tissues. The glycolytic phenotype is one of the hallmarks of cancer cells and is considered to be one of the crucial features of malignant cancers. Here, we show glycolytic oscillations in the concentrations of metabolites in the glycolytic pathway in two types of cancer cells, HeLa cervical cancer cells and DU145 prostate cancer cells, and in two types of cellular morphologies, spheroids and monolayers. Autofluorescence from nicotinamide adenine dinucleotide (NADH) in cells was used for monitoring the glycolytic oscillations at the single-cell level. The frequencies of NADH oscillations were different among the cellular types and morphologies, indicating that more glycolytic cancer cells tended to exhibit oscillations with higher frequencies than less glycolytic cells. A mathematical model for glycolytic oscillations in cancer cells reproduced the experimental results quantitatively, confirming that the higher frequencies of oscillations were due to the higher activities of glycolytic enzymes. Thus, glycolytic oscillations are expected as a medical indicator to evaluate the malignancy of cancer cells with glycolytic phenotypes.

The Mitochondrial ATP Synthase/IF1 Axis in Cancer Progression: Targets for Therapeutic Intervention.

Cancer poses a significant global health problem with profound personal and economic implications on National Health Care Systems. The reprograming of metabolism is a major trait of the cancer phenotype with a clear potential for developing effective therapeutic strategies to combat the disease. Herein, we summarize the relevant role that the mitochondrial ATP synthase and its physiological inhibitor, ATPase Inhibitory Factor 1 (IF1), play in metabolic reprogramming to an enhanced glycolytic phenotype. We stress that the interplay in the ATP synthase/IF1 axis has additional functional roles in signaling mitohormetic programs, pro-oncogenic or anti-metastatic phenotypes depending on the cell type. Moreover, the same axis also participates in cell death resistance of cancer cells by restrained mitochondrial permeability transition pore opening. We emphasize the relevance of the different post-transcriptional mechanisms that regulate the specific expression and activity of ATP synthase/IF1, to stimulate further investigations in the field because of their potential as future targets to treat cancer. In addition, we review recent findings stressing that mitochondria metabolism is the primary altered target in lung adenocarcinomas and that the ATP synthase/IF1 axis of OXPHOS is included in the most significant signature of metastatic disease. Finally, we stress that targeting mitochondrial OXPHOS in pre-clinical mouse models affords a most effective therapeutic strategy in cancer treatment.

Heterogeneity of Glycolytic Phenotype Determined by 18F-FDG PET/CT Using Coefficient of Variation in Patients with Advanced Non-Small Cell Lung Cancer.

We investigated the role of Coefficient of Variation (CoV), a first-order texture parameter derived from 18F-FDG PET/CT, in the prognosis of Non-Small Cell Lung Cancer (NSCLC) patients. Eighty-four patients with advanced NSCLC who underwent 18F-FDG PET/CT before therapy were retrospectively studied. SUVmax, SUVmean, CoV, total Metabolic Tumor Volume (MTVTOT) and whole-body Total Lesion Glycolysis (TLGWB) were determined by an automated contouring program (SUV threshold at 2.5). We analyzed 194 lesions: primary tumors (n = 84), regional (n = 48) and non-regional (n = 17) lymph nodes and metastases in liver (n = 9), bone (n = 23) and other sites (n = 13); average CoVs were 0.36 ± 0.13, 0.36 ± 0.14, 0.42 ± 0.18, 0.30 ± 0.14, 0.37 ± 0.17, 0.34 ± 0.13, respectively. No significant differences were found between the CoV values among the different lesion categories. Survival analysis included age, gender, histology, stage, MTVTOT, TLGWB and imaging parameters derived from primary tumors. At univariate analysis, CoV (p = 0.0184), MTVTOT (p = 0.0050), TLGWB (p = 0.0108) and stage (p = 0.0041) predicted Overall Survival (OS). At multivariate analysis, age, CoV, MTVTOT and stage were retained in the model (p = 0.0001). Patients with CoV > 0.38 had significantly better OS than those with CoV ≤ 0.38 (p = 0.0143). Patients with MTVTOT ≤ 89.5 mL had higher OS than those with MTVTOT > 89.5 mL (p = 0.0063). Combining CoV and MTVTOT, patients with CoV ≤ 0.38 and MTVTOT > 89.5 mL had the worst prognosis. CoV, by reflecting the heterogeneity of glycolytic phenotype, can predict clinical outcomes in NSCLC patients.

MiR-494 induces metabolic changes through G6pc targeting and modulates sorafenib response in hepatocellular carcinoma

BackgroundMetabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker.MethodsBioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats.ResultsMiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells.ConclusionsMiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy.

Colorectal polyps increase the glycolytic activity.

In colorectal cancer (CRC) energy metabolism research, the precancerous stage of polyp has remained rather unexplored. By now, it has been shown that CRC has not fully obtained the glycolytic phenotype proposed by O. Warburg and rather depends on mitochondrial respiration. However, the pattern of metabolic adaptations during tumorigenesis is still unknown. Understanding the interplay between genetic and metabolic changes that initiate tumor development could provide biomarkers for diagnosing cancer early and targets for new cancer therapeutics. We used human CRC and polyp tissue material and performed high-resolution respirometry and qRT-PCR to detect changes on molecular and functional level with the goal of generally describing metabolic reprogramming during CRC development. Colon polyps were found to have a more glycolytic bioenergetic phenotype than tumors and normal tissues. This was supported by a greater GLUT1, HK, LDHA, and MCT expression. Despite the increased glycolytic activity, cells in polyps were still able to maintain a highly functional OXPHOS system. The mechanisms of OXPHOS regulation and the preferred substrates are currently unclear and would require further investigation. During polyp formation, intracellular energy transfer pathways become rearranged mainly by increasing the expression of mitochondrial adenylate kinase (AK) and creatine kinase (CK) isoforms. Decreased glycolysis and maintenance of OXPHOS activity, together with the downregulation of the CK system and the most common AK isoforms (AK1 and AK2), seem to play a relevant role in CRC development.

Lactate-induced lactylation circularly regulates glucose metabolism enzymes in breast cancer.

e13092 Background: Breast cancer is the most common malignant tumor in the female population worldwide and has become the disease with the highest cancer-related mortality. Among the molecular subtypes of breast cancer, triple-negative breast cancer accounts for only 15% of primary malignancies, but due to its increasing recurrence, metastasis, and mortality, we need to find new therapeutic targets to address this phenomenon. Analysis from the field of metabolism found that the level of glycolysis in triple-negative breast cancer was significantly higher than that in other cancers. Glycolysis is a common malignant phenotype of cancer, namely the famous Warburg effect. LDHA is the last metabolic enzyme in the glycolytic pathway, and its expression is increased in triple-negative breast cancer. Lactate is the final product of glycolysis, which converts pyruvate to lactate and NADH to NAD+ under the catalysis of LDHA. The acidic tumor microenvironment formed by the accumulation of lactate will not only affect the progression of tumors and the therapeutic effect of anticancer drugs, but also inhibit the function of immune cells. Lactic acid groups can bind to histones and non-histone proteins to produce a new post-translational modification, lactylation. Methods: Breast cancer cells were infected with lentivirus vectors overexpressing or knocking down LDHA, and cell proliferation and cytotoxicity assay, transwell assay and Lactic Acid Content Assay were performed. To further explore the presence of lactylation of LDHA, immunoprecipitation experiments were performed and the modification sites were mutated to examine the enzymatic activity and function of LDHA. Results: In triple negative breast cancer with strong glycolytic metabolic phenotype, altering LDHA level can affect the biological function of breast cancer cells. Secondly, RNA-seq sequencing results showed that LDHA can affect the protein and transcription levels of MEST and CSTA. Finally, the lactylation of histones was also significantly changed after LDHA dysregulated. Reduced levels of LDHA were accompanied by reduced levels of MEST, CSTA, and histone lactylation. In addition, LDHA itself also has lactic acid modification, and mutations in the modification sites can change the enzymatic activity and function of LDHA. Conclusions: The regulatory axis of LDHA-lactate-lactylation has important value and function in the occurrence and development of breast cancer. LDHA regulates the function of some genes by lactate-induced lactylation of histones. In addition, the lactylation of LDHA can alter the enzyme activity, which may be a new therapeutic target for breast cancer.