Lactate Dehydrogenase A Expression Research Articles

Regulating the Warburg effect on metabolic stress and myocardial fibrosis remodeling and atrial intracardiac waveform activity induced by atrial fibrillation

Atrial fibrillation (AF) is associated with metabolic stress and induces myocardial fibrosis reconstruction by increasing glycolysis. One goal in the treatment of paroxysmal AF (p-AF) is to improve myocardial fibrosis reconstruction and myocardial metabolic stress caused by the Warburg effect. Adopted male canine that rapid right atrial pacing (RAP) for 6 days to establish a p-AF model. The canines were pre-treated with phenylephrine (PE) or dichloroacetic acid (DCA) before exposure to p-AF or non-p-AF. P-wave duration (Pmax), minimum P-wave duration (Pmin), P wave dispersion (PWD), atrial effective refractory period (AERP) and AERP dispersion (AERPd) were measured in canine atrial cardiomyocytes. Pyruvate dehydrogenase kinase-1 (PDK-1), PDK-4, lactate dehydrogenase A (LDHA), pyruvate dehydrogenase (PDH), citrate synthase (CS), isocitrate dehydrogenase (IDH), and matrix metalloproteinase 9 (MMP-9) were evaluated by western blotting and reverse transcription polymerase chain reaction (RT-PCR), content of adenosine monophosphate (AMP), adenosine triphosphate (ATP), lactic acid and glycogen, and activity of LDHA, PDK-1 and PDK-4 were evaluated by enzyme-linked immunosorbent assay (ELISA), myocardial tissue glycogen content was evaluated by PAS, myocardial fibrosis remodeling was evaluated by hematoxylin and eosin (H&E) and Masson staining. Our findings demonstrated that p-AF increases the Warburg effect-related metabolic stress and myocardial fibrosis remodeling by increasing the expression and activity of PDK-1, PDK-4, and LDHA, content of AMP and lactic acid, and the ratio of AMP/ATP and decreasing the expression of PDH, CS, and IDH, and glycogen content. In addition, p-AF can induce cardiomyocyte fibrosis remodeling and increase MMP-9 expression, and p-AF also increases atrial intracardiac waveform activity by prolonging Pmax, Pmin, PWD, and AERPd and shortening AERP. PDK isoforms agonists (PE) produce a similar p-AF pathological effect and can produce synergistic effects with p-AF, further increasing Warburg effect-related metabolic stress, myocardial fibrosis remodeling, and atrial intracardiac waveform activity. In contrast, the use of PDK-specific inhibitors (DCA) completely reverses these pathophysiological changes induced by p-AF. We demonstrate that p-AF can induce the Warburg effect in canine atrial cardiomyocytes and significantly improve p-AF-induced metabolic stress, myocardial fibrosis remodeling, and atrial intracardiac waveform activity by inhibiting the Warburg effect.

Metabolic adaptation to glycolysis is a basic defense mechanism of macrophages for Mycobacterium tuberculosis infection.

Macrophages are major components of tuberculosis (TB) granulomas and are responsible for host defenses against the intracellular pathogen, Mycobacterium tuberculosis. We herein showed the strong expression of hypoxia-inducible factor-1α (HIF-1α) in TB granulomas and more rapid death of HIF-1α-conditional knockout mice than wild-type (WT) mice after M. tuberculosis infection. Although interferon-γ (IFN-γ) is a critical host-protective cytokine against intracellular pathogens, HIF-1-deficient macrophages permitted M. tuberculosis growth even after activation with IFN-γ. These results prompted us to investigate the role of HIF-1α in host defenses against infection. We found that the expression of lactate dehydrogenase-A (LDH-A) was controlled by HIF-1α in M. tuberculosis-infected macrophages IFN-γ independently. LDH-A is an enzyme that converts pyruvate to lactate and we found that the intracellular level of pyruvate in HIF-1α-deficient bone marrow-derived macrophages (BMDMs) was significantly higher than in WT BMDMs. Intracellular bacillus replication was enhanced by an increase in intracellular pyruvate concentrations, which were decreased by LDH-A. Mycobacteria in phagosomes took up exogenous pyruvate more efficiently than glucose, and used it as the feasible carbon source for intracellular growth. These results demonstrate that HIF-1α prevents the hijacking of pyruvate in macrophages, making it a fundamental host-protective mechanism against M. tuberculosis.

MicroRNA 34b inhibits cell proliferation in pediatric acute myeloid leukemia via regulating LDHA.

To elucidate the regulatory effect of microRNA-34b on the occurrence of pediatric acute myeloid leukemia and the underlying mechanism. The expression of microRNA-34b in the bone marrow of 72 children with newly diagnosed acute myeloid leukemia (AML) was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between microRNA-34b expression and pathological characteristics was analyzed. Kaplan-Meier curve was introduced for evaluating the prognostic value of microRNA-34b in pediatric AML. The regulatory effects of microRNA-34b on proliferation, cell cycle, and apoptosis of leukemia cells were accessed by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Bioinformatics prediction and dual-luciferase reporter gene assay were conducted to evaluate the binding between microRNA-34b and lactate dehydrogenase A (LDHA). LDHA expression after overexpression of microRNA-34b was determined by qRT-PCR and Western blot. Rescue experiments were conducted to verify whether microRNA-34b could regulate proliferative and apoptotic behaviors of leukemia cells by suppressing LDHA expression. MicroRNA-34b was markedly downregulated in AML children. Low expression of microRNA-34b was correlated to FAB typing, cytogenetic abnormality, and day 7 response to the treatment of pediatric AML. By collecting the follow-up data, it was found that low expression of microRNA-34b was correlated to the poor prognosis of AML. Overexpression of microRNA-34b inhibited proliferative ability and cell cycle progression, but accelerated apoptosis of AML cells. Dual-luciferase reporter gene assay verified that microRNA-34b could bind to LDHA, thereafter inhibiting LDHA expression. Overexpression of LDHA reversed the regulatory effects of microRNA-34b on proliferation, cell cycle, and apoptosis of AML cells. We found that microRNA-34b is lowly expressed in pediatric AML patients, and low expression of microRNA-34b may serve as an indicator of malignant progression and poor prognosis of pediatric AML. MicroRNA-34b may affect the proliferation and apoptosis of leukemia cells by regulating the expression of LDHA.

A Novel Lactate Dehydrogenase Inhibitor, 1-(Phenylseleno)-4-(Trifluoromethyl) Benzene, Suppresses Tumor Growth through Apoptotic Cell Death

The Warburg effect, wherein cancer cells prefer glycolysis rather than oxidative phosphorylation even under normoxic conditions, is a major characteristic of malignant tumors. Lactate dehydrogenase A (LDHA) is the main enzyme regulating the Warburg effect, and is thus, a major target for novel anti-cancer drug development. Through our ongoing screening of novel inhibitors, we found that several selenobenzene compounds have inhibitory effects on LDHA activity. Among them, 1-(phenylseleno)-4-(trifluoromethyl) benzene (PSTMB) had the most potent inhibitory effect on the enzymatic activity of LDHA. The results from biochemical assays and computational modeling showed that PSTMB inhibited LDHA activity. In addition, PSTMB inhibited the growth of several tumor cell lines, including NCI-H460, MCF-7, Hep3B, A375, HT29, and LLC. In HT29 human colon cancer cells, PSTMB dose-dependently inhibited the viability of the cells and activity of LDHA, without affecting the expression of LDHA. Under both normoxic and hypoxic conditions, PSTMB effectively reduced LDHA activity and lactate production. Furthermore, PSTMB induced mitochondria-mediated apoptosis of HT29 cells via production of reactive oxygen species. These results suggest that PSTMB may be a novel candidate for development of anti-cancer drugs by targeting cancer metabolism.

Response of AMP-activated protein kinase and lactate metabolism of largemouth bass (Micropterus salmoides) under acute hypoxic stress

To study adaptation of largemouth bass (Micropterus salmoides) to hypoxic stress, we investigated physiological responses and lactate metabolism of the fish under acute hypoxia. The objectives of this study were to (a) observe changes in glucose, glycogen, and lactate content; (b) detect the activity of lactate dehydrogenase (LDH) in serum, brain, heart, and liver tissues; and (c) quantify the dynamic gene expression of AMP activated protein kinase alpha (AMPKα), hypoxia-inducible factor-1 alpha (HIF-1α), monocarboxylate transporter 1 (MCT1), monocarboxylate transporter 4 (MCT4), and lactate dehydrogenase-a (LDHa) following exposure to hypoxia. The fish were subjected to two hypoxia stresses (dissolved oxygen [DO] 1.20 ± 0.2 mg/L and 3.50 ± 0.3 mg/L, respectively) for 24 h. Our results showed that hypoxic stress significantly increased the decomposition of liver glycogen and significantly increased the concentration of blood glucose; however, the muscle glycogen content was not significantly decreased, which indicates that liver glycogen was the main energy source under acute hypoxia. Moreover, hypoxia led to accumulation of a large amount of lactic acid in tissues, possibly due to the activity of lactic acid dehydrogenase, but this process was delayed in the heart and brain relative to the liver. Additionally, hypoxia induced the expression of AMPKα, HIF-1α, MCT1, MCT4, and LDHa, suggesting that glycometabolism had switched from aerobic to anaerobic. Our results contribute to a better understanding of the molecular mechanisms of the response to hypoxia in largemouth bass.

Lamc1 promotes the Warburg effect in hepatocellular carcinoma cells by regulating PKM2 expression through AKT pathway

ABSTRACTHepatocellular carcinoma (HCC), the most common aggressive malignancy of liver, is the third leading cause of cancer death across the world. Laminin gamma 1 (Lamc1), encodes laminin-γ1, an extracellular matrix protein involved in various progresses such as tumor cell proliferation and metabolism. In the present study, high expression of Lamc1 and PKM2 was observed in tumor tissues of HCC patients. In vitro, down-regulation of Lamc1 inhibited proliferation of HCC cells by promoting cell death, reduced glucose consumption and lactate production, accompanied by a decrease in the expression of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA), and PTEN increased, as well as PTEN S380 and AKT S473/T308 phosphorylation decreased, while Lamc1 up-regulation had the opposite effect. The effects of PKM2 were similar to that of Lamc1 and markedly counteracted the effects of Lamc1 down-regulation. In addition, Lamc1-induced increase in PKM2 expression was strongly attenuated by a PI3K inhibitor, LY294002 or a si-p110 PI3K, with a significant decrease in GLUT1 and LDHA expression, as well as decreased AKT T308 phosphorylation. Thus, we speculated that Lamc1 was implicated in the progression of HCC probably by regulating PKM2 expression through PTEN/AKT pathway.

Abstract A089: The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors

Abstract Objective: Determine whether LDH-A knockdown enhances CAR T-cell tumor-targeting and treatment response.Background: It has been shown that T-cells are restricted from entering many human and murine solid tumors, including both murine and human prostate tumors. CD3(+) T-cells are restricted from Myc-CaP murine prostate tumors and localize around the periphery of the tumor nodules (1). Human prostate specific membrane antigen (hPSMA) targeted CAR T-cells are also restricted from hPSMA(+) Myc-CaP tumors and this is only partially reversed by anti-PD1 treatment (1). This report describes the benefit of LDH-A depletion and a greater response of CAR T-cells targeting Myc-CaP tumors with low expression of LDH-A. Methods: LDH-A depletion in hPSMA(+) Myc-CaP cells bearing a bioluminescence reporter (Renilla Luciferase), was achieved by shRNA knockdown (KD) (1). A scrambled IgG shRNA was used as a control (NC). LDH-A expression was quantified by digital droplet PCR (ddPCR), Western blotting and LDH enzyme activity. The glycolytic activity of KD and NC cells was measured using Seahorse XF96 and XFp analyzers. Intratumoral lactate levels were monitored by magnetic resonance spectroscopy (MRS). The preparation and characterization of “second generation” hPSMA-directed CAR T-cells and the hPSMA(+) Myc-CaP tumor models in SCID mice have been described (1). To monitor CAR T-cell trafficking, T-cells were transduced with a Click Beetle Red luciferase reporter to enable efficient visualization by bioluminescence imaging (BLI) of CAR T-cell trafficking, persistence and viability within the hPSMA(+)Myc-CaP tumor mass. Tumor volume was calculated from caliper measurements. CD31 and PD-L1 expression was quantified with immunofluorescent staining and Metamorph Offline image analysis. Results and Discussion: These studies demonstrated that LDH-A KD was the dominant factor in reducing tumor growth. The difference in tumor doubling time (DT) between KD and NC tumors in the presence of CAR T-cell therapy was significant (p<0.0001): DT= 5.0±0.3 vs 2.9±0.5 days, respectively. A significant difference was also found in the absence of CAR T-cell therapy. Although LDH-A KD produced the larger single-treatment effect, the addition of CAR T-cells was additive. To explain these observations and study the effect of LDH-A KD on tumor metabolism and microenvironment, Seahorse and MRS experiments were performed. LDH-A depletion (KD) resulted in: lower rates of basal (NC: 24.0±1.9 vs KD: 6.9±1.9 pmol H+/min/µg protein; p<0.0001) and compensatory glycolysis (NC: 43.0±2.3 vs KD: 18.3±3.9 pmol H+/min/µg protein, p<0.0001), and lactate production. In contrast, basal and maximal respiration increased following LDH-A KD (NC: 4.0±0.8 vs KD: 8.3±0.6, p=0.0011, and NC: 6.7±1.4 vs KD: 13.8±0.6 pmol O2/min/µg protein, p=0.0003, respectively). However, total tumor lactate, measured in vivo by MRS, showed only marginal differences between KD and NC tumors. Nevertheless, there were significant differences in CAR T-cell trafficking-to and persistence-in KD compared to NC tumors, and the increase in tumor doubling time was directly related to the CAR T-cell expansion ratio. Interestingly, tumor vascularity (CD31), but not PD-L1, was significantly lower in KD compared to NC tumors. Necrosis was minimal in both KD and NC tumors, with or without CAR T-cell treatment. Conclusions: LDHA knockdown (KD) in the Myc-CaP murine prostate tumor model has a significant effect on tumor cell metabolism resulting in reduced glycolysis, reduced lactate production and increased oxidative phosphorylation that impacts the tumor microenvironment. The LDH-A KD induced changes in tumor metabolism and the microenvironment diminish Myc-CaP tumor growth rate and impact CAR T-cell trafficking and persistence in the tumor. Future studies will compare the effects of anti-PD1 and anti-CTLA4 therapy in combination with LDH-A knockdown. Reference: 1. Serganova I, et al. Molecular Therapy: Oncolytics 2017;4:41. Citation Format: Mayuresh M. Mane, Khalid Shalaby, Ivan Cohen, Avi Albeg, Jenny Ijoma, Myat Ko, Masatomo Maeda, Kiranmayi Vemuri, Jaya Satagopan, Anna Moroz, Juan Zurita, Larissa Shenker, Ellen Ackerstaff, Masahiro Shindo, Ekaterina Moroz, Maxim A. Moroz, Inna Serganova, Jason Koutcher, Vladimir Ponomarev, Ronald G. Blasberg. The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A089.

P53/Lactate dehydrogenase A axis negatively regulates aerobic glycolysis and tumor progression in breast cancer expressing wild-type p53.

Tumor suppressor p53 is a master regulator of apoptosis and plays key roles in cell cycle checkpoints. p53 responds to metabolic changes and alters metabolism through several mechanisms in cancer. Lactate dehydrogenase A (LDHA), a key enzyme in glycolysis, is highly expressed in a variety of tumors and catalyzes pyruvate to lactate. In the present study, we first analyzed the association and clinical significance of p53 and LDHA in breast cancer expressing wild‐type p53 (wt‐p53) and found that LDHA mRNA levels are negatively correlated with wt‐p53 but not with mutation p53 mRNA levels, and low p53 and high LDHA expression are significantly associated with poor overall survival rates. Furthermore, p53 negatively regulates LDHA expression by directly binding its promoter region. Moreover, a series of LDHA gain‐of‐function and rescore experiments were carried out in breast cancer MCF7 cells expressing endogenous wt‐p53, showing that ectopic expression of p53 decreases aerobic glycolysis, cell proliferation, migration, invasion and tumor formation of breast cancer cells and that restoration of the expression of LDHA in p53‐overexpressing cells could abolish the suppressive effect of p53 on aerobic glycolysis and other malignant phenotypes. In conclusion, our findings showed that repression of LDHA induced by wt‐p53 blocks tumor growth and invasion through downregulation of aerobic glycolysis in breast cancer, providing new insights into the mechanism by which p53 contributes to the development and progression of breast cancer.

Combined Aberrant Expression of NDRG2 and LDHA Predicts Hepatocellular Carcinoma Prognosis and Mediates the Anti-tumor Effect of Gemcitabine.

The Warburg effect is one of the important hallmarks of cancer. The activation of oncogene and inactivation of tumor suppressor gene contribute to the enhancement of glycolytic enzymes and the Warburg effect. The N-myc downstream regulated gene 2 (NDRG2) is a tumor suppressor gene and is frequently lost in various types of cancer. However, little is known about glycolytic function and therapeutic value of NDRG2 in hepatocellular carcinoma (HCC). In this study, we found that NDRG2 and lactate dehydrogenase A (LDHA) were aberrantly expressed in HCC and were closely related to the Warburg effect. The correlation between NDRG2 and LDHA expression predicted HCC prognosis and the clinical response to chemotherapy. NDRG2 expression was significantly decreased while LDHA expression was increased in HCC specimens. NDRG2 and LDHA expression was significantly correlated with differentiation status, vascular invasion, and TNM stage of HCC. NDRG2 inhibited LDHA expression, the Warburg effect and the growth of HCC cells. Furthermore, NDRG2 mediated gemcitabine-induced inhibition of LDHA expression and the Warburg effect in HCC cells. Taken together, our data suggest that NDRG2 plays an important role in inhibiting the Warburg effect and the malignant growth of HCC via LDHA. NDRG2 combined with LDHA might be powerful prognostic biomarkers and targets for chemotherapy treatment of HCC.

C-Myc-Induced Long Non-Coding RNA Small Nucleolar RNA Host Gene 7 Regulates Glycolysis in Breast Cancer.

PurposeRecent studies have shown that long non-coding RNA (lncRNA) play an important role in cancer metabolism and development. The lncRNA small nucleolar RNA host gene 7 (SNHG7) was reported to be upregulated in colorectal cancer and contribute to its progression. In the current study, we investigated the role of lncRNA-SNHG7 in breast cancer and explored the underlying mechanism.MethodsWe monitored the expression of lncRNA-SNHG7 in breast cancer tissues and breast cancer cell lines. We evaluated the effects of lncRNA-SNHG7 on cell proliferation and glycolysis in breast cancer cells by knocking down or overexpressing lncRNA-SNHG7. We searched for the potential microRNA (miRNA) target of lncRNA-SNHG7 and evaluated the effects of the target miRNA on glycolysis. We evaluated the potential regulation of lncRNA-SNHG7 by c-Myc.ResultsLncRNA-SNHG7 was up-regulated in both breast cancer tissues and breast cancer cell lines. Knocking down lncRNA-SNHG7 inhibited breast cancer cell proliferation while overexpressing lncRNA-SNHG7 enhanced cell proliferation. Knocking down lncRNA-SNHG7 resulted in decreased expression of lactate dehydrogenase A (LDHA) and decreased glycolysis. LncRNA-SNHG7 targeted miR-34a-5p to regulate LDHA expression and glycolysis. c-Myc bound to promoter of lncRNA-SNHG7 and positively regulated lncRNA-SNHG7 expression.ConclusionWe demonstrated that c-Myc regulated glycolysis through the lncRNA-SNHG7/miR-34a-5p/LDHA axis in breast cancer cells.

LDHA in Neuroblastoma Is Associated with Poor Outcome and Its Depletion Decreases Neuroblastoma Growth Independent of Aerobic Glycolysis.

Purpose: To investigate whether lactate dehydrogenase A (LDHA), an important component of the LDH tetramer crucial for aerobic glycolysis, is associated with patient outcome and constitutes a therapeutic target in neuroblastoma (NB).Experimental Design: Expression of LDHA mRNA and protein was determined in 709 and 110 NB patient samples, respectively, and correlated with survival and risk factors. LDHA and LDHB were depleted in human NB cell lines by CRISPR/Cas9 and shRNA, respectively, and aerobic glycolysis, clonogenicity, and tumorigenicity were determined. Expression of LDHA in relation to MYCN was measured in NB cell lines and in the TH-MYCN NB mouse model.Results: Expression of LDHA, both on the mRNA and the protein level, was significantly and independently associated with decreased patient survival. Predominant cytoplasmic localization of LDHA protein was associated with poor outcome. Amplification and expression of MYCN did not correlate with expression of LDHA in NB cell lines or TH-MYCN mice, respectively. Knockout of LDHA inhibited clonogenicity, tumorigenicity, and tumor growth without abolishing LDH activity or significantly decreasing aerobic glycolysis. Concomitant depletion of LDHA and the isoform LDHB ablated clonogenicity while not abrogating LDH activity or decreasing aerobic glycolysis. The isoform LDHC was not expressed.Conclusions: High expression of LDHA is independently associated with outcome of NB, and NB cells can be inhibited by depletion of LDHA or LDHB. This inhibition appears to be unrelated to LDH activity and aerobic glycolysis. Thus, investigations of inhibitory mechanisms beyond attenuation of aerobic glycolysis are warranted, both in NB and normal cells. Clin Cancer Res; 24(22); 5772-83. ©2018 AACR.

Downregulation of miR-33b promotes non-small cell lung cancer cell growth through reprogramming glucose metabolism miR-33b regulates non-small cell lung cancer cell growth.

Glucose metabolism is a common target for cancer regulation and microRNAs (miRNAs) are important regulators of this process. Here we aim to investigate a tumor‐suppressing miRNA, miR‐33b, in regulating the glucose metabolism of non‐small cell lung cancer (NSCLC). In our study, quantitative real‐time polymerase chain reaction (qRT‐PCR) showed that miR‐33b was downregulated in NSCLC tissues and cell lines, which was correlated with increased cell proliferation and colony formation. Overexpression of miR‐33b through miR‐33b mimics transfection suppressed NSCLC proliferation, colony formation, and induced cell‐cycle arrest and apoptosis. Meanwhile, miR‐33b overexpression inhibited glucose metabolism in NSCLC cells. Luciferase reporter assay confirmed that miR‐33b directly binds to the 3′‐untranslated region of lactate dehydrogenase A (LDHA). qRT‐PCR and Western blot analysis showed that miR‐33b downregulated the expression of LDHA. Moreover, introducing LDHA mRNA into cells over‐expressing miR‐33b attenuated the inhibitory effect of miR‐33b on the growth and glucose metabolism in NSCLC cells. Taken together, these results confirm that miR‐33b is an anti‐oncogenic miRNA, which inhibits NSCLC cell growth by targeting LDHA through reprogramming glucose metabolism.

Iron Depletion Affects Genes Encoding Mitochondrial Electron Transport Chain and Genes of Non-Oxidative Metabolism, Pyruvate Kinase and Lactate Dehydrogenase, in Primary Human Cardiac Myocytes Cultured upon Mechanical Stretch.

(1) Background: Oxidative energy metabolism is presumed to rely on the optimal iron supply. Primary human cardiac myocytes (HCM) exposed to different iron availability conditions during mechanical stretch are anticipated to demonstrate expression changes of genes involved in aerobic and anaerobic metabolic pathways. (2) Methods: HCM were cultured for 48 h either in static conditions and upon mechanical stretch at the optimal versus reduced versus increased iron concentrations. We analyzed the expression of pyruvate kinase (PKM2), lactate dehydrogenase A (LDHA), and mitochondrial complexes I–V at the mRNA and protein levels. The concentration of l-lactate was assessed by means of lactate oxidase method-based kit. (3) Results: Reduced iron concentrations during mechanical work caused a decreased expression of complexes I–V (all p < 0.05). The expression of PKM2 and LDHA, as well as the medium concentration of l-lactate, was increased in these conditions (both p < 0.05). HCM exposed to the increased iron concentration during mechanical effort demonstrated a decreased expression of mitochondrial complexes (all p < 0.01); however, a decrement was smaller than in case of iron chelation (p < 0.05). The iron-enriched medium caused a decrease in expression of LDHA and did not influence the concentration of l-lactate. (4) Conclusions: During mechanical effort, the reduced iron availability enhances anaerobic glycolysis and extracellular lactate production, whilst decreasing mitochondrial aerobic pathway in HCM. Iron enrichment during mechanical effort may be protective in the context of intracellular protein machinery of non-oxidative metabolism with no effect on the extracellular lactate concentration.

Double genetic disruption of lactate dehydrogenases A and B is required to ablate the “Warburg effect” restricting tumor growth to oxidative metabolism

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.

Targeting the Warburg effect via LDHA inhibition engages ATF4 signaling for cancer cell survival.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

Non-Invasive Assessment of Lactate Production and Compartmentalization in Renal Cell Carcinomas Using Hyperpolarized 13C Pyruvate MRI.

Optimal treatment selection for localized renal tumors is challenging due to their variable biological behavior and limited ability to pre-operatively assess their aggressiveness. We investigated hyperpolarized (HP) 13C pyruvate MRI to noninvasively assess tumor lactate production and compartmentalization, which are strongly associated with renal tumor aggressiveness. Orthotopic tumors were created in mice using human renal cell carcinoma (RCC) lines (A498, 786-O, UOK262) with varying expression of lactate dehydrogenase A (LDHA) which catalyzes the pyruvate-to-lactate conversion, and varying expression of monocarboxylate transporter 4 (MCT4) which mediates lactate export out of the cells. Dynamic HP 13C pyruvate MRI showed that the A498 tumors had significantly higher 13C pyruvate-to-lactate conversion than the UOK262 and 786-O tumors, corresponding to higher A498 tumor LDHA expression. Additionally, diffusion-weighted HP 13C pyruvate MRI showed that the A498 tumors had significantly higher 13C lactate apparent diffusion coefficients compared to 786-O tumors, with corresponding higher MCT4 expression, which likely reflects more rapid lactate export in the A498 tumors. Our data demonstrate the feasibility of HP 13C pyruvate MRI to inform on tumor lactate production and compartmentalization, and provide the scientific premise for future clinical investigation into the utility of this technique to noninvasively interrogate renal tumor aggressiveness and to guide treatment selection.

The Downregulation of miR-200c Promotes Lactate Dehydrogenase A Expression and Non-Small Cell Lung Cancer Progression.

This study was aimed to investigate the function and mechanism of microRNA-200c (miR-200c) in the progression of non-small cell lung cancer (NSCLC). A total of 76 patients diagnosed as having NSCLC were enrolled in this study. The expression level of miR-200c in NSCLC tissues and cell lines was investigated using the quantitative real-time polymerase chain reaction (RT-qPCR) method. We found that the expression of miR-200c was significantly reduced in NSCLC tissues and cell lines compared with normal lung tissues and the human bronchial epithelial cell line. Overexpression of miR-200c using the miR-200c mimic significantly suppressed cell proliferation and migration of NSCLC cell lines. The results of the luciferase reporter assay identified lactate dehydrogenase A (LDHA) as a direct target of miR-200c. The expression of LDHA was shown to be suppressed in NSCLC cell lines with miR-200c mimic transfection. Furthermore, the transfection of small interfering RNA (siRNA) targeting LDHA suppressed the proliferation and migration of NSCLC cell lines. In summary, our results presented in this study suggested that miR-200c was able to inhibit the proliferation and migration of NSCLC cells by downregulating LDHA. Therefore, miR-200c may be considered as a potential candidate for the treatment of NSCLC.

MiR-323a-3p suppressed the glycolysis of osteosarcoma via targeting LDHA.

Accumulating evidence has demonstrated that there is critical involvement of miRNAs in the initiation and progression of cancers. Here, we showed that miR-323a-3p was significantly down-regulated in osteosarcoma (OS) tissues and cell lines. Overexpression of miR-323a-3p decreased the cell viability, colon formation and induced the apoptosis of OS cells. Using bioinformatics analysis, lactate dehydrogenase A (LDHA) was predicted as one of the down-steam targets of miR-323a-3p. Highly expressed miR-323a-3p significantly decreased both the mRNA and protein levels of LDHA. Inverse correlation between the expression of LDHA and miR-323a-3p was observed in OS tissues. Consistent with the function of LDHA in glycolysis of cancer cells, overexpression of miR-323a-3p attenuated the lactate production of OS cells. These results demonstrated that miR-323a-3p suppressed the growth of OS cells via targeting LDHA and inhibited the glycolysis of OS. This study provides insight into the molecular mechanism of miR-323a-3p in regulating OS.

MiR‑199a‑3p/Sp1/LDHA axis controls aerobic glycolysis in testicular tumor cells.

Aerobic glycolysis is one of the characteristics of tumor metabolism and contributes to the development of tumors. Studies have identified that microRNA (miRNA/miR) serves an important role in glucose metabolism of tumors. miR‑199a‑3p is a member of the miR‑199a family that controls the outcomes of cell survival and death processes, and previous studies have indicated that the expression of miR‑199a‑3p is low and may be an inhibitor in several cancer types, including testicular tumors. The present study discussed the role and underlying mechanism of miR‑199a‑3p in aerobic glycolysis of Ntera‑2 cells and identified its downstream factors. Firstly, miR‑199a‑3p exhibited an inhibitory effect on lactic acid production, glucose intake, and reactive oxygen species (ROS) and adenosine 5'‑triphosphate (ATP) levels in Ntera‑2 cells. Then, using bioinformatics, recombinant construction and a dual luciferase reporter gene system, transcription factor Specificity protein 1 (Sp1) was determined as the direct target of miR‑199a‑3p. Also, downregulation of Sp1 by RNA interference decreased lactic acid production, glucose intake, and ROS and ATP levels in Ntera‑2 cells. Subsequently, through a functional rescue experiment, it was identified that the overexpression of Sp1 may abate the inhibition of miR‑199a‑3p on glucose metabolism, with the exception of ATP level, suggesting a reciprocal association between Sp1 and miR‑199a‑3p. Finally, it was determined that miR‑199a‑3p overexpression and Sp1 knockdown decreased lactate dehydrogenaseA (LDHA) protein expression, which indicated that LDHA is a downstream target of the miR‑199a‑3p/Sp1 signaling pathway. To additionally verify the regulation of LDHA expression by 199a‑3p/Sp1, a LDHA promoter reporter plasmid was generated and the high activity of the promoter, which contained 3 potential Sp1 binding elements, was confirmed. In addition, the overexpression of Sp1 led to the increased activity of the LDHA promoter, whereas knockdown of Sp1 exhibited the opposite effect. Therefore, the results of the present study demonstrated that miR‑199a‑3p can inhibit LDHA expression by downregulating Sp1, and provided mechanistic evidence supporting the existence of a novel miR‑199a‑3p/Sp1/LDHA axis and its critical contribution to aerobic glycolysis in testicular cancer cells.

Abstract 4110: Metabolic imaging of glioblastoma using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model

Abstract OBJECTIVES: Since the cancer stem-like cells (CSCs) play an important role in the resistance against radiation and/or chemotherapy due to its different metabolic profile, to investigate the specific metabolic characteristics of CSCs is valuable. Dynamic nuclear polarization (DNP) technique using 13C-labeled substrates enables magnetic resonance imaging (MRI) to monitor specific enzymatic reactions in tumors. In this study, DNP-MRI along with hyperpolarized [1-13C] pyruvate was conducted to evaluate the difference in glycolytic profile between a glioblastoma cell line and a CSC-derived glioma using orthotopic xenograft mouse model. The imaging findings were compared to those in immunohistology and cell cultures. METHODS: A 3T MRI scanner along with a custom-made head coil was used for imaging. To obtain hyperpolarized [1-13C] pyruvate, 30 μL of [1-13C] pyruvic acid containing 15 mM of OX063 (trityl radical compound) and 2.5 mM of the gadolinium chelate were polarized at 3.35 T and 1.4 K in the Hypersense DNP polarizer. The hyperpolarized sample dissolved in a buffered medium was injected via the tail vein followed by a 13C-two-dimensional spectroscopic imaging. During the acquisition, the body temperature was kept in the range of 35.0 to 36.9 Celsius degrees. Orthotopic brain tumor model was developed in nude mice by intracranial implantation of glioblastoma cell lines, U251 and glioma stem-like cells, NSC11. DNP-MRI was performed when the tumor size ranged 50 + 10 mm3 on T2-weighted MRI. Independently, immunofluorescent analyses of monocarboxylate transporter 1(MCT1) and MCT4 was performed using paraffin-embedded tumor tissue. Expressed protein level of lactate dehydrogenase A (LDHA), LDHB, MCT1 and MCT4 under the normal cell culture conditions were evaluated by Western blotting. RESULTS: 13C-MRI showed an increase in lactate-to-pyruvate ratio (Lac/Pyr) both in U251 and NSC11 compared to the contralateral normal brain tissue, suggested elevated anaerobic glycolysis (P&amp;lt;0.05). The extent of the increase in Lac/Pyr compared to the normal brain tissue was higher in U251 than NSC11 except for one U251 case. Western blotting showed both LDHA and LHDB expression in U251 was lower than NSC11. The expression of MCT4 was higher in U251 than NSC11, whereas they were almost the same for MCT1. Immunohistochemistry showed relative weak staining for MCT4 in NSC11, consistent with the result of Western blotting. CONCLUSION: The quality of 13C-DNP-MRI using [1-13C] pyruvate was validated and considered to be feasible to apply to the brain tumors. The result was reasonable in that malignant tumors enhance anaerobic glycolytic pathway even in the aerobic environment. 13C-DNP-MRI also demonstrated a difference in the extent of Lac/Pyr in relation to the normal brain between the two tumor models probably related to expression levels of glycolysis-related proteins. Citation Format: Tatsuya Kawai, Murali C. Krishna, Jeffery R. Brender, Jennifer Lee, Tamalee Kramp, Kazu Yamamoto, Shun Kishimoto, Tomohiro Seki, Kevin A. Camphausen. Metabolic imaging of glioblastoma using hyperpolarized 13C-MRI: Glycolytic metabolism in cancer stem cell-like cell-derived tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4110.