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Purpose: Hyperuricemia (HUA) is an important factor leading to chronic kidney disease (CKD). The kidney tubular inflammatory response is activated in HUA. This study aimed to investigate whether lactate dehydrogenase A (LDHA) is involved in mediating uric acid-induced kidney tubular inflammatory response., Methods: In vivo, an HUA mouse model was established by continuous intraperitoneal injection of potassium oxonate (PO) for one week. A total of 18 C57BL/6J male adult mice were divided into three groups: control group, HUA group, and HUA+oxamate group, with six mice in each group. Oxamate was intraperitoneally injected into the mice one hour after PO injection. In vitro, an HUA model was simulated by stimulating HK-2 cells with uric acid. Oxamate and tempol inhibited LDHA and reactive oxygen species (ROS) in HK-2 cells., Results: In HUA mice, blood uric acid levels were significantly elevated. LDHA in kidney tubular cells was significantly increased in both in vivo and in vitro HUA models, accompanied by an increase in kidney tubular inflammation and ROS. Mechanistically, LDHA mediates uric acid-induced inflammation to kidney tubular cells through the ROS/NLRP3 pathway. Pharmacologic inhibition of LDHA or ROS in kidney tubular cells can significantly ameliorate inflammation response caused by uric acid., Conclusions: LDHA in kidney tubular cells significantly was increased in HUA models. LDHA mediates kidney inflammation response induced by uric acid through the ROS/NLRP3 pathway. This study may provide a new intervention target for preventing kidney tubular inflammation caused by uric acid.

Osteoarthritis (OA) is a worldwide joint disease, leading to the physical pain, stiffness, and even disability. Lactate dehydrogenase A (LDHA) is known as a lactylation mediator that can regulate histone lactylation of its target genes. However, the role of LDHA-mediated histone H3 lysine 18 lactylation (H3K18la) in OA progression is yet to be clarified. Our study aims at revealing the role and mechanism of LDHA-mediated histone lactylation in the glycolysis of chondrocytes. In this study, we determined at first that the H3K18la level was enhanced in OA. Energy metabolism such as glycolysis is often altered in OA progress. Therefore, we further explored the mechanism mediating glycolysis and thus promoting OA progress. Moreover, glycolysis was enhanced in LPS-induced OA cell model, as evidenced by the increased glucose consumption and lactate production. Furthermore, we silenced LDHA for loss-of-function assays. The results showed that knockdown of LDHA suppressed glycolysis of LPS-induced chondrocytes. In vivo animal study demonstrated that knockout of LDHA recovered cartilage injury of OA mice. Mechanistically, we uncovered that LDHA-mediated H3K18la in TPI1 promoter enhanced the transcription activity of TPI1. Mutation of K69 site was found to ameliorate LPS-induced glycolysis in OA cell model. In conclusion, our study reveals the role of LDHA-mediated H3K18la of TPI1 promoter in OA progress.

Lactate dehydrogenase A (LDHA) is a key enzyme in Warburg's effect, a characteristic of cancer cells. LDHA is a target of anticancer agents that inhibit the metabolism of cancer cells. Gossypol is a known cancer therapeutic agent that inhibits LDHA by competitive inhibition. However, the mechanisms of inhibition of LDHA by gossypol is unknown. Here, we elucidate the binding of gossypol and LDHA using biochemical and biophysical methods. The crystal structure of the complex between LDHA and gossypol is presented. The binding of gossypol affects LDHA activity by a conformational change in the active-site loop. Our research contributes to the structural insight into LDHA with gossypol and approaches gossypol as a novel therapeutic candidate targeting the metabolic pathways for cancer cells., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Current cancer immunotherapy predominately focuses on eliciting type 1 immune responses fighting cancer; however, long-term complete remission remains uncommon 1,2 . A pivotal question arises as to whether type 2 immunity can be orchestrated alongside type 1-centric immunotherapy to achieve enduring response against cancer 3,4 . Here we show that an interleukin-4 fusion protein (Fc-IL-4), a typical type 2 cytokine, directly acts on CD8 + T cells and enriches functional terminally exhausted CD8 + T (CD8 + T TE ) cells in the tumour. Consequently, Fc-IL-4 enhances antitumour efficacy of type 1 immunity-centric adoptive T cell transfer or immune checkpoint blockade therapies and induces durable remission across several syngeneic and xenograft tumour models. Mechanistically, we discovered that Fc-IL-4 signals through both signal transducer and activator of transcription 6 (STAT6) and mammalian target of rapamycin (mTOR) pathways, augmenting the glycolytic metabolism and the nicotinamide adenine dinucleotide (NAD) concentration of CD8 + T TE cells in a lactate dehydrogenase A-dependent manner. The metabolic modulation mediated by Fc-IL-4 is indispensable for reinvigorating intratumoural CD8 + T TE cells. These findings underscore Fc-IL-4 as a potent type 2 cytokine-based immunotherapy that synergizes effectively with type 1 immunity to elicit long-lasting responses against cancer. Our study not only sheds light on the synergy between these two types of immune responses, but also unveils an innovative strategy for advancing next-generation cancer immunotherapy by integrating type 2 immune factors., (© 2024. The Author(s).)

Lactate dehydrogenase A (LDHA) is highly expressed in various tumors. However, the role of LDHA in the pathogenesis of B-cell lymphoma remains unclear. Analysis of data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases revealed an elevated LDHA expression in diffuse large B-cell lymphoma (DLBC) tissues compared with normal tissues. Similarly, our results demonstrated a significant increase in LDHA expression in tumor tissues from the patients with B-cell lymphoma compared with those with lymphadenitis. To further elucidate potential roles of LDHA in B-cell lymphoma pathogenesis, we silenced LDHA in the Raji cells (a B-cell lymphoma cell line) using shRNA techniques. Silencing LDHA led to reduced mitochondrial membrane integrity, adenosine triphosphate (ATP) production, glycolytic activity, cell viability and invasion. Notably, LDHA knockdown substantially suppressed in vivo growth of Raji cells and extended survival in mice bearing lymphoma (Raji cells). Moreover, proteomic analysis identified feline sarcoma-related protein (FER) as a differential protein positively associated with LDHA expression. Treatment with E260, a FER inhibitor, significantly reduced the metabolism, proliferation and invasion of Raji cells. In summary, our findings highlight that LDHA plays multiple roles in B-cell lymphoma pathogenesis via FER pathways, establishing LDHA/FER may as a potential therapeutic target., (© 2024 International Union of Biochemistry and Molecular Biology.)

Regulatory cell death is an important way to eliminate the DNA damage that accompanies the rapid proliferation of neural stem cells during cortical development, including pyroptosis, apoptosis, and so on. Here, the study reports that the absence of GSDMD-mediated pyroptosis results in defective DNA damage sensor pathways accompanied by aberrant neurogenesis and autism-like behaviors in adult mice. Furthermore, GSDMD is involved in organizing the mitochondrial electron transport chain by regulating the AMPK/PGC-1α pathway to target Aifm3. This process promotes a switch from oxidative phosphorylation to glycolysis. The perturbation of metabolic homeostasis in neural progenitor cells increases lactate production which acts as a signaling molecule to regulate the p38MAPK pathway. And activates NF-𝜿B transcription to disrupt cortex development. This abnormal proliferation of neural progenitor cells can be rescued by inhibiting glycolysis and lactate production. Taken together, the study proposes a metabolic axis regulated by GSDMD that links pyroptosis with metabolic reprogramming. It provides a flexible perspective for the treatment of neurological disorders caused by genotoxic stress and neurodevelopmental disorders such as autism., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Tumor cells undergo uncontrolled proliferation driven by enhanced anabolic metabolism including glycolysis and glutaminolysis. Targeting these pathways to inhibit cancer growth is a strategy for cancer treatment. Critically, however, tumor-responsive T cells share metabolic features with cancer cells, making them susceptible to these treatments as well. Here, we assess the impact on anti-tumor T cell immunity and T cell exhaustion by genetic ablation of lactate dehydrogenase A (LDHA) and glutaminase1 (GLS1), key enzymes in aerobic glycolysis and glutaminolysis. Loss of LDHA severely impairs expansion of T cells in response to tumors and chronic infection. In contrast, T cells lacking GLS1 can compensate for impaired glutaminolysis by engaging alternative pathways, including upregulation of asparagine synthetase, and thus efficiently respond to tumor challenge and chronic infection as well as immune checkpoint blockade. Targeting GLS1-dependent glutaminolysis, but not aerobic glycolysis, may therefore be a successful strategy in cancer treatment, particularly in combination with immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: High levels of lactate are positively associated with prognosis and mortality in pulmonary hypertension (PH). Lactate dehydrogenase A (LDHA) is a key enzyme for the production of lactate. This study is undertaken to investigate the role and molecular mechanisms of lactate and LDHA in PH., Methods: Lactate levels were measured by a lactate assay kit. LDHA expression and localization were detected by western blot and Immunofluorescence. Proliferation and migration were determined by CCK8, western blot, EdU assay and scratch-wound assay. The right heart catheterization and right heart ultrasound were measured to evaluate cardiopulmonary function., Results: In vitro, we found that lactate promoted proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) in an LDHA-dependent manner. In vivo, we found that LDHA knockdown reduced lactate overaccumulation in the lungs of mice exposed to hypoxia. Furthermore, LDHA knockdown ameliorated hypoxia-induced vascular remodeling and right ventricular dysfunction. In addition, the activation of Akt signaling by hypoxia was suppressed by LDHA knockdown both in vivo and in vitro. The overexpression of Akt reversed the inhibitory effect of LDHA knockdown on proliferation in PASMCs under hypoxia. Finally, LDHA inhibitor attenuated vascular remodeling and right ventricular dysfunction in Sugen/hypoxia mouse PH model, Monocrotaline (MCT)-induced rat PH model and chronic hypoxia-induced mouse PH model., Conclusions: Thus, LDHA-mediated lactate production promotes pulmonary vascular remodeling in PH by activating Akt signaling pathway, suggesting the potential role of LDHA in regulating the metabolic reprogramming and vascular remodeling in PH., (© 2024. The Author(s).)

Podocytes are crucial for regulating glomerular permeability. They have foot processes that are integral to the renal filtration barrier. Understanding their energy metabolism could shed light on the pathogenesis of filtration barrier injury. Lactate has been increasingly recognized as more than a waste product and has emerged as a significant metabolic fuel and reserve. The recent identification of lactate transporters in podocytes, the expression of which is modulated by glucose levels and lactate, highlights lactate's relevance. The present study investigated the impact of lactate on podocyte respiratory efficiency and mitochondrial dynamics. We confirmed lactate oxidation in podocytes, suggesting its role in cellular energy production. Under conditions of glucose deprivation or lactate supplementation, a significant shift was seen toward oxidative phosphorylation, reflected by an increase in the oxygen consumption rate/extracellular acidification rate ratio. Notably, lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB) isoforms, which are involved in lactate conversion to pyruvate, were detected in podocytes for the first time. The presence of lactate led to higher intracellular pyruvate levels, greater LDH activity, and higher LDHB expression. Furthermore, lactate exposure increased mitochondrial DNA-to-nuclear DNA ratios and resulted in upregulation of the mitochondrial biogenesis markers peroxisome proliferator-activated receptor coactivator-1α and transcription factor A mitochondrial, regardless of glucose availability. Changes in mitochondrial size and shape were observed in lactate-exposed podocytes. These findings suggest that lactate is a pivotal energy source for podocytes, especially during energy fluctuations. Understanding lactate's role in podocyte metabolism could offer insights into renal function and pathologies that involve podocyte injury., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Oligodendrocytes and astrocytes are metabolically coupled to neuronal compartments. Pyruvate and lactate can shuttle between glial cells and axons via monocarboxylate transporters. However, lactate can only be synthesized or used in metabolic reactions with the help of lactate dehydrogenase (LDH), a tetramer of LDHA and LDHB subunits in varying compositions. Here we show that mice with a cell type-specific disruption of both Ldha and Ldhb genes in oligodendrocytes lack a pathological phenotype that would be indicative of oligodendroglial dysfunctions or lack of axonal metabolic support. Indeed, when combining immunohistochemical, electron microscopical, and in situ hybridization analyses in adult mice, we found that the vast majority of mature oligodendrocytes lack detectable expression of LDH. Even in neurodegenerative disease models and in mice under metabolic stress LDH was not increased. In contrast, at early development and in the remyelinating brain, LDHA was readily detectable in immature oligodendrocytes. Interestingly, by immunoelectron microscopy LDHA was particularly enriched at gap junctions formed between adjacent astrocytes and at junctions between astrocytes and oligodendrocytes. Our data suggest that oligodendrocytes metabolize lactate during development and remyelination. In contrast, for metabolic support of axons mature oligodendrocytes may export their own glycolysis products as pyruvate rather than lactate. Lacking LDH, these oligodendrocytes can also "funnel" lactate through their "myelinic" channels between gap junction-coupled astrocytes and axons without metabolizing it. We suggest a working model, in which the unequal cellular distribution of LDH in white matter tracts facilitates a rapid and efficient transport of glycolysis products among glial and axonal compartments., (© 2024 The Authors. GLIA published by Wiley Periodicals LLC.)

Cardiac fibrosis is a typical feature of cardiac pathological remodeling, which is associated with adverse clinical outcomes and has no effective therapy. Nicotine is an important risk factor for cardiac fibrosis, yet its underlying molecular mechanism remains poorly understood. This study aimed to identify its potential molecular mechanism in nicotine-induced cardiac fibrosis. Our results showed nicotine exposure led to the proliferation and transformation of cardiac fibroblasts (CFs) into myofibroblasts (MFs) by impairing autophagy flux. Through the use of drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) technology, it was discovered that nicotine directly increased the stability and protein levels of lactate dehydrogenase A (LDHA) by binding to it. Nicotine treatment impaired autophagy flux by regulating the AMPK/mTOR signaling pathway, impeding the nuclear translocation of transcription factor EB (TFEB), and reducing the activity of cathepsin B (CTSB). In vivo, nicotine treatment exacerbated cardiac fibrosis induced in spontaneously hypertensive rats (SHR) and worsened cardiac function. Interestingly, the absence of LDHA reversed these effects both in vitro and in vivo. Our study identified LDHA as a novel nicotine-binding protein that plays a crucial role in mediating cardiac fibrosis by blocking autophagy flux. The findings suggest that LDHA could potentially serve as a promising target for the treatment of cardiac fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Aim: Although lactate supplementation at the reperfusion stage of ischemic stroke has been shown to offer neuroprotection, whether the role of accumulated lactate at the ischemia phase is neuroprotection or not remains largely unknown. Thus, in this study, we aimed to investigate the roles and mechanisms of accumulated brain lactate at the ischemia stage in regulating brain injury of ischemic stroke. Methods and Results: Pharmacological inhibition of lactate production by either inhibiting LDHA or glycolysis markedly attenuated the mouse brain injury of ischemic stroke. In contrast, additional lactate supplement further aggravates brain injury, which may be closely related to the induction of neuronal death and A1 astrocytes. The contributing roles of increased lactate at the ischemic stage may be related to the promotive formation of protein lysine lactylation (Kla), while the post-treatment of lactate at the reperfusion stage did not influence the brain protein Kla levels with neuroprotection. Increased protein Kla levels were found mainly in neurons by the HPLC-MS/MS analysis and immunofluorescent staining. Then, pharmacological inhibition of lactate production or blocking the lactate shuttle to neurons showed markedly decreased protein Kla levels in the ischemic brains. Additionally, Ldha specific knockout in astrocytes ( Aldh1l1 CreERT2 ; Ldha fl/fl mice, cKO) mice with MCAO were constructed and the results showed that the protein Kla level was decreased accompanied by a decrease in the volume of cerebral infarction in cKO mice compared to the control groups. Furthermore, blocking the protein Kla formation by inhibiting the writer p300 with its antagonist A-485 significantly alleviates neuronal death and glial activation of cerebral ischemia with a reduction in the protein Kla level, resulting in extending reperfusion window and improving functional recovery for ischemic stroke. Conclusion: Collectively, increased brain lactate derived from astrocytes aggravates ischemic brain injury by promoting the protein Kla formation, suggesting that inhibiting lactate production or the formation of protein Kla at the ischemia stage presents new therapeutic targets for the treatment of ischemic stroke., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Breast cancer is the most prevalent malignancy and the most significant contributor to mortality in female oncology patients. Potassium Two Pore Domain Channel Subfamily K Member 1 (KCNK1) is differentially expressed in a variety of tumors, but the mechanism of its function in breast cancer is unknown. In this study, we found for the first time that KCNK1 was significantly up-regulated in human breast cancer and was correlated with poor prognosis in breast cancer patients. KCNK1 promoted breast cancer proliferation, invasion, and metastasis in vitro and vivo. Further studies unexpectedly revealed that KCNK1 increased the glycolysis and lactate production in breast cancer cells by binding to and activating lactate dehydrogenase A (LDHA), which promoted histones lysine lactylation to induce the expression of a series of downstream genes and LDHA itself. Notably, increased expression of LDHA served as a vicious positive feedback to reduce tumor cell stiffness and adhesion, which eventually resulted in the proliferation, invasion, and metastasis of breast cancer. In conclusion, our results suggest that KCNK1 may serve as a potential breast cancer biomarker, and deeper insight into the cancer-promoting mechanism of KCNK1 may uncover a novel therapeutic target for breast cancer treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The initiation of fibroblast growth factor 1 (FGF1) expression coincident with the decrease of FGF2 expression is a well-documented event in prostate cancer (PCa) progression. Lactate dehydrogenase A (LDHA) and LDHB are essential metabolic products that promote tumor growth. However, the relationship between FGF1/FGF2 and LDHA/B-mediated glycolysis in PCa progression is not reported. Thus, we aimed to explore whether FGF1/2 could regulate LDHA and LDHB to promote glycolysis and explored the involved signaling pathway in PCa progression., Methods: In vitro studies used RT‒qPCR, Western blot, CCK-8 assays, and flow cytometry to analyze gene and protein expression, cell viability, apoptosis, and cell cycle in PCa cell lines. Glycolysis was assessed by measuring glucose consumption, lactate production, and extracellular acidification rate (ECAR). For in vivo studies, a xenograft mouse model of PCa was established and treated with an FGF pathway inhibitor, and tumor growth was monitored., Results: FGF1, FGF2, and LDHA were expressed at high levels in PCa cells, while LDHB expression was low. FGF1/2 positively modulated LDHA and negatively modulated LDHB in PCa cells. The depletion of FGF1, FGF2, or LDHA reduced cell proliferation, induced cell cycle arrest, and inhibited glycolysis. LDHB overexpression showed similar inhibitory effect on PCa cells. Mechanistically, we found that FGF1/2 positively regulated STAT1 and STAT1 transcriptionally activated LDHA expression while suppressed LDHB expression. Furthermore, the treatment of an FGF pathway inhibitor suppressed PCa tumor growth in mice., Conclusion: The FGF pathway facilitates glycolysis by activating LDHA and suppressing LDHB in a STAT1-dependent manner in PCa., (© 2024. The Author(s).)

As one of the key metabolic enzymes in the glycolytic pathway, lactate dehydrogenase A (LDHA) might be linked to tumor proliferation by driving the Warburg effect. Circular RNAs (circRNAs) are widely implicated in tumor progression. Here, we report that circTATDN3, a circular RNA that interacts with LDHA, plays a critical role in proliferation and energy metabolism in CRC. We found that circTATDN3 expression was increased in CRC cells and tumor tissues and that high circTATDN3 expression was positively associated with poor postoperative prognosis in CRC patients. Additionally, circTATDN3 promoted the proliferation of CRC cells in vivo and vitro. Mechanistically, circTATDN3 was shown to function as an adaptor molecule that enhances the binding of LDHA to FGFR1, leading to increased LDHA phosphorylation and consequently promoting the Warburg effect. Moreover, circTATDN3 increased the expression of LDHA by sponging miR-511-5p, which synergistically promoted CRC progression and the Warburg effect. In conclusion, circTATDN3 may be a target for the treatment of CRC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Genotoxic therapy triggers reactive oxygen species (ROS) production and oxidative tissue injury. S-nitrosylation is a selective and reversible posttranslational modification of protein thiols by nitric oxide (NO), and 5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for NO synthesis. However, the mechanism by which BH4 affects protein S-nitrosylation and ROS generation has not been determined. Here, we showed that ionizing radiation disrupted the structural integrity of BH4 and downregulated GTP cyclohydrolase I (GCH1), which is the rate-limiting enzyme in BH4 biosynthesis, resulting in deficiency in overall protein S-nitrosylation. GCH1-mediated BH4 synthesis significantly reduced radiation-induced ROS production and fueled the global protein S-nitrosylation that was disrupted by radiation. Likewise, GCH1 overexpression or the administration of exogenous BH4 protected against radiation-induced oxidative injury in vitro and in vivo. Conditional pulmonary Gch1 knockout in mice (Gch1 fl/fl ; Sftpa1-Cre +/- mice) aggravated lung injury following irradiation, whereas Gch1 knock-in mice (Gch1 lsl/lsl ; Sftpa1-Cre +/- mice) exhibited attenuated radiation-induced pulmonary toxicity. Mechanistically, lactate dehydrogenase (LDHA) mediated ROS generation downstream of the BH4/NO axis, as determined by iodoacetyl tandem mass tag (iodoTMT)-based protein quantification. Notably, S-nitrosylation of LDHA at Cys163 and Cys293 was regulated by BH4 availability and could restrict ROS generation. The loss of S-nitrosylation in LDHA after irradiation increased radiosensitivity. Overall, the results of the present study showed that GCH1-mediated BH4 biosynthesis played a key role in the ROS cascade and radiosensitivity through LDHA S-nitrosylation, identifying novel therapeutic strategies for the treatment of radiation-induced lung injury., (© 2024. The Author(s).)

Lactate dehydrogenase A (LDHA) primarily catalyzes the conversion between lactic acid and pyruvate, serving as a key enzyme in the aerobic glycolysis pathway of sugar in tumor cells. LDHA plays a crucial role in the occurrence, development, progression, invasion, metastasis, angiogenesis, and immune escape of tumors. Consequently, LDHA not only serves as a biomarker for tumor diagnosis and prognosis but also represents an ideal target for tumor therapy. Although LDHA inhibitors show great therapeutic potential, their development has proven to be challenging. In the development of LDHA inhibitors, the key active sites of LDHA are emphasized. Nevertheless, there is a relative lack of research on the amino acid residues around the active center of LDHA. Therefore, in this study, we investigated the amino acid residues around the active center of LDHA. Through structure comparison analysis, five key amino acid residues (Ala30, Met41, Lys131, Gln233, and Ala259) were identified. Subsequently, the effects of these five residues on the enzymatic properties of LDHA were investigated using site-directed mutagenesis. The results revealed that the catalytic activities of the five mutants varied to different degrees in both the reaction from lactic acid to pyruvate and pyruvate to lactic acid. Notably, the catalytic activities of LDHA M41G and LDHA K131I were improved, particularly in the case of LDHA K131I . The results of the molecular dynamics analysis of LDHA K131I explained the reasons for this phenomenon. Additionally, the optimum temperature of LDHA M41G and LDHA Q233M increased from 35 °C to 40 °C, whereas in the reverse reaction, the optimum temperature of LDHA M41G and LDHA K131I decreased from 70 °C to 60 °C. These findings indicate that Ala30, Met41, Lys131, Gln233, and Ala259 exert diverse effects on the catalytic activity and optimum temperature of LHDA. Therefore, these amino acid residues, in addition to the key catalytic site of the active center, play a crucial role. Considering these residues in the design and screening of LDHA inhibitors may lead to the development of more effective inhibitors.

Lactate dehydrogenase A (LDHA) serves as a key regulator of the Warburg Effect by catalyzing the conversion of pyruvate to lactate in the final step of glycolysis. Both the expression level and enzyme activity of LDHA are upregulated in cancers, however, the underlying mechanism remains incompletely understood. Here, we show that LDHA is post-translationally palmitoylated by ZDHHC9 at cysteine 163, which promotes its enzyme activity, lactate production, and reduces reactive oxygen species (ROS) generation. Replacement of endogenous LDHA with a palmitoylation-deficient mutant leads to reduced pancreatic cancer cell proliferation, increased T-cell infiltration, and limited tumor growth; it also affects pancreatic cancer cell response to chemotherapy. Moreover, LDHA palmitoylation is upregulated in gemcitabine resistant pancreatic cancer cells. Clinically, ZDHHC9 is upregulated in pancreatic cancer and correlated with poor prognoses for patients. Overall, our findings identify ZDHHC9-mediated palmitoylation as a positive regulator of LDHA, with potentially significant implications for cancer etiology and targeted therapy for pancreatic cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

SET domain bifurcated 1 (SETDB1), a pivotal histone lysine methyltransferase, is transported to the cytoplasm via a chromosome region maintenance 1 (CMR1)‑dependent pathway, contributing to non‑histone methylation. However, the function and underlying mechanism of cytoplasmic SETDB1 in breast cancer remain elusive. In the present study, immunohistochemistry revealed that elevated cytoplasmic SETDB1 was correlated with lymph node metastasis and more aggressive breast cancer subtypes. Functionally, wound healing and Transwell assays showed that cytoplasmic SETDB1 is key for cell migration and invasion, as well as induction of epithelial‑mesenchymal transition (EMT), which was reversed by leptomycin B (LMB, a CMR1 inhibitor) treatment. Furthermore, RNA‑seq and metabolite detection revealed that cytoplasmic SETDB1 was associated with metabolism pathway and elevated levels of metabolites involved in the Warburg effect, including glucose, pyruvate, lactate and ATP. Immunoblotting and reverse transcription‑quantitative PCR verified that elevation of cytoplasmic SETDB1 contributed to elevation of c‑MYC expression and subsequent upregulation of lactate dehydrogenase A (LDHA) expression. Notably, gain‑ and loss‑of‑function approaches revealed that LDHA overexpression in T47D cells enhanced migration and invasion by inducing EMT, while its depletion in SETDB1‑overexpressing MCF7 cells reversed SETDB1‑induced migration and invasion, as well as the Warburg effect and EMT. In conclusion, subcellular localization of cytoplasmic SETDB1 may be a pivotal factor in breast cancer progression. The present study offers valuable insight into the novel functions and mechanisms of cytoplasmic SETDB1.

Metabolic reprogramming, a hallmark of cancer, is closely associated with tumor development and progression. Changes in glycolysis play a crucial role in conferring radiation resistance to tumor cells. How radiation changes the glycolysis status of cancer cells is still unclear. Here we revealed the role of TAB182 in regulating glycolysis and lactate production in cellular response to ionizing radiation. Irradiation can significantly stimulate the production of TAB182 protein, and inhibiting TAB182 increases cellular radiosensitivity. Proteomic analysis indicated that TAB182 influences several vital biological processes, including multiple metabolic pathways. Knockdown of TAB182 results in decreased lactate production and increased pyruvate and ATP levels in cancer cells. Moreover, knocking down TAB182 reverses radiation-induced metabolic changes, such as radioresistant-related lactate production. TAB182 is necessary for activating LDHA transcription by affecting transcription factors SP1 and c-MYC; its knockdown attenuates the upregulation of LDHA by radiation, subsequently suppressing lactate production. Targeted suppression of TAB182 significantly enhances the sensitivity of murine xenograft tumors to radiotherapy. These findings advance our understanding of glycolytic metabolism regulation in response to ionizing radiation, which may offer significant implications for developing new strategies to overcome tumor radioresistance., (© 2024. The Author(s).)

Background and Aims: Aerobic glycolysis reprogramming occurs during HSC activation, but how it is initiated and sustained remains unknown. We investigated the mechanisms by which canonical Wnt signaling regulated HSC glycolysis and the therapeutic implication for liver fibrosis., Approach and Results: Glycolysis was examined in HSC-LX2 cells upon manipulation of Wnt/β-catenin signaling. Nuclear translocation of lactate dehydrogenase A (LDH-A) and its interaction with hypoxia-inducible factor-1α (HIF-1α) were investigated using molecular simulation and site-directed mutation assays. The pharmacological relevance of molecular discoveries was intensified in primary cultures, rodent models, and human samples. HSC glycolysis was enhanced by Wnt3a but reduced by β-catenin inhibitor or small interfering RNA (siRNA). Wnt3a-induced rapid transactivation and high expression of LDH-A dependent on TCF4. Wnt/β-catenin signaling also stimulated LDH-A nuclear translocation through importin β2 interplay with a noncanonical nuclear location signal of LDH-A. Mechanically, LDH-A bound to HIF-1α and enhanced its stability by obstructing hydroxylation-mediated proteasome degradation, leading to increased transactivation of glycolytic genes. The Gly28 residue of LDH-A was identified to be responsible for the formation of the LDH-A/HIF-1α transcription complex and stabilization of HIF-1α. Furthermore, LDH-A-mediated glycolysis was required for HSC activation in the presence of Wnt3a. Results in vivo showed that HSC activation and liver fibrosis were alleviated by HSC-specific knockdown of LDH-A in mice. β-catenin inhibitor XAV-939 mitigated HSC activation and liver fibrosis, which were abrogated by HSC-specific LDH-A overexpression in mice with fibrosis., Conclusions: Inhibition of HSC glycolysis by targeting Wnt/β-catenin signaling and LDH-A had therapeutic promise for liver fibrosis., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Renal cell carcinoma (RCC) is one of the three major malignant tumors of the urinary system and originates from proximal tubular epithelial cells. Clear cell renal cell carcinoma (ccRCC) accounts for approximately 80% of RCC cases and is recognized as a metabolic disease driven by genetic mutations and epigenetic alterations. Through bioinformatic analysis, we found that FK506 binding protein 10 (FKBP10) may play an essential role in hypoxia and glycolysis pathways in ccRCC progression. Functionally, FKBP10 promotes the proliferation and metastasis of ccRCC in vivo and in vitro depending on its peptidyl-prolyl cis-trans isomerase (PPIase) domains. Mechanistically, FKBP10 binds directly to lactate dehydrogenase A (LDHA) through its C-terminal region, the key regulator of glycolysis, and enhances the LDHA-Y10 phosphorylation, which results in a hyperactive Warburg effect and the accumulation of histone lactylation. Moreover, HIFα negatively regulates the expression of FKBP10, and inhibition of FKBP10 enhances the antitumor effect of the HIF2α inhibitor PT2385. Therefore, our study demonstrates that FKBP10 promotes clear cell renal cell carcinoma progression and regulates sensitivity to HIF2α blockade by facilitating LDHA phosphorylation, which may be exploited for anticancer therapy., (© 2024. The Author(s).)

The dynamic changes of RNA N6-methyladenosine (m 6 A) during cancer progression participate in various cellular processes. However, less is known about a possible direct connection between upstream regulator and m 6 A modification, and therefore affects oncogenic progression. Here, we have identified that a key enzyme in N4-acetylcytidine (ac4C) acetylation NAT10 is highly expressed in human osteosarcoma tissues, and its knockdown enhanced m 6 A contents and significantly suppressed osteosarcoma cell growth, migration and invasion. Further results revealed that NAT10 silence inhibits mRNA stability and translation of m 6 A reader protein YTHDC1, and displayed an increase in glucose uptake, a decrease in lactate production and pyruvate content. YTHDC1 recognizes differential m 6 A sites on key enzymes of glycolysis phosphofructokinase (PFKM) and lactate dehydrogenase A (LDHA) mRNAs, which suppress glycolysis pathway by increasing mRNA stability of them in an m 6 A methylation-dependent manner. YTHDC1 partially abrogated the inhibitory effect caused by NAT10 knockdown in tumor models in vivo, lentiviral overexpression of YTHDC1 partially restored the reduced stability of YTHDC1 caused by lentiviral depleting NAT10 at the cellular level. Altogether, we found ac4C driven RNA m 6 A modification can positively regulate the glycolysis of cancer cells and reveals a previously unrecognized signaling axis of NAT10/ac4C-YTHDC1/m 6 A-LDHA/PFKM in osteosarcoma. Video Abstract., (© 2024. The Author(s).)

Osteoclasts are multinucleated, specializes bone-resorbing cells that are derived from the monocyte/macrophage lineage. Excessive resorbing activities of osteoclasts are involved in destructive bone diseases. The detailed mechanism of acidification at the bone adhesion surface during the bone resorption process of osteoclasts remains to be defined. During glycolysis, pyruvate proceeds to the tricarboxylic cycle under aerobic conditions and pyruvate is converted to lactate via lactate dehydrogenase A (LDHA) under anaerobic conditions. However, tumor cells produce ATP during aerobic glycolysis and large amounts of pyruvate are converted to lactate and H + by LDHA. Lactate and H + are excreted outside the cell, whereby they are involved in invasion of tumor cells due to the pH drop around the cell. In this study, we focused on aerobic glycolysis and investigated the production of lactate by LDHA in osteoclasts. Expression of LDHA and monocarboxylate transporter 4 (MCT4) was upregulated during osteoclast differentiation. Intracellular and extracellular lactate levels increased with upregulation of LDHA and MCT4, respectively. FX11 (an LDHA inhibitor) inhibited osteoclast differentiation and suppressed the bone-resorbing activity of osteoclasts. We propose that inhibition of LDHA may represent a novel therapeutic strategy for controlling excessive bone resorption in osteoporosis and rheumatoid arthritis., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Lung adenocarcinoma (LUAD) is one of the most prevalent and aggressive types of lung cancer. Metabolic reprogramming plays a critical role in the development and progression of LUAD. Pyruvate dehydrogenase kinase 1 (PDK1) and lactate dehydrogenase A (LDHA) are two key enzymes involved in glucose metabolism, whilst their aberrant expressions are often associated with tumorigenesis. Herein, we investigated the anticancer effects of combined inhibition of PDK1 and LDHA in LUAD in vitro and in vivo and its underlying mechanisms of action. The combination of a PDK1 inhibitor, 64, and a LDHA inhibitor, NHI-Glc-2, led to a synergistic growth inhibition in 3 different LUAD cell lines and more than additively suppressed tumor growth in the LUAD xenograft H1975 model. This combination also inhibited cellular migration and colony formation, while it induced a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) resulting in mitochondrial depolarization and apoptosis in LUAD cells. These effects were related to modulation of multiple cell signaling pathways, including AMPK, RAS/ERK, and AKT/mTOR. Our findings demonstrate that simultaneous inhibition of multiple glycolytic enzymes (PDK1 and LDHA) is a promising novel therapeutic approach for LUAD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cancer is the second leading cause of death worldwide, with a dramatic impact due to the acquired resistance of cancers to used chemotherapeutic drugs and treatments. The enzyme lactate dehydrogenase (LDH-A) is responsible for cancer cell proliferation. Recently the development of selective LDH-A inhibitors as drugs for cancer treatment has been reported to be an efficient strategy aiming to decrease cancer cell proliferation and increase the sensitivity to traditional chemotherapeutics. This study aims to obtain a stable and active biocatalyst that can be utilized for such drug screening purposes. It is conceived by adopting human LDH-A enzyme ( h LDH-A) and investigating different immobilization techniques on porous supports to achieve a stable and reproducible biosensor for anticancer drugs. The h LDH-A enzyme is covalently immobilized on mesoporous silica (MCM-41) functionalized with amino and aldehyde groups following two different methods. The mesoporous support is characterized by complementary techniques to evaluate the surface chemistry and the porous structure. Fluorescence microscopy analysis confirms the presence of the enzyme on the support surface. The tested immobilizations achieve yields of ≥80%, and the best retained activity of the enzyme is as high as 24.2%. The optimal pH and temperature of the best immobilized h LDH-A are pH 5 and 45 °C for the reduction of pyruvate into lactate, while those for the free enzyme are pH 8 and 45 °C. The stability test carried out at 45 °C on the immobilized enzyme shows a residual activity close to 40% for an extended time. The inhibition caused by NHI-2 is similar for free and immobilized h LDH-A, 48% and 47%, respectively. These findings are significant for those interested in immobilizing enzymes through covalent attachment on inorganic porous supports and pave the way to develop stable and active biocatalyst-based sensors for drug screenings that are useful to propose drug-based cancer treatments.

We have previously reported the toxicity of microcystin-LR (MC-LR) to the male reproductive system, which results in functional changes in mouse testes. In this study, mice were orally exposed to MC-LR at 1, 7.5, 15, or 30 μg/L daily for 180 days. We found an increase in germ cell apoptosis in the seminiferous tubules and low-quality sperm in the epididymis. A decrease in lactate dehydrogenase A (Ldha) expression in testes through high-throughput sequencing was observed. We validated that MC-LR disrupted lactate production in Sertoli cells by suppressing the expression of Ldha. Further studies identified that methyltransferase 3 (Mettl3) catalysed N 6 -methyladenosine (m 6 A) methylation of Ldha mRNA. Mettl3 was downregulated in Sertoli cells following exposure to MC-LR, decreasing m 6 A levels of Ldha. The stability of Ldha mRNA decreased when m 6 A levels of Ldha were inhibited. In conclusion, these results showed that MC-LR inhibits the expression of Ldha in an m 6 A-dependent manner, which might result in the apoptosis of spermatogenic cells and a decline in sperm quality. Our work provides a new perspective to understanding MC-LR-induced male infertility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia and hypovascular tumor microenvironment. Nucleolar and spindle associated protein 1 (NUSAP1) is a microtubule-associated protein that is known to be involved in cancer biology. Our study aimed to investigate the role of NUSAP1 in glycolytic metabolism and metastasis in PDAC. Expression and prognostic value of NUSAP1 in PDAC and common gastrointestinal tumors was evaluated. The function of NUSAP1 in PDAC progression was clarified by single-cell RNA-seq and further experiments in vitro, xenograft mouse model, spontaneous PDAC mice model and human tissue microarray. The downstream genes and signaling pathways regulated by NUSAP1 were explored by RNA-Seq. And the regulation of NUSAP1 on Lactate dehydrogenase A (LDHA)-mediated glycolysis and its underlying mechanism was further clarified by CHIP-seq. NUSAP1 was an independent unfavorable predictor of PDAC prognosis that playing a critical role in metastasis of PDAC by regulating LDHA-mediated glycolysis. Mechanically, NUSAP1 could bind to c-Myc and HIF-1α that forming a transcription regulatory complex localized to LDHA promoter region and enhanced its expression. Intriguingly, lactate upregulated NUSAP1 expression by inhibiting NUSAP1 protein degradation through lysine lactylated (Kla) modification, thus forming a NUSAP1-LDHA-glycolysis-lactate feedforward loop. The NUSAP1-LDHA-glycolysis-lactate feedforward loop is one of the underlying mechanisms to explain the metastasis and glycolytic metabolic potential in PDAC, which also provides a novel insights to understand the Warburg effect in cancer. Targeting NUSAP1 would be an attractive paradigm for PDAC treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The effect of aerobic glycolysis remains elusive in pediatric T-cell acute lymphoblastic leukemia (T-ALL). Increasing evidence has revealed that dysregulation of deubiquitination is involved in glycolysis, by targeting glycolytic rate-limiting enzymes. Here, we demonstrated that upregulated deubiquitinase ubiquitin-specific peptidase 1 (USP1) expression correlated with poor prognosis in pediatric primary T-ALL samples. USP1 depletion abolished cellular proliferation and attenuated glycolytic metabolism. In vivo experiments showed that USP1 suppression decreased leukemia progression in nude mice. Inhibition of USP1 caused a decrease in both mRNA and protein levels in lactate dehydrogenase A (LDHA), a critical glycolytic enzyme. Moreover, USP1 interacted with and deubiquitinated polo-like kinase 1 (PLK1), a critical regulator of glycolysis. Overexpression of USP1 with upregulated PLK1 was observed in most samples of patients with T-ALL. In addition, PLK1 inhibition reduced LDHA expression and abrogated the USP1-mediated increase of cell proliferation and lactate level. Ectopic expression of LDHA can rescue the suppressive effect of USP1 silencing on cell growth and lactate production. Pharmacological inhibition of USP1 by ML323 exhibited cell cytotoxicity in human T-ALL cells. Taken together, our results demonstrated that USP1 may be a promising therapeutic target in pediatric T-ALL., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Objective: In 1930, Otto Warburg reported that "aerobic glycolysis" is the intrinsic property of all tumor cells' fermentation of glucose to L-Lactate by lactate dehydrogenase A (LDHA) activity. This only produces per mole of glucose two moles of adenosine triphosphate (ATP), compared with 32 moles of ATP in a normal cell. Thus, tumor cells have to uptake 30 folds more glucose, the resulting accumulated lactate are then transported by a monocarboxylate transporter (MCT) with the participation of a CD147 molecule. Inhibition of MCT1 by RNA interference (RNAi) disrupted the unique metabolism of the tumor and caused tumor cell death. However, the effectiveness of the strategies depends on the targeted delivery of the therapeutics., Materials and Methods: In this study, a synergistic approach was used to target LDHA and MCT1 with small molecule inhibitors FX11 and AR-C155858, respectively. Cell cytotoxicity assays (AlamarBlue assay), and Mitochondria Membrane Potential (JC-1) dye assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. To achieve this aim, the following objectives were proposed: the effect of metabolic inhibitors on tumor glycolytic metabolite environment, and the efficacy of metabolite inhibitors on human breast and colorectal cancer cells in vitro. Then, gene expression analysis was performed using Qiagen RT2 Profiler PCR array for apoptosis. All these assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. Normal human fibroblasts were used as control cells under normal and hypoxic culture conditions., Results: In this study, the use of FX-11 inhibitors under normoxia or hypoxia in two or more cancer and normal cell lines has a direct effect on LDHA, whereby it inhibits its production, and this reduces the growth and cell proliferation of tumors. One of the more significant findings to emerge from this study is that using AR-C155858 inhibitor alone has increased the cell proliferation and showed no significant changes compared with the control. The other major finding was that combination of the two inhibitors, FX-11 and AR-C155858, under normoxia or hypoxia in two different cell lines MCF-7 and HCT-116 measured a decrease in the cells proliferative and red/green ratio., Conclusions: We successfully demonstrated that a combination of MCT1 inhibitor and LDHA inhibitor led to better outcomes. Indeed, this makes LDHA an ideal metabolic therapeutic target.

It has been demonstrated that hair follicle stem cells (HFSCs) can contribute to wound closure and repair. However, the specific mechanism remains unclear due to the complexity of the wound repair process. Lysine-specific demethylase 1 (LSD1), an important gene for the regulation of stem cell differentiation, has been reported to participate in wound healing regulation. Heat shock protein 90 (HSP90), a chaperone protein, is recently discovered to be a driver gene for wound healing. This study explored the molecular mechanisms by which the binding between LSD1 and HSP90 affects the role of HFSCs during skin wound healing. Following bioinformatics analysis, the key genes acting on HFSCs were identified. The expression of LSD1, HSP90, and c-MYC was found to be upregulated in differentiated HFSCs. Analysis of their binding affinity revealed that LSD1 interacted with HSP90 to enhance the stability of the transcription factor c-MYC. Lactate dehydrogenase A (LDHA) has been documented to be essential for HFSC activation. Therefore, we speculate that LDHA may induce the differentiation of HFSCs through glucose metabolism reprogramming. The results showed that c-MYC activated LDHA activity to promote glycolytic metabolism, proliferation, and differentiation of HFSCs. Finally, in vivo animal experiments further confirmed that LSD1 induced skin wound healing in mice via the HSP90/c-MYC/LDHA axis. From our data, we conclude that LSD1 interacting with HSP90 accelerates skin wound healing by inducing HFSC glycolytic metabolism, proliferation, and differentiation via c-MYC/LDHA axis., (© 2023 Federation of American Societies for Experimental Biology.)

Introduction: Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late stages, which decreases the chance of recovery. Molecular markers can improve the prediction of clinical outcome. Glycolysis is altered in 70% of CCs, so molecular markers of this pathway associated with the aggressiveness of CC can be identified., Methods: The expression of 14 glycolytic genes was analyzed in 97 CC and 29 healthy cervical tissue (HCT) with microarray; only LDHA and PFKP were validated at the mRNA and protein levels in 36 of those CC samples and in 109 new CC samples, and 31 HCT samples by qRT-PCR, Western blotting, or immunohistochemistry. A replica analysis was performed on 295 CC from The Cancer Genome Atlas (TCGA) database., Results: The protein expression of LDHA and PFKP was associated with poor overall survival [OS: LDHA HR = 4.0 (95% CI = 1.4-11.1); p = 8.0 × 10 -3 ; PFKP HR = 3.3 (95% CI = 1.1-10.5); p = 4.0 × 10 -2 ] and disease-free survival [DFS: LDHA HR = 4.5 (95% CI = 1.9-10.8); p = 1.0 × 10 -3 ; PFKP HR = 3.2 (95% CI = 1.2-8.2); p = 1.8 × 10 -2 ] independent of FIGO clinical stage, and the results for mRNA expression were similar. The risk of death was greater in patients with overexpression of both biomarkers than in patients with advanced FIGO stage [HR = 8.1 (95% CI = 2.6-26.1; p = 4.3 × 10 -4 ) versus HR = 7 (95% CI 1.6-31.1, p = 1.0 × 10 -2 )] and increased exponentially as the expression of LDHA and PFKP increased., Conclusions: LDHA and PFKP overexpression at the mRNA and protein levels was associated with poor OS and DFS and increased risk of death in CC patients regardless of FIGO stage. The measurement of these two markers could be very useful for evaluating clinical evolution and the risk of death from CC and could facilitate better treatment decision making., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Osteoarthritis (OA) is the most common form of arthritis and joint disorder worldwide. Metabolic reprogramming of osteoarthritic chondrocytes from oxidative phosphorylation to glycolysis results in the accumulation of lactate from glycolytic metabolite pyruvate by lactate dehydrogenase A (LDHA), leading to cartilage degeneration. In the present study, we investigated the protective effects of the intra-articular administration of oxamate (LDHA inhibitor) against OA development and glycolysis-related protein expression in experimental OA rats. The animals were randomly allocated into four groups: Sham, anterior cruciate ligament transection (ACLT), ACLT + oxamate (0.25 and 2.5 mg/kg). Oxamate-treated groups received an intra-articular injection of oxamate once a week for 5 weeks. Intra-articular oxamate significantly reduced the weight-bearing defects and knee width in ACLT rats. Histopathological analyses showed that oxamate caused significantly less cartilage degeneration in the ACLT rats. Oxamate exerts hypertrophic effects in articular cartilage chondrocytes by inhibiting glucose transporter 1, glucose transporter 3, hexokinase II, pyruvate kinase M2, pyruvate dehydrogenase kinases 1 and 2, pyruvate dehydrogenase kinase 2, and LHDA. Further analysis revealed that oxamate significantly reduced chondrocyte apoptosis in articular cartilage. Oxamate attenuates nociception, inflammation, cartilage degradation, and chondrocyte apoptosis and possibly attenuates glycolysis-related protein expression in ACLT-induced OA rats. The present findings will facilitate future research on LDHA inhibitors in prevention strategies for OA progression.

Cerebral ischemia (CI), as the cerebrovascular disease with the highest incidence rate, is treated by limited intravenous thrombolysis and intravascular therapy to recanalize the embolized vessels. Recently, the discovery of histone lactylation proposes a potential molecular mechanism for the role of lactate in physiological and pathological processes. This study aimed to analyze the lactate dehydrogenase A (LDHA) mediated histone lactylation in CI reperfusion (CI/R) injury. Oxygen-glucose deprivation/reoxygenation (OGD/R) treated N2a cells and middle cerebral artery occlusion (MCAO) treated rats was used as the CI/R model in vivo and in vitro. Cell viability and pyroptosis was assessed using CCK-8 and flow cytometry. RT-qPCR was performed to detect the relative expression. The relationship between histone lactylation and HMGB1 was verified by CHIP assay. LDHA, HMGB1, lactate and histone lactylation was up-regulated in the OGD/R treated N2a cells. Additionally, LDHA knockdown decreased HMGB1 levels in vitro, and relieved CI/R injury in vivo. Besides, LDHA silencing declined the histone lactylation mark enrichment on HMGB1 promoter, and lactate supplement rescued it. What?s more, LDHA knockdown decreased the IL-18 and IL-1β contents, and the cleaved-caspase-1 and GSDMD-N protein levels in the OGD/R treated N2a cells, which was reversed by HMGB1 overexpression. Knockdown of LDHA suppressed the pyroptosis in the N2a cells induced by OGD/R, which was reversed by HMGB1 overexpression. Mechanistically, LDHA mediated the histone lactylation induced pyroptosis through targeting HMGB1 in the CI/R injury., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Dysregulation of long noncoding RNAs (lncRNAs) has been associated with the development and progression of many human cancers. Lactate dehydrogenase A (LDHA) enzymatic activity is also crucial for cancer development, including the development of papillary thyroid cancer (PTC). However, whether specific lncRNAs can regulate LDHA activity during cancer progression remains unclear. Through screening, we identified an LDHA-interacting lncRNA, GLTC, which is required for the increased aerobic glycolysis and cell viability in PTC. GLTC was significantly upregulated in PTC tissues compared with nontumour thyroid tissues. High expression of GLTC was correlated with more extensive distant metastasis, a larger tumour size, and poorer prognosis. Mass spectrometry revealed that GLTC, as a binding partner of LDHA, promotes the succinylation of LDHA at lysine 155 (K155) via competitive inhibition of the interaction between SIRT5 and LDHA, thereby promoting LDHA enzymatic activity. Overexpression of the succinylation mimetic LDHA K155E mutant restored glycolytic metabolism and cell viability in cells in which metabolic reprogramming and cell viability were ceased due to GLTC depletion. Interestingly, GLTC inhibition abrogated the effects of K155-succinylated LDHA on radioiodine (RAI) resistance in vitro and in vivo. Taken together, our results indicate that GLTC plays an oncogenic role and is an attractive target for RAI sensitisation in PTC treatment., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Background: LSECs (Liver sinusoidal endothelial cells) are the portal of liver, their pathological angiogenesis plays a constructive role in etiopathogenesis of liver fibrosis by affecting liver tissue repair and inflammatory drive. Although intervention in angiogenesis can effectively inhibit abnormal activation of LSEC, no effective drugs have been found to treat liver fibrosis., Purpose: We investigated the effect of the natural compound Curcumol on LSEC angiogenesis and elucidated the novel underlying mechanism, expecting to provide a scientific basis for exploring potential therapeutic drugs for liver fibrosis., Methods: Various cellular and molecular assays, as well as genetic assays, were used to detect pathological angiogenesis and changes in glycolysis levels in cultured rat LSECs and mouse liver fibrosis models., Results: Transcription factor KLF5 is able to influence the angiogenic properties of LSEC by regulating the glycolytic process, and affect the expression of LDH-A by transcriptionally binding to its promoter. In our study, we were surprised to find that LDH-A (the final step of glycolysis) has a strong regulatory effect on the glycolytic process of LSEC. Through in-depth study, we found that LDH-A could affect the transcriptional activity of KLF5, thus forming a positive feedback loop. Curcumol could break this positive feedback loop and inhibit the glycolysis-dependent angiogenic nature of LSEC, thus alleviating liver fibrosis. Curcumol reduced extracellular matrix (ECM) deposition, attenuated pathological angiogenesis in LSEC, and decreased the level of CCl 4 -induced liver fibrosis in mice., Conclusion: Our results demonstrated the great utilization potentiality of KLF5 in liver fibrosis, and the innovative discovery that LDH-A regulates the glycolytic process and forms a malignant feedback loop by exerting non-enzymatic effects. It also reveals the prospect of Curcumol-regulated KLF5/LDH-A feedback loop in the treatment of liver fibrosis, providing a new option for the future medicine of liver fibrosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier GmbH.)

Lactate dehydrogenase A (LDHA) is a glycolytic enzyme catalysing the reversible conversion of pyruvate to lactate. It has been implicated as a substrate for PIM kinases, yet the relevant target sites and functional consequences of phosphorylation have remained unknown. Here, we show that all three PIM family members can phosphorylate LDHA at serine 161. When we investigated the physiological consequences of this phosphorylation in PC3 prostate cancer and MCF7 breast cancer cells, we noticed that it suppressed ubiquitin-mediated degradation of nuclear LDHA and promoted interactions between LDHA and 14-3-3 proteins. By contrast, in CRISPR/Cas9-edited knock-out cells lacking all three PIM family members, ubiquitination of nuclear LDHA was dramatically increased followed by its decreased expression. Our data suggest that PIM kinases support nuclear LDHA expression and activities by promoting phosphorylation-dependent interactions of LDHA with 14-3-3ε, which shields nuclear LDHA from ubiquitin-mediated degradation., (© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Polycystic ovary syndrome (PCOS) is a disease characterized by metabolic disorders. This study aimed to examine the effects of resveratrol treatment on ovulation in the PCOS rat model. Quantitative real-time PCR and immunohistochemistry were used to determine the mRNA and protein expression levels. TNUEL assay was used to evaluate cell apoptosis in ovary. The metabolites were evaluated by liquid chromatography with tandem mass spectrometry. Resveratrol alleviated disrupted estrous cycle and improved granular cell layers, and reversed the decreased proliferation and increased cell apoptosis of granulosa cells in the ovarian tissues of PCOS rats. Resveratrol restored the changes in the mRNA expression levels in the rate-limiting genes of glycolysis in the PCOS ovary. The expression of lactate dehydrogenase A (LDH-A), pyruvate kinase isozyme M2 (PKM2), and sirtuin 1 (SIRT1) was significantly downregulated in ovarian tissues of the PCOS rats; while the resveratrol treatment significantly increased the expression of LDH-A, PKM2, and SIRT1 in the ovarian tissues of PCOS rats. Collectively, the protective effects of resveratrol in the PCOS rats may be associated with the regulation of glycolysis-related mediators including PKM2, LDH-A, and SIRT1. Resveratrol may represent a good candidate in alleviating the development of PCOS.

Isoenzyme divergence is a prevalent mechanism governing tissue-specific and developmental stage-specific metabolism in mammals. The lactate dehydrogenase (LDH) isoenzyme spectrum reflects the tissue-specific metabolic status. We found that three tetrameric isoenzymes composed of LDHA and LDHB (LDH-3/4/5) comprise the LDH spectrum in T cells. Genetically deleting LDHA or LDHB altered the isoenzyme spectrum by removing all heterotetramers and leaving T cells with LDH-1 (the homotetramer of LDHB) or LDH-5 (the homotetramer of LDHA), respectively. Accordingly, deleting LDHA suppressed glycolysis, cell proliferation, and differentiation. Unexpectedly, deleting LDHB enhanced glycolysis but suppressed T cell differentiation, indicating that an optimal zone of glycolytic activity is required to maintain cell fitness. Mechanistically, the LDH isoenzyme spectrum imposed by LDHA and LDHB is necessary to optimize glycolysis to maintain a balanced nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide hydrogen pool. Our results suggest that the LDH isoenzyme spectrum enables "Goldilocks levels" of glycolytic and redox activity to control T cell differentiation.

Alterations in energy metabolism are associated with depression. However, the role of glycolysis in the pathogenesis of depression and the underlying molecular mechanisms remain unexplored. Through an unbiased proteomic screen coupled with biochemical verifications, we show that the levels of glycolysis and lactate dehydrogenase A (LDHA), a glycolytic enzyme that catalyzes L-lactate production, are reduced in the dorsomedial prefrontal cortex (dmPFC) of stress-susceptible mice in chronic social defeat stress (CSDS) model. Conditional knockout of LDHA from the brain promotes depressive-like behaviors in both male and female mice, accompanied with reduced L-lactate levels and decreased neuronal excitability in the dmPFC. Moreover, these phenotypes could be duplicated by knockdown of LDHA in the dmPFC or specifically in astrocytes. In contrast, overexpression of LDHA reverses these phenotypic changes in CSDS-susceptible mice. Mechanistic studies demonstrate that L-lactate promotes neuronal excitability through monocarboxylic acid transporter 2 (MCT2) and by inhibiting large-conductance Ca 2+ -activated potassium (BK) channel. Together, these results reveal a role of LDHA in maintaining neuronal excitability to prevent depressive-like behaviors., (© 2023. The Author(s).)

Increased glycolysis is one of the key metabolic hallmarks of cancer cells. However, the roles of lncRNAs in energy metabolism and cancer metastasis remain unclear. Here, the expression of TMEM105 associated with glycolysis was dramatically elevated from normal to breast cancer to breast cancer liver metastasis tissues, and the survival analysis revealed that high TMEM105 expression was related to poor survival, especially in patients with liver metastasis. Moreover, TMEM105 facilitated the glycolysis of breast cancer cells and induced cell invasion and breast cancer liver metastasis (BCLM). Mechanistically, TMEM105 regulated LDHA expression by sponging miR-1208, which further promoted cell glycolysis and BCLM. Importantly, glycolytic production of lactate enhanced TMEM105 expression in breast cancer cells by activating the SHH-MAZ signaling pathway. These findings suggested that the lactate-responsive TMEM105 acted as a miRNA sponge, inducing BCLM via a glycolysis-mediated positive feedback loop, which might be a rational target for the treatment of BCLM patients., (© 2023. The Author(s).)

The study was performed to evaluate the effects of the reduced lactate production by sodium oxamate (SO) on growth performance, lactate and glucose and lipid metabolism, and glucose tolerance of Micropterus salmoides fed high-carbohydrate (CHO) diets. In in vitro study, primary hepatocytes were incubated for 48 h in a control medium (5.5 mM glucose), a high-glucose medium (25 mM glucose, HG), or a SO-containing high-glucose medium (25 mM glucose + 50 mM SO, HG-SO). Results indicated lactate and triglyceride (TG) levels, and lactate dehydrogenase a (LDH-a) expression in the HG-SO group were remarkably lower than those of the HG group. In in vivo study, M. salmoides (5.23 ± 0.03 g) were fed four diets containing a control diet (10% CHO, C) and three SO contents [0 (HC), 100 (HC-SO1), and 200 (HC-SO2) mg·kg -1 , respectively] of high-CHO diets (20% CHO) for 11 wk. High-CHO diets significantly reduced weight gain rate (WGR), specific growth rate (SGR), p-AMPK-to-t-AMPK ratio, and expression of insulin receptor substrate 1 (IRS1), insulin-like growth factor I (IGF-I), insulin-like growth factor I receptor (IGF-IR), fructose-1,6-biphosphatase (FBPase), peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyl transferase 1α (CPT1α) compared with the C group, whereas the opposite was true for plasma levels of glucose, TG, lactate, tissue glycogen, and lipid contents, and expression of LDH-a, monocarboxylate transporter 1 and 4 (MCT1 and MCT4), insulin, glucokinase (GK), pyruvate dehydrogenase E1 subunit (PDH), sterol-regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FAS). The HC-SO2 diets remarkably increased WGR, SGR, p-AMPK-to-t-AMPK ratio, and expression of IRS1, IGF-I, IGF-IR, GK, PDHα, PDHβ, FAS, acetyl-CoA carboxylase 1 (ACC1), PPARα, and CPT1α compared with the HC group. Besides, HC-SO2 diets also enhanced glucose tolerance of fish after a glucose loading. Overall, the reduced lactate production by SO benefits growth performance and glucose homeostasis of high-CHO-fed M. salmoides through the enhancement of glycolysis, lipogenesis, and fatty acid β-oxidation coupled with the suppression of glycogenesis and gluconeogenesis.

Background: Irritable bowel syndrome (IBS) is a known brain-gut disorder. Currently, the molecular and cellular mechanisms of IBS remain unclear. Atractylenolide-I (ATL-I) is a majorly bioactive component extracted from Rhizoma Atractylodes Macrocephalae ., Methods: Studies have revealed that ATL-I functioned as an anti-tumor drug in various cancers. However, the effects and molecular mechanisms of ATL-I on the pathological processes of colonic mucosal epithelial cells (CMECs) during IBS remain unclear. This study reports ATL-I effectively alleviated the oxidative stress-induced colonic mucosal epithelial cell dysfunction. In colonic mucosal tissues from IBS patients, we detected upregulated miR-34a-5p and suppressed glucose metabolism enzyme expressions. Under H 2 O 2 treatment which mimics in vitro oxidative stress, miR-34a-5p was induced and glucose metabolism was inhibited in the colon mucosal epithelial cell line, NCM460. Meanwhile, ATL-I treatment effectively overcame the oxidative stress-induced miR-34a- 5p expression and glucose metabolism in NCM460 cells., Result: By bioinformatics analysis, Western blot and luciferase assay, we illustrated that miR-34a-5p directly targeted the 3'UTR region of glucose metabolism key enzyme, lactate dehydrogenase-A (LDHA) in colonic mucosal epithelial cells. Rescue experiments validated that miR-34a-5p inhibited glucose metabolism by targeting LDHA. Finally, we demonstrated that ATL-I treatment reversed the miR-34a-5p-inhibited glucose metabolism and -exacerbated colonic mucosal epithelial cell dysfunction under oxidative stress by modulating the miR-34a-5p-LDHA pathway., Conclusion: Summarily, our study reports the roles and mechanisms of ATL-I in the oxidative stress-induced colonic mucosal epithelial cell dysfunction during IBS through regulating the miR-34a-5p-LDHA-glucose metabolism axis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The incidence of breast cancer ranks at the top of female malignant tumors in China. Metastasis remains the main cause of death among breast cancer patients. The overexpression of ErbB2 is closely related to the metastasis and poor prognosis of breast cancer patients. Therefore, ErbB2 is an important clinical therapeutic target of breast cancer. However, the molecular mechanism of ErbB2 promoting breast cancer metastasis has not been studied clearly. Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme in catalyzing the conversion of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). SCD1 is overexpressed in breast cancer, and its overexpression is an indicator of poor prognosis in breast cancer patients. However, the role of SCD1 in ErbB2-overexpressing breast cancer metastasis has not been reported. In this study, we investigated the role of SCD1 in the migration and invasion of ErbB2-overexpressing breast cancer cells and its molecular mechanism. First, we demonstrated that ErbB2 upregulates the expression of SCD1. Second, we found that SCD1 and its catalytic product oleic acid played crucial roles in migration and invasion of ErbB2-overexpressing breast cancer cells. Finally, we found that in breast cancer cells, ErbB2 upregulated SCD1 through lactate dehydrogenase A (LDHA). To sum up, upregulation of SCD1 by ErbB2 via LDHA promotes the migration and invasion of breast cancer cells., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Aim: Cancer cells exhibit Warburg effect, characterized by increased glycolysis followed by fermentative conversion of pyruvate to lactate. Upregulation of Lactate Dehydrogenase-A (LDH-A) is elucidated to be a dominant molecular mediator of the phenomenon. Also, microRNA (miRNA) dysregulation participates in malignant progression and dissemination in several cancers. miR-16-5p is considerably reduced in lung cancers (LC), suggesting its tumor-suppressive role. However, its role in the regulation of aerobic glycolysis remains unknown. Our study aims to identify the regulatory roles of miR-16-5p/LDH-A in Non-small cell lung cancer (NSCLC)., Main Methods: We evaluated the differential expression of LDH-A and its prognostic potential in NSCLC tissues using online databases. We performed Tissue analysis using Immunohistochemistry (IHC); In-vitro cellular analysis including transient transfection, cellular proliferation, migration, and colony forming analysis. We also performed cell survival, metabolic, cell cycle, apoptotic, ROS generation and Immunocytochemistry (ICC) analyses to identify the role of miR-16-5p/LDH-A in aerobic glycolysis and tumorigenesis of NSCLC., Key Findings: We have identified that miR-16-5p directly targets LDH-A by binding to the complementary binding regions present in its 3'-UTR region, leading to degradation, sequentially leading to reduced lactate accumulation, glucose uptake and ATP levels. Our study also demonstrated the role of lactate accumulation in promoting NSCLC tumorigenesis via activation of NF-κB signaling pathway. However, miR-16-5p mediated targeting of LDH-A downregulates the expression of NF-κB associated genes, along with increased ROS generation, apoptosis, and cell cycle arrest., Significance: In conclusion, our findings identify miR-16-5p/LDH-A/lactate/NF-κB as an important link between metabolism and NSCLC cells tumorigenesis., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: T cell-mediated antitumor immunity has a vital role in cancer prevention and treatment; however, the immune-suppressive tumor microenvironment (TME) constitutes a significant contributor to immune evasion that weakens antitumor immunity. Here, we explore the relationship between nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the BTB (broad-complex, Tramtrack, bric a brac)/POZ (Poxvirus, and Zinc finger) gene family, and the TME., Methods: Adoptive cell transfer (ACT) of mouse or human tumor antigen (Ag)-specific CD8 + cytotoxic T lymphocytes (CTLs) was tested in an immunocompetent or immunodeficient mouse model of melanoma with or without expression of NAC1. The effects of NAC1 expression on immune evasion in tumor cells were assessed in vitro and in vivo. CRISPR/Cas9, glycolysis analysis, retroviral transduction, quantitative real-time PCR, flow cytometric analysis, immunoblotting, database analyses were used to screen the downstream target and underlying mechanism of NAC1 in tumor cells., Results: Tumorous expression of NAC1 negatively impacts the CTL-mediated antitumor immunity via lactate dehydrogenase A (LDHA)-mediated suppressive TME. NAC1 positively regulated the expression of LDHA at the transcriptional level, which led to higher accumulation of lactic acid in the TME. This inhibited the cytokine production and induced exhaustion and apoptosis of CTLs, impairing their cell-killing ability. In the immunocompetent and immunodeficient mice, NAC1 depleted melanoma tumors grew significantly slower and had an elevated infiltration of tumor Ag-specific CTLs following ACT, compared with the control groups., Conclusions: Tumor expression of NAC1 contributes substantially to immune evasion through its regulatory role in LDHA expression and lactic acid production. Thus, therapeutic targeting of NAC1 warrants further exploration as a potential strategy to reinforce cancer immunotherapy, such as the ACT of CTLs., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Breast cancer is the most prevalent cancer diagnosed in women and the major malignancy that threatens women health, thus we explored the role of long noncoding RNA LINC01605 in triple-negative breast cancer (TNBC). We collected tissue samples from TNBC patients and cultured breast cancer cells to detect LINC01605 levels by RT-PCR. We then constructed LINC01605 knockdown and LINC01605 overexpressed TNBC cell lines, cell proliferation was measured by CCK-8 and colony formation assays, cell migration and invasion were measured by Transwell assay, and aerobic glycolysis of cells was detected. Furthermore, a downstream target gene was found, and its role was confirmed by mouse allogeneic tumor formation. It discovered that LINC01605 expression was significantly increased in TNBC patients, and its high expression predicted a low survival prognosis for TNBC patients. Stable knockdown of LINC01605 remarkably inhibited cell proliferation, migration, and invasion, as well as aerobic glycolysis by inhibiting lactate dehydrogenase A in TNBC cell lines. Notably, knockdown of LINC01605 suppressed in vivo tumor formation and migration in TNBC transplanted mice. In conclusion, targeting long noncoding RNA LINC01605 might serve as a therapeutic candidate strategy to treat patients with TNBC., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Aortic dissection (AD) is a disease with high mortality and lacks effective drug treatment. Recent studies have shown that the development of AD is closely related to glucose metabolism. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme and plays an important role in cardiovascular disease. However, the role of LDHA in the progression of AD remains to be elucidated. Here, we found that the level of LDHA was significantly elevated in AD patients and the mouse model established by BAPN combined with Ang II. In vitro, the knockdown of LDHA reduced the growth of human aortic vascular smooth muscle cells (HAVSMCs), glucose consumption, and lactate production induced by PDGF-BB. The overexpression of LDHA in HAVSMCs promoted the transformation of HAVSMCs from contractile phenotype to synthetic phenotype, and increased the expression of MMP2/9. Mechanistically, LDHA promoted MMP2/9 expression through the LDHA-NDRG3-ERK1/2-MMP2/9 pathway. In vivo, Oxamate, LDH and lactate inhibitor, reduced the degradation of elastic fibers and collagen deposition, inhibited the phenotypic transformation of HAVSMCs from contractile phenotype to synthetic phenotype, reduced the expression of NDRG3, p-ERK1/2, and MMP2/9, and delayed the progression of AD. To sum up, the increase of LDHA promotes the production of MMP2/9, stimulates the degradation of extracellular matrix (ECM), and promoted the transformation of HAVSMCs from contractile phenotype to synthetic phenotype. Oxamate reduced the progression of AD in mice. LDHA may be a therapeutic target for AD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Multiple myeloma (MM) is the second most common hematologic malignancy, which is characterized by clonal proliferation of neoplastic plasma cells in the bone marrow. This microenvironment is characterized by low oxygen levels (1-6% O 2 ), known as hypoxia. For MM cells, hypoxia is a physiologic feature that has been described to promote an aggressive phenotype and to confer drug resistance. However, studies on hypoxia are scarce and show little conformity. Here, we analyzed the mRNA expression of previously determined hypoxia markers to define the temporal adaptation of MM cells to chronic hypoxia. Subsequent analyses of the global proteome in MM cells and the stromal cell line HS-5 revealed hypoxia-dependent regulation of proteins, which directly or indirectly upregulate glycolysis. In addition, chronic hypoxia led to MM-specific regulation of nine distinct proteins. One of these proteins is the cysteine protease legumain (LGMN), the depletion of which led to a significant growth disadvantage of MM cell lines that is enhanced under hypoxia. Thus, herein, we report a methodologic strategy to examine MM cells under physiologic hypoxic conditions in vitro and to decipher and study previously masked hypoxia-specific therapeutic targets such as the cysteine protease LGMN.