LDHA as a predictive biomarker and its association with the infiltration of immune cells in pancreatic adenocarcinoma.

Background: Lactate dehydrogenase A (LDHA) plays a crucial role in the final step of anaerobic glycolysis, converting L-lactate and NAD+ to pyruvate and nicotinamide adenine dinucleotide (NADH). Its high expression has been linked to tumorigenesis and patient survival in various human cancers. However, the full implications of LDHA's role and its correlation with clinicopathological features in pancreatic adenocarcinoma (PAAD) remain to be fully understood. This study was thus conducted to elucidate the specific functions of LDHA in PAAD, with the aim of providing more robust evidence for clinical diagnosis and treatment. Methods: In an extensive systems analysis, we searched through numerous databases, including The Cancer Genome Atlas (TCGA) and Oncomine. Our objective was to clarify the clinical implications and functional role of LDHA in PAAD. Bioinformatics was used to identify the biological function of LDHA expression and its correlation with tumor immune status. Results: Our analysis revealed that the LDHA gene is overexpressed in PAAD and that this upregulation was associated with a worse patient prognosis. Through gene set enrichment analysis, we found that LDHA's influence on PAAD is linked to signaling pathways involving Kirsten rat sarcoma viral oncogene homolog (K-Ras), transforming growth factor-ß (TGF-ß), and hypoxia inducible factor-1 (HIF-1). Mutation of K-Ras could upregulate its own expression and was positively correlated with LDHA expression. Moreover, our data demonstrated that LDHA expression was linked to immune infiltration and poor prognosis in PAAD, indicating its role in disease pathogenesis. Overexpression of LDHA may suppress tumor immunity, suggesting it as a potential target for the diagnosis and treatment of PAAD, thus providing new insights into managing this aggressive cancer. Conclusions: Overall, our results showed that LDHA as a prognostic biomarker could serve as a novel target for future PAAD immunotherapy.

The mRNA and protein levels of the glycolytic enzymes lactate dehydrogenase A (LDHA) and phosphofructokinase platelet (PFKP) are good predictors of survival time, recurrence, and risk of death in cervical cancer patients.

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.

[Effect of Liangfang Wenjing Decoction on expression of key glycolytic enzymes in uterus and ovaries of rats with coagulating cold and blood stasis syndrome].

This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.

LDHA as a regulator of T cell fate and its mechanisms in disease.

T cells are the main force of anti-infection and antitumor and are also involved in autoimmune diseases. During the development of these diseases, T cells need to rapidly produce large amounts of energy to satisfy their activation, proliferation, and differentiation. In this review, we introduced lactate dehydrogenase A(LDHA), predominantly involved in glycolysis, which provides energy for T cells and plays a dual role in disease by mediating lactate production, non-classical enzyme activity, and oxidative stress. Mechanistically, the signaling molecule can interact with the LDHA promoter or regulate LDHA activity through post-translational modifications. These latest findings suggest that modulation of LDHA may have considerable therapeutic effects in diseases where T-cell activation is an important pathogenesis.

Effect of Liangfang Wenjing Decoction on expression of key glycolytic enzymes in uterus and ovaries of rats with coagulating cold and blood stasis syndrome / 中国中药杂志

This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.

STUB1 directs FOXQ1-mediated transactivation of Ldha gene and facilitates lactate production in mouse Sertoli cells.

Sertoli cells (SCs) preferentially use glucose to convert to lactate. As an energy source, lactate is essential for survival of developed germ cells (GCs) due to its anti-apoptotic effect. Failure to maintain lactate metabolism homeostasis leads to infertility or germ cell apoptosis. Several Sertoli cell-expressed genes, such as Foxq1 and Gata4, have been identified as critical regulators for lactate synthesis, but the pathways that potentially modulate their expression remain ill defined. Although recent work from our collaborators pointed to an involvement of STIP1 homology and U-box-containing protein 1 (STUB1) in the modulation of Sertoli cell response to GCs-derived IL-1α, a true physiological function of STUB1 signaling in SCs has not been demonstrated. We therefore conditionally ablated Stub1 in SCs using Amh-Cre. Stub1 knockout males exhibited impaired fertility due to oligozoospermia and asthenospermia, possibly caused by lactate deficiency. Furthermore, by means of chromatin immunoprecipitation, in vivo ubiquitination, and luciferase reporter assays, we showed that STUB1 directed forkhead box Q1 (FOXQ1)-mediated transactivation of the lactate dehydrogenase A (Ldha) gene via K63-linked non-proteolytic polyubiquitination, thus facilitating lactate production in follicle-stimulating hormone (FSH)-stimulated SCs. In agreement, overexpression of LDHA by lentivirus infection effectively rescued the lactate production in TM4Stub1-/- cells. Our results collectively identify STUB1-mediated transactivation of FOXQ1 signaling as a post-translationally modified transcriptional regulatory network underlying nursery function in SCs, which may nutritionally contribute to Sertoli cell dysfunction of male infertility.

Epstein-Barr Virus-Encoded MicroRNA-BART18-3p Promotes Colorectal Cancer Progression by Targeting De Novo Lipogenesis.

The Epstein-Barr virus (EBV) genome encodes a cluster of 22 viral microRNAs, called miR-BamHI-A rightward transcripts (miR-BARTs), which are shown to promote the development of cancer. Here, this study reports that EBV-miR-BART18-3p is highly expressed in colorectal cancer (CRC) and is closely associated with the pathological and advanced clinical stages of CRC. Ectopic expression of EBV-miR-BART18-3p leads to increased migration and invasion capacities of CRC cells in vitro and causes tumor metastasis in vivo. Mechanistically, EBV-miR-BART18-3p activates the hypoxia inducible factor 1 subunit alpha/lactate dehydrogenase A axis by targeting Sirtuin, which promotes lactate accumulation and acetyl-CoA production in CRC cells under hypoxic condition. Increased acetyl-CoA utilization subsequently leads to histone acetylation of fatty acid synthase and fatty acid synthase-dependent fat synthesis, which in turn drives de novo lipogenesis. The oncogenic role of EBV-miR-BART18-3p is confirmed in the patient-derived tumor xenograft mouse model. Altogether, the findings define a novel mechanism of EBV-miR-BART18-3p in CRC development through the lipogenesis pathway and provide a potential clinical intervention target for CRC.

Protein arginine methyltransferase 3 promotes glycolysis and hepatocellular carcinoma growth by enhancing arginine methylation of lactate dehydrogenase A.

BACKGROUND: Protein arginine methylation has emerged a pivotal role in cancer progression. However, the role of protein arginine methyltransferase 3 (PRMT3) in hepatocellular carcinoma (HCC) remains unknown. METHODS: The expression pattern of PRMT3 in HCC was analysed using quantitative real-time-polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry assays. Loss- and gain-of-function experiments were carried out to determine the oncogenic role of PRMT3 in HCC. Glucose consumption and lactate production assays, seahorse bioscience, mass spectrometry, co-immunoprecipitation, metabonomic analysis and site-specific mutation experiments were used to explore the underlying molecular mechanisms. Furthermore, a xenograft mouse model was established to investigate the effects of PRMT3 and its inhibitor, SGC707, treatment on tumour growth in vivo. RESULTS: The expression of PRMT3 was significantly upregulated in HCC, with high expression of which correlated with poor prognosis. PRMT3 knockdown led to the decrease in proliferation, glycolysis of HCC cells and tumour growth, whilst its overexpression showed opposite results. The catalytic activity of PRMT3 was important in mediating these biological processes. Mechanistically, our data showed that PRMT3 interacted with and mediated asymmetric dimethylarginine (ADMA) modification of lactate dehydrogenase A (LDHA) at arginine 112 (R112). Compared with LDHA-wild-type (LDHA-WT) cells, LDHA-R112K-mutant-expressing HCC cells exhibited a decrease in lactate dehydrogenase (LDH) activity, HCC cell glycolysis and proliferation. Furthermore, the administration of SGC707, a selective inhibitor of PRMT3, disrupted the PRMT3-mediated LDHA methylation and abolished PRMT3-induced HCC glycolysis and tumour growth. CONCLUSIONS: Our results suggested a novel oncogenic role of PRMT3 in HCC, and it could be a promising therapeutic target for HCC by linking post-translational modification and cancer metabolism.

Targeting glucose metabolism to develop anticancer treatments and therapeutic patents.

INTRODUCTION: One of the most distinctive hallmarks of cancer cells is increased glucose consumption for aerobic glycolysis, which is called the Warburg effect. In recent decades, extensive research has been carried out to exploit this famous phenomenon, trying to detect promising targetable vulnerabilities in altered metabolism to fight cancer. Targeting aberrant glucose metabolism can perturb cancer malignant proliferation and even induce programmed cell death. AREAS COVERED: This review covered the recent patents which focused on targeting key glycolytic enzymes, including hexokinase, pyruvate dehydrogenase kinases, and lactate dehydrogenase for cancer treatment. EXPERT OPINION: Compared with the conventional cancer treatment, specifically targeting the well-known Achilles heel, the Warburg effect has attracted considerable attention. Although there is still no single glycolytic agent for clinical cancer treatment, the combination of glycolytic inhibitor with conventional anticancer drugs or the combined use of multiple glycolytic inhibitors are being investigated extensively in recent years, which could emerge as attractive anticancer strategies.

LDH-A negatively regulates dMMR in colorectal cancer.

Although immune checkpoint inhibitors (ICIs) have achieved unprecedented success in dMMR tumors, pMMR tumors accounting for 85% of colorectal cancer (CRC) cases remain unresponsive. Lactate dehydrogenase A (LDH-A) is the rate-limiting enzyme that catalyzes the transformation of pyruvate to lactate in the process of glycolysis. We investigated the relationship between LDH-A and dMMR with the purpose of exploring the treatment strategy for pMMR CRC patients. We here show that LDH-A can promote the proliferation of dMMR and pMMR CRC cells by positively regulating MMR proteins both in vitro and in vivo. LDH-A inhibition can improve the efficacy of PD-1 blockade in a pMMR CRC xenograft model. A statistical analysis of 186 CRC specimens showed a significant correlation between LDH-A and dMMR status. Moreover, patients with both low LDH-A expression and dMMR exhibited better disease-free survival compared with patients with other combinations. The close correlation of LDH-A and dMMR may offer a promising therapeutic strategy in which the combination of LDH-A inhibitor and ICIs may improve the clinical benefit for pMMR CRC patients.

Unappreciated Role of LDHA and LDHB to Control Apoptosis and Autophagy in Tumor Cells.

Tumor cells possess a high metabolic plasticity, which drives them to switch on the anaerobic glycolysis and lactate production when challenged by hypoxia. Among the enzymes mediating this plasticity through bidirectional conversion of pyruvate and lactate, the lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB), are indicated. LDHA has a higher affinity for pyruvate, preferentially converting pyruvate to lactate, and NADH to NAD+ in anaerobic conditions, whereas LDHB possess a higher affinity for lactate, preferentially converting lactate to pyruvate, and NAD+ to NADH, when oxygen is abundant. Apart from the undisputed role of LDHA and LDHB in tumor cell metabolism and adaptation to unfavorable environmental or cellular conditions, these enzymes participate in the regulation of cell death. This review presents the latest progress made in this area on the roles of LDHA and LDHB in apoptosis and autophagy of tumor cells. Several examples of how LDHA and LDHB impact on these processes, as well as possible molecular mechanisms, will be discussed in this article. The information included in this review points to the legitimacy of modulating LDHA and/or LDHB to target tumor cells in the context of human and veterinary medicine.

Lactate dehydrogenase C is required for the protein expression of a sperm-specific isoform of lactate dehydrogenase A.

Metabolites are sensitive indicators of moment-to-moment cellular status and activity. Expecting that tissue-specific metabolic signatures unveil a unique function of the tissue, we examined metabolomes of mouse liver and testis and found that an unusual metabolite, 2-hydroxyglutarate (2-HG), was abundantly accumulated in the testis. 2-HG can exist as D- or L-enantiomer, and both enantiomers interfere with the activities of 2-oxoglutarate (2-OG)-dependent dioxygenases, such as the Jumonji family of histone demethylases. Whereas D-2-HG is produced by oncogenic mutants of isocitrate dehydrogenases (IDH) and known as an oncometabolite, L-2-HG was the major enantiomer detected in the testis, suggesting that a distinct mechanism underlies the testicular production of this metabolite. We clarified that lactate dehydrogenase C (LDHC), a testis-specific lactate dehydrogenase, is responsible for L-2-HG accumulation by generating and analysing Ldhc-deficient mice. Although the inhibitory effects of 2-HG on 2-OG-dependent dioxygenases were barely observed in the testis, the LDHA protein level was remarkably decreased in Ldhc-deficient sperm, indicating that LDHC is required for LDHA expression in the sperm. This unique functional interaction between LDH family members supports lactate dehydrogenase activity in the sperm. The severely impaired motility of Ldhc-deficient sperm suggests a substantial contribution of glycolysis to energy production for sperm motility.

MiR-33b inhibits osteosarcoma cell proliferation through suppression of glycolysis by targeting Lactate Dehydrogenase A (LDHA).

Osteosarcoma (OS) is one of the most common types of malignant bone tumor in adolescent. MicroRNAs (miRNAs) are widely studied regulatory molecules which play important roles in tumor development, differentiation, growth, invasion, chemosensitivity and cellular metabolism. Recently, miR-33b has been reported to act as a tumor suppressor in osteosarcoma. However, the detailed mechanism of miR-33b in regulating osteosarcoma cell proliferation remains unclear. In this study, we detected miR-33b was significantly downregulated in osteosarcoma tissues compared to their matched adjacent nontumor tissues. The decreased expressions of miR-33b were also found in multiple osteosarcoma cell lines, including MG63, Saos-2, U2OS and SOSP-9607 when compared to normal osteoblast cell line hFOB. Overexpression of miR-33b suppressed U2OS cell proliferation and anaerobic glycolysis. We identified Lactate dehydrogenase-A (LDHA) as a direct target of miR-33b in osteosarcoma tumors and cells by Western blot and luciferase assay. Moreover, inhibition of LDHA significantly suppressed glycolysis and cell proliferation of osteosarcoma cells. Restoration of LDHA in miR-33b-overexpressing osteosarcoma cells reversed the suppressive effect of miR-33b on cell proliferation. In addition, we report a significantly negative correlation between LDHA mRNA and miR-33b expression in osteosarcoma tumors: miR-33b is downregulated in OS tumors with high levels of LDHA (92.9%). Meanwhile, high miR-33b expressions were found majorly in OS tumors with low LDHA mRNA levels (82.4%). This study reveals that miR-33b plays a suppressive role in the regulation of osteosarcoma cell proliferation through direct targeting LDHA. The miR-33b/glycolysis/LDHA axis may contribute to development of therapeutic anti-tumor agents for osteosarcoma.

MiR-34b-3 and miR-449a inhibit malignant progression of nasopharyngeal carcinoma by targeting lactate dehydrogenase A.

MicroRNA expression profiling assays have shown that miR-34b/c and miR-449a are down-regulated in nasopharyngeal carcinoma (NPC); however, the targets and functions of miR-34b/c and miR-449a in the pathologenesis of NPC remain elusive. In this study, we verified miR-34b/c and miR-449a were significantly reduced with the advance of NPC. Overexpression of miR-34b-3 and miR-449a suppressed the growth of NPC cells in culture and mouse tumor xenografts. Using tandem mass tags for quantitative labeling and LC-MS/MS analysis to investigate protein changes after restoring expression of miR-34b-3, 251 proteins were found to be down-regulated after miR-34b-3 transfection. Through 3 replicate experiments, we found that miR-34b-3 regulated the expression of 15 potential targeted genes mainly clustered in the key enzymes of glycolysis metabolism, including lactate dehydrogenase A (LDHA). Further investigation revealed that miR-34b-3 and miR-449a negatively regulated LDHA by binding to the 3' untranslated regions of LDHA. Furthermore, LDHA overexpression rescued the miR-34b-3 and miR-449a induced tumor inhibition effect in CNE2 cells. In addition, miR-34b-3 and miR-449a suppressed LDH activity and reduced LD content, which were directly induced by downregulation of the LDHA. Our findings suggest that miR-34b-3 and miR-449a suppress the development of NPC through regulation of glycolysis via targeting LDHA and may be potential therapeutic targets for the treatment of NPC.

The Actual Role of LDH as Tumor Marker, Biochemical and Clinical Aspects.

Lactate dehydrogenase (LDH) among many biochemical parameters represents a very valuable enzyme in patients with cancer with possibility for easy routine measurement in many clinical laboratories. Previous studies where mostly based on investigated LDH in serum of patients with cancer with aims to estimate their clinical significance. The new directions in investigation of LDH where based on the principle that tumor cells release intracellular enzymes trough damaged cell membrane, that is mostly consequence in intracellular mitochondrial machinery alteration, and apoptosis deregulation. This consideration can be used not only in-vitro assays, but also in respect to clinical characteristics of tumor patients. Based on new techniques of molecular biology it is shown that intracellular characteristics of LDH enzyme are very sensitive indicators of the cellular metabolic state, aerobic or anaerobic direction of glycolysis, activation status and malignant transformation. Using different molecular analyses it is very useful to analyzed intracellular LDH activity in different cell line and tumor tissues obtained from patients, not only to understanding complexity in cancer biochemistry but also in early clinical diagnosis. Based on understandings of the LDH altered metabolism, new therapy option is created with aims to blocking certain metabolic pathways and stop tumors growth.