Targeting Lactate Dehydrogenase A Research Articles

Long noncoding RNA HAGLR sponges miR-338-3p to promote 5-Fu resistance in gastric cancer through targeting the LDHA-glycolysis pathway.

Gastric cancer (GC) is one of the most common human malignancies due to its invasiveness and metastasis. 5‐Fu is a widely applied chemotherapeutic agent against GC. Although 5‐Fu therapy has achieved improvements in GC treatment, a large fraction of patients developed drug resistance which significantly limited its clinical applications. Recent studies revealed the pivotal roles of long noncoding RNAs (lncRNAs) in tumorigenesis and progressions of various tumors, including GC. However, the biological roles and molecular mechanisms of lncRNA HAGLR in GC remain unclear. Here, we report HAGLR was upregulated in both GC tissues and cell lines. In addition, HAGLR was associated with a poorly survival rate of GC patients. Blocking HAGLR inhibited GC cells proliferation and sensitized GC cells to 5‐Fu. Bioinformatical analysis and luciferase assay demonstrated that HAGLR sponged microRNA (miR)‐338‐3p, which functions as a tumor suppressor in GC to downregulate its expressions. Moreover, from the established 5‐Fu resistant GC cell line (HGC27 5‐Fu R), we detected significantly elevated HAGLR, downregulated miR‐338‐3p, and glucose metabolism compared with parental HGC27 cells. We identified lactate dehydrogenase‐A (LDHA), a glucose metabolism key enzyme, was the direct target of miR‐338‐3p in GC cells. Rescue experiments demonstrated that restoration of miR‐338‐3p in HAGLR‐overexpressing HGC27 5‐Fu R cells successfully overrode the HAGLR‐promoted 5‐Fu resistance through targeting LDHA. Taken together, this study revealed essential roles and molecular mechanisms for the HAGLR‐mediated 5‐Fu resistance in GC, contributing to the development of new noncoding RNA‐based therapeutic strategies against chemoresistant GC.

Influences of the lncRNA TUG1-miRNA-34a-5p network on fibroblast-like synoviocytes (FLSs) dysfunction in rheumatoid arthritis through targeting the lactate dehydrogenase A (LDHA).

BackgroundRheumatoid arthritis (RA) is a systemic and chronic inflammatory disease. The cellular glucose metabolism of fibroblast‐like synoviocytes (FLSs) of RA has been revealed to be essential to the pathogenesis and development of RA. To date, the precise roles and molecular mechanisms of long noncoding RNA TUG1 in RA have not been elucidated.MethodsTUG1 and miR‐34a‐5p were detected by qRT‐PCR. Interactions between lncRNA‐miRNA and miRNA‐mRNA were validated by RNA pull‐down assay and luciferase assay. The glucose metabolism was evaluated by glucose uptake and extracellular acidification rate (ECAR). Cell viability was determined by MTT assay and Annexin V assay.ResultsTUG1 expression was significantly upregulated in synovial fibroblast‐like synoviocytes (FLSs) compared with normal FLSs. Functional assays uncovered that silence of TUG1 suppressed FLSs‐RA invasion, migration, glucose metabolism, and increased apoptosis. Bioinformatics analysis indicated that TUG1 interacted with miR‐34a‐5p. RNA pull‐down assay and luciferase assay validated that TUG1 sponged miR‐34a‐5p in FLSs‐RA. Overexpression of miR‐34a‐5p effectively inhibited glucose metabolism of FLSs‐RA. Furthermore, the glucose metabolism of FLSs‐RA was significantly elevated compared with normal FLSs. The glucose metabolism enzyme, LDHA, was directly targeted by miR‐34a‐5p in FLSs. Rescue experiments validated that the miR‐34a‐5p‐inhibited glucose metabolism of FLSs‐RA was through targeting LDHA. Finally, we showed restoration of miR‐34a‐5p in TUG1‐overexpressing FLSs‐RA successfully overcame the TUG1‐promoted glucose metabolism and apoptosis resistance via targeting LDHA.ConclusionThe present study uncovered critical roles and molecular mechanisms underlying the TUG1‐mediated glucose metabolism and apoptosis of FLSs‐RA through modulating the miR‐34a‐5p‐LDHA pathway in fibroblast‐like synoviocytes of rheumatoid arthritis.

Targeting lactate dehydrogenase A (LDHA) exerts antileukemic effects on T-cell acute lymphoblastic leukemia.

BackgroundT‐cell acute lymphoblastic leukemia (T‐ALL) is an uncommon and aggressive subtype of acute lymphoblastic leukemia (ALL). In the serum of T‐ALL patients, the activity of lactate dehydrogenase A (LDHA) is increased. We proposed that targeting LDHA may be a potential strategy to improve T‐ALL outcomes. The current study was conducted to investigate the antileukemic effect of LDHA gene‐targeting treatment on T‐ALL and the underlying molecular mechanism.MethodsPrimary T‐ALL cell lines Jurkat and DU528 were treated with the LDH inhibitor oxamate. MTT, colony formation, apoptosis, and cell cycle assays were performed to investigate the effects of oxamate on T‐ALL cells. Quantitative real‐time PCR (qPCR) and Western blotting analyses were applied to determine the related signaling pathways. A mitochondrial reactive oxygen species (ROS) assay was performed to evaluate ROS production after T‐ALL cells were treated with oxamate. A T‐ALL transgenic zebrafish model with LDHA gene knockdown was established using CRISPR/Cas9 gene‐editing technology, and then TUNEL, Western blotting, and T‐ALL tumor progression analyses were conducted to investigate the effects of LDHA gene knockdown on T‐ALL transgenic zebrafish.ResultsOxamate significantly inhibited proliferation and induced apoptosis of Jurkat and DU528 cells. It also arrested Jurkat and DU528 cells in G0/G1 phase and stimulated ROS production (all P < 0.001). Blocking LDHA significantly decreased the gene and protein expression of c‐Myc, as well as the levels of phosphorylated serine/threonine kinase (AKT) and glycogen synthase kinase 3 beta (GSK‐3β) in the phosphatidylinositol 3′‐kinase (PI3K) signaling pathway. LDHA gene knockdown delayed disease progression and down‐regulated c‐Myc mRNA and protein expression in T‐ALL transgenic zebrafish.ConclusionTargeting LDHA exerted an antileukemic effect on T‐ALL, representing a potential strategy for T‐ALL treatment.

Inhibition of glycolysis by targeting lactate dehydrogenase A facilitates hyaluronan synthase 2 synthesis in synovial fibroblasts of temporomandibular joint osteoarthritis.

Although associations between dysregulated glucose metabolism and human rheumatoid arthritis have been reported, the disturbance and influence of glycolytic metabolism on temporomandibular joint osteoarthritis remains unclear. This study aimed to investigate the expression level and metabolite profile of the critical glycolytic enzyme, lactate dehydrogenase A (LDHA) in synovial fibroblasts (SFs) of TMJOA, assess the effect of glycolytic inhibition on synthesis of hyaluronan synthase 2 (HAS2) and inflammation progression in these cells. Immunohistochemistry and western blotting were performed to detect the expression of LDHA in the lining and sub-lining layers of synovial tissue and SFs. MTT and EdU assays were used to measure the cell proliferation. The cell apoptosis were demonstrated by TUNEL staining and Annexin V/PI double staining. A potent and specific inhibitor of LDHA, GSK2837808A, was administrated to suppress the activity of LDHA and detect the potential efficacy on HAS2. LDHA expression was dramatically higher in the synovial tissue and SFs from TMJOA patients compared to control groups. LDHA inhibition impaired active LDHA performance, suppressed the glucose uptake and decreased lactate concentration. Furthermore, GSK2837808A reversed the occurrence of low ratio of ATP/AMP, high level of Adenosine Monophosphate-activated Protein Kinase (AMPK) activation, disturbed HAS2 synthesis and hyaluronic acid (HA) production by inhibiting LDHA. The cellular viability and cell cycle were not affected by GSK2837808A at the working concentration. Targeting LDHA using its specific suppressant GSK2837808A impeded lactate secretion and contributed to HAS2 and HA synthesis in TMJOA SFs, providing the vital role of LDHA associated with TMJOA pathogenesis and a novel therapeutic approach for TMJOA.

MiR-34a suppresses osteoblast differentiation through glycolysis inhibition by targeting lactate dehydrogenase-A (LDHA).

Bone formation (osteogenesis) is mediated through recruitment of bone marrow mesenchymal stem cells (MSCs) with capacity to differentiate into osteoblasts, a process which is regulated by transcriptional and post-transcriptional mechanisms. Multiple studies have suggested that miRNAs might have important roles in osteoblast differentiation. Expressions of miR-34a were detected by qRT-PCR. Cellular glucose metabolism was assessed by measurements of glucose uptake and lactate production. mRNA expressions of glycolysis enzymes were detected by qRT-PCR. Osteogenic differentiation of human MSCs (hMSCs) was analyzed by alkaline phosphatase (ALP) activity and Alizarin red staining. Here, we report that microRNA-34a is upregulated during the osteoblast differentiation from hMSCs. miR-34a overexpressing inhibited late osteoblast differentiation of hMSCs in vitro. The ALP activity and Alizarin red staining were significantly decreased by miR-34a in hMSCs. Target prediction analysis reveals that the lactate dehydrogenase-A (LDHA) is a potential target of miR-34a. We hypothesized that miR-34a inhibits osteoblast differentiation through targeting the LDHA-mediated cellular glycolysis. Results from Western blotting and luciferase assay validated that miR-34a could directly target 3'UTR of LDHA mRNA. In addition, we demonstrated that overexpression of miR-34a inhibits cellular anaerobic glycolysis through targeting LHDA. The protein and mRNA expressions of glycolysis enzymes, Hexokinase 2 (HK2), glucose transporter 1 (GLUT1), and LDHA were significantly downregulated by miR-34a overexpression in hMSCs. Furthermore, we showed that LDHA restoration in miR-34a overexpressing hMSCs successfully rescued the osteoblast differentiation of hMSCs. This study demonstrated the roles of miR-34a in regulating osteoblast differentiation, suggesting that miR-34a inhibition could be a new therapeutic strategy for improving bone formation.

Chiral Pyridine-3,5-bis- (L-phenylalaninyl-L-leucinyl) Schiff Base Peptides as Potential Anticancer Agents: Design, Synthesis, and Molecular Docking Studies Targeting Lactate Dehydrogenase-A.

A series of branched tetrapeptide Schiff bases 3–6 were designed and synthesized from corresponding tetrapeptide hydrazide 2 as a starting material.In vitroevaluation of the synthesized compounds 4–6 against breast MCF-7 carcinoma cells identified their excellent anticancer potency, with IC50 ranging from 8.12 ± 0.14 to 17.55 ± 0.27 μM in comparison with the references, cisplatin and milaplatin (IC50= 13.34 ± 0.11and 18.43 ± 0.13 μM, respectively). Furthermore, all derivatives demonstrated promising activity upon evaluation of theirin vitroandin vivosuppression of p53 ubiquitination and inhibition assessment for LDHA kinase. Finally, molecular docking studies were performed to predict the possible binding features of the potent derivatives within the ATP pocket of LDHA in an attempt to get a lead for developing a more potent LDHA inhibitor with anti-proliferative potency.

MiR-638 Repressed Vascular Smooth Muscle Cell Glycolysis by Targeting LDHA.

BackgroundAbnormal proliferation and migration of vascular smooth muscle cells (VSMCs) accelerated vascular diseases progression, like atherosclerosis and restenosis. MicroRNAs were reported to participate in modulating diverse cellular processes. Here, we focused on exploring the role of miR-638 in VSMCs glycolysis and underlying mechanism.MethodsCell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Western blot assay was conducted to determine the expression of cell proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67, as well as Lactate dehydrogenase A (LDHA). VSMCs migration and invasion were evaluated by Transwell assay. Luciferase reporter gene assay and RNA immunoprecipitation were performed to validate the target relationship between miR-638 and LDHA. LDHA and miR-638 expression were also determined. Glycolysis of VSMCs was tested by corresponding Kits.ResultsPlatelet-derived growth factor-bb (PDGF-bb) promoted the VSMCs viability and down-regulated miR-638. Overexpression of miR-638 inhibited cell proliferation, migration and invasion of VSMCs. LDHA was identified as a target of miR-638, and counter-regulated by miR-638. Loss of miR-638 attenuated the suppressor effects on the proliferation, migration and invasion of VSMCs induced by LDHA down-regulation. MiR-638 inhibited the glycolysis of VSMCs by targeting LDHA.ConclusionMiR-638 is down-regulated by PDGF-bb treatment and suppressed the glycolysis of VSMCs via targeting LDHA.

New avenues in pancreatic cancer: exploiting microRNAs as predictive biomarkers and new approaches to target aberrant metabolism

ABSTRACTIntroduction: Most pancreatic cancer patients are diagnosed at advanced-stages and first-line regimens (FOLFIRINOX and gemcitabine/nab-paclitaxel) provide limited survival advantage and are associated with considerable toxicities. In this grim scenario, novel treatments and biomarkers are warranted.Areas covered: MicroRNAs (miRNAs) emerged as biomarkers for cancer prognosis and chemoresistance and blood-based miRNAs are being evaluated as indicators of therapeutic activity. Moreover, aberrant metabolism, such as aerobic glycolysis, has been correlated to tumor aggressiveness and poor prognosis. Against this background, innovative approaches to tackle metabolic aberrations are being implemented and glycolytic inhibitors targeting lactate dehydrogenase-A (LDH-A) showed promising effects in preclinical models. A PubMed search was used to compile relevant publications until February 2019.Expert opinion: Analysis of tissue/circulating miRNA might improve selection for optimal treatment regimens. For instance, miR-181a modulation seems to predict response to FOLFIRINOX. However, we need further studies to validate predictive miRNA profiles, as well as to exploit miRNAs for treatment-tailoring. Several miRNAs have also a key role in regulating metabolic aberrations. Since preliminary evidence supports the development of new agents targeting these aberrations, such as LDH-A inhibitors, the identification of biomarkers for these treatments, including the above-mentioned miRNAs, should shorten the gap between preclinical studies and personalized therapies.

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.

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

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

Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells

Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells

Abstract SY02-02: Exploring the lung cancer metabolome, in vivo and ex vivo, for individualized medicine

Abstract Lung cancer is a leading cause of cancer death worldwide. This disease is of serious concern in Kentucky, which leads the nation in both lung cancer incidence and mortality. The past decade of research in cancer metabolism reveals the untapped value of exploring human metabolome for the discovery of novel therapeutic and diagnostic biomarkers for human cancers and other diseases. To better understand metabolic reprogramming in individual tumors of lung cancer patients, we have developed stable isotope tracers (e.g., [13C6]-glucose) coupled with NMR and MS-based stable isotope-resolved metabolomics (SIRM) analysis directly in patient and in patient-derived ex vivo and in vivo models. In the in vivo patient study, we mapped differential metabolic network between paired cancerous (CA) and noncancerous (NC) tissues procured from non–small cell lung cancer (NSCLC) patients infused with [13C6]-glucose and snap-frozen immediately after resection (1). In particular, we uncovered elevated anaplerotic pyruvate carboxylase (PC) activity in CA versus paired NC tissues. Proliferating cancer cells require active Krebs cycle for generating anabolic precursors, in addition to energy production. Diversion of the Krebs cycle intermediates to meet anabolic demands cannot be sustained without anaplerosis. Pyruvate carboxylation represents one of the two major anaplerotic pathways that replenish the Krebs cycle intermediates; the other involves glutaminolysis initiated by glutaminase (GLS). We also found that PC but not GLS protein was overexpressed (median 8-10 fold; n=86) in CA tissues relative to paired NC tissues and that PC expression was functionally important to NSCLC cell growth both in vitro and in vivo (1). We also utilize the “Warburg slice” concept to systematically define metabolic distinctions between thinly sliced paired CA versus NC lung tissues freshly resected from individual NSCLC patients and cultured in stable isotope tracers. This ex vivo tissue slice culture system is excellently suited for delineating reprogrammed metabolic pathways in CA tissues without systemic interferences (2-4). These lung tissue slices were metabolically viable for up to 72 hr, while maintaining their 3D architecture and microenvironment (4). We found that the reprogrammed metabolic network in the cultured human tissue slices recapitulated that in vivo (1). These advantages make the ex vivo human tissue slice systems a unique preclinical model for exploring human target tissue metabolism and how it underlies the response to anticancer agents such as chemopreventive Se compounds, enzyme inhibitors (5), and the immune modular β-glucan (4). We found that selenite blocked PC anaplerosis and elicited massive necrosis in CA but not in NC lung tissues. CA lung tissue slices also responded to β-glucan with perturbed metabolic activity and histopathologic changes, which were consistent with polarization towards M1-type macrophages. We were intrigued to find that the metabolism and histopathology of these CA lung tissue slices from different patients responded distinctly to these anticancer agents, which could be translated into individual patients' response to drugs. Furthermore, to delineate systemic and microenvironmental influences on cancer metabolism, we compared and found that the ex vivo tissue slice cultures incorporated to a higher extent 13C from [13C6]-glucose into glycolytic, PPP, and purine nucleotide products than the corresponding mouse PDX in vivo, illustrating the high metabolic viability of the ex vivo tissue slice cultures. These new developments in preclinical models and mechanistic metabolic interrogations promise to provide rigorous prediction for individual patients' response to therapeutics while revealing new and exciting targets for the next generation of personalized therapeutics. Supported by 1R01CA118434-01A2, 1P01CA163223-01A1, 1R01ES022191-01, 3R01ES022191-04S1, 3R01CA118434-02S1, 1U24DK097215-01A1, P30CA177558; KLCRP, and the KY Challenge for Excellence.

Abstract 4400: Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells

Abstract Introduction and objectives: It is well known as Warburg effect that anaerobic glycolytic pathway is activated in various type of advanced cancers including prostate cancer (PC). Lactate dehydrogenase-A (LDH-A) controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. Since LDH-A pathways have been implicated in chemoresistance in various cancers, we investigated whether inhibition of LDH-A pathway could mediate the sensitivity to docetaxel (DOC) in human PC cells. Materials and Method: Four PC cell lines (PC3, DU145, LNCaP, LN-CSS ) were used. LN-CSS is one of the LNCaP-derived castration-resistant PC (CRPC) cell lines established in our laboratories. Sodium oxamate (SO) was used as a specific LDH-A inhibitor. The protein expression was detected by western blot analysis using specific antibodies. Cell growth and survival were evaluated by WST-1 assays. Cell cycle progression and apoptotic inducibility were evaluated by flow cytometry using propidium iodide and Annexin V. The cytotoxicity of SO/DOC combination on PC cells was evaluated using the Chou-Talalay combination index (CI) method which offers quantitative definition for additive effect (CI = 1), synergism (CI &amp;lt; 1), and antagonism (CI &amp;gt; 1) in drug combinations. Result: Western blot analysis showed that LDH-A protein was highly expressed in LN-CSS cells compared with other PC cell lines including the parental LNCaP. WST-1 assays showed that treatment with SO (50 mM) for 72 hours in PC cells resulted in growth inhibition (PC3; ~30%, DU145; ~55%, LNCaP; ~20%,LN-CSS; ~55% ), while SO has little growth inhibitory effects on normal lymphocytes in the concentrations between 1-100 mM. IC50 to DOC in PC cells were showed to be 4 nM, 1 nM, 1nM, and 4.5 nM in PC3, DU145, LNCaP and LN-CSS, respectively, suggesting that both PC3 and LN-CSS were relatively resistant to DOC compared with DU145 and LNCaP. Synergistic cytotoxicity was observed after the combination therapy with DOC and SO in LN-CSS (CI: 0.5) but not in PC3 (CI: 1.9), DU145 (CI: 2.0), or LNCaP (CI: 6.5). Cell cycle analyses revealed that the combination with DOC and SO for 72 hours resulted in the accumulation of cells in G2-M phase followed by sub-G1 accumulation in LN-CSS cells. Annexin V assays showed that 43% apoptosis was induced by the combination therapy in LN-CSS cells, while only 12% by DOC only in LN-CSS cells. Conclusion: Our results strongly suggest that LDH-A plays an important role in DOC resistance in advanced PC cells and inhibition of LDH-A promotes DOC-sensitivity especially in CRPC cells. Our study may provide valuable information for the future development of targeted therapies in patients with CRPC. Citation Format: Hiroyuki Muramatsu, Makoto Sumitomo, Shingo Morinaga, Hiroshi Saiki, Ikuo Kobayashi, Keishi Kajikawa, Genya Nishikawa, Yoshiharu Kato, Masahito Watanabe, Kent Kanao, Kogenta Nakamura, kazuhiro Yoshikawa. Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity predominantly in castration-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4400. doi:10.1158/1538-7445.AM2017-4400

MiR-30a-5p suppresses breast tumor growth and metastasis through inhibition of LDHA-mediated Warburg effect

Lactate dehydrogenase A (LDHA), a key enzyme regulating aerobic glycolysis, is overexpressed in many human cancers, and correlates with poor clinical outcomes. Aerobic glycolysis is a hallmark of cancer, and drugs targeting its regulators, including LDHA, are being developed. However, the mechanisms of LDHA inhibition and the physiological significance of the LDHA inhibitors in cancer cells are unclear. Here, we show that microRNA-30a-5p (miR-30a-5p) suppresses LDHA expression by directly targeting its 3′-UTR. Through inhibition of LDHA, miR-30a-5p dampens glycolysis by decreasing glucose uptake, lactate production, ATP generation, and extracellular acidification rate (ECAR), and increasing oxygen consumption rate (OCR) in breast cancer cells. Importantly, glycolysis regulated by miR-30a-5p is critical for its regulating breast tumor growth and metastasis both in vitro and in vivo. In breast cancer patients, miR-30a-5p expression is negatively correlated with LDHA expression. Moreover, patients who had increased glucose uptake in tumors assessed by PET scans showed decreased miR-30a-5p expression and increased expression of LDHA. Our findings provide clues regarding the role of miR-30a-5p as a tumor suppressor in breast cancer through the inhibition of LDHA. Targeting LDHA through miR-30a-5p could be a potential therapeutic strategy in breast cancer.

Proteomic identification of the lactate dehydrogenase A in a radioresistant prostate cancer xenograft mouse model for improving radiotherapy

Radioresistance is a major challenge for prostate cancer (CaP) metastasis and recurrence after radiotherapy. This study aimed to identify potential protein markers and signaling pathways associated with radioresistance using a PC-3 radioresistant (RR) subcutaneous xenograft mouse model and verify the radiosensitization effect from a selected potential candidate. PC-3RR and PC-3 xenograft tumors were established and differential protein expression profiles from two groups of xenografts were analyzed using liquid chromatography tandem-mass spectrometry. One selected glycolysis marker, lactate dehydrogenase A (LDHA) was validated, and further investigated for its role in CaP radioresistance. We found that 378 proteins and 51 pathways were significantly differentially expressed between PC-3RR and PC-3 xenograft tumors, and that the glycolysis pathway is closely linked with CaP radioresistance. In addition, we also demonstrated that knock down of LDHA with siRNA or inhibition of LDHA activity with a LDHA specific inhibitor (FX-11), could sensitize PC-3RR cells to radiotherapy with reduced epithelial-mesenchymal transition, hypoxia, DNA repair ability and autophagy, as well as increased DNA double strand breaks and apoptosis. In summary, we identified a list of potential RR protein markers and important signaling pathways from a PC-3RR xenograft mouse model, and demonstrate that targeting LDHA combined with radiotherapy could increase radiosensitivity in RR CaP cells, suggesting that LDHA is an ideal therapeutic target to develop combination therapy for overcoming CaP radioresistance.

Abstract 4271: Beta-glucan activates macrophages in human NSCLC demonstrated by Stable Isotope Resolved Metabolomics (SIRM)

Abstract Introduction: We use SIRM to evaluate metabolic reprogramming of lung cancer cells in monoculture, in mouse xenograft/explant models, and in NSCLC patients in situ (1,2). We have now extended the range of models to fresh human tissue slices, which retain the original tissue architecture and heterogeneity with a paired benign versus cancer tissue design under defined cell culture conditions. β-glucan is a polysaccharide that repolarizes tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype in mice (3). Here we report the activation of TAMs in human NSCLC tissue slices. Experimental: Freshly resected paired tissue slices from individual patients (approx. 1 mm or less thick and 5-40 mg wet weight) were incubated ± particulate β-glucan in standard cell culture conditions, with gentle rocking to enable efficient gas, nutrient, and waste product exchange. Tissue slices could be maintained metabolically viable for at least 48 h of incubation. The metabolic activity was determined by measuring the uptake and transformation of 13C and/or 15N-enriched common nutrient tracers such as glucose and glutamine, using high resolution mass spectrometry, GC-MS, and NMR after a period of incubation (2). Findings: Time-course analysis of the slices by NMR, MS, and histology revealed that NSCLC tissue slices, both benign and tumorous, retained their architecture and a broad spectrum of metabolic activities. Glucose and glutamine metabolism was reprogrammed in the tumor relative to the paired benign tissues ex vivo, as the in vivo case. The paired tissues from different patients showed significantly different metabolic responses to β-glucan, as expected Conclusions: This platform offers a human tissue model for preclinical studies on metabolic reprogramming of human cancer and stromal cells in their tissue context, and response to drug treatment (4). As the microenvironment of the target human tissue is maintained, including the resident immune cells, individualized response to immune-active agents can be determined in a clinically relevant setting. Supported by NCI P01CA163223-01 and NIEHS 1R01ES022191-01 1. Lane, A.N., Fan, T.W.-M., Bousamra II, M., et al. (2011) Clinical Applications of Stable Isotope-Resolved Metabolomics (SIRM) in Non-Small Cell Lung Cancer. Omics, 15, 173-182. 2. Sellers, K., Fox, M.P., Bousamra, M., II, et al. (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. Journal of Clinical Investigation, 125, 687-698. 3. Liu, M., Luo, F., Ding, C., et al. (2015) Particulate β-Glucan Converts Immunosuppressive Macrophages into M1 Phenotype Through Dectin-1-induced Syk-Card9-Erk Pathway and Raf-1-c-Maf Pathway. J. Immunol., 195, 5055-5065. 4. Xie, H., Hanai, J., Ren, et al. (2014) Targeting lactate dehydrogenase-A (LDH-A) inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor initiating cells. Cell Metabolism 19, 795-809 Citation Format: Teresa W-M Fan, Andrew N. Lane, Jun Yan, Richard M. Higashi, Jeremiah T. Martin, Michael Bousamra. Beta-glucan activates macrophages in human NSCLC demonstrated by Stable Isotope Resolved Metabolomics (SIRM). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4271.

MiR-200b is a key regulator of tumor progression and metabolism targeting lactate dehydrogenase A in human malignant glioma.

Lactate dehydrogenase A (LDHA) is involved in various cancers. In this study, we investigated the expression and function of LDHA in glioma. We found that LDHA was up-regulated in glioma samples. Furthermore, we found that overexpression of LDHA promoted proliferation, invasion and glycolysis in glioma cells. Luciferase reporter assays confirmed that LDHA was a direct target of miR-200b. miR-200b was found to be down-regulated in glioma samples, which was inversely correlated with LDHA expression. Repression of LDHA by miR-200b suppressed the glycolysis, cell proliferation and invasion of glioma cells. These results provide evidence that miR-200b acts as a tumor suppressor in glioma through the inhibition of LDHA both in vitro and in vivo. Targeting LDHA through miR-200b could be a potential therapeutic strategy in glioma.

Abstract 3199: Stable isotope resolved metabolomics (SIRM) on fresh human tissues as a preclinical drug testing platform

Abstract Introduction: All preclinical drug testing models have advantages and drawbacks. We have been using SIRM to evaluate metabolic reprogramming of lung cancer cells in monoculture, in mouse xenograft/explant models, and in NSCLC patients in situ (1), and to determine the influence of the tumor microenvironment using these models. We have now extended the range of models to fresh human tissue slices, similar to those originally described by Warburg (2), which retain the original tissue architecture and heterogeneity with a paired benign versus cancer design under controlled cell culture conditions. Experimental: Freshly resected tissue slices from individuals (ca. 1 mm or less thick and 5-40 mg wet weight) were incubated in standard cell culture conditions with gentle rocking for efficient gas, nutrient and waste product exchange. The metabolic activity was determined by measuring the uptake and transformation of 13C and/or 15N-enriched nutrient tracers such as glucose and glutamine, using high-resolution MS, GC-MS, and NMR after a period of incubation. Acute metabolic and histologic response to inhibitors or drugs was readily detected in treated tissue slices. Findings: Analysis at different time points by NMR, MS and histology shows that the NSCLC tissue slices, both benign and tumorous, retained their architecture and remained metabolically viable for at least 48 h of incubation. Glucose and glutamine metabolism was reprogrammed in the tumor relative to the paired benign tissues. The paired tissue also showed very different responses to Se-containing compounds when incubated at the IC50 established for cell lines. After 24 h of incubation, large scale necrosis was evident in the tumor, but not in the benign slices, which was accompanied by large changes in metabolic activities observed by SIRM analysis. Conclusions: This platform offers a human tissue model for preclinical studies on metabolic reprogramming of human cancer cells in their tissue context, and response to drug treatment (3). As the microenvironment of the target human tissue is retained and individualized response to drugs is obtained, this platform promises to transcend current limitations of drug selection for clinical trials or treatments. Supported by NCI P01CA163223-01A1 and NIEHS 1R01ES022191-01 1. Lane, A.N., Fan, T.W.-M., Bousamra II, M., Higashi, R.M., Yan, J. and Miller, D.M. (2011) Clinical Applications of Stable Isotope-Resolved Metabolomics (SIRM) in Non-Small Cell Lung Cancer. Omics, 15, 173-182. 2. Warburg, O. (1923) Versuche an überlebendem Carcinomgewebe (Methoden). Biochem. Zeitschr., 142, 317-333. 3. Xie, H., Hanai, J., Ren, J.-G., Kats, L., Burgess, K., Bhargava, P., Signoretti, S., Billiard, J., Duffy, K.J., Grant, A. et al. (2014) Targeting lactate dehydrogenase-A (LDH-A) inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor initiating cells. Cell Metabolism 19, 795-809. Citation Format: Andrew N. Lane, Teresa W-M Fan, Alexander C. Belshoff, Richard M. Higashi, Jeremiah Martin, Michael Bousamra. Stable isotope resolved metabolomics (SIRM) on fresh human tissues as a preclinical drug testing platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3199. doi:10.1158/1538-7445.AM2015-3199

408 Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity in prostate cancer cells

408 Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A promotes docetaxel induced cytotoxicity in prostate cancer cells

Targeting Lactate Dehydrogenase-A Inhibits Tumorigenesis and Tumor Progression in Mouse Models of Lung Cancer and Impacts Tumor-Initiating Cells

The lactate dehydrogenase-A (LDH-A) enzyme catalyzes the interconversion of pyruvate and lactate, is upregulated in human cancers, and is associated with aggressive tumor outcomes. Here we use an inducible murine model and demonstrate that inactivation of LDH-A in mouse models of NSCLC driven by oncogenic K-RAS or EGFR leads to decreased tumorigenesis and disease regression in established tumors. We also show that abrogation of LDH-A results in reprogramming of pyruvate metabolism, with decreased lactic fermentation invitro, invivo, and exvivo. This was accompanied by reactivation of mitochondrial function invitro, but not invivo or exvivo. Finally, using a specific small molecule LDH-A inhibitor, we demonstrated that LDH-A is essential for cancer-initiating cell survival and proliferation. Thus, LDH-A can be a viable therapeutic target for NSCLC, including cancer stem cell-dependent drug-resistant tumors.