Lactate Production Assays Research Articles

Effect of transcription factor WT1 on triple-negative breast cancer metastasis through PFKFB4-mediated glycolysis.

e13145 Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high probability of metastasis as well as a lack of specific targets and targeted therapeutics. Preliminary study suggested that Wilms' tumor gene 1 (WT1) is highly expressed in breast cancer patients with poor prognosis and may promote TNBC metastasis, but the underlying mechanism remains poorly defined. Methods: WT1 expression was evaluated by immunohistochemistry (IHC) in breast cancer patients. Kaplan–Meier survival analysis was performed to assess the prognostic significance of WT1 expression. WT1 was silenced in MDA-MB-231 and BT549 cells or overexpressed in HCC1806 cells. qRT-PCR and Western blot were used to detect the WT1 expression in tissues and cells. Wound healing assays, transwell assays and 3D spheroid assays were used to examine the migration and invasion abilities in TNBC cells. The lung metastasis model of mice was used to evaluate metastasis of TNBC in vivo. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome sequencing (RNA-seq) were performed to find PFKFB4, a downstream target gene for WT1. ChIP-PCR and dual-luciferase reporter assays were used to explore the transcriptional regulation of PFKFB4 by WT1. Seahorse XF glycolysis stress assay, glucose uptake assay, and lactate production assay were used to investigate the role of WT1 in regulating glycolysis metabolites. Results: WT1 was highly expressed in TNBC and correlated to poor prognosis in TNBC patients. Functional assays revealed that WT1 promoted TNBC cell metastasis in vitro and in vivo. Our mechanistic investigations demonstrated that WT1 promoted TNBC cells migration and invasion by transcriptionally activating the expression of PFKFB4.This action leaded to increased glycolytic capacity, glucose uptake, and lactate production in cancer cells, therefore promoting metastasis of TNBC. Clinically, the combined expression of WT1 and PFKFB4 provides a reliable predictive biomarker for the prognosis of TNBC patients. Conclusions: Our findings reveal a molecular mechanism of WT1 promoting TNBC metastasis, which provides new targets for the precision treatment of TNBC and new perspectives for the development of targeted metabolic anticancer drugs.

Electroporation enhances cell death in 3D scaffold-based MDA-MB-231 cells treated with metformin

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer lacks estrogen, progesterone, and HER2 receptors and hence, is therapeutically challenging. Towards this, we studied an alternate therapy by repurposing metformin (FDA-approved type-2 diabetic drug with anticancer properties) in a 3D-scaffold culture, with electrical pulses. 3D cell culture was used to simulate the tumor microenvironment more closely and MDA-MB-231, human TNBC cells, treated with both 5 mM metformin (Met) and 8 electrical pulses at 2500 V/cm, 10 µs (EP1) and 800 V/cm, 100 µs (EP2) at 1 Hz were studied in 3D and 2D. They were characterized using cell viability, reactive oxygen species (ROS), glucose uptake, and lactate production assays at 24 h. Cell viability, as low as 20 % was obtained with EP1 + 5 mM Met. They exhibited 1.65-fold lower cell viability than 2D with EP1 + 5 mM Met. ROS levels indicated a 2-fold increase in oxidative stress for EP1 + 5 mM Met, while the glucose uptake was limited to only 9 %. No significant change in the lactate production indicated glycolytic arrest and a non-conducive environment for MDA-MB-231 growth. Our results indicate that 3D cell culture, with a more realistic tumor environment that enhances cell death using metformin and electrical pulses could be a promising approach for TNBC therapeutic intervention studies.

Withaferin A Inhibits Liver Cancer Tumorigenesis by Suppressing Aerobic Glycolysis through the p53/IDH1/HIF-1α Signaling Axis.

The energy supply of certain cancer cells depends on aerobic glycolysis rather than oxidative phosphorylation. Our previous studies have shown that withaferin A (WA), a lactone compound derived from Withania somnifera, suppresses skin carcinogenesis at least partially by stabilizing IDH1 and promoting oxidative phosphorylation. Here, we have extended our studies to evaluate the anti-tumor effect of WA in liver cancer. Differential expression of glycolysis-related genes between liver cancer tissues and normal tissues and prognosis were verified using an online database. Glycolysis-related protein expression was detected using western blot after overexpression and knockdown of IDH1 and mitochondrial membrane potential assay based on JC-1, and mitochondrial complex I activity was also detected. The inhibitory effect of WA on the biological functions of HepG2 cells was detected along with cell viability using MTT assay, scratch assay, clone formation assay, glucose consumption and lactate production assay. Western blot and qRT-PCR were used to detect the expression of proteins and genes related to IDH1, p53 and HIF1α signaling pathways. We first identified that IDH1 expression was downregulated in human liver cancer cells compared to normal liver cells. Next, we found that treatment of HepG2 cells with WA resulted in significantly increased protein levels of IDH1, accompanied by decreased levels of several glycolytic enzymes. Furthermore, we found that WA stabilized IDH1 proteins by inhibiting the degradation by the proteasome. The tumor suppressor p53 was also upregulated by WA treatment, which played a critical role in the upregulation of IDH1 and downregulation of the glycolysis-related genes. Under hypoxic conditions, glycolysis-related genes were induced, which was suppressed by WA treatment, and IDH1 expression was still maintained at higher levels under hypoxia. Taken together, our results indicated that WA suppresses liver cancer tumorigenesis by p53-mediated IDH1 upregulation, which promotes mitochondrial respiration, thereby inhibiting the HIF-1α pathway and blocking aerobic glycolysis.

Gastric cancer mesenchymal stem cells promote tumor glycolysis and chemoresistance by regulating B7H3 in gastric cancer cells.

Despite surgical treatment combined with multidrug therapy having made some progress, chemotherapy resistance is the main cause of recurrence and death of gastric cancer (GC).Gastric cancer mesenchymal stem cells (GCMSCs)have been reported to be correlated with the limited efficacy of chemotherapy in GC, but the mechanism of GCMSCs regulating GC resistance needs to be further studied. The gene set enrichment analysis (GSEA)was performed to explore the glycolysis-related pathways heterogeneity across different cell subpopulations. Glucose uptake and lactate production assays were used to evaluate the importance of B7H3 expression in GCMSCs-treated GCcells. The therapeutic efficacy of oxaliplatin (OXA)and paclitaxel (PTX)was determined using CCK-8and colony formation assays. Signaling pathways altered by GCMSCs-CMwere revealed by immunoblotting. The expression of TNF-αin GCMSCs and bone marrow mesenchymal stem cells (BMMSCs)was detected by western blot analysis and qPCR. Our results showed that the OXA and PTX resistance of GC cells were significantly enhanced in the GCMSCs-CMtreated GC cells. Acquired OXA and PTX resistance was characterized by increased cell viability for OXA and PTX, the formation of cell colonies, and decreased levels of cell apoptosis, which were accompanied by reduced levels of cleaved caspase-3 and Bax expression, and increased levels of Bcl-2, HK2, MDR1, and B7H3 expression. Blocking TNF-αin GCMSCs-CM,B7H3 knockdown or the use of 2-DG,a key enzyme inhibitor of glycolysis in GC cells suppressed the OXA and PTX resistance of GC cells that had been treated with GCMSCs-CM. This study shows that GCMSCs-CMderived TNF-α could upregulate the expression of B7H3 of GC cells to promote tumor chemoresistance. Our results provide a new basis for the treatment of GC.

Hypoxia-induced SHMT2 protein lactylation facilitates glycolysis and stemness of esophageal cancer cells.

Esophageal cancer (EC) is a familiar digestive tract tumor with highly lethal. The hypoxic environment has been demonstrated to be a significant factor in modulating malignant tumor progression and is strongly associated with the abnormal energy metabolism of tumor cells. Serine hydroxymethyl transferase 2 (SHMT2) is one of the most frequently expressed metabolic enzymes in human malignancies. The study was designed to investigate the biological functions and regulation mechanisms of SHMT2 in EC under hypoxia. We conducted RT-qPCR to assess SHMT2 levels in EC tissues and cells (TE-1 and EC109). EC cells were incubated under normoxia and hypoxia, respectively, and altered SHMT2 expression was evaluated through RT-qPCR, western blot, and immunofluorescence. The biological functions of SHMT2 on EC cells were monitored by performing CCK-8, EdU, transwell, sphere formation, glucose uptake, and lactate production assays. The SHMT2 protein lactylation was measured by immunoprecipitation and western blot. In addition, SHMT2-interacting proteins were analyzed by bioinformatics and validated by rescue experiments. SHMT2 was notably upregulated in EC tissues and cells. Hypoxia elevated SHMT2 protein expression, augmenting EC cell proliferation, migration, invasion, stemness, and glycolysis. In addition, hypoxia triggered lactylation of the SHMT2 protein and enhanced its stability. SHMT2 knockdown impeded the malignant phenotype of EC cells. Further mechanistic studies disclosed that SHMT2 is involved in EC progression by interacting with MTHFD1L. Hypoxia-induced SHMT2 protein lactylation and upregulated its protein level, which in turn enhanced MTHFD1L expression and accelerated the malignant progression of EC cells.

Verteporfin Suppresses YAP-Induced Glycolysis in Breast Cancer Cells

Objective The inhibition of the Hippo pathway through targeting the Yes-associated protein (YAP) presents a novel and promising approach for treating tumors. However, the efficacy of YAP inhibitors in the context of breast cancer (BC) remains incompletely understood. Here, we aimed to investigate the involvement of YAP in BC's metabolic reprogramming and reveal the potential underlying mechanisms. To this end, we assessed the function of verteporfin (VP), a YAP-TEAD complex inhibitor, on the glycolytic activity of BC cells. Methods We evaluated the expression of YAP by utilizing immunohistochemistry (IHC) in BC patients who have undergone 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) prior to biopsy/surgery. We employed RNA immunoprecipitation (RIP) and fluorescent in situ hybridization (FISH) assays to assess the interaction between YAP mRNA and human antigen R (HuR) in BC cells. The biological importance of YAP in the metabolism and malignancy of BC was evaluated in vitro. Finally, the effect of VP on glycolysis was determined by using 18F-FDG uptake, glucose consumption, and lactate production assays. Results Our studies revealed that high expression of YAP was positively correlated with the maximum uptake value (SUVmax) determined by 18F-FDG PET/CT imaging in BC samples. Inhibition of YAP activity suppressed glycolysis in BC. The mechanism underlying this phenomenon could be the binding of YAP to HuR, which promotes glycolysis in BC cells. Treatment with VP effectively suppressed glycolysis induced by YAP overexpression in BC cells. Conclusion VP exhibited anti-glycolytic effect on BC cells, indicating its therapeutic value as an FDA-approved drug.

LncRNA AP000695.2 promotes glycolysis of lung adenocarcinoma via the miR-335-3p/TEAD1 axis.

AP000695.2 is a novel long non-coding RNA (lncRNA). Its aberrant high expression is remarkably associated with poor prognosis of patients with lung adenocarcinoma (LUAD). However, its role and underlying mechanism in LUAD remains unclear. Previous bioinformatics analysis indicated that AP000695.2 may be closely related to the glycolysis of LUAD. This study aims to verify and explore the mechanism of AP000695.2 in glycolysis of LUAD. Overexpression plasmid and siRNA are used to construct cell models of upregulation and downregulation of AP000695.2, respectively. AP000695.2 is highly expressed in lung cancer cell lines as revealed by qPCR. Western blot analysis, FDG uptake, lactate production assay and ECAR determination results show that high expression of AP000695.2 facilitates glycolysis of LUAD cells. CCK-8, EdU staining, Transwell and wound healing assays show that high expression of AP000695.2 promotes cell growth and migration of LUAD. The relationship between AP000695.2 and miR-335-3p is confirmed by bioinformatics analysis and dual-luciferase reporter assays. Through the dual-luciferase reporter assay, TEA domain transcription factor 1 (TEAD1) is identified as a target gene of miR-335-3p. Rescue experiments are applied to verify the relationship among AP000695.2, miR-335-3p and TEAD1. Our study indicates that AP000695.2 is involved in the mechanism of LUAD through functioning as a ceRNA to competitively sponge miR-335-3p, thereby regulating the expression of TEAD1. In the in vivo models, AP000695.2 depletion restrains tumor growth and glycolysis. AP000695.2 promotes the glycolysis of LUAD by regulating the miR-335-3p/TEAD1 axis, and it may serve as a potential target of anti-tumor energy metabolism therapy.

CircAGFG1 absence decreases PKM2 expression to enhance oxaliplatin sensitivity in colorectal cancer in a miR-7-5p-dependent manner

Circular RNA (circRNA) ArfGAP with FG repeats 1 (circAGFG1) contributes to colorectal cancer (CRC) development. However, whether circAGFG1 regulates the resistance of CRC to oxaliplatin (L-OHP) remains unknown. CircAGFG1, microRNA-7-5p (miR-7-5p) and pyruvate kinase M2 (PKM2) RNA expression were quantified by quantitative real-time polymerase chain reaction. Protein expression was detected by western blot assay and immunohistochemistry assay. Glycolysis was analyzed through glucose uptake, lactate production and adenosine triphosphate (ATP) concentration assays. 50% inhibitory concentration of L-OHP was determined by cell counting kit-8 assay. Cell proliferation and apoptotic rate were analyzed by cell colony formation and flow cytometry analysis, respectively. Dual-luciferase reporter assay was used to identify the relationship among circAGFG1, miR-7- 5p and PKM2. The effect of circAGFG1 on L-OHP sensitivity in vivo was further evaluated by a xenograft model assay. CircAGFG1 and PKM2 expression were significantly increased, while miR-7-5p was decreased in L-OHP-resistant CRC tissues and cells. High circAGFG1 expression predicted a poor prognosis of CRC. CircAGFG1 knockdown or PKM2 depletion decreased glycolysis and cell proliferation and increased L-OHP sensitivity and cell apoptosis. PKM2 introduction rescued circAGFG1 silencing-induced effects in CRC cells. In terms of mechanism, circAGFG1 bound to miR-7-5p, which was identified to target PKM2. Also, circAGFG1 regulated PKM2 expression by interacting with miR-7-5p. Further, circAGFG1 knockdown improved the sensitivity of tumors to L-OHP in vivo. CircAGFG1 depletion inhibited L-OHP resistance by regulating the miR-7-5p/PKM2 pathway.

Matrine suppresses liver cancer progression and the Warburg effect by regulating the circROBO1/miR-130a-5p/ROBO1 axis.

Matrine, an effective component extracted from the traditional Chinese herb, Sophora flavescens, has been indicated to exert antitumor activity in different types of cancer. However, the role and precise mechanism of matrine in the progression of liver cancer remains largely unclear. Cell viability, cell proliferation, cell apoptosis, and Warburg effect were estimated by cell counting kit-8 assay, colony formation assay, flow cytometry assay, and glucose uptake and lactate production assay, respectively. The candidate Circular RNAs (circRNAs) were screened by integrating the Gene Expression Omnibus database (GSE155949) analysis with the online program GEO2R. A quantitative real-time polymerase chain reaction was employed to test the expression of circRNA circROBO1, microRNA miR-130a-5p, and roundabout homolog 1 (ROBO1). The interaction of circROBO1/miR-130a-5p/ROBO1 axis was predicted and confirmed by bioinformatics analysis, a dual-luciferase reporter assay, and an RNA pull-down assay. A xenograft mouse model was employed to reveal the role of matrine in vivo. Matrine repressed liver cancer cell viability, proliferation, and Warburg effect, but increased cell apoptosis in vitro. CircROBO1 and ROBO1 were upregulated, but miR-130a-5p was downregulated in liver cancer tissues. Additionally, matrine could reduce the expression of circROBO1 and ROBO1, and increase the expression of miR-130a-5p. Mechanically, overexpression of circROBO1 partly recovered the effect of matrine on liver cancer cell viability, proliferation, apoptosis, and Warburg effect by regulating the miR-130a-5p/ROBO1 axis. Matrine impeded liver cancer development by mediating the circROBO1/miR-130a-5p/ROBO1 axis, which provided a theoretical basis for the application of matrine as an effective anticancer drug for liver cancer.

The SIX1/LDHA Axis Promotes Lactate Accumulation and Leads to NK Cell Dysfunction in Pancreatic Cancer.

Pancreatic cancer (PC) is a malignant cancer with poor prognosis and high mortality rate. Sine oculis homeobox homolog 1 (SIX1) participates in the development of many cancers. However, the function of SIX1 in PC is not fully understood. SIX1 expression was determined using immunohistochemistry in PC tissues and cell lines. Glucose consumption, lactate production, and ATP assays were used to detect the function of SIX1. PC cells and NK cells were cocultured to study the effect of SIX1 overexpression in PC cells on NK cell function. Chromatin immunoprecipitation (ChIP) assays were used to study the relationship between SIX1 and lactate dehydrogenase A (LDHA). A series of in vitro and in vivo assays were further applied to elucidate the important role of the SIX1/LDHA axis in metabolism and NK cell dysfunction in PC. SIX1 was significantly upregulated in PC tissue; SIX1 overexpression promoted the glycolysis capacity of PANC-1 and CFPAC-1 cells and resulted in NK cell dysfunction after the NK cells had been cultured with PC cells. LDHA inhibitor partially restored the promotion of PC caused by SIX1 overexpression. According to ChIP assays, SIX1 directly binds to the LDHA promoter region. Moreover, LDHA inhibitor and lactate transporter blocker treatment promoted the function of NK cells cocultured with PC cells. In vivo experiments yielded the same results. The SIX1/LDHA axis promotes lactate accumulation and leads to NK cell dysfunction in PC.

ASPP2 suppresses tumour growth and stemness characteristics in HCC by inhibiting Warburg effect via WNT/β-catenin/HK2 axis.

Abnormal energy metabolism is one of the characteristics of tumours. In the last few years, more and more attention is being paid to the role and regulation of tumour aerobic glycolysis. Cancer cells display enhanced aerobic glycolysis, also known as the Warburg effect, whereby tumour cells absorb glucose to produce a large amount of lactic acid and energy under aerobic conditions to favour tumour proliferation and metastasis. In this study, we report that the haploinsufficient tumour suppressor ASPP2, can inhibit HCC growth and stemness characteristics by regulating the Warburg effect through the WNT/β-catenin pathway. we performed glucose uptake, lactate production, pyruvate production, ECAR and OCR assays to verify ASPP2 can inhibit glycolysis in HCC cells. The expression of ASPP2 and HK2 was significantly inversely correlated in 80 HCC tissues. Our study reveals downregulation of ASPP2 can promote the aerobic glycolysis metabolism pathway, increasing HCC proliferation, glycolysis metabolism, stemness and drug resistance. This ASPP2-induced inhibition of glycolysis metabolism depends on the WNT/β-catenin pathway. ASPP2-regulated Warburg effect is associated with tumour progression and provides prognostic value. and suggest that may be promising as a new therapeutic strategy in HCC.

Circular RNA, circular RARS, promotes aerobic glycolysis of non-small-cell lung cancer by binding with LDHA.

Accumulating evidence has highlighted the critical roles of circular RNAs (circRNAs) in non-small-cell lung cancer (NSCLC). This study aims to unveil the roles of circRARS (circular RARS) (hsa_circ_0001551) in NSCLC. Quantitative real-time PCR was used to determine the expression of circRARS in NSCLC tissues and cells. Kaplan-Meier analysis was used to determine the prognostic value of circRARS expression. CCK8, transwell, and wound healing assays were used to assess the proliferation, invasion, and migration abilities of NSCLC cells. RNA pull-down, cell fraction, glucose consumption, lactate production, and lactate dehydrogenase activity assays were conducted to explore the potential mechanisms of circRARS in NSCLC. circRARS is upregulated in NSCLC tissues and positively correlated with smoking status, lymph node metastasis, and higher tumor stages. NSCLC patients with high expression of circRARS have poor overall survival. Functional assays demonstrated that circRARS accelerated the proliferation, invasion, and migration of NSCLC cells in vitro. The cell fraction suggested that circRARS mainly accumulated in cytoplasm and the RNA pull-down assay showed lactate dehydrogenase (LDHA) could bind with circRARS. Furthermore, circRARS positively regulates LDHA activity and LDHA expression at the transcription level. Moreover, downregulated circRARS decreases glucose consumption and lactate production and compromises aerobic glycolysis in NSCLC cells. Finally, rescue assays showed circRARS could promote NSCLC cell proliferation by regulating LDHA activity. This study shows that circRARS can promote glycolysis and tumor progression in NSCLC by regulating LDHA.

MiR-194-5p suppresses the warburg effect in ovarian cancer cells through the IGF1R/PI3K/AKT axis

Background: The Warburg effect is considered as a hallmark of various types of cancers, while the regulatory mechanism is poorly understood. Our previous study demonstrated that miR-194-5p directly targets and regulates insulin-like growth factor1 receptor (IGF1R). In this study, we aimed to investigate the role of miR-194-5p in the regulation of the Warburg effect in ovarian cancer cells. Methods: The stable ovarian cell lines with miR-194-5p overexpression or silencing IGF1R expression were established by lentivirus infection. ATP generation, glucose uptake, lactate production and extracellular acidification rate (ECAR) assay were used to analyze the effects of aerobic glycolysis in ovarian cancer cells. Gene expression was analyzed by quantitative polymerase chain reaction (qPCR) and western blot. Immunohistochemistry assays were performed to assess the expression of the IGF1R protein in ovarian cancer tissues. Results: Overexpression of miR-194-5p or silencing IGF1R expression in ovarian cancer cells decreases ATP generation, glucose uptake, lactate production, and ECAR and inhibits both the mRNA and protein expression of PKM2, LDHA, GLUT1, and GLUT3. While the knockdown of miR-194-5p expression led to opposite results. Overexpression of miR-194-5p or silencing IGF1R expression suppressed the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway, whose activation can sustain aerobic glycolysis in cancer cells, and the knockdown of miR-194-5p expression promoted the activation of the PI3K/AKT pathway. Conclusion: Our results suggest that miR- 194-5p can inhibit the Warburg effect by negative regulation of IGF1R and further repression of the PI3K/AKT pathway, which provides a theoretical basis for further test of miR-194-5p as a target in the treatment of ovarian cancer.

CircTUBGCP5 promotes liver cancer progression and glycolysis by up-regulating the expression of ACSL4

A large number of researches have shown that circular RNA (circRNA) is new hope for the diagnosis or treatment of tumors, including liver cancer (LCa). However, it remains largely unclear the role of circRNA in the progression of LCa and its molecular mechanism. GSE164803 microarray dataset was applied to identify dysregulated circRNAs in LCa and noncancerous tissues. CircTUBGCP5 (hsa_circ_0034049) was selected for further research. Biological functions of circTUBGCP5 were investigated by EdU, colony formation, flow cytometry, glucose consumption and lactate production assay, and in vivo tumorigenesis. RNA pull-down assay and dual-luciferase reporter assay were used to investigate the interaction between circTUBGCP5, miR-144-3p, and ACSL4. We demonstrated that circTUBGCP5 was significantly up-regulated in LCa tissues and cells. CircTUBGCP5 promoted LCa cell proliferation, anti-apoptotic ability, glycolysis, and tumorigenesis at least partially by sponging miR-144-3p to regulate ACSL4 protein level. In conclusion, circTUBGCP5 is a forceful contributor to malignant behaviors and glycolysis of LCa via modulating the circTUBGCP5/miR-144-3p/ACSL4 axis, which has provided a target for the diagnosis and treatment of LCa patients.

MiR-532–3p suppresses proliferation and invasion of ovarian cancer cells via GPNMB/HIF-1α/HK2 axis

ObjectiveIdentifying a new target of miR-532–3p and studying its functional mechanism to explore the detailed anti-tumor mechanism of miR-532–3p in ovarian cancer. MethodsBiological and molecular methods including real-time quantitative PCR (RT-qPCR), Western blotting, colony formation, in vitro migration and invasion assays, glucose consumption and lactate production assays, RNA interference and tumor xenograft mouse models were used to study the role of miR-532–3p and its target in ovarian cancer. mRNA sequencing, dual-luciferase reporter assay and immunohistochemistry (IHC) were used to identify miR-532–3p target. STRING dataset analysis, qPCR and Western blotting were used to investigate the downstream pathway of the target of miR-532–3p. ResultsForced expression of miR-532–3p inhibited the proliferation, migration and invasion of ovarian cancer cells in vitro and the tumor growth in nude mice. RNA sequencing found 299 mRNAs were downregulated in miR-532–3p-overexpressed ovarian cancer cells, and bioinformatic analysis indicated Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB), a type I membrane glycoprotein, was the potential target of miR-532–3p. GPNMB was reduced at both RNA and protein levels in miR-532–3p-overexpressed ovarian cancer cells. Dual-luciferase reporter assay determined GPNMB as the target of miR-532–3p. Interference of GPNMB inhibited the proliferation, migration, invasion, glucose consumption and lactate production of ovarian cancer cells. Knocking down of GPNMB reduced the protein level of HIF-1α without affecting HIF-1α mRNA level. Overexpression of GPNMB reversed the antitumor effect of miR-532–3p. ConclusionmiR-532–3p exerted the anti-cancer effect by targeting GPNMB/ HIF-1α/ HK2 pathway to inhibit aerobic glycolysis in ovarian cancer.

Disulfiram/copper induces antitumor activity against gastric cancer cells in vitro and in vivo by inhibiting S6K1 and c-Myc.

Disulfiram (DSF) is an approved drug for the treatment of alcohol dependence. Accumulating evidence indicates that DSF, alone or in combination with copper (Cu), possesses strong antitumor activity in various malignancies. This study investigated the effects of DSF on gastric cancer (GC) and the potential mechanisms involved. GC cell proliferation and apoptosis upon treatment with DSF with or without copper were analyzed using CCK-8 assay, colony formation assay, and flow cytometry. Glucose metabolism was investigated using glucose consumption and lactate production assays. The expression of caspase-3, Bcl-2, LC-3, P62, S6K1, c-Myc, GLUT1, PKM2, and LDHA was analyzed using western blot assay. In vivo nude mice studies were performed to verify the findings from in vitro analyses. Our study showed that DSF was highly toxic to GC cells in a Cu-dependent manner. Nontoxic concentrations of Cu enhanced the inhibitory effects of DSF on cell viability and colony formation. DSF also induced apoptotic and autophagic cell death in the presence of Cu. In addition, DSF/Cu inhibited glycolysis and xenograft growth of GC cells by suppressing the expression of S6K1, c-Myc, and their downstream molecules, including GLUT1, PKM2, and LDHA. Our study demonstrated that DSF/Cu exerted antitumor activity against GC cells both in vitro and in vivo. DSF/Cu may represent a promising therapeutic strategy for the treatment of GC.

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

BackgroundProtein 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.MethodsThe 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.ResultsThe 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.ConclusionsOur 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.

Hsa_circ_0023990 Promotes Tumor Growth and Glycolysis in Dedifferentiated TC via Targeting miR-485-5p/FOXM1 Axis

During the transformation to dedifferentiated thyroid cancer (TC) types, the ability of papillary thyroid carcinomas (PTCs) to concentrate radioactive iodine might be lost, raising difficulty for the current therapy. circRNAs were proved to be implicated in the progression of various cancers. In this study, we aimed to investigate the functional role and mechanism of hsa_circ_0023990 in dedifferentiated TC. The expression pattern of genes were detected using quantitative PCR or western blot assays. Cell proliferation was determined by CCK8, colony formation, EdU, and cell-cycle assays. Glycolysis was assessed using glucose uptake and lactate production assays. Luciferase reporter assay was performed to examine the interactions between miR-485-5p and hsa_circ_0023990 or FOXM1. Xenograft assay was allowed for observation of tumor growth in vivo. Hsa_circ_0023990 and FOXM1 were upregulated in dedifferentiated TC tissues and cell lines. The higher level of hsa_circ_0023900 could stimulate the proliferation and glycolysis of dedifferentiated TC cells via positively regulating FOXM1. Mechanistically, miR-485-5p was demonstrated to interact with hsa_circ_0023990 and FOXM1 and involved in the regulation of has_circ_0023990 and FOXM1 in TC biological processes. Our results discovered the functional network of hsa_circ_0023990 in dedifferentiated TC development by facilitating cell proliferation and glycolysis via miR-485-5p/FOXM1 axis, implying that hsa_circ_0023990 might be a potential therapeutic target for the dedifferentiated TC treatment.

Downregulation of GLUT3 impairs STYK1/NOK-mediated metabolic reprogramming and proliferation in NIH-3T3 cells.

Serine threonine tyrosine kinase 1 (STYK1)/novel oncogene with kinase domain (NOK) has been demonstrated to promote cell carcinogenesis and tumorigenesis, as well as to strengthen cellular aerobic glycolysis, which is considered to be a defining hallmark of cancer. As the carriers of glucose into cells, glucose transporters (GLUTs) are important participants in cellular glucose metabolism and even tumorigenesis. However, to the best of our knowledge, the role of GLUTs in biological events caused by STYK1/NOK has not yet been reported. The present study assessed GLUT3 as a key transporter, and glucose consumption and lactate production assays revealed that downregulation of GLUT3 impaired STYK1/NOK-induced augmented glucose uptake and lactate production, and RT-qPCR and western blotting confirmed that GLUT3 knockdown attenuated the STYK1/NOK-induced increase in the expression levels of key enzymes implicated in glycolysis. Furthermore, MTT and Transwell assays demonstrated that STYK1/NOK-triggered cell proliferation and migration were also markedly decreased following knockdown of GLUT3. To the best of our knowledge, the present study is the first to demonstrate that GLUT3 serves a prominent role in STYK1/NOK-driven aerobic glycolysis and cell proliferation characteristics. These findings may provide a clue for the investigation of the oncogenic activity of STYK1/NOK and for the identification of potential tumor therapy targets associated with GLUT3.

Increased M2 Isoform of Pyruvate Kinase in Fibroblasts Contributes to the Growth, Aggressiveness, and Osteoclastogenesis of Odontogenic Keratocysts

To investigate the role of glycolysis and the M2 isoform of pyruvate kinase (PKM2) in odontogenic keratocysts (OKCs), the glycolytic flux of primary odontogenic keratocyst fibroblasts (OKC-Fs) and normal oral mucosa fibroblasts (OM-Fs) was determined by glucose uptake, lactate production, and cell proliferation assays. Wound healing assay and Matrigel-coated chamber system were used to investigate the effects of PKM2 on migration and invasion capacities of OKC-Fs. Co-culture of OKC-Fs with osteoclast precursors (RAW264.7 cells) was used to clarify the role of glycolysis in the osteoclastogenic effects of OKC-Fs. In addition, hypoxia-inducible factor 1α and some key enzymes related to glycolysis, including PKM2, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A, were detected to assess the activation of glycolysis in OKC stroma by immunohistochemistry. Results showed that the glucose uptake and lactate production were significantly higher in OKC-Fs than OM-Fs. PKM2 was elevated in OKC-Fs compared with that in OM-Fs. PKM2 significantly regulated glycolysis, proliferation, migration, invasion, and osteoclastogenic effects of OKC-Fs. Additionally hypoxia-inducible factor 1α, 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3, hexokinase 2, and lactate dehydrogenase A were markedly overexpressed in OKC stroma, and correlated with PKM2. Moreover, the expression of PKM2 was regulated by oxygen concentration invitro. In sum, PKM2-mediated glycolysisregulated the growth, aggressiveness, and osteoclastogenesis of OKC.