TSPAN31 regulates the proliferation,migration, and apoptosis of gastric cancer cells through the METTL1/CCT2 pathway

Gastric cancer (GC) is one of the most common human malignancies worldwide, but the molecular mechanism of GC has not been fully elucidated. Tetraspanin 31 (TSPAN31) has been rarely studied in human malignant tumors. This study aimed to investigate the effects of TSPAN31 on GC. We analyzed GC tissues through high-throughput sequencing technology and chose TSPAN31 with high expression. The expression of TSPAN31 in GC was analyzed through bioinformatics website and qRT-PCR. The protein level of TSPAN31 in GC tissues was determined by western blot and immunochemistry. The proliferation, migration, and apoptosis of GC cells were detected by the cell counting kit-8, transwell, and apoptosis experiments. METTL1 and CCT2 that may co-express with TSPAN31 were predicted by the GEPIA database, and analyzed the correlation between the expression levels of TSPAN31, METTL1 and CCT2. The results shows TSPAN31 was highly expressed in GC tissues, and high expression of TSPAN31 was found to result in poor prognosis of patients with GC. TSPAN31 could regulate the proliferation, migration and apoptosis of GC cells. The relative expression levels of TSPAN31, METTL1 and CCT2 in GC were positively correlated. Low expression of TSPAN31 could partially reverse the effect of high expression of METTL1 and CCT2 on the tumor progression of GC cells. In conclusion, TSPAN31 was highly expressed in GC tissues and led to poor prognosis of patients with GC. TSPAN31 may regulate the proliferation, migration, and apoptosis of GC cells. This regulatory mechanism may be achieved through co-expression with METTL1 and CCT2.     

激活Toll样受体2可以促进胃癌细胞中的OXPHOS和糖酵解

目的:调查TLR家族中哪种TLR受体的配体依赖性激活可引起胃癌细胞的代谢重编程。方法:通过实时荧光定量PCR(RT-qPCR)和蛋白质印迹(WB)在一组人GC细胞中测量TLR家族成员的表达。通过进行Seahorse生物能测定以及测量L-乳酸和活性氧(ROS)的产生,确定激动剂对不同TLR(TLR2、4、9)诱导的人GC细胞的代谢变化;通过RT-qPCR在被刺激的GC细胞中分析了涉及氧化磷酸化和糖酵解的基因的表达;通过Western印迹表征SOD2的表达。结果:由合成分子或全病原体抗原激活的TLR2信号传导增强了胃癌细胞中高表达TLR2的细胞株的糖酵解活性和线粒体呼吸,而配体诱导的TLR4和TLR9活化抑制了线粒体呼吸或细胞外酸化率。同时,涉及葡萄糖代谢和氧化还原系统调节的基因,例如HIF1A,PFKFB3和SOD2,在TLRs下游被上调。结论:由配体诱导的特定TLRs的激活介导了人类GC细胞中不同的代谢表型。TLR2是唯一同时促进OXPHOS和糖酵解的家族成员,这可能导致肿瘤进展。     

METTL16 promotes cell proliferation by up-regulating cyclin D1 expression in gastric cancer

N6-methyladenosine (m6A) is a well-known modification of RNA. However, as a key m6A methyltransferase, METTL16 has not been thoroughly studied in gastric cancer (GC). Here, the biological role of METTL16 in GC and its underlying mechanism was studied. Immunohistochemistry was used to detect the expression of METTL16 and relationship between METTL16 level and prognosis of GC was analysed. CCK8, colony formation assay, EdU assay and xenograft mouse model were used to study the effect of METTL16. Regulatory mechanism of METTL16 in the progression of GC was studied through flow cytometry analysis, RNA degradation assay, methyltransferase inhibition assay, RT-qPCR and Western blotting. METTL16 was highly expressed in GC cells and tissues and was associated with prognosis. In vitro and in vivo experiments confirmed that METTL16 promoted proliferation of GC cells and tumour growth. Furthermore, down-regulation of METTL16 inhibited proliferation by G1/S blocking. Significantly, we identified cyclin D1 as a downstream effector of METTL16. Knock-down METTL16 decreased the overall level of m6A and the stability of cyclin D1 mRNA in GC cells. Meanwhile, inhibition of methyltransferase activity reduced the level of cyclin D1. METTL16-mediated m6A methylation promotes proliferation of GC cells through enhancing cyclin D1 expression.     

m6A methylation mediates LHPP acetylation as a tumour aerobic glycolysis suppressor to improve the prognosis of gastric cancer

LHPP, a histidine phosphatase, has been implicated in tumour progression. However, its role, underlying mechanisms, and prognostic significance in human gastric cancer (GC) are elusive. Here, we obtained GC tissues and corresponding normal tissues from 48 patients and identified LHPP as a downregulated gene via RNA-seq. qRT-PCR and western blotting were applied to examine LHPP levels in normal and GC tissues. The prognostic value of LHPP was elucidated using tissue microarray and IHC analyses in two independent GC cohorts. The functional roles and mechanistic insights of LHPP in GC growth and metastasis were evaluated in vitro and in vivo. The results showed that LHPP expression was significantly decreased in GC tissues at both the mRNA and protein levels. Multivariate Cox regression analysis revealed that LHPP was an independent prognostic factor and effective predictor in patients with GC. The low expression of LHPP was significantly related to the poor prognosis and chemotherapy sensitivity of gastric cancer patients. Moreover, elevated LHPP expression effectively suppressed GC growth and metastasis in vitro and in vivo. Mechanistically, the m6A modification of LHPP mRNA by METTL14 represses its expression; LHPP inhibits the phosphorylation of GSK3b through acetylation and mediates HIF1A to inhibit glycolysis, proliferation, invasion and metastasis of gastric cancer cells. Together, our findings suggest that LHPP is regulated by m6A methylation and regulates the metabolism of GC by changing the acetylation level. Thus, LHPP is a potential predictive biomarker and therapeutic target for GC.Subject terms: Gastric cancer, Predictive markers     

Bioenergetic Analysis of Ovarian Cancer Cell Lines: Profiling of Histological Subtypes and Identification of a Mitochondria-Defective Cell Line

Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines'' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1α in response to 1% O₂ hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs.     

NDUFA4L2 promotes glioblastoma progression,is associated with poor survival,and can be effectively targeted by apatinib

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) is a subunit of Complex I of the mitochondrial respiratory chain, which is important in metabolic reprogramming and oxidative stress in multiple cancers. However, the biological role and molecular regulation of NDUFA4L2 in glioblastoma (GBM) are poorly understood. Here, we found that NDUFA4L2 was significantly upregulated in GBM; the elevated levels were correlated with reduced patient survival. Gene knockdown of NDUFA4L2 inhibited tumor cell proliferation and enhanced apoptosis, while tumor cells initiated protective mitophagy in vitro and in vivo. We used lentivirus to reduce expression levels of NDUFA4L2 protein in GBM cells exposed to mitophagy blockers, which led to a significant enhancement of tumor cell apoptosis in vitro and inhibited the development of xenografted tumors in vivo. In contrast to other tumor types, NDUFA4L2 expression in GBM may not be directly regulated by hypoxia-inducible factor (HIF)-1α, because HIF-1α inhibitors failed to inhibit NDUFA4L2 in GBM. Apatinib was able to effectively target NDUFA4L2 in GBM, presenting an alternative to the use of lentiviruses, which currently cannot be used in humans. Taken together, our data suggest the use of NDUFA4L2 as a potential therapeutic target in GBM and demonstrate a practical treatment approach.Subject terms: CNS cancer, Mitophagy     

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.     

Energy metabolism in tumor cells

In early studies on energy metabolism of tumor cells, it was proposed that the enhanced glycolysis was induced by a decreased oxidative phosphorylation. Since then it has been indiscriminately applied to all types of tumor cells that the ATP supply is mainly or only provided by glycolysis, without an appropriate experimental evaluation. In this review, the different genetic and biochemical mechanisms by which tumor cells achieve an enhanced glycolytic flux are analyzed. Furthermore, the proposed mechanisms that arguably lead to a decreased oxidative phosphorylation in tumor cells are discussed. As the O(2) concentration in hypoxic regions of tumors seems not to be limiting for the functioning of oxidative phosphorylation, this pathway is re-evaluated regarding oxidizable substrate utilization and its contribution to ATP supply versus glycolysis. In the tumor cell lines where the oxidative metabolism prevails over the glycolytic metabolism for ATP supply, the flux control distribution of both pathways is described. The effect of glycolytic and mitochondrial drugs on tumor energy metabolism and cellular proliferation is described and discussed. Similarly, the energy metabolic changes associated with inherent and acquired resistance to radiotherapy and chemotherapy of tumor cells, and those determined by positron emission tomography, are revised. It is proposed that energy metabolism may be an alternative therapeutic target for both hypoxic (glycolytic) and oxidative tumors.     

Aerobic Glycolysis by Proliferating Cells: Protection against Oxidative Stress at the Expense of Energy Yield

Primary cultures of mitogen-activated rat thymocytes were used to study energy metabolism, gene expression of glycolytic enzymes, and production of reactive oxygen species during cell cycle progression. During transition from the resting to the proliferating state a 7- to 10-fold increase of glycolytic enzyme induction occurs which enables the cells to meet the enhanced energy demand by increased aerobic glycolysis. Cellular redox changes have been found to regulate gene expression of glycolytic enzymes by reversible oxidative inactivation of Spl-binding to the cognate DNA-binding sites in the promoter region. In contrast to nonproliferating cells, production of phorbol 12-myristate 13-acetate (PMA)-primed reactive oxygen species (ROS) in proliferating rat thymocytes and HL-60 cells is nearly abolished. Pyruvate, a product of aerobic glycolysis, is an effective scavenger of ROS, which could be shown to be generated mainly at the site of complex III of the mitochondrial respiratory chain. Aerobic glycolysis by proliferating cells is discussed as a means to minimize oxidative stress during the phases of the cell cycle when maximally enhanced biosynthesis and cell division do occur.     

Neurofilament Heavy Polypeptide Regulates the Akt-β-Catenin Pathway in Human Esophageal Squamous Cell Carcinoma

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Increase in mitochondrial biogenesis,oxidative stress,and glycolysis in murine lymphomas

Lymphomas adapt to their environment by undergoing a complex series of biochemical changes that are currently not well understood. To better define these changes, we examined the gene expression and gene ontology profiles of thymic lymphomas from a commonly used model of carcinogenesis, the p53^?/? mouse. These tumors show a highly significant upregulation of mitochondrial biogenesis, mitochondrial protein translation, mtDNA copy number, reactive oxygen species, antioxidant defenses, proton transport, ATP synthesis, hypoxia response, and glycolysis, indicating a fundamental change in the bioenergetic profile of the transformed T cell. Our results suggest that T cell tumorigenesis involves a simultaneous upregulation of mitochondrial biogenesis, mitochondrial respiration, and glycolytic activity. These processes would allow cells to adapt to the stressful tumor environment by facilitating energy production and thereby promote tumor growth. Understanding these adaptations is likely to result in improved therapeutic strategies for this tumor type.     

In vitro identification of mitochondrial oxidative stress production by time-resolved fluorescence imaging of glioma cells

Oxidative phosphorylation and glycolysis are important features, by which cells could bypass oxidative stress. The level of oxidative stress, and the ability of cells to promote oxidative phosphorylation or glycolysis, significantly determined proliferation or cell demise. In the present work, we have employed selective mitochondrial probe MitoTracker? Orange CMTM/Ros (MTO) to estimate the level of oxidative stress in cancer cells at different stressed conditions. MTO is partially sensitive to decrease of mitochondrial membrane potential and to reactive oxygen species (ROS) generated in mitochondria. We have demonstrated, that fluorescence lifetime of MTO is much more sensitive to oxidative stress than intensity-based approaches. This method was validated in different cancer cell lines. Our approach revealed, at relatively low ROS levels, that Gö 6976, a protein kinase C (PKC) α inhibitor, and rottlerin, an indirect PKCδ inhibitor, increased mitochondrial ROS level in glioma cell. Their involvement in oxidative phosphorylation and apoptosis was investigated with oxygen consumption rate estimation, western blot and flow-cytometric analysis. Our study brings new insight to identify feeble differences in ROS production in living cells.