Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.     

H19 promotes aerobic glycolysis,proliferation, and immune escape of gastric cancer cells through the microRNA-519d-3p/lactate dehydrogenase A axis

Long noncoding RNAs (lncRNAs) have been investigated in multiple human cancers including gastric cancer (GC). Our research aims to explore the role of H19 in aerobic glycolysis, proliferation, and immune escape of GC cells. The expression of H19 in GC samples was analyzed using Gene Expression Profiling Interactive Analysis, Gene Expression Omnibus data, and real-time quantitative PCR analysis. Relative quantification of glucose consumption and lactate production from cell supernatant were applied to assess the aerobic glycolysis of GC cells. Subcellular fractionation, luciferase reporter, and western blot assays certified the binding between genes. Cell Counting Kit-8 and colony formation assays were used to determine GC cell proliferation. Flow cytometry, ELISA, and real-time quantitative PCR assays were applied to analyze the immunosuppressive effect of H19. H19 was highly expressed in samples of patients with GC, and associated with tumor growth in vivo. H19 knockdown suppressed glucose consumption, lactate production, and proliferation of GC cells by regulating the microRNA (miR)-519d-3p/lactate dehydrogenase A (LDHA) axis. Both miR-519d-3p depletion and LDHA overexpression could reverse the H19 knockdown-induced decrease in aerobic glycolysis and proliferation. Moreover, conditioned medium from stable knockdown H19 GC cells modulated the activity of immune cells including γδT cells, Jurkat cells, and tumor-associated macrophages in a miR-519d-3p/LDHA/lactate axis-dependent manner. The H19/miR-519d-3p/LDHA axis mainly contributed to aerobic glycolysis, proliferation, and immune escape of GC cells.     

The actin-associating protein Tm5NM1 blocks mesenchymal motility without transition to amoeboid motility

Cell migration is an integral component of metastatic disease. The ability of cells to transit between mesenchymal and amoeboid modes of migration has complicated the development of successful therapies designed to target cell migration as a means of inhibiting metastasis. Therefore, investigations of the mechanisms that regulate cell migration and render cells stationary are necessary. Tropomyosins are actin-associating proteins that regulate the activity of several effectors of actin filament dynamics. Previously, we have shown that the tropomyosin isoform Tm5NM1 stabilizes actin filaments and inhibits cell migration in a two-dimensional culture system. Here, we show that Tm5NM1 inhibits the mesenchymal migration of multiple cell lines in an isoform-specific manner. Tm5NM1 stimulates the downregulation of Src kinase activity and a rounded or elliptical morphology in three-dimensional collagen gels, and cells have dramatically reduced capacity to form pseudopodia. Importantly, we find that Tm5NM1 inhibits both the mesenchymal to amoeboid and amoeboid to mesenchymal transitions. Collectively, our data suggest that mimicking the action of Tm5NM1 overexpression represents an approach for effectively inhibiting the mesenchymal mode of migration.     

Development of a rational strategy for integration of lactate dehydrogenase A suppression into therapeutic algorithms for head and neck cancer

Background Lactate dehydrogenase (LDH) is a critical metabolic enzyme. LDH A (LDHA) overexpression is a hallmark of aggressive malignancies and has been linked to tumour initiation, reprogramming and progression in multiple tumour types. However, successful LDHA inhibition strategies have not materialised in the translational and clinical space. We sought to develop a rational strategy for LDHA suppression in the context of solid tumour treatment.Methods We utilised a doxycycline-inducible short hairpin RNA (shRNA) system to generate LDHA suppression. Lactate and LDH activity levels were measured biochemically and kinetically using hyperpolarised ¹³C-pyruvate nuclear magnetic resonance spectroscopy. We evaluated effects of LDHA suppression on cellular proliferation and clonogenic survival, as well as on tumour growth, in orthotopic models of anaplastic thyroid carcinoma (ATC) and head and neck squamous cell carcinoma (HNSCC), alone or in combination with radiation.Results shRNA suppression of LDHA generated a time-dependent decrease in LDH activity with transient shifts in intracellular lactate levels, a decrease in carbon flux from pyruvate into lactate and compensatory shifts in metabolic flux in glycolysis and the Krebs cycle. LDHA suppression decreased cellular proliferation and temporarily stunted tumour growth in ATC and HNSCC xenografts but did not by itself result in tumour cure, owing to the maintenance of residual viable cells. Only when chronic LDHA suppression was combined with radiation was a functional cure achieved.Conclusions Successful targeting of LDHA requires exquisite dose and temporal control without significant concomitant off-target toxicity. Combinatorial strategies with conventional radiation are feasible as long as the suppression is targeted, prolonged and non-toxic.Subject terms: Cancer therapy, Thyroid cancer, Cancer metabolism, Head and neck cancer     

p53/Lactate dehydrogenase A axis negatively regulates aerobic glycolysis and tumor progression in breast cancer expressing wild‐type p53

Tumor suppressor p53 is a master regulator of apoptosis and plays key roles in cell cycle checkpoints. p53 responds to metabolic changes and alters metabolism through several mechanisms in cancer. Lactate dehydrogenase A (LDHA), a key enzyme in glycolysis, is highly expressed in a variety of tumors and catalyzes pyruvate to lactate. In the present study, we first analyzed the association and clinical significance of p53 and LDHA in breast cancer expressing wild‐type p53 (wt‐p53) and found that LDHA mRNA levels are negatively correlated with wt‐p53 but not with mutation p53 mRNA levels, and low p53 and high LDHA expression are significantly associated with poor overall survival rates. Furthermore, p53 negatively regulates LDHA expression by directly binding its promoter region. Moreover, a series of LDHA gain‐of‐function and rescore experiments were carried out in breast cancer MCF7 cells expressing endogenous wt‐p53, showing that ectopic expression of p53 decreases aerobic glycolysis, cell proliferation, migration, invasion and tumor formation of breast cancer cells and that restoration of the expression of LDHA in p53‐overexpressing cells could abolish the suppressive effect of p53 on aerobic glycolysis and other malignant phenotypes. In conclusion, our findings showed that repression of LDHA induced by wt‐p53 blocks tumor growth and invasion through downregulation of aerobic glycolysis in breast cancer, providing new insights into the mechanism by which p53 contributes to the development and progression of breast cancer.     

Profilin-1 versus profilin-2: two faces of the same coin?

Proteins belonging to the profilin family of actin-binding proteins are considered to be important control elements for actin polymerization and have been linked to a broad spectrum of cellular functions, including cell migration. An intriguing paper recently published in Cancer Cell unveils differential effects of profilin-1 and profilin-2, the two major isoforms of profilin, on actin cytoskeletal regulation, motility, and invasion of breast cancer cells, and further establishes a mechanism underlying profilin-2''s suppressive effect on breast cancer cell migration. This viewpoint discusses the implications of these findings in the context of how profilins might regulate breast cancer cell motility.     

Lactate dehydrogenase A is overexpressed in pancreatic cancer and promotes the growth of pancreatic cancer cells

The prognosis for pancreatic cancer is very poor, and developing new therapeutic strategies for this cancer is needed. Recently, the Warburg effect (aerobic glycolysis) has attracted much attention for its function in the tumorigenesis. Lactate dehydrogenase A (LDHA) executes the final step of aerobic glycolysis and has been reported to be involved in the tumor progression. However, the function of LDHA in pancreatic cancer has not been studied. Here, we found that the expression of LDHA was elevated in the clinical pancreatic cancer samples. Forced expression of LDHA promoted the growth of pancreatic cancer cells, while knocking down the expression of LDHA inhibited cell growth dramatically. Moreover, silencing the expression of LDHA inhibited the tumorigenicity of pancreatic cancer cells in vivo. Mechanistically, knocking down the expression of LDHA activated apoptosis pathway. Taken together, our study revealed the oncogenic role of LDHA in pancreatic cancer and suggested that LDHA might be a potential therapeutic target.     

MUC16-mediated activation of mTOR and c-MYC reprograms pancreatic cancer metabolism

MUC16, a transmembrane mucin, facilitates pancreatic adenocarcinoma progression and metastasis. In the current studies, we observed that MUC16 knockdown pancreatic cancer cells exhibit reduced glucose uptake and lactate secretion along with reduced migration and invasion potential, which can be restored by supplementing the culture media with lactate, an end product of aerobic glycolysis. MUC16 knockdown leads to inhibition of mTOR activity and reduced expression of its downstream target c-MYC, a key player in cellular growth, proliferation and metabolism. Ectopic expression of c-MYC in MUC16 knockdown pancreatic cancer cells restores the altered cellular physiology. Our LC-MS/MS based metabolomics studies indicate global metabolic alterations in MUC16 knockdown pancreatic cancer cells, as compared to the controls. Specifically, glycolytic and nucleotide metabolite pools were significantly decreased. We observed similar metabolic alterations that correlated with MUC16 expression in primary tumor tissue specimens from human pancreatic adenocarcinoma cancer patients. Overall, our results demonstrate that MUC16 plays an important role in metabolic reprogramming of pancreatic cancer cells by increasing glycolysis and enhancing motility and invasiveness.     

LDHA is necessary for the tumorigenicity of esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal tumors in the world, and the development of new therapeutic targets is needed. Recent studies have shown that aerobic glycolysis, also known as the Warburg effect, mediated the anti-apoptotic effects in cancer cells. Lactate dehydrogenase A (LDHA) which executed the final step of aerobic lactate production has been reported to be involved in the tumor progression. However, the function of LDHA in ESCC has not been investigated. In this study, it was found that LDHA was up-regulated in ESCC clinical samples. Knockdown of the expression of LDHA inhibited cell growth and cell migration in vitro as well as tumorigenesis in vivo. With regard to the molecular mechanism, silencing the expression of LDHA was related to decreased AKT activation and cyclin D1 expression and increased cleavage of PARP and caspase 8. Taken together, our findings suggest that LDHA plays an important role in the progression of ESCC by modulating cell growth, and LDHA might be a potential therapeutic target in ESCC.     

Increased expression of the E3 ubiquitin ligase RNF5 is associated with decreased survival in breast cancer

The selective ubiquitination of proteins by ubiquitin E3 ligases plays an important regulatory role in control of cell differentiation, growth, and transformation and their dysregulation is often associated with pathologic outcomes, including tumorigenesis. RNF5 is an E3 ubiquitin ligase that has been implicated in motility and endoplasmic reticulum stress response. Here, we show that RNF5 expression is up-regulated in breast cancer tumors and related cell lines. Elevated expression of RNF5 was seen in breast cancer cell lines that became more sensitive to cytochalasin D- and paclitaxel-induced apoptosis following its knockdown with specific short interfering RNA. Inhibition of RNF5 expression markedly decreased cell proliferation and caused a reorganization of the actin cytoskeleton in response to stress in MCF-7 but not in p53 mutant breast cancer cells, suggesting a p53-dependent function. Significantly, high levels of RNF5 were associated with decreased survival in human breast cancer specimens. Similarly, RNF5 levels were higher in metastatic melanoma specimens and in melanoma, leukemia, ovarian, and renal tumor-derived cell lines, suggesting that increased RNF5 expression may be a common event during tumor progression. These results indicate that RNF5 is a novel regulator of breast cancer progression through its effect on actin cytoskeletal alterations, which also affect sensitivity of breast cancer cells to cytoskeletal targeting antineoplastic agents.     

Suppression of mitochondrial respiration with auraptene inhibits the progression of renal cell carcinoma: involvement of HIF-1α degradation

Renal cell carcinoma (RCC) progression resulting from the uncontrolled migration and enhanced angiogenesis is an obstacle to effective therapeutic intervention. Tumor metabolism has distinctive feature called Warburg effect, which enhances the aerobic glycolysis rapidly supplying the energy for migration of tumor. To manipulate this metabolic change characteristic of aggressive tumors, we utilized the citrus extract, auraptene, known as a mitochondrial inhibitor, testing its anticancer effects against the RCC4 cell line. We found that auraptene impaired RCC4 cell motility through reduction of mitochondrial respiration and glycolytic pathway-related genes. It also strongly disrupted VEGF-induced angiogenesis in vitro and in vivo. Hypoxia-inducible factor 1a (HIF-1a), a key regulator of cancer metabolism, migration and angiogenesis that is stably expressed in RCCs by virtue of a genetic mutation in the von Hippel–Lindau (VHL) tumor-suppressor protein, was impeded by auraptene, which blocked HIF-1a translation initiation without causing cytotoxicity. We suggest that blockade HIF-1a and reforming energy metabolism with auraptene is an effective approach for suspension RCC progression.     

Podocalyxin‐like protein,linked to poor prognosis of pancreatic cancers,promotes cell invasion by binding to gelsolin

The cell‐adhesion glycoprotein PODXL is associated with an aggressive tumor phenotype in several forms of cancer. Here, we report that high PODXL expression was an independent predictor of worse overall survival of pancreatic cancer patients, and that PODXL promoted pancreatic cancer cell motility and invasion by physically binding to the cytoskeletal protein gelsolin. Suppression of PODXL or gelsolin decreased membrane protrusions with abundant peripheral actin structures, and in turn inhibited cell motility and invasion. Transfection of a PODXL‐rescue construct renewed the expression of gelsolin bound to peripheral actin structures in cell protrusions, and abrogated the decreased cell protrusions caused by the knockdown of PODXL. Furthermore, transfection of a PODXL‐rescue construct into pancreatic cancer cells in which both PODXL and gelsolin were suppressed failed to increase the formation of the protrusions. Thus, PODXL enhances motility and invasiveness through an increase in gelsolin–actin interactions in cell protrusions.     

miR-372 inhibits p62 in head and neck squamous cell carcinoma in vitro and in vivo

Here we showed that exogenous miR-372 expression and knockdown of p62 (sequestosome1 or SQSTM1), both increased migration of head and neck squamous cell carcinoma (HNSCC) cells. p62 induced phase II detoxification enzyme NADPH quinone oxidoreductase 1 (NQO1), which decreased ROS levels and cell migration. Also, miR-372 decreased p62 during hypoxia, thus increasing cell migration. Levels of miR-372 and p62 inversely correlated in human HNSCC tissues. Plasma levels of miR-372 was associated with advanced tumor stage and patient mortality. Both plasma and salivary miR-372 levels were decreased after tumor resection. We conclude that miR-372 decreases p62, thus increasing ROS and motility in HNSCC cells.     

LINC01605 promotes aerobic glycolysis through lactate dehydrogenase A in triple‐negative breast cancer

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

lncRNA MAFG-AS1通过调控miR-11181-3p/GLG1轴促进胃癌AGS细胞迁移、侵袭和有氧糖酵解

目的：探讨lncRNA MAFG-AS1/miR-11181-3p/GLG1分子轴对胃癌（gastric cancer,GC）细胞迁移、侵袭和有氧糖酵解的影响及其可能的机制。方法：选取 MAFG-AS1 相对高表达的 GC 细胞系 AGS 作为研究对象,采用 qPCR 法检测其 MAFGAS1、miR-11181-3p、GLG1的RNA表达水平,Transwell实验、糖酵解分析等检测细胞迁移、侵袭和有氧糖酵解的变化,利用生物信息学分析及双荧光素酶报告基因验证MAFG-AS1、miR-11181-3p、GLG1之间的相互作用关系。结果：敲减MAFG-AS1显著上调miR-11181-3p及下调GLG1的表达（均P<0.01）,并可显著抑制GC细胞迁移、侵袭和有氧糖酵解（均P<0.01）;荧光素酶报告基因证实MAFG-AS1竞争性吸附miR-11181-3p（P<0.01）;抑制miR-11181-3p或过表达GLG1可部分逆转敲减MAFG-AS1对GC细胞迁移、侵袭和有氧糖酵解的抑制作用（均P<0.05或P<0.01）。结论：MAFG-AS1通过miR-11181-3p/GLG1分子轴增强GC迁移、侵袭和有氧糖酵解,可能是GC诊疗的潜在分子靶点。     

RPL35 promotes neuroblastoma progression via the enhanced aerobic glycolysis

Neuroblastoma (NB) is an rare type of tumor that almost affects children age 5 or younger due to its rapid proliferation ability. The overall survival rate of patients with advanced NB is not satisfactory. Ribosomal proteins (RPs) play a critical role in the development and progress of cancer. However, the contribution of RPL35 in NB has not been proven. In this study, we reveal that RPL35 is upregulated in NB tissues and the upregulation of RPL35 promotes proliferation and migration of NB while RPL35 knockdown significantly restrained the proliferation of NB cells. In terms of mechanism, glycolysis was decreased and the mitochondrial respiration was increased with knockdown of RPL35 in NB cells, indicating that RPL35 function as a positive regulator in aerobic glycolysis. Importantly, our data indicated that RPL35 deficiency decreased HIF1α expression both in mRNA and protein levels. Western blot analysis showed that RPL35 knockdown has a negative regulatory effect on the ERK pathway, and RPL35 modulated aerobic glycolysis in part through its regulation of the RPL35/ERK/HIF1α axis. Overall, RPL35 functions as a positive regulator of aerobic glycolysis, and the RPL35/ERK/HIF1α axis could be a potential therapeutic target for the therapy of NB.     

Lactate dehydrogenase A silencing in IDH mutant gliomas

Background

Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects.

Methods

We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant (^mt) and IDH wild-type (^wt) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas.

Results

We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH^mt gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH^mt derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH^wt), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH^mt glioblastomas.

Conclusion

To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH^mt gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.     

Lactate dehydrogenase A negatively regulated by miRNAs promotes aerobic glycolysis and is increased in colorectal cancer

Reprogramming metabolism of tumor cells is a hallmark of cancer. Lactate dehydrogenase A (LDHA) is frequently overexpressed in tumor cells. Previous studies has shown higher levels of LDHA is related with colorectal cancer (CRC), but its role in tumor maintenance and underlying molecular mechanisms has not been established. Here, we investigated miRNAs-induced changes in LDHA expression. We reported that colorectal cancer express higher levels of LDHA compared with adjacent normal tissue. Knockdown of LDHA resulted in decreased lactate and ATP production, and glucose uptake. Colorectal cancer cells with knockdown of LDHA had much slower growth rate than control cells. Furthermore, we found that miR-34a, miR-34c, miR-369-3p, miR-374a, and miR-4524a/b target LDHA and regulate glycolysis in cancer cells. There is a negative correlation between these miRNAs and LDHA expression in colorectal cancer tissues. More importantly, we identified a genetic loci newly associated with increased colorectal cancer progression, rs18407893 at 11p15.4 (in 3′-UTR of LDHA), which maps to the seed sequence recognized by miR-374a. Cancer cells overexpressed miR-374a has decreased levels of LDHA compared with miR-374a-MUT (rs18407893 at 11p15.4). Taken together, these novel findings provide more therapeutic approaches to the Warburg effect and therapeutic targets of cancer energy metabolism.     

三阴性乳腺癌细胞中线粒体功能障碍对糖代谢途径的影响

目的:探讨线粒体功能损伤和膜通透性转换与乳腺癌细胞中葡萄糖有氧糖酵解途径的相关性。方法:分别采用MitoTracker Green、TMRE和钙黄绿素（Calcein AM）荧光染色检测乳腺上皮细胞MCF-10A和乳腺癌细胞MDA-MB-231、BT-20的线粒体形态、线粒体膜电位和膜通透性;通过检测试剂盒分析其葡萄糖消耗量、乳酸生成量、ATP水平,以及三羧酸循环中琥珀酸脱氢酶（SDH）和线粒体异柠檬酸脱氢酶（ICDHm）的活性;采用Western blot检测三组细胞中糖酵解相关蛋白葡萄糖转运受体1（GLUTI）、甘油醛-3-磷酸脱氢酶（GAPDH）、M2型丙酮酸激酶（PKM2）和乳酸脱氢酶A（LDHA）的表达情况。采用2.5 μmol/L环孢菌素A（CsA）处理MDA-MB-231和BT-20细胞24 h后检测其线粒体形态及糖代谢的相关指标。结果:与MCF-10A细胞比较,乳腺癌MDA-MB-231和BT-20细胞的线粒体呈短管状或点状,线粒体膜电位下降而线粒体膜通透性增加,葡萄糖消耗量和乳酸生成量均升高（P＜0.05）;细胞内GLUT1、GAPDH、PKM2和LDHA蛋白表达均显著升高而SDH和ICDHm酶活性降低（P＜0.05）;CsA处理后MDA-MB-231和BT-20细胞中线粒体呈长杆状,葡萄糖消耗量和乳酸生成量下降而ATP水平、SDH和ICDHm酶活性均升高（P＜0.05）。结论:乳腺癌MDA-MB-231和BT-20细胞中有氧糖酵解与线粒体异常相关,CsA可恢复线粒体结构和功能,重塑葡萄糖代谢途径。