PBRM1 deficiency oncogenic addiction is associated with activated AKT–mTOR signalling and aerobic glycolysis in clear cell renal cell carcinoma cells

The PBRM1 (PB1) gene which encodes the specific subunit BAF180 of the PBAF SWI/SNF complex, is highly mutated (~ 40%) in clear cell renal cell carcinoma (ccRCC). However, its functions and impact on cell signalling are still not fully understood. Aerobic glycolysis, also known as the ‘Warburg Effect’, is a hallmark of cancer, whether PB1 is involved in this metabolic shift in clear cell renal cell carcinoma remains unclear. Here, with established stable knockdown PB1 cell lines, we performed functional assays to access the effects on 786‐O and SN12C cells. Based on the RNA‐seq data, we selected some genes encoding key glycolytic enzymes, including PFKP, ENO1, PKM and LDHA, and examined the expression levels. The AKT–mTOR signalling pathway activity and expression of HIF1α were also analysed. Our data demonstrate that PB1 deficiency promotes the proliferation, migration, Xenograft growth of 786‐O and SN12C cells. Notably, knockdown of PB1 activates AKT–mTOR signalling and increases the expression of key glycolytic enzymes at both mRNA and protein levels. Furthermore, we provide evidence that deficient PB1 and hypoxic conditions exert a synergistic effect on HIF 1α expression and lactate production. Thus, our study provides novel insights into the roles of tumour suppressor PB1 and suggests that the AKT–mTOR signalling pathway, as well as glycolysis, is a potential drug target for ccRCC patients with deficient PB1.     

The real face of HIF1α in the tumor process

It is well known that the hypoxia-inducible factor 1 α (HIF1α) is detectable as adaptive metabolic response to hypoxia. However, HIF1/HIF1α is detectable even under normoxic conditions, if the metabolism is altered, e.g., high proliferation index. Importantly, both hypoxic metabolism and the Warburg effect have in common a decrease of the intracellular pH value.

In our interpretation, HIF1α is not directly accumulated by hypoxia, but by a process which occurs always under hypoxic conditions, a decrease of the intracellular pH value because of metabolic imbalances. We assume that HIF1α is a sensitive controller of the intracellular pH value independently of the oxygen concentration. Moreover, HIF1α has its major role in activating genes to eliminate toxic metabolic waste products (e.g., NH₃/NH₄+) generated by the tumor-specific metabolism called glutaminolysis, which occur during hypoxia, or the Warburg effect. For that reason, HIF1α appears as a potential target for tumor therapy to disturb the pH balance and to inhibit the elimination of toxic metabolic waste products in the tumor cells.     

Metabolic Responses of Primary and Transformed Cells to Intracellular Listeria monocytogenes

The metabolic response of host cells, in particular of primary mammalian cells, to bacterial infections is poorly understood. Here, we compare the carbon metabolism of primary mouse macrophages and of established J774A.1 cells upon Listeria monocytogenes infection using ¹³C-labelled glucose or glutamine as carbon tracers. The ¹³C-profiles of protein-derived amino acids from labelled host cells and intracellular L. monocytogenes identified active metabolic pathways in the different cell types. In the primary cells, infection with live L. monocytogenes increased glycolytic activity and enhanced flux of pyruvate into the TCA cycle via pyruvate dehydrogenase and pyruvate carboxylase, while in J774A.1 cells the already high glycolytic and glutaminolytic activities hardly changed upon infection. The carbon metabolism of intracellular L. monocytogenes was similar in both host cells. Taken together, the data suggest that efficient listerial replication in the cytosol of the host cells mainly depends on the glycolytic activity of the hosts.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

Tumour necrosis factor-α suppresses the hypoxic response by NF-κB-dependent induction of inhibitory PAS domain protein in PC12 cells

Inflammation is often accompanied by hypoxia. However, crosstalk between signalling pathways activated by inflammation and signalling events that control adaptive response to hypoxia is not fully understood. Here we show that exposure to tumour necrosis factor-α (TNF-α) activates expression of the inhibitory PAS domain protein (IPAS) to suppress the hypoxic response caused by hypoxia-inducible factor (HIF)-1 and HIF-2 in rat pheochromocytoma PC12 cells but not in human hepatoma Hep3B cells. This induction of IPAS was dependent on the nuclear factor-κB (NF-κB) pathway and attenuated hypoxic induction of HIF-1 target genes such as tyrosine hydroxylase (TH) and vascular endothelial growth factor (VEGF). HIF-dependent reporter activity in hypoxia was also decreased following TNF-α treatment. Knockdown of IPAS mRNA by small interfering RNA (siRNA) restored the TNF-α-suppressed hypoxic response. These results indicate that TNF-α is a cell-type specific suppressor of HIFs and suggest a novel crosstalk between stimulation by inflammatory mediators and HIF-dependent hypoxic response.     

Glycolysis/gluconeogenesis specialization in microbes is driven by biochemical constraints of flux sensing

Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic reorganization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis, and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida. We combined growth dynamics and dynamic changes in intracellular metabolite levels with a coarse‐grained model that integrates fluxes, regulation, protein synthesis, and growth and uncovered fundamental limitations of the regulatory network: After nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade‐offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.     

Characterization of a new liver- and kidney-specific pfkfb3 isozyme that is downregulated by cell proliferation and dedifferentiation

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2) catalyzes the synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P₂), a signalling molecule that controls the balance between glycolysis and gluconeogenesis in several cell types. Four genes, designated Pfkfb1-4, code several PFK-2 isozymes that differ in their kinetic properties, molecular masses, and regulation by protein kinases. In rat tissues, Pfkfb3 gene accounts for eight splice variants and two of them, ubiquitous and inducible PFK-2 isozymes, have been extensively studied and related to cell proliferation and tumour metabolism. Here, we characterize a new kidney- and liver-specific Pfkfb3 isozyme, a product of the RB2K3 splice variant, and demonstrate that its expression, in primary cultured hepatocytes, depends on hepatic cell proliferation and dedifferentiation. In parallel, our results provide further evidence that ubiquitous PFK-2 is a crucial isozyme in supporting growing and proliferant cell metabolism.     

Regulation of a truncated isoform of AMP-activated protein kinase α (AMPKα) in response to hypoxia in the muscle of Pacific oyster Crassostrea gigas

AMP-activated protein kinase α (AMPKα) is a key regulator of energy balance in many model species during hypoxia. In a marine bivalve, the Pacific oyster Crassostrea gigas, we analyzed the protein content of adductor muscle in response to hypoxia during 6 h. In both smooth and striated muscles, the amount of full-length AMP-activated protein kinase α (AMPKα) remained unchanged during hypoxia. However, hypoxia induced a rapid and muscle-specific response concerning truncated isoforms of AMPKα. In the smooth muscle, a truncated isoform of AMPKα was increased from 1 to 6 h of hypoxia, and was linked with accumulation of AKT kinase, a key enzyme of the insulin signaling pathway which controls intracellular glucose metabolism. In this muscle, aerobic metabolism was maintained over the 6 h of hypoxia, as mitochondrial citrate synthase activity remained constant. In contrast, in striated muscle, hypoxia did not induce any significant modification of neither truncated AMPKα nor AKT protein content, and citrate synthase activity was altered after 6 h of hypoxia. Together, our results demonstrate that hypoxia response is specific to muscle type in Pacific oyster, and that truncated AMPKα and AKT proteins might be involved in maintaining aerobic metabolism in smooth muscle. Such regulation might occur in vivo during tidal intervals that cause up to 6 h of hypoxia.     

The tumour suppressor C/EBPδ inhibits FBXW7 expression and promotes mammary tumour metastasis

Inflammation and hypoxia are known to promote the metastatic progression of tumours. The CCAAT/enhancer-binding protein-δ (C/EBPδ, CEBPD) is an inflammatory response gene and candidate tumour suppressor, but its physiological role in tumourigenesis in vivo is unknown. Here, we demonstrate a tumour suppressor function of C/EBPδ using transgenic mice overexpressing the Neu/Her2/ERBB2 proto-oncogene in the mammary gland. Unexpectedly, this study also revealed that C/EBPδ is necessary for efficient tumour metastasis. We show that C/EBPδ is induced by hypoxia in tumours in vivo and in breast tumour cells in vitro, and that C/EBPδ-deficient cells exhibit reduced glycolytic metabolism and cell viability under hypoxia. C/EBPδ supports CXCR4 expression. On the other hand, C/EBPδ directly inhibits expression of the tumour suppressor F-box and WD repeat-domain containing 7 gene (FBXW7, FBW7, AGO, Cdc4), encoding an F-box protein that promotes degradation of the mammalian target of rapamycin (mTOR). Consequently, C/EBPδ enhances mTOR/AKT/S6K1 signalling and augments translation and activity of hypoxia-inducible factor-1α (HIF-1α), which is necessary for hypoxia adaptation. This work provides new insight into the mechanisms by which metastasis-promoting signals are induced specifically under hypoxia.     

Dynamics of Glycolytic Regulation during Adaptation of Saccharomyces cerevisiae to Fermentative Metabolism

The ability of baker's yeast (Saccharomyces cerevisiae) to rapidly increase its glycolytic flux upon a switch from respiratory to fermentative sugar metabolism is an important characteristic for many of its multiple industrial applications. An increased glycolytic flux can be achieved by an increase in the glycolytic enzyme capacities (V_max) and/or by changes in the concentrations of low-molecular-weight substrates, products, and effectors. The goal of the present study was to understand the time-dependent, multilevel regulation of glycolytic enzymes during a switch from fully respiratory conditions to fully fermentative conditions. The switch from glucose-limited aerobic chemostat growth to full anaerobiosis and glucose excess resulted in rapid acceleration of fermentative metabolism. Although the capacities (V_max) of the glycolytic enzymes did not change until 45 min after the switch, the intracellular levels of several substrates, products, and effectors involved in the regulation of glycolysis did change substantially during the initial 45 min (e.g., there was a buildup of the phosphofructokinase activator fructose-2,6-bisphosphate). This study revealed two distinct phases in the upregulation of glycolysis upon a switch to fermentative conditions: (i) an initial phase, in which regulation occurs completely through changes in metabolite levels; and (ii) a second phase, in which regulation is achieved through a combination of changes in V_max and metabolite concentrations. This multilevel regulation study qualitatively explains the increase in flux through the glycolytic enzymes upon a switch of S. cerevisiae to fermentative conditions and provides a better understanding of the roles of different regulatory mechanisms that influence the dynamics of yeast glycolysis.