Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism

NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/β-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability

Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), encoded by the nuclear PCK2 gene, links TCA cycle intermediates and glycolytic pools through the conversion of mitochondrial oxaloacetate into phosphoenolpyruvate. In the liver PEPCK-M adjoins its profusely studied cytosolic isoform (PEPCK-C) potentiating gluconeogenesis and TCA flux. However, PEPCK-M is present in a variety of non-gluconeogenic tissues, including tumors of several origins. Despite its potential relevance to cancer metabolism, the mechanisms responsible for PCK2 gene regulation have not been elucidated. The present study demonstrates PEPCK-M overexpression in tumorigenic cells as well as the mechanism for the modulation of PCK2 abundance under several stress conditions. Amino acid limitation and ER stress inducers, conditions that activate the amino acid response (AAR) and the unfolded protein response (UPR), stimulate PCK2 gene transcription. Both the AAR and UPR lead to increased synthesis of ATF4, which mediates PCK2 transcriptional up-regulation through its binding to a putative ATF/CRE composite site within the PCK2 promoter functioning as an amino acid response element. In addition, activation of the GCN2-eIF2α-ATF4 and PERK-eIF2α-ATF4 signaling pathways are responsible for increased PEPCK-M levels. Finally, PEPCK-M knockdown using either siRNA or shRNA were sufficient to reduce MCF7 mammary carcinoma cell growth and increase cell death under glutamine deprivation or ER stress conditions. Our data demonstrate that this enzyme has a critical role in the survival program initiated upon stress and shed light on an unexpected and important role of mitochondrial PEPCK in cancer metabolism

USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination

The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRCs), resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs

Akt-dependent activation of the heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB2) isoenzyme by amino acids

Reciprocal regulation of metabolism and signaling allows cells to modulate their activity in accordance with their metabolic resources. Thus, amino acids could activate signal transduction pathways that control cell metabolism. To test this hypothesis, we analyzed the effect of amino acids on fructose-2,6-bisphosphate (Fru-2,6-P2) metabolism. We demonstrate that amino acids increase Fru-2,6-P2 concentration in HeLa and in MCF7 human cells. In conjunction with this, 6-phosphofructo-2-kinase activity, glucose uptake, and lactate concentration were increased. These data correlate with the specific phosphorylation of heart 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB2) isoenzyme at Ser-483. This activation was mediated by the PI3K and p38 signaling pathways. Furthermore, Akt inactivation blocked PFKFB2 phosphorylation and Fru-2,6-P2 production, thereby suggesting that the above signaling pathways converge at Akt kinase. In accordance with these results, kinase assays showed that amino acid-activated Akt phosphorylated PFKFB2 at Ser-483 and that knockdown experiments confirmed that the increase in Fru-2,6-P2 concentration induced by amino acids was due to PFKFB2. In addition, similar effects on Fru-2,6-P2 metabolism were observed in freshly isolated rat cardiomyocytes treated with amino acids, which indicates that these effects are not restricted to human cancer cells. In these cardiomyocytes, the glucose consumption and the production of lactate and ATP suggest an increase of glycolytic flux. Taken together, these results demonstrate that amino acids stimulate Fru-2,6-P2 synthesis by Akt-dependent PFKFB2 phosphorylation and activation and show how signaling and metabolism are inextricably linked

PFKFB3 activation in cancer cells by the p38/MK2 pathway in response to stress stimuli.

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase) catalyses the synthesis and degradation of Fru-2,6-P2 (fructose 2,6-bisphosphate), a key modulator of glycolysis and gluconeogenesis. The PFKFB3 gene is involved in cell proliferation owing to its role in carbohydrate metabolism. In the present study we analysed the mechanism of regulation of PFKFB3 as an immediate early gene controlled by stress stimuli that activates the p38/MK2 [MAPK (mitogen-activated protein kinase)-activated protein kinase 2] pathway. We report that exposure of HeLa and T98G cells to different stress stimuli (NaCl, H2O2, UV radiation and anisomycin) leads to a rapid increase (15-30 min) in PFKFB3 mRNA levels. The use of specific inhibitors in combination with MK2-deficient cells implicate control by the protein kinase MK2. Transient transfection of HeLa cells with deleted gene promoter constructs allowed us to identify an SRE (serum-response element) to which SRF (serum-response factor) binds and thus transactivates PFKFB3 gene transcription. Direct binding of phospho-SRF to the SRE sequence (-918 nt) was confirmed by ChIP (chromatin immunoprecipiation) assays. Moreover, PFKFB3 isoenzyme phosphorylation at Ser461 by MK2 increases PFK-2 activity. Taken together, the results of the present study suggest a multimodal mechanism of stress stimuli affecting PFKFB3 transcriptional regulation and kinase activation by protein phosphorylation, resulting in an increase in Fru-2,6-P2 concentration and stimulation of glycolysis in cancer cells

Identifying strategies to target the metabolic flexibility of tumours

Plasticity of cancer metabolism can be a major obstacle to efficient targeting of tumour-specific metabolic vulnerabilities. Here, we identify the compensatory mechanisms following the inhibition of major pathways of central carbon metabolism in c-MYC-induced liver tumours. We find that, while inhibition of both glutaminase isoforms (Gls1 and Gls2) in tumours considerably delays tumourigenesis, glutamine catabolism continues, owing to the action of amidotransferases. Synergistic inhibition of both glutaminases and compensatory amidotransferases is required to block glutamine catabolism and proliferation of mouse and human tumour cells in vitro and in vivo. Gls1 deletion is also compensated for by glycolysis. Thus, co-inhibition of Gls1 and hexokinase 2 significantly affects Krebs cycle activity and tumour formation. Finally, the inhibition of biosynthesis of either serine (Psat1-KO) or fatty acid (Fasn-KO) is compensated for by uptake of circulating nutrients, and dietary restriction of both serine and glycine or fatty acids synergistically suppresses tumourigenesis. These results highlight the high flexibility of tumour metabolism and demonstrate that either pharmacological or dietary targeting of metabolic compensatory mechanisms can improve therapeutic outcomes

Role of Akt/PKB and PFKFB isoenzymes in the control of glycolysis, cell proliferation and protein synthesis in mitogen-stimulated thymocytes

Proliferating cells depend on glycolysis mainly to supply precursors for macromolecular synthesis. Fructose 2,6-bisphosphate (Fru-2,6-P2) is the most potent positive allosteric effector of 6-phosphofructo-1-kinase (PFK-1), and hence of glycolysis. Mitogen stimulation of rat thymocytes with concanavalin A (ConA) led to time-dependent increases in lactate accumulation (6-fold), Fru-2,6-P2 content (4-fold), 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase isoenzyme 3 and 4 (PFKFB3 and PFKFB4) protein levels (~ 2-fold and ~ 15-fold, respectively) and rates of cell proliferation (~ 40-fold) and protein synthesis (10-fold) after 68 h of incubation compared with resting cells. After 54 h of ConA stimulation, PFKFB3 mRNA levels were 45-fold higher than those of PFKFB4 mRNA. Although PFKFB3 could be phosphorylated at Ser461 by protein kinase B (PKB) in vitro leading to PFK-2 activation, PFKFB3 Ser461 phosphorylation was barely detectable in resting cells and only increased slightly in ConA-stimulated cells. On the other hand, PFKFB3 and PFKFB4 mRNA levels were decreased (90% and 70%, respectively) by exposure of ConA-stimulated cells to low doses of PKB inhibitor (MK-2206), suggesting control of expression of the two PFKFB isoenzymes by PKB. Incubation of thymocytes with ConA resulted in increased expression and phosphorylation of the translation factors eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) and ribosomal protein S6 (rpS6). Treatment of ConA-stimulated thymocytes with PFK-2 inhibitor (3PO) or MK-2206 led to significant decreases in Fru-2,6-P2 content, medium lactate accumulation and rates of cell proliferation and protein synthesis. These data were confirmed by using siRNA knockdown of PFKFB3, PFKFB4 and PKB α/β in the more easily transfectable Jurkat E6-1 cell line. The findings suggest that increased PFKFB3 and PFKFB4 expression, but not increased PFKFB3 Ser461 phosphorylation, plays a role in increasing glycolysis in mitogen-stimulated thymocytes and implicate PKB in the upregulation of PFKFB3 and PFKFB4. The results also support a role for Fru-2,6-P2 in coupling glycolysis to cell proliferation and protein synthesis in this model