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• W2548993203 abstract "A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer. A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer. Multiple metabolic compartments exist in human tumors including breast cancer (1.DeNicola G.M. Cantley L.C. Cancer's fuel choice: new flavors for a picky eater.Mol. Cell. 2015; 60: 514-523Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). There is metabolic coupling with lactate transfer between highly glycolytic carcinoma cells and carcinoma cells with reduced glycolysis in models of vulvar and colon cancer (2.Sonveaux P. Végran F. Schroeder T. Wergin M.C. Verrax J. Rabbani Z.N. De Saedeleer C.J. Kennedy K.M. Diepart C. Jordan B.F. Kelley M.J. Gallez B. Wahl M.L. Feron O. Dewhirst M.W. Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice.J. Clin. Invest. 2008; 118: 3930-3942PubMed Google Scholar). High stromal glycolysis with low cancer cell glycolysis have also been described in breast, ovarian, prostate, bladder, head and neck carcinomas, and sarcomas (3.Martinez-Outschoorn U.E. Sotgia F. Lisanti M.P. Caveolae and signalling in cancer.Nat. Rev. Cancer. 2015; 15: 225-237Crossref PubMed Scopus (147) Google Scholar). The interactions between carcinoma cells and fibroblasts in breast cancer play an important role in tumor progression. Fibroblasts have been shown to promote breast cancer tumor growth (4.Orimo A. Gupta P.B. Sgroi D.C. Arenzana-Seisdedos F. Delaunay T. Naeem R. Carey V.J. Richardson A.L. Weinberg R.A. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.Cell. 2005; 121: 335-348Abstract Full Text Full Text PDF PubMed Scopus (2909) Google Scholar) and metastasis (5.Karnoub A.E. Dash A.B. Vo A.P. Sullivan A. Brooks M.W. Bell G.W. Richardson A.L. Polyak K. Tubo R. Weinberg R.A. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis.Nature. 2007; 449: 557-563Crossref PubMed Scopus (2526) Google Scholar). Carcinoma cell invasiveness and resistance to chemotherapy in breast cancer are induced by fibroblasts (6.Shekhar M.P. Santner S. Carolin K.A. Tait L. Direct involvement of breast tumor fibroblasts in the modulation of tamoxifen sensitivity.Am. J. Pathol. 2007; 170: 1546-1560Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). It is unknown how cancer cell metabolism in breast cancer modulates adjacent fibroblast metabolism. TP53-induced glycolysis and apoptosis regulator (TIGAR) 2The abbreviations used are: TIGAR, TP53-induced glycolysis and apoptosis regulator; PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Fru-2,6-P2, fructose 2,6-bisphosphate; PFK-1, phosphofructokinase-1; OXPHOS, oxidative phosphorylation; MCT1 and -2, monocarboxylate transporter 1 and 2; LDH-B, lactate dehydrogenase B; GLS1, glutaminase 1; PGC1, peroxisome proliferator-activated receptor coactivator 1; NRF1, nuclear respiratory factor 1; OCR, oxygen consumption rate; TOMM20, transporter of the outer mitochondrial membrane member 20; 3PO, 3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; 2-NBDG, 2-(N-(7-nitrobenz-2- oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (fluorescent 2-deoxyglucose); HIF, hypoxia-inducible factor; RFP, red fluorescent protein; PI, propidium iodide. 2The abbreviations used are: TIGAR, TP53-induced glycolysis and apoptosis regulator; PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; Fru-2,6-P2, fructose 2,6-bisphosphate; PFK-1, phosphofructokinase-1; OXPHOS, oxidative phosphorylation; MCT1 and -2, monocarboxylate transporter 1 and 2; LDH-B, lactate dehydrogenase B; GLS1, glutaminase 1; PGC1, peroxisome proliferator-activated receptor coactivator 1; NRF1, nuclear respiratory factor 1; OCR, oxygen consumption rate; TOMM20, transporter of the outer mitochondrial membrane member 20; 3PO, 3-(3- pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; 2-NBDG, 2-(N-(7-nitrobenz-2- oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (fluorescent 2-deoxyglucose); HIF, hypoxia-inducible factor; RFP, red fluorescent protein; PI, propidium iodide. is an inhibitor of glycolysis and is highly expressed in the majority of human breast-invasive ductal carcinomas (7.Bensaad K. Tsuruta A. Selak M.A. Vidal M.N. Nakano K. Bartrons R. Gottlieb E. Vousden K.H. TIGAR, a p53-inducible regulator of glycolysis and apoptosis.Cell. 2006; 126: 107-120Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar, 8.Won K.Y. Lim S.J. Kim G.Y. Kim Y.W. Han S.A. Song J.Y. Lee D.K. Regulatory role of p53 in cancer metabolism via SCO2 and TIGAR in human breast cancer.Hum. Pathol. 2012; 43: 221-228Crossref PubMed Scopus (85) Google Scholar). TIGAR is the only known phosphatase glycolytic modulator regulated by TP53. It is unknown if TIGAR induces aggressive breast cancer. The human TIGAR gene is similar to the bisphosphatase domain of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) (7.Bensaad K. Tsuruta A. Selak M.A. Vidal M.N. Nakano K. Bartrons R. Gottlieb E. Vousden K.H. TIGAR, a p53-inducible regulator of glycolysis and apoptosis.Cell. 2006; 126: 107-120Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar). TIGAR decreases glycolysis by functioning as a bisphosphatase that reduces levels of intracellular fructose-2,6-bisphosphate (Fru-2,6-P2) and 2,3-bisphosphoglycerate (7.Bensaad K. Tsuruta A. Selak M.A. Vidal M.N. Nakano K. Bartrons R. Gottlieb E. Vousden K.H. TIGAR, a p53-inducible regulator of glycolysis and apoptosis.Cell. 2006; 126: 107-120Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar, 9.Gerin I. Noël G. Bolsée J. Haumont O. Van Schaftingen E. Bommer G.T. Identification of TP53-induced glycolysis and apoptosis regulator (TIGAR) as the phosphoglycolate-independent 2,3-bisphosphoglycerate phosphatase.Biochem. J. 2014; 458: 439-448Crossref PubMed Scopus (32) Google Scholar), which are regulators of glycolysis. Phosphofructokinase-1 (PFK-1) is a key glycolytic enzyme that converts fructose 6-phosphate to fructose 1,6-bisphosphate. PFK-1 is allosterically activated by Fru-2,6-P2 (10.Okar D.A. Manzano A. Navarro-Sabatè A. Riera L. Bartrons R. Lange A.J. PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate.Trends Biochem. Sci. 2001; 26: 30-35Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Also, Fru-2,6-P2 is an inhibitor of fructose-1,6-bisphosphatase, which opposes the activity of PFK-1 by converting fructose 1,6-bisphosphate to fructose 6-phosphate (10.Okar D.A. Manzano A. Navarro-Sabatè A. Riera L. Bartrons R. Lange A.J. PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate.Trends Biochem. Sci. 2001; 26: 30-35Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The synthesis and breakdown of Fru-2,6-P2 depends on the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoenzymes, products of four genes (PFKFB1–4) that code for the different PFKFB isoenzymes and that have distinct cell expression patterns and display different kinase/bisphosphatase activity ratios and control by different protein kinases (10.Okar D.A. Manzano A. Navarro-Sabatè A. Riera L. Bartrons R. Lange A.J. PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate.Trends Biochem. Sci. 2001; 26: 30-35Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 11.Bartrons R. Caro J. Hypoxia, glucose metabolism and the Warburg's effect.J. Bioenerg. Biomembr. 2007; 39: 223-229Crossref PubMed Scopus (193) Google Scholar12.Novellasdemunt L. Bultot L. Manzano A. Ventura F. Rosa J.L. Vertommen D. Rider M.H. Navarro-Sabate À. Bartrons R. PFKFB3 activation in cancer cells by the p38/MK2 pathway in response to stress stimuli.Biochem. J. 2013; 452: 531-543Crossref PubMed Scopus (57) Google Scholar). In tumor cells, the concentration of Fru-2,6-P2 is generally elevated due to overexpression and activation of PFKFB3, which has opposite effects of TIGAR (11.Bartrons R. Caro J. Hypoxia, glucose metabolism and the Warburg's effect.J. Bioenerg. Biomembr. 2007; 39: 223-229Crossref PubMed Scopus (193) Google Scholar, 12.Novellasdemunt L. Bultot L. Manzano A. Ventura F. Rosa J.L. Vertommen D. Rider M.H. Navarro-Sabate À. Bartrons R. PFKFB3 activation in cancer cells by the p38/MK2 pathway in response to stress stimuli.Biochem. J. 2013; 452: 531-543Crossref PubMed Scopus (57) Google Scholar13.Atsumi T. Chesney J. Metz C. Leng L. Donnelly S. Makita Z. Mitchell R. Bucala R. High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers.Cancer Res. 2002; 62: 5881-5887PubMed Google Scholar). Conversely, TIGAR depletion increases glycolytic flux by increasing the activity of PFK-1 and the glycolytic flux by increasing Fru-2,6-P2 levels (7.Bensaad K. Tsuruta A. Selak M.A. Vidal M.N. Nakano K. Bartrons R. Gottlieb E. Vousden K.H. TIGAR, a p53-inducible regulator of glycolysis and apoptosis.Cell. 2006; 126: 107-120Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar, 14.Peña-Rico M.A. Calvo-Vidal M.N. Villalonga-Planells R. Martínez-Soler F. Giménez-Bonafé P. Navarro-Sabaté À. Tortosa A. Bartrons R. Manzano A. TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells.Radiother. Oncol. 2011; 101: 132-139Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). It is unknown if TIGAR modulates catabolism of other substrates such as lactate and glutamine, which have been shown to be alternate catabolites to glucose for carcinoma cells (1.DeNicola G.M. Cantley L.C. Cancer's fuel choice: new flavors for a picky eater.Mol. Cell. 2015; 60: 514-523Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). TIGAR expression has been shown to be inversely associated with glycolysis as TIGAR expression was inversely associated with 2-deoxyglucose uptake on PET scans in subjects with non-small cell lung cancer (15.Zhou X. Xie W. Li Q. Zhang Y. Zhang J. Zhao X. Liu J. Huang G. TIGAR is correlated with maximal standardized uptake value on FDG-PET and survival in non-small cell lung cancer.PLoS ONE. 2013; 8: e80576Crossref PubMed Scopus (19) Google Scholar). TIGAR regulates hexokinase 2 (HK2) activity and increases mitochondrial membrane potential, but the effect of TIGAR on mitochondrial metabolism, oxygen consumption rates, and ATP generation is unknown (16.Cheung E.C. Ludwig R.L. Vousden K.H. Mitochondrial localization of TIGAR under hypoxia stimulates HK2 and lowers ROS and cell death.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 20491-20496Crossref PubMed Scopus (158) Google Scholar). In sum, TIGAR reduces glycolysis, but its effects on the catabolism of other substrates and mitochondrial metabolism is poorly characterized. TIGAR has been reported to mediate human cancer aggressiveness, although the mechanism is unclear, and its effect in breast cancer is unknown. In addition to reducing glycolysis, TIGAR reduces apoptosis (7.Bensaad K. Tsuruta A. Selak M.A. Vidal M.N. Nakano K. Bartrons R. Gottlieb E. Vousden K.H. TIGAR, a p53-inducible regulator of glycolysis and apoptosis.Cell. 2006; 126: 107-120Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar). TIGAR is overexpressed in nasopharyngeal carcinoma, and genetic overexpression increases tumor growth with carcinoma cell growth, colony formation, migration, and invasion with NFκB activation in carcinoma cells (17.Zhao M. Fan J. Liu Y. Yu Y. Xu J. Wen Q. Zhang J. Fu S. Wang B. Xiang L. Feng J. Wu J. Yang L. Oncogenic role of the TP53-induced glycolysis and apoptosis regulator in nasopharyngeal carcinoma through NF-κB pathway modulation.Int. J. Oncol. 2016; 48: 756-764Crossref PubMed Scopus (15) Google Scholar). Knockdown of this bisphosphatase induces apoptosis of HepG2 hepatocellular carcinoma cells (18.Ye L. Zhao X. Lu J. Qian G. Zheng J.C. Ge S. Knockdown of TIGAR by RNA interference induces apoptosis and autophagy in HepG2 hepatocellular carcinoma cells.Biochem. Biophys. Res. Commun. 2013; 437: 300-306Crossref PubMed Scopus (37) Google Scholar). TIGAR down-regulation in HepG2 carcinoma cells reduces the size of hepatocellular carcinoma xenografts (19.Xie J.M. Li B. Yu H.P. Gao Q.G. Li W. Wu H.R. Qin Z.H. TIGAR has a dual role in cancer cell survival through regulating apoptosis and autophagy.Cancer Res. 2014; 74: 5127-5138Crossref PubMed Scopus (61) Google Scholar). TIGAR reduces apoptosis rates of non-small cell lung cancer H-1299 and osteosarcoma U2OS cells (7.Bensaad K. Tsuruta A. Selak M.A. Vidal M.N. Nakano K. Bartrons R. Gottlieb E. Vousden K.H. TIGAR, a p53-inducible regulator of glycolysis and apoptosis.Cell. 2006; 126: 107-120Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar). TIGAR is also overexpressed in the majority of glioblastomas, protecting cells from starvation-induced cell death by up-regulating respiration and improving cellular redox homeostasis (20.Wanka C. Steinbach J.P. Rieger J. Tp53-induced glycolysis and apoptosis regulator (TIGAR) protects glioma cells from starvation-induced cell death by up-regulating respiration and improving cellular redox homeostasis.J. Biol. Chem. 2012; 287: 33436-33446Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Radiosensitization of glioma cells also occurs with TIGAR knockdown (14.Peña-Rico M.A. Calvo-Vidal M.N. Villalonga-Planells R. Martínez-Soler F. Giménez-Bonafé P. Navarro-Sabaté À. Tortosa A. Bartrons R. Manzano A. TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown results in radiosensitization of glioma cells.Radiother. Oncol. 2011; 101: 132-139Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, another study has shown that TIGAR decreases cell viability in glioblastoma (21.Sinha S. Ghildiyal R. Mehta V.S. Sen E. ATM-NFκB axis-driven TIGAR regulates sensitivity of glioma cells to radiomimetics in the presence of TNFα.Cell Death Dis. 2013; 4: e615Crossref PubMed Scopus (34) Google Scholar). TIGAR is required for proliferation of small intestine cells (22.Cheung E.C. Athineos D. Lee P. Ridgway R.A. Lambie W. Nixon C. Strathdee D. Blyth K. Sansom O.J. Vousden K.H. TIGAR is required for efficient intestinal regeneration and tumorigenesis.Dev. Cell. 2013; 25: 463-477Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). TIGAR null mice have decreased tumorigenesis and increased survival in a model in which the tumor suppressor adenomatous polyposis coli (APC) is deleted in LGR5+ intestinal stem cells (22.Cheung E.C. Athineos D. Lee P. Ridgway R.A. Lambie W. Nixon C. Strathdee D. Blyth K. Sansom O.J. Vousden K.H. TIGAR is required for efficient intestinal regeneration and tumorigenesis.Dev. Cell. 2013; 25: 463-477Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). TIGAR is highly expressed in the majority of breast cancers and carcinoma cells in breast cancer frequently have low rates of glycolysis and apoptosis (7.Bensaad K. Tsuruta A. Selak M.A. Vidal M.N. Nakano K. Bartrons R. Gottlieb E. Vousden K.H. TIGAR, a p53-inducible regulator of glycolysis and apoptosis.Cell. 2006; 126: 107-120Abstract Full Text Full Text PDF PubMed Scopus (1512) Google Scholar, 8.Won K.Y. Lim S.J. Kim G.Y. Kim Y.W. Han S.A. Song J.Y. Lee D.K. Regulatory role of p53 in cancer metabolism via SCO2 and TIGAR in human breast cancer.Hum. Pathol. 2012; 43: 221-228Crossref PubMed Scopus (85) Google Scholar, 24.Bensaad K. Cheung E.C. Vousden K.H. Modulation of intracellular ROS levels by TIGAR controls autophagy.EMBO J. 2009; 28: 3015-3026Crossref PubMed Scopus (296) Google Scholar, and 25.Li L. Ishdorj G. Gibson S.B. Reactive oxygen species regulation of autophagy in cancer: implications for cancer treatment.Free Radic Biol. Med. 2012; 53: 1399-1410Crossref PubMed Scopus (121) Google Scholar). However, no reports have described the effect of TIGAR on breast cancer tumor growth in vivo. Also, the role of TIGAR in the low rates of glycolysis and apoptosis with high proliferation observed in human breast cancer is unknown. Breast tumors have high expression of mitochondrial biogenesis transcription factors and mitochondrial markers of oxidative phosphorylation (OXPHOS) (26.Wallace D.C. Mitochondria and cancer.Nat. Rev. Cancer. 2012; 12: 685-698Crossref PubMed Scopus (1456) Google Scholar) (27.Sotgia F. Whitaker-Menezes D. Martinez-Outschoorn U.E. Salem A.F. Tsirigos A. Lamb R. Sneddon S. Hulit J. Howell A. Lisanti M.P. Mitochondria “fuel” breast cancer metabolism: fifteen markers of mitochondrial biogenesis label epithelial cancer cells, but are excluded from adjacent stromal cells.Cell Cycle. 2012; 11: 4390-4401Crossref PubMed Scopus (131) Google Scholar). Multiple metabolic compartments exist in human malignancies (28.Martinez-Outschoorn U.E. Lisanti M.P. Sotgia F. Catabolic cancer-associated fibroblasts transfer energy and biomass to anabolic cancer cells, fueling tumor growth.Semin. Cancer Biol. 2014; 25: 47-60Crossref PubMed Scopus (284) Google Scholar). Metabolic compartmentalization occurs in breast cancer with highly glycolytic stromal cells and cancer cells with high OXPHOS (28.Martinez-Outschoorn U.E. Lisanti M.P. Sotgia F. Catabolic cancer-associated fibroblasts transfer energy and biomass to anabolic cancer cells, fueling tumor growth.Semin. Cancer Biol. 2014; 25: 47-60Crossref PubMed Scopus (284) Google Scholar). There are no data on the metabolic effects of TIGAR expression in breast cancer and if it plays a role in metabolic compartmentalization or heterogeneity in tumors. We hypothesize that TIGAR expression in carcinoma cells induces aggressive disease with utilization of alternative catabolites to glucose and metabolic compartmentalization. To investigate the effects of TIGAR on markers of lactate and glutamine utilization, we overexpressed TIGAR in breast carcinoma cells. TIGAR overexpression up-regulated moderately monocarboxylate transporter 2 (MCT2) protein and mRNA expression (Fig. 1, A and B). MCT2 is a lactate importer. TIGAR also up-regulates lactate dehydrogenase B (LDH-B), which is the enzyme that converts lactate into pyruvate for mitochondrial metabolism (Fig. 1C) and TIGAR up-regulates glutaminase 1 (GLS1) modestly, which is the rate-limiting enzyme in glutamine catabolism and converts glutamine to glutamate (Fig. 1D). Conversely, glutamate ammonia ligase (GLUL), which is the enzyme that mediates glutamine synthesis from glutamate and ammonia, was down-regulated with TIGAR overexpression (Fig. 1E). TIGAR markedly decreased the NADP+/NADPH ratio (Fig. 1, F and G), which is consistent with increasing flux through the pentose phosphate pathway. To investigate the effects of TIGAR on mitochondrial biogenesis, we studied the expression of peroxisome proliferator-activated receptor γ coactivator 1 (PGC1) and nuclear respiratory factor 1 (NRF1). TIGAR overexpression induced these markers of mitochondrial biogenesis (Fig. 2A). To investigate the effects of TIGAR and exposure to glutamine and lactate on OXPHOS, we studied oxygen consumption rates (OCR) in control and TIGAR-overexpressing carcinoma cells. Oxygen consumption rates were higher for TIGAR overexpressing carcinoma cells exposed to glutamine, lactate, or glutamine and lactate compared with carcinoma cells expressing the control vector (p < 0.05) (Fig. 2B). TIGAR overexpressing carcinoma cells exposed to glutamine have a 1.2-fold greater OCR than control carcinoma cells exposed to glutamine (p < 0.05) and 1.5-fold greater OCR when exposed to lactate or glutamine and lactate than the control carcinoma cells exposed to the same conditions (p < 0.05). OCRs were not increased by glutamine or lactate in the absence of TIGAR overexpression. TIGAR overexpressing carcinoma cells had lower OCR than control cells (0.8-fold) when cultured with glucose but without glutamine and lactate (p < 0.05). Next, we studied the markers of OXPHOS metabolism MITONEET, and Transporter of the Outer Mitochondrial Membrane Member 20 (TOMM20). Both MITONEET and TOMM20 are up-regulated by TIGAR (Fig. 2C). TOMM20 overexpression in carcinoma cells also increased expression of TIGAR (Fig. 2D). Exposure of carcinoma cells to lactate, glutamine, or glutamine and lactate increased ATP generation (Fig. 2E). We then exposed control and TIGAR-overexpressing carcinoma cells to 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), which is a PFKFB3 inhibitor that reduces Fru-2,6-P2 levels. Control cells exposed to 3PO do not have a significant change in ATP levels (Fig. 2F). However, TIGAR-overexpressing cells exposed to 3PO increased ATP levels (Fig. 2F). To investigate stromal-epithelial interactions, we used a coculture model system composed of 1) human fibroblasts immortalized with the telomerase catalytic domain (hTERT-BJ1 fibroblasts) and 2) T47D or MCF7 cells, which are well established breast cancer cell lines. TIGAR and PFKFB3 are markers of glycolysis, although they have opposite effects. TIGAR mRNA expression was increased 1.5-fold in T47D cells in coculture (p < 0.01) (Fig. 3A) and 1.6-fold in MCF7 cells in coculture (p < 0.05), whereas PFKFB3 mRNA expression was reduced 1.9-fold in MCF7 cells in coculture (p < 0.05) (Fig. 3B). No statistically significant difference in PFKFB3 mRNA expression was noted in T47D cells in coculture (Fig. 3A). Fibroblasts induced TIGAR expression in carcinoma cells (Fig. 3C), and TIGAR induced aggressive carcinoma cells with increased proliferation and reduced apoptosis. Carcinoma cells cocultured with fibroblasts had a 4.7-fold increase in DNA synthesis at the expense of G0-G1 and G2-M (p < 0.01) (Fig. 3D). Carcinoma cells also had a 2-fold decreased apoptosis rate when in coculture with fibroblasts (p < 0.05) (Fig. 3E). We studied 2-deoxyglucose uptake in T47D cells overexpressing TIGAR in coculture with fibroblasts by measuring fluorescence of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), which is a green fluorescent 2-deoxyglucose compound. 2-NBDG uptake correlates with glycolysis with higher uptake indicating increased glycolysis. T47D cells overexpressing TIGAR had a 1.3-fold reduced 2-NBDG uptake compared with control T47D cells in coculture (p < 0.05) (Fig. 4A). T47D cells overexpressing TIGAR in homotypic culture had no statistically significant difference in 2-NBDG uptake compared with control T47D cells (data not shown). T47D cells overexpressing TIGAR had a 1.9-fold reduced lactate production compared with control T47D cells (p < 0.05) (Fig. 4B). TOMM20 overexpression in carcinoma cells also reduced lactate production 1.9-fold (p < 0.05) (Fig. 4C). Lactate production is a measure of glycolysis with lower lactate levels indicating lower glycolysis. Hence the effect of TIGAR on glycolysis can be phenocopied by expression of TOMM20. Monocarboxylate transporter 1 (MCT1) is the main importer of lactate into cells and is inversely related to rates of glycolysis. TIGAR overexpression in carcinoma cells up-regulated MCT1 protein expression (Fig. 4D). Conversely TIGAR down-regulation in carcinoma cells using CRISPR-Cas9 reduced MCT1 expression (Fig. 4E–F). Next we studied the effect of TIGAR overexpression in T47D cells on fibroblast metabolism. Fibroblasts cocultured with T47D cells overexpressing TIGAR had a 1.2-fold increase in 2-NBDG uptake compared with fibroblasts in control cocultures (p < 0.05) (Fig. 5A). Glucose uptake in fibroblasts was measured by quantifying green fluorescence of the 2-deoxyglucose compound 2-NBDG. 2-Deoxyglucose and 2-NBDG uptake is an additional measure of glycolysis with lower uptake indicating reduced glycolysis. PFKFB3 and LDH-A expression increased in fibroblasts cocultured with TIGAR overexpressing carcinoma cells (Fig. 5, B and C). PFKFB3 and LDH-A are markers of glycolysis. LDH-B expression is reduced with coculture with TIGAR overexpressing carcinoma cells (Fig. 5C). LDH-B has opposite effects to LDH-A, converting lactate to pyruvate. We next assessed the effect of TIGAR on activation of hypoxia-inducible factor (HIF) in fibroblasts by using a HIF1A luciferase reporter as HIF1A is one of the main glycolytic transcription factors. NIH3T3 fibroblasts stably transfected with a HIF1A luciferase reporter were cultured with control T47D cells or TIGAR-overexpressing T47D cells. Activation of HIF1A is increased by 1.6-fold (p < 0.05) in fibroblasts cocultured with T47D cells overexpressing TIGAR cells in 0.5% O2 hypoxia compared with control coculture conditions (Fig. 5D). Note that there was no significant change in HIF1A activation in normoxia between control coculture and coculture with T47D cells overexpressing TIGAR. Fibroblasts exposed to carcinoma cells have reduced TIGAR expression, but overexpression of TIGAR in fibroblasts led to 1.2-fold reduced glucose uptake compared with control fibroblasts (Fig. 5E). In sum, TIGAR overexpression in carcinoma cells induced a glycolytic phenotype in fibroblasts. T47D cells were cultured alone or with fibroblasts and exposed to tamoxifen. TIGAR overexpression in carcinoma cells exposed to tamoxifen induced apoptosis resistance with a 1.2-fold reduction in homotypic culture and a 1.4-fold reduction in coculture with fibroblasts (p < 0.05) (Fig. 6A). In contrast, TIGAR-overexpressing carcinoma cells were more sensitive to apoptosis by mitochondrial modulators. Metformin, which is an OXPHOS complex I inhibitor, doxycycline, which inhibits mitochondrial translation, and ABT-199, which inhibits BCL2 as a single agent or in combination induced higher rates of apoptosis in TIGAR-overexpressing T47D cells than controls (Fig. 6, B–E). Exposure to lactate in MCF7 cells led to increased TIGAR and BCL2 expression with reduced MCT4 expression (Fig. 6F). Exposure to tamoxifen in MCF7 cells decreased expression of TIGAR, BCL-XL, BCL2, MITONEET, and labile subunits of OXPHOS (Fig. 6, G–H). Overexpression of MITONEET in MCF7 cells, which is a marker of OXPHOS, reduced apoptosis rates in coculture with tamoxifen 1.3-fold (p < 0.05) (Fig. 6I). MDA-MB-231, T47D, and MCF7 cells overexpressing TIGAR and control cells were injected into the mammary gland of nude female mice. MDA-MB-231 cells overexpressing TIGAR" @default.
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• W2548993203 date "2016-12-01" @default.
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• W2548993203 title "TP53-inducible Glycolysis and Apoptosis Regulator (TIGAR) Metabolically Reprograms Carcinoma and Stromal Cells in Breast Cancer" @default.
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• W2548993203 doi "https://doi.org/10.1074/jbc.m116.740209" @default.
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