Browsing by Author "Clem, Brian F."
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Item A novel small molecule antagonist of choline kinase-alpha that simultaneously suppresses MAPK and PI3K/AKT signaling(Springernature, 2011-07) Clem, Brian F.; Clem, Amy L.; Goswami, Umesh; Arumugam, Sengodagounder; Telang, Sucheta; Trent, John O.; Chesney, Jason A.; Yalçın, Abdullah; Uludağ Üniversitesi/Veterinerlik Fakültesi/Biyokimya Anabilim Dalı.; 0000-0001-8519-8375; ABI-4164-2020; A-5261-2016; 36857831000Choline kinase-alpha expression and activity are increased in multiple human neoplasms as a result of growth factor stimulation and activation of cancer-related signaling pathways. The product of choline kinase-alpha, phosphocholine, serves as an essential metabolic reservoir for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the production of lipid second messengers. Using in silico screening for small molecules that may interact with the choline kinase-alpha substrate binding domain, we identified a novel competitive inhibitor, N-(3,5-dimethylphenyl)-2-[[5-(4-ethylphenyl)-1H-1,2,4-triazol-3-yl] sulfanyl] acetamide (termed CK37) that inhibited purified recombinant human choline kinase-alpha activity, reduced the steady-state concentration of phosphocholine in transformed cells, and selectively suppressed the growth of neoplastic cells relative to normal epithelial cells. Choline kinase-alpha activity is required for the downstream production of phosphatidic acid, a promoter of several Ras signaling pathways. CK37 suppressed mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling, disrupted actin cytoskeletal organization, and reduced plasma membrane ruffling. Finally, administration of CK37 significantly decreased tumor growth in a lung tumor xenograft mouse model, suppressed tumor phosphocholine, and diminished activating phosphorylations of extracellular signal-regulated kinase and AKT in vivo. Together, these results further validate choline kinase-alpha as a molecular target for the development of agents that interrupt Ras signaling pathways, and indicate that receptor-based computational screening should facilitate the identification of new classes of choline kinase-alpha inhibitors.Item Nuclear targeting of 6-Phosphofructo-2-kinase (PFKFB3) increases proliferation via cyclin-dependent kinases(American Society of Biochemistry Molecular Biology, 2009-09-04) Clem, Brian F.; Simmons, Alan J.; Lane, Andrew N.; Nelson, Kristin; Clem, Amy L.; Brock, Erin; Wattenberg, Brinks W.; Telang, Sucheta; Chesney, Jason; Yalçın, Abdullah; Uludağ Üniversitesi/Veterinerlik Fakültesi/Biyokimya Anabilim Dalı.; 0000-0001-8519-8375; ABI-4164-2020; A-5261-2016; 36857831000The regulation of metabolism and growth must be tightly coupled to guarantee the efficient use of energy and anabolic substrates throughout the cell cycle. Fructose 2,6-bisphosphate (Fru-2,6-BP) is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in glycolysis. The concentration of Fru-2,6-BP in mammalian cells is set by four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4), which interconvert fructose 6-phosphate and Fru-2,6-BP. The relative functions of the PFKFB3 and PFKFB4 enzymes are of particular interest because they are activated in human cancers and increased by mitogens and low oxygen. We examined the cellular localization of PFKFB3 and PFKFB4 and unexpectedly found that whereas PFKFB4 localized to the cytoplasm (i.e. the site of glycolysis), PFKFB3 localized to the nucleus. We then overexpressed PFKFB3 and observed no change in glucose metabolism but rather a marked increase in cell proliferation. These effects on proliferation were completely abrogated by mutating either the active site or nuclear localization residues of PFKFB3, demonstrating a requirement for nuclear delivery of Fru-2,6-BP. Using protein array analyses, we then found that ectopic expression of PFKFB3 increased the expression of several key cell cycle proteins, including cyclin-dependent kinase (Cdk)-1, Cdc25C, and cyclinD3 and decreased the expression of the cell cycle inhibitor p27, a universal inhibitor of Cdk-1 and the cell cycle. We also observed that the addition of Fru-2,6-BP to HeLa cell lysates increased the phosphorylation of the Cdk-specific Thr-187 site of p27. Taken together, these observations demonstrate an unexpected role for PFKFB3 in nuclear signaling and indicate that Fru-2,6-BP may couple the activation of glucose metabolism with cell proliferation.Item Regulation of glucose metabolism by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases in cancer(Academic Press Inc Elsevier Science, 2009-06) Telang, Sucheta; Clem, Brian F.; Chesney, Jason; Yalçın, Abdullah; Uludağ Üniversitesi/ Veterinerlik Fakültesi/ Biyokimya Anabilim Dalı.; 0000-0001-8519-8375; ABI-4164-2020; A-5261-2016; 36857831000A high rate of glycolytic flux, even in the presence of oxygen, is a central metabolic hallmark of neoplastic tumors. Cancer cells preferentially utilize glycolysis in order to satisfy their increased energetic and biosynthetic requirements. This metabolic phenotype has been confirmed in human studies using positron emission tomography (PET) with F-18-2-fluoro-deoxy-glucose which have demonstrated that tumors take up 10-fold more glucose than adjacent normal tissues in vivo. The high glucose metabolism of cancer cells is caused by a combination of hypoxia-responsive transcription factors, activation of oncogenic proteins and the loss of tumor suppressor function. Over-expression of HIF-1 alpha and myc, activation of ras and loss of p53 function each have been found to stimulate glycolysis in part by activating a family of regulatory bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB). The PFKFB enzymes synthesize fructose-2,6-bisphosphate (F2,6BP) which allosterically activates 6-phosphofructo-1-kinase (PFK-1), a rate-limiting enzyme and essential control point in the glycolytic pathway. PFK-1 is inhibited by ATP when energy stores are abundant and F2,6BP can override this inhibition and enhance glucose uptake and glycolytic flux. It is therefore not surprising that F2,6BP synthesis is stimulated by several oncogenic alterations which simultaneously cause both enhanced consumption of glucose and growth. Importantly, these studies suggest that selective depletion of intracellular F2,6BP in cancer cells may suppress glycolytic flux and decrease their survival, growth and invasiveness. This review will summarize the requirement of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases for the regulation of glycolysis in tumor cells and their potential utility as targets for the development of antineoplastic agents.Item Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling(Springernature, 2010-01-07) Clem, Brian F.; Makoni, S.; Clem, Amy L.; Nelson, Kristin K.; Thornburg, Joshua M.; Siow, Deanna L.; Lane, Andrew N.; Brock, Stephanie E.; Goswami, Umesh; Eaton, John W.; Telang, Sucheta; Chesney, Jason A.; Yalçın, Abdullah; Uludağ Üniversitesi/Veterinerlik Fakültesi/Temel Bilimler Bölümü.; 0000-0001-8519-8375; ABI-4164-2020; 36857831000Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras(V12) in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.