title: Mass spectrometry analysis shows the biosynthetic pathways supported by pyruvate carboxylase in highly invasive breast cancer cells.
abstract: We recently showed that the anaplerotic enzyme pyruvate carboxylase (PC) is up-regulated in human breast cancer tissue and its expression is correlated with the late stages of breast cancer and tumor size [Phannasil et al., PloS One 10, e0129848, 2015]. In the current study we showed that PC enzyme activity is much higher in the highly invasive breast cancer cell line MDA-MB-231 than in less invasive breast cancer cell lines. We generated multiple stable PC knockdown cell lines from the MDA-MB-231 cell line and used mass spectrometry with13C6-glucose and13C5-glutamine to discern the pathways that use PC in support of cell growth. Cells with severe PC knockdown showed a marked reduction in viability and proliferation rates suggesting the perturbation of pathways that are involved in cancer invasiveness. Strong PC suppression lowered glucose incorporation into downstream metabolites of oxaloacetate, the product of the PC reaction, including malate, citrate and aspartate. Levels of pyruvate, lactate, the redox partner of pyruvate, and acetyl-CoA were also lower suggesting the impairment of mitochondrial pyruvate cycles. Serine, glycine and 5-carbon sugar levels and flux of glucose into fatty acids were decreased. ATP, ADP and NAD(H) levels were unchanged indicating that PC suppression did not significantly affect mitochondrial energy production. The data indicate that the major metabolic roles of PC in invasive breast cancer are primarily anaplerosis, pyruvate cycling and mitochondrial biosynthesis of precursors of cellular components required for breast cancer cell growth and replication.
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Phatchariya
authors.initials: P
authors.lastname: Phannasil
authors.affiliation: Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
authors.forename: Israr-Ul H
authors.initials: IH
authors.lastname: Ansari
authors.affiliation: Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: El Azzouny
authors.affiliation: Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Longacre
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: Khanti
authors.initials: K
authors.lastname: Rattanapornsompong
authors.affiliation: Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Charles F
authors.initials: CF
authors.lastname: Burant
authors.affiliation: Childrens Diabetes Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: MacDonald
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand. Electronic address: sarawut.jit@mahidol.ac.th.
authors.forename: Sarawut
authors.initials: S
authors.lastname: Jitrapakdee
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D053898:Biosynthetic Pathways; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019343:Citric Acid / Q000378:metabolism; D005260:Female; D055785:Gene Knockdown Techniques; D005998:Glycine / Q000378:metabolism; D006019:Glycolysis; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology*; D009711:Nucleotides / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: D008293:Malates; D009711:Nucleotides; D019343:Citric Acid; D001224:Aspartic Acid; D019344:Lactic Acid; D012694:Serine; D000105:Acetyl Coenzyme A; C030298:malic acid; D019289:Pyruvic Acid; D011766:Pyruvate Carboxylase; D005998:Glycine
keywords: Anaplerosis; Breast cancer; Cancer biology; Cell proliferation rate; Gene knockout; Mitochondrial biosynthesis; Mitochondrial metabolism; Pyruvate carboxylase (PC)