title: Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells.
abstract: BACKGROUND
  The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as "methionine dependence" or "methionine stress sensitivity" in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.
  
  
  RESULTS
  Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential.
  
  
  CONCLUSIONS
  This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions.
authors.affiliation: Department of Biological Chemistry, University of California, Irvine, Irvine, CA USA.
authors.forename: Stacey L
authors.initials: SL
authors.lastname: Borrego
authors.affiliation: West Coast Metabolomics Center, University of California, Davis, Davis, CA USA.|Present Address: University of Texas M.D. Anderson Cancer Center, Houston, TX USA.
authors.forename: Johannes
authors.initials: J
authors.lastname: Fahrmann
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, CA USA.
authors.forename: Rupsa
authors.initials: R
authors.lastname: Datta
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, CA USA.|Present Address: Laboratory for Optics and Biosciences, Ecole polytechnique, CNRS, INSERM, Université Paris-Saclay, 91128 Palaiseau cedex, France.
authors.forename: Chiara
authors.initials: C
authors.lastname: Stringari
authors.affiliation: CDS Creative Solutions, Ballwin, MO USA.|Present Address: CDS Creative Data Solutions, Ballwin, MO USA.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Grapov
authors.affiliation: Department of Computer Science, University of California, Irvine, Irvine, CA USA.|Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, CA USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Zeller
authors.affiliation: Department of Biological Chemistry, University of California, Irvine, Irvine, CA USA.
authors.forename: Yumay
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Biological Chemistry, University of California, Irvine, Irvine, CA USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Computer Science, University of California, Irvine, Irvine, CA USA.|Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, CA USA.
authors.forename: Pierre
authors.initials: P
authors.lastname: Baldi
authors.affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, CA USA.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.affiliation: West Coast Metabolomics Center, University of California, Davis, Davis, CA USA.|Biochemistry Department, King Abdulaziz University, Jeddah, Saudi-Arabia.
authors.forename: Oliver
authors.initials: O
authors.lastname: Fiehn
authors.affiliation: Department of Biological Chemistry, University of California, Irvine, Irvine, CA USA.|Institute for Genomics and Bioinformatics, University of California, Irvine, Irvine, CA USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Kaiser
keywords: Cancer metabolism; Homocysteine; Methionine; Methionine stress; S-adenosylmethionine; SAM