Altered Glutamine Metabolism Exposes EMT Derived Mesenchymal Cells to PI3K/Akt/mTOR Pathway Inhibition

doi:10.21203/rs.3.rs-100299/v1

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Altered Glutamine Metabolism Exposes EMT Derived Mesenchymal Cells to PI3K/Akt/mTOR Pathway Inhibition

https://doi.org/10.21203/rs.3.rs-100299/v1

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Background: Epithelial-to-mesenchymal transition (EMT) is a fundamental developmental process with strong implications in cancer progression. Understanding the metabolic alterations associated with EMT may open new avenues of treatment and prevention.

Methods: We utilize ¹³C carbon analogs of glucose and glutamine to examine difference in internal flux patterns of metabolites in central carbon and lipid metabolism following EMT in breast epithelial cell lines. Furthermore, an isotopomer spectral analysis, ¹³C-metabolic flux analysis and weighted correlation network analysis are utilized for investigation of the alterations in metabolic functionality following EMT in breast.

Results: There are inherent differences in metabolic profiles before and after EMT. We observed EMT-dependent re-routing of TCA-cycle flux characterized by increased mitochondrial IDH2 -mediated reductive carboxylation of glutamine to lipid biosynthesis with a concomitant lowering of glycolytic rates and glutamine-dependent glutathione (GSH) generation. Our network approach identified specific subtype of cancer drugs that are significantly associated with GSH abundance and we confirmed this in vitro.

Conclusions: EMT-linked alterations in GSH synthesis modulate the sensitivity of breast epithelial cells to PI3K/Akt/mTOR inhibitors.

Cancer Biology

Oncology

Metabolomics

Epithelial-to-mesenchymal transition

Fluxomics

Network analysis

Breast cancer

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13CMFARESULTS.xlsx
Results from 13C-metabolic flux analysis This file is an Microsoft Excel file (.xlsx) containing information about the reactions, their formulas and the calculated flux values (and errors) in the 13C-MFA of D492 and D492M.
12Glucose6hoursIDH2AUGUST2020.png
UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with 1,2-13C-glucose for 6 hours
12Glucose6hoursWTAUGUST2020.png
UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with 1-13C-glutamine for 6 hours
1Glutamine6hoursWTAUGUST2020.png
UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with 1-13C-glutamine for 6 hours
1glutamine6hoursIDH2AUGUST2020.png
UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with 1-13C-glutamine for 6 hours
5Glutamine6hoursWTAUGUST2020.png
UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with 5-13C-glutamine for 6 hours
5glutamine6hoursIDH2AUGUST2020.png
UPLC-MS label enrichment in various metabolites after culturing of D492 and D492M with 5-13C-glutamine for 6 hours, either with or without shRNA-lentiviral silencing of IDH2 expression
DrugnetworkanalysisFORSUBMISSIONAUGUST2020.R
An R-script for the weighted correlation network analysis This file is a .R file which can be opened in RStudio.
GlucoseglutaminedependencyAUGUST2020.png
Dependency of D492 and D492M on glucose and glutamine A) Cell number of D492 and D492M in H14 medium, H14 without glucose and H14 without glutamine. B) Growth rate calculations from numbers used in A. Values are presented as mean + standard error (n=3). Asterisks represent significance of pairwise comparison between full medium and the depleted medium.
NMRFINALAUGUST2020.png
NMR data from 1H and 13C labelling experiments of D492 and D492M A) PC 1 scores after Principal Component Analysis of 1H NMR data and B) Corresponding loading plot from PC 1*. C) NMR peak area after incubation with 1,2-13C-glucose for 6 hours. Integrated peak areas of 4-13C glutamate. Results are presented as mean + std (n=3). D) NMR peak area after incubation with 1-13C-glutamine for 6 hours. The metabolites show integrated peak areas of 1-13C glutamate, 1-13C glutathione and 1-13C proline. Results are presented as mean + std (n=2). *: Peak assignment: a: isoleucine, b: leucine, c: valine, d: lactate, e: alanine, f: arginine, g: glutamine, h: glutamate, i: glutathione, j: phosphocholine (10 times higher than shown), k: myo-inositol, l: glycine, m: asparagine, n: proline, o: amp, p: adp, q: atp: r: threonine, s: glucose, t: fumarate, u: tyrosine, v: phenylalanine, w: NAD+, x: NADP, y: NADH.
Primersequences.xlsx
Primers used for quantitative Real-Time PCR A table showing nucleotide sequences of IDH2 and IDH1 primers used in this study. ’FWD’ and ’REV’ are abbreviations for forward and reverse, respectively.

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Altered Glutamine Metabolism Exposes EMT Derived Mesenchymal Cells to PI3K/Akt/mTOR Pathway Inhibition

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