DOI: 10.5061/dryad.931zcrjqk
title: The PLOD2/Succinate axis regulates the epithelial-mesenchymal plasticity and cancer cell stemness

description: Aberrant accumulation of succinate has been detected in many cancers.
 However, the cellular function and regulation of succinate in cancer
 progression is not completely understood. Using stable isotope-resolved
 metabolomics (SIRM) analysis, we showed that the epithelial mesenchymal
 transition (EMT) was associated with profound changes in metabolites,
 including the elevation of cytoplasmic succinate levels. Treatment with
 cell-permeable succinate induced mesenchymal phenotypes in mammary
 epithelial cells and enhanced cancer cell stemness. Chromatin
 immunoprecipitation (ChIP) and sequence analysis showed that elevated
 cytoplasmic succinate levels were sufficient to reduce global
 5-hydroxymethylcytosinene (5hmC) accumulation and induce transcriptional
 repression of EMT-related genes. We showed that expression of
 procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2) was associated
 with an elevation of cytoplasmic succinate during the EMT process. Silence
 of PLOD2 expression in breast cancer cells reduced succinate levels and
 inhibited cancer cell mesenchymal phenotypes and stemness, which was
 accompanied by elevated 5hmC levels in chromatin. Importantly, exogenous
 succinate rescued cancer cell stemness and 5hmC levels in PLOD2-silenced
 cells, suggesting that PLOD2 promotes cancer progression at least
 partially through succinate. These results reveal the previously
 unidentified function of succinate in enhancing cancer cell plasticity and
 stemness.
description: Polar extracts of cells treated with [U-13C]-glucose were
 resolved and detected using an ion-chromatogrphay (Dionex) system
 interfaced to an Orbitrap mass spectrometer (Thermo).
 The polar extracts were reconstituted in 20 μL Nanopure water,
 and analyzed by a Dionex ICS-5000+ ion chromatograph interfaced to an
 Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San
 Jose, CA, USA) operating at a resolution setting of 500,000 (FWHM at m/z
 200) on MS1 acquisition to capture all <sup>13</sup>C
 isotopologues. The chromatograph was outfitted with a Dionex IonPac
 AG11-HC-4 µm RFIC&HPIC (2 x 50 mm) guard column upstream of a
 Dionex IonPac AS11-HC-4 µm RFIC&HPIC (2 x 250 mm) column.
 Chromatography and mass spectrometric settings were the same as described
 previously (Fan, T., S. El-Amouri, J. Macedo, Q. Wang, H. Song, T. Cassel,
 and A. Lane. 2018. Stable Isotope-Resolved Metabolomics Shows Metabolic
 Resistance to Anti-Cancer Selenite in 3D Spheroids versus 2D Cell
 Cultures. <em>Metabolites</em> 8: 40.) with an m/z range of 80
 to 700. Metabolites and their isotopologues were identified and their peak
 areas were integrated and exported to Excel via the TraceFinder 3.3
 (Thermo) software package. Peak areas were corrected for natural abundance
 as previously described (Moseley, H. N. 2010. Correcting for the effects
 of natural abundance in stable isotope resolved metabolomics experiments
 involving ultra-high resolution mass spectrometry. <em>BMC
 Bioinformatics</em> 11: 139), after which fractional enrichment and
 µmoles metabolites/g protein were calculated to quantify
 <sup>13</sup>C incorporation into various
 metabolites.