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.

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 ¹³C 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. Metabolites 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. BMC Bioinformatics 11: 139), after which fractional enrichment and µmoles metabolites/g protein were calculated to quantify ¹³C incorporation into various metabolites.