Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial-Mesenchymal Transition and Responds to Oxidative Stress

Funding Information: com. This work was funded by Icelandic Center for Research (RANNÍS, 163254-052) and Göngum Saman 2018. Funding Information: We are grateful for the contributions from Douglas Lamont and Amy Tavendale at the "Finger- Prints" Proteomics Facility, College of Life Sciences, MSI/ WTB/JBC Complex, University of Dundee, for their help with the proteomic and phosphoproteomic data collection and analysis. The graphical abstract was created with BioRender.com. This work was funded by Icelandic Center for Research (RANNIS, 163254-052) and Göngum Saman 2018. Publisher Copyright: © 2021 THE AUTHORS.Breast cancer cells that have undergone partial epithelial- mesenchymal transition (EMT) are believed to be more invasive than cells that have completed EMT. To study metabolic reprogramming in different mesenchymal states, we analyzed protein expression following EMT in the breast epithelial cell model D492 with single-shot LFQ supported by a SILAC proteomics approach. The D492 EMT cell model contains three cell lines: the epithelial D492 cells, the mesenchymal D492M cells, and a partial mesenchymal, tumorigenic variant of D492 that overexpresses the oncogene HER2. The analysis classified the D492 and D492M cells as basal-like and D492HER2 as claudin-low. Comparative analysis of D492 and D492M to tumorigenic D492HER2 differentiated metabolic markers of migration from those of invasion. Glutamine-fructose-6- phosphate transaminase 2 (GFPT2) was one of the top dysregulated enzymes in D492HER2. Gene expression analysis of the cancer genome atlas showed that GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA-mediated knockdown of GFPT2 influenced the EMT marker vimentin and both cell growth and invasion in vitro and was accompanied by lowered metabolic flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide: quinone oxidoreductase (SQOR) in the transsulfuration pathway that regulates H2S production and mitochondrial homeostasis. Moreover, GFPT2 was within the regulation network of insulin and EGF, and its expression was regulated by reduced glutathione (GSH) and suppressed by the oxidative stress regulator GSK3-β. Our results demonstrate that GFPT2 controls growth and invasion in the D492 EMT model, is a marker for oxidative stress, and associated with poor prognosis in claudin-low breast cancer.Peer reviewe

UDP-glucose dehydrogenase expression is upregulated following EMT and differentially affects intracellular glycerophosphocholine and acetylaspartate levels in breast mesenchymal cell lines

Funding Information: The study received funding from Icelandic centre for research award number: 163254‐051; Recipient: Ottar Rolfsson and Doktorsstyrkir Háskóla Ísland; Doktorsstyrkir 2020; Recipient: Qiong Wang. Publisher Copyright: © 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.Metabolic rewiring is one of the indispensable drivers of epithelial–mesenchymal transition (EMT) involved in breast cancer metastasis. In this study, we explored the metabolic changes during spontaneous EMT in three separately established breast EMT cell models using a proteomic approach supported by metabolomic analysis. We identified common proteomic changes, including the expression of CDH1, CDH2, VIM, LGALS1, SERPINE1, PKP3, ATP2A2, JUP, MTCH2, RPL26L1 and PLOD2. Consistently altered metabolic enzymes included the following: FDFT1, SORD, TSTA3 and UDP-glucose dehydrogenase (UGDH). Of these, UGDH was most prominently altered and has previously been associated with breast cancer patient survival. siRNA-mediated knock-down of UGDH resulted in delayed cell proliferation and dampened invasive potential of mesenchymal cells and downregulated expression of the EMT transcription factor SNAI1. Metabolomic analysis revealed that siRNA-mediated knock-down of UGDH decreased intracellular glycerophosphocholine (GPC), whereas levels of acetylaspartate (NAA) increased. Finally, our data suggested that platelet-derived growth factor receptor beta (PDGFRB) signalling was activated in mesenchymal cells. siRNA-mediated knock-down of PDGFRB downregulated UGDH expression, potentially via NFkB-p65. Our results support an unexplored relationship between UGDH and GPC, both of which have previously been independently associated with breast cancer progression.Peer reviewe

UDP-glucose dehydrogenase expression is upregulated following EMT and differentially affects intracellular glycerophosphocholine and acetylaspartate levels in breast mesenchymal cell lines

Metabolic rewiring is one of the indispensable drivers of epithelial-mesenchymal transition (EMT) involved in breast cancer metastasis. In this study, we explored the metabolic changes during spontaneous EMT in three separately established breast EMT cell models using a proteomics approach supported by metabolomic analysis. We identified common proteomic changes, including in the expression of CDH1, CDH2, VIM, LGALS1, SERPINE1, PKP3, ATP2A2, JUP, MTCH2, RPL26L1 and PLOD2. Consistently altered metabolic enzymes included: FDFT1, SORD, TSTA3 and UDP-glucose dehydrogenase (UGDH). Of these, UGDH was most prominently altered and has previously been associated with breast cancer patient survival. siRNA-mediated knockdown of UGDH resulted in delayed cell proliferation and dampened invasive potential of mesenchymal cells, and downregulated expression of the EMT transcription factor SNAI1. Metabolomic analysis revealed that siRNA-mediated knockdown of UGDH decreased intracellular glycerophosphocholine (GPC), whereas levels of acetyl aspartate (NAA) increased. Finally, our data suggested that platelet-derived growth factor receptor beta (PDGFRB) signaling was activated in mesenchymal cells. siRNA-mediated knockdown of PDGFRB downregulated UGDH expression, potentially via NFkB-p65. Our results support an unexplored relationship between UGDH and GPC, both of which have previously been independently associated with breast cancer progression