Defining the metabolic phenotype of epithelial-mesenchymal transition in breast epithelium

Abstract

Breast cancer is the leading cause of cancer deaths among women in Iceland and worldwide. Epithelial-mesenchymal transition (EMT) is a cellular developmental process where epithelial cells assume mesenchymal-like phenotypes through degradation of the extracellular matrix, loss of adhesions, and increased mobility. It is believed that dissemination of cancer cells occurs partly following EMT. EMT contains a spectrum of epithelial-mesenchymal intermediate cell states that impart different degrees of malignancy. The ability of cells to assume these states is termed epithelial-mesenchymal plasticity (EMP). This project aimed at characterizing the metabolic phenotypes of EMT and EMP to obtain knowledge of breast cancer progression and to identify biomarkers and potential therapeutic targets for breast cancer treatment. Proteomics analysis of cell models of EMT and EMP revealed changes to enzymes involved in glycan metabolism. UDP-glucose dehydrogenase (UGDH) and glutamine-fructose-6-phosphate transaminase 2 (GFPT2) were identified as the topmost altered glycan metabolic enzymes in EMT and EMP, respectively. UGDH converts UDP-glucose into UDP-glucuronic acid and is involved in the formation of hyaluronan in the extracellular matrix. GFPT2 influences the downstream formation of UDP-N-acetylglucosamine (UDP-GlcNAc) and protein O-GlycNAcylation. UGDH was associated with patient survival, affected cell proliferation, cell invasion, and the expression of the EMT marker SNAI1. siRNA-mediated knockdown of UGDH influenced glycerophosphocholine (GPC) and increased N-acetylaspartate (NAA) levels. GFPT2 similiarly influenced cell proliferation, migration, invasion, and expression of the EMT marker vimentin and was associated with claudin-low breast cancer. GFPT2 was shown to be a marker of oxidative stress, and knockdown of GFPT2 affected cystathionine levels and the mitochondrial enzyme sulfide quinone oxidoreductase (SQOR). Phosphoproteomics analysis indicated distinct phosphorylation profiles of epithelial versus mesenchymal cells. Specifically, pyruvate dehydrogenase kinases (PDHKs), serine/threonine-protein kinase (PAK1), and protein kinase A catalytic subunit α (PKACA) were differentially regulated across the mesenchymal cell lines tested. In conclusion, these results suggest that UGDH and GFPT2 are central to changes that occur within glycan metabolism following EMT and EMP, respectively. Both enzymes were associated with cancer progression and GFPT2, specifically, may serve as a biomarker for cellular oxidative stress and claudin-low breast cancer. The work furthermore implicates UGDH in GPC and NAA metabolism.