Glutamine-fructose-6-phosphate transaminase 2 (GFPT2) is upregulated in breast epithelial-mesenchymal transition and involved in oxidative stress regulation.

Epithelial-mesenchymal transition (EMT) is a diverse and dynamic biological process which is involved in cancer progression. It is important for carcinoma cells during invasion and metastasis. EMT has been in the spotlight for cancer cells to disseminate to distant organs by gaining partial EMT phenotype. Cancer cells with partial EMT are believed to be more cancerogenic/invasive than cells that have undergone complete EMT. The proteomic changes that occur following EMT in breast epithelial cells and how these relate to changes in cellular metabolism are incompletely understood. To study metabolic reprogramming in different mesenchymal states, we analyzed proteomic changes following EMT in the breast epithelial cell model D492, with single-shot LFQ supported by SILAC proteomic approach. The D492 EMT cell model contains three isogenic cell lines: epithelial D492 cells, mesenchymal D492M cells, and partial mesenchymal, HER2 overexpressing, tumorigenic D492HER2 cells. Proteomic analysis positioned the D492 and D492M cells as basal-like while D492HER2 as claudin-low. Further comparison of the non-tumorigenic D492 and D492M cells to tumorigenic D492HER2 differentiated the metabolic EMT markers of migration from those of invasion. Among these were markers of glycan metabolism. We identified glutamine-fructose-6-phosphate transaminase [isomerizing] 2 (GFPT2) as the top dysregulated enzyme in glycan metabolism and found increased GFPT2 expression was a characteristic of claudin-low breast cancer. siRNA knockdown of GFPT2 influenced both cell growth and invasion in vitro and was accompanied by lowered flux through the hexosamine biosynthesis pathway (HBP). Knockdown of GFPT2 decreased cystathionine and sulfide:quinone oxidoreductase (SQOR) in the transsulfuration pathway which regulates H2S production and mitochondrial homeostasis. Moreover, GFPT2 expression was regulated by the level of reduced glutathione (GSH) and suppressed by the oxidative stress regulator, GSK3-β. Our results demonstrate that GFPT2 is a marker for oxidative stress. It is upregulated and controls growth and invasion in the D492 EMT model and is associated with claudin-low and poor prognosis in breast cancer.

上皮间质转化（Epithelial-mesenchymal transition, EMT）是一类多样且动态的生物学过程，参与肿瘤进展进程。其对于癌细胞的侵袭与转移至关重要。EMT因癌细胞可通过获得部分EMT表型向远处器官播散，而成为研究热点。相较于完全经历EMT的细胞，携带部分EMT表型的癌细胞被认为具有更强的致瘤性与侵袭能力。目前，乳腺癌上皮细胞发生EMT后的蛋白质组学变化，以及这些变化与细胞代谢改变的关联尚未完全阐明。为研究不同间质状态下的代谢重编程，我们依托稳定同位素标记氨基酸细胞培养（Stable Isotope Labeling by Amino acids in Cell culture, SILAC）蛋白质组学方法辅助的单针无标记定量（single-shot LFQ）技术，对乳腺癌上皮细胞模型D492在EMT发生后的蛋白质组学变化进行了分析。D492 EMT细胞模型包含三株同基因细胞系：上皮型D492细胞、间质型D492M细胞，以及部分间质型、人表皮生长因子受体2（HER2）过表达且具备致瘤性的D492HER2细胞。蛋白质组学分析显示，D492与D492M细胞属于基底样亚型，而D492HER2细胞则归类为低紧密连接蛋白（claudin-low）亚型。进一步将非致瘤性的D492与D492M细胞与致瘤性的D492HER2细胞进行比较，我们区分出了与迁移相关的代谢性EMT标志物，以及与侵袭相关的代谢性EMT标志物，其中涵盖了糖代谢相关标志物。我们鉴定出谷氨酰胺-果糖-6-磷酸转氨酶[异构化]2（GFPT2）是糖代谢中表达差异最显著的酶，且发现GFPT2表达升高是低紧密连接蛋白型乳腺癌的特征。通过小干扰RNA（siRNA）敲低GFPT2可在体外同时影响细胞生长与侵袭能力，同时伴随己糖胺生物合成途径（hexosamine biosynthesis pathway, HBP）代谢流的降低。敲低GFPT2还会减少转硫途径中的胱硫醚与硫化物：醌氧化还原酶（sulfide:quinone oxidoreductase, SQOR）的表达，而该途径可调控硫化氢（H₂S）的生成与线粒体稳态。此外，GFPT2的表达受还原型谷胱甘肽（reduced glutathione, GSH）水平调控，并可被氧化应激调控因子糖原合成激酶3β（glycogen synthase kinase 3β, GSK3-β）所抑制。本研究结果表明，GFPT2是氧化应激的标志物。在D492 EMT模型中，GFPT2表达上调并调控细胞生长与侵袭能力，且与低紧密连接蛋白型乳腺癌及不良预后密切相关。