Project Summary – Project Leader Chenguang Fan Breast cancer is one of the top threats to women’s health. About 1 in 8 US women (~12%) will develop invasive breast cancer over the course of her lifetime. Previous studies have shown the important role of a key tricarboxylic acid cycle (TCA) enzyme isocitrate dehydrogenase (IDH) in breast cancer. A variety of IDH alternations including somatic mutations, aberrant expression, and altered activities have been observed in breast cancer patients. Recently, phosphoproteomic studies of breast cancer cells have identified several unique phosphorylation sites in IDH isoforms, and those sites are specific for different types of breast cancer. Such heterogeneity poses challenges for effective diagnosis and treatment, so there is a critical need to discover specific mechanisms of IDH phosphorylation for breast cancer. The overall goal of this project is to identify the role of IDH phosphorylation in breast cancer. As IDH homologues in bacteria are known to be regulated by phosphorylation, the hypothesis is that phosphorylation of IDH in human cells alters IDH functions and cellular metabolism, thus facilitating cancerous transformations. To test this hypothesis, three specific aims are proposed: Aim 1 is to identify the effects of phosphorylation on IDH enzyme functions at the molecular level. Aim 2 is to identify the influences of IDH phosphorylation on cellular metabolism and redox status at the metabolic level. Aim 3 is to identify mechanisms or pathways in which IDH phosphorylation plays roles in breast cancer at the cellular level. The oncogenic transformation test on normal human mammary epithelial cells will be performed to identify whether IDH phosphorylation is an initiator for breast cancer or a cellular adaption to facilitate cancer formation. This proposal is innovative because: 1) the functions of IDH phosphorylation in human remain largely unknown, and this proposal will be the first to systematically study the phosphorylation of all the three IDH isoforms in human cells; 2) To address the problem that the classic glutamate-substitution method for phosphorylation studies is not always effective, the genetic code expansion technique will be applied in this proposal to co-translationally incorporate phosphoamino acid (pAA) at a controlled site in target proteins in order to produce site-specifically phosphorylated IDH variants which have been identified in breast cancer. This proposal will be the first to introduce pAA-incorporation systems into human cell lines to study phosphorylation in living cells. The proposed research is significant because it will provide key evidence for the influences and roles of IDH phosphorylation in breast cancer. Given that protein phosphorylation is usually involved in signaling pathways through different kinase cascades, the role of IDH phosphorylation in breast cancer could be different from other IDH alternations. Ultimately, the proposed studies on IDH phosphor...