The biology of high grade serous ovarian cancer (OvCa) is distinct from that of most epithelial tumors, in that hematogenous metastases are rare. Ovarian tumors remain confined to the peritoneal cavity and primarily seed to the omentum and peritoneum. There, cancer cells interact with cancer associated fibroblasts (CAFs) which promote cancer cell proliferation, invasion, and metastasis. To study bidirectional signaling between the two cell types, we co-cultured them and used SILAC coupled with quantitative, label-free phosphoproteomics to identify phospho-tyrosine signaling events in both OvCa cells and fibroblasts. We identified activation of phosphoglucomutase 1 (PGM1) in the cancer cells, pointing towards regulation of glycogen metabolism in OvCa cells by CAFs. Further, preliminary experiments showed that glycogenolysis provides energy to cancer cells and regulates protein glycosylation and histone methylation. Based on these data, the primary hypothesis underlying this application is that CAF-mediated glycogenolysis promotes metastasis through several different mechanisms, including glycolysis, protein glycosylation, and epigenetics.
In Aim I, we will explore the metabolic consequences of PGM1 activation or inhibition in both OvCa cells and normal fallopian tube epithelial cells using untargeted metabolomics and metabolic flux studies. We will use PGM knockdown cells and investigate the effects of PGM1 inhibition on adhesion/invasion/proliferation using a 3D model and a syngeneic mouse model of metastasis. We will then utilize a high-throughput RNAi library with the goal of identifying the upstream signaling pathways regulating glycogenolysis in response to CAFs.
In Aim II, we will systematically study glycogen-mediated glycosylation events using human protein glycosylation arrays to identify which proteins are specifically glycosylated by CAF-mediated glycogenolysis. This investigation will be complemented by studies determining how glycosyltransferase enzymes regulate CAF- mediated glycosylation. We will then study the role of glycosyltransferases on in vivo metastasis assays using primary CAF/OvCa cells and a syngeneic mouse model of OvCa metastasis. Using targeted metabolomics data and flux analysis of CAFs co-cultured with OvCa cells, we discovered that glycogenolysis induced ?- ketoglutarate (KG). We therefore propose, in Aim III, to study how glycogenolysis alters epigenetic changes in cancer cells. We will characterize genome-wide changes in histone methylation (ChIP-seq) and DNA hydroxymethylation (Nano-hmC-Seal) to identify genes epigenetically regulated by glycogenolysis. The proposed experiments aim to define the link between glycogenolysis and epigenetics to identify functional regulators of OvCa metastasis. By understanding glycogen metabolism in the tumor organ, we may be able to elucidate novel metabolic mechanisms important for metastasis, which could result in the identification of a new and clinically relevant approach to the treatment of metastatic ovarian cancer.
Ovarian cancer metastasis involves the interplay between tumor cells and the tumor microenvironment. We propose that cancer-associated fibroblasts (CAFs) regulate glycogen metabolism in ovarian cancer cells, thereby promoting tumor growth and metastasis. Experiments are planned to understand the novel mechanisms through which CAFs induce glycogen release in cancer cells and glycogen fuels tumor progression.
