Breast cancer cells secrete glycosylated protein to facilitate blood-brain barrier opening and brain metastasis - Our overall goal of this project is to reveal the comprehensive mechanism of how metastatic breast cancer induces permeability of the blood-brain barrier (BBB), thereby facilitating breast cancer brain metastases (BCBM). BCBM is a leading cause of death in late-stage breast cancer patients. BCBM is particularly difficult to treat because drugs cannot easily cross the BBB, a protective barrier around the brain. Despite the critical role of the BBB in safeguarding the brain, it remains unclear how cancer cells can pass through it and metastasize to the brain. Based on our new findings, brain-metastatic breast cancer cells highly express and secret fetuin-A (FetA). Cancer-derived FetA transiently opened the endothelial barrier in vitro and the BBB in mice. Although FetA is known as a serum protein released from the liver, human serum FetA did not open the barrier in vitro and in vivo. These data clearly showed a significant biological difference between cancer-derived FetA and serum FetA. Furthermore, de-glycosylation of cancer-derived FetA abolished its activity on the endothelial barrier, suggesting glycosylation of cancer-derived FetA is critical for opening the endothelial barrier. The uptake of cancer-derived FetA by Annexin A2 (AnxA2) induced Ca2+ influx and activation of calmodulin-dependent protein kinase II (CaMKII) in the brain endothelial cells (ECs). The peptide inhibitor designed based on the docking model of FetA and AnxA2 inhibited cancer-derived FetA-induced brain endothelial permeability. Based on these solid supporting data, we hypothesize that brain-metastatic breast cancer cells secrete FetA to facilitate brain metastases by transiently opening the BBB. Thus, inhibition of FetA-induced BBB permeability helps develop a novel strategy to prevent BCBM. We will examine our hypothesis with two specific aims. AIM1: To elucidate a mechanism of how cancer-derived FetA induces BBB permeability. We will investigate glycosylation patterns of cancer-derived FetA by comparing them with human serum FetA. We will examine the roles of glycosylation of FetA in inducing brain endothelial permeability and the effects on each cell type of the BBB (brain ECs, pericytes, and astrocytes). AIM2: To examine how cancer-derived FetA facilitates breast cancer brain metastasis. We will determine the roles of cancer-derived FetA and its glycosylation by inducing knockout or glycosylation-defective mutant FetA in breast cancer cells and test their brain metastasis in mice. Further, we will employ peptide-based inhibitors for FetA uptake and test the effects on the FetA-induced BBB permeability and breast cancer brain metastasis in animal models. In summary, our project will reveal essential insights into how metastatic breast cancer opens the BBB to invade the brain to metastasize. Also, gaining a deeper understanding of the unique biological action involving FetA will contribute to developing a new concept for brain metastasis.
