THE ROLE OF NEUROPEPTIDE Y AND ITS Y5 RECEPTOR IN RHOA-MEDIATED REGULATION OF CELL MOTILITY AND CYTOKINESIS: IMPLICATIONS FOR CANCER METASTASIS

Abstract

Neuropeptide Y (NPY) and its Y5 receptor (Y5R) are expressed in a number of malignancies, including pediatric tumors neuroblastoma (NB) and Ewing sarcoma (ES). Our previous clinical and experimental data implicated NPY/Y5R axis in NB and ES metastasis. We have shown that Y5R is highly expressed in angioinvasive NB cells, while elevated release of NPY from NB tumors associates with metastatic disease and poor clinical outcome. Additionally, hypoxia-induced over- activation of NPY/Y5R pathway in ES tumors, which express particularly high levels of Y5R, led to the formation of polyploid cells that gave rise to chromosomally unstable, metastatic progeny. Altogether, our data suggested a role for the NPY/Y5R pathway in the regulation of cell migration and cytokinesis, processes essential in tumor growth and dissemination. Thus, the goal of our study was to determine mechanisms of NPY/Y5R actions. We have found that Y5R stimulation activated an essential cytoskeleton regulator, RhoA, activity of which is tightly controlled in a spatial and temporal manner during both cell migration and cytokinesis. In migratory cells cultured in vitro, Y5R localized to the regions of high RhoA activity and dynamic cytoskeleton remodeling, including leading and trailing edges, cell-cell junctions, filopodia and the leader cells in cell clusters. Y5R stimulation resulted in an increased RhoA activity and Y5R-RhoA-GTP interactions, as shown by pull-down and proximity ligation assays, respectively. Consequently, activation of iii the NPY/Y5R/RhoA axis stimulated cell motility and chemotaxis. However, over-activation of this pathway, such as this observed in hypoxic ES cells, led to perturbations in normal RhoA functions. In mitotic cells, overexpression of Y5R led to the accumulation of RhoA in the narrow zone of the cleavage furrow at the abscission phase of cytokinesis. Such RhoA activation at this stage was previously shown to trigger cytokinesis failure. These mitotic defects resulted in the formation of polyploid ES cells, the progeny of which exhibited high chromosomal instability, an ability to invade and colonize bone, and resistance to chemotherapy. To our knowledge, this is the first demonstration of the role of the NPY/Y5R axis in RhoA activation and subsequent cytoskeleton remodeling facilitating cell movement and cytokinesis defects. These findings implicate Y5R as a potential target in anti-metastatic therapies in cancer types expressing this receptor.