Studies of signaling pathways that regulate Ewing tumour cell growth in vitro

The Ewing family of peripheral primitive neuroectodermal tumours (ET or pPNET) comprises a genetically related group of bone and soft tissue tumours that primarily affects children and adolescents. All members of this tumour family are characterized by tumour specific translocations involving the EWS gene from chromosome llq23 and a second gene contributed by a variety of chromosomal partners. In all cases, the partner gene is a member of the ETS family of transcription factors and the resultant EWS-ETS gene fusions encode novel chimeric proteins comprised of the EWS-amino-terminal domain and an ETS-DNA binding domain. These fusion proteins are oncogenic and the accumulated evidence suggests that they are the primary pathologic lesions contributing to the development of ET. Despite its genetic characterization, little is known about the mechanisms of EWS-ETS mediated oncogenesis specifically, or in fact, the biologic features that contribute to the malignant nature of ET cells. We have endeavoured to study proliferative signaling in ET cells in an effort to better characterize the biology of these aggressive and often fatal tumours. Using differential-display PCR, our laboratory had previously identified differential expression of the human gastrin-releasing peptide (GRP) gene among EWS-ETS expressing tumour cell lines. Further studies confirmed that 100% of ET cell lines and approximately 50% of primary ET samples express the GRP gene and the gene encoding its receptor. Protein and functional studies demonstrated that the mature GRP peptide is secreted by ET cells and that GRP acts as an autocrine growth factor in ET both in vitro and in vivo. The proliferative pathways initiated in response to GRP stimulation are still not completely understood but are known to be both cell-type and situation specific. Our attempts at characterizing GRP-mediated proliferative signaling in ET suggest that the effects of this peptide may not be primarily mitogenic and we hypothesize that involvement of cytoskeletal proteins and differentiation pathways may be more important to the autocrine growth-factor capability of GRP in ET cells. Furthermore, studies of ET cell proliferation grown as multi-cellular spheroids indicate that in vivo growth and proliferation may be better represented by this anchorage-independent cell culture model. In comparing adherent and anchorage-independent proliferation, we also observed that fundamental differences exist in the regulation of cyclin DI between these two models. Moreover, the PI3K—AKT pathway was shown to be of key importance to the regulation of both cyclin DI expression and proliferation in ET cells. Our results suggest that an autocrine growth factor pathway mediated by GRP exists in ET and that proliferative signaling in these tumours is critically dependent on cell-cell and/or cell-matrix adhesion and the PI3K—AKT pathway.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat