Abstract 106: Growth-associated protein 43 (GAP-43): the link to Ewing sarcoma bone metastasis

Abstract Ewing Sarcoma (ES) is an aggressive pediatric tumor that arises in bone or soft tissues. ES bone metastases do not respond well to routine therapies and their frequency markedly increases upon disease recurrence, which results in a lower survival rate in patients with osseous metastases as compared to those with pulmonary dissemination. Our previous studies have determined that ES bone metastasis is initiated by hypoxia in the primary tumor site, which leads to the formation of polyploid tumor cells, the progeny of which, named HYP-4n cells, exhibit high levels of chromosomal instability. These cells metastasize almost exclusively to bone and are less sensitive to cytostatic agents. In this study, we demonstrate that HYP-4n cells exhibit an upregulation in several neuronal proteins, specifically growth-associated protein 43 (GAP-43), a membrane protein promoting axon growth in neurons and formation of microtubes connecting glioblastoma cells into invasive and therapy-resistant cellular network. Similar phenotype characterized by the presence of microtube-like cell-cell connections was also observed in ES HYP-4n cells. GAP-43 immunostaining in HYP-4n cells revealed a punctuated pattern along the protrusions that is similar to its localization in neurons. Western Blot analysis performed on HYP-4n cells revealed a membranous localization of GAP-43, which was not detectable in their normoxic diploid counterparts. The functional role of the GAP-43-positive protrusions in connecting HYP-4n cells was confirmed by the gap-junction permeable dye transfer between the cells and ion transfer detected by a patch clamp assay. DAPI staining also displayed nuclear fragments within the protrusions, suggesting the transfer of genetic material. The suppression of GAP-43 expression via siRNA was further shown to inhibit HYP-4n cell migration measured via transwell assay. In immunostained ES xenografts, GAP-43 expression was shown in HYP-4n primary and osseous metastatic tumors, with the highest membranous expression in the ES cells on the invading edge of the tumors. A similar pattern of expression of GAP-43 was noted in the human samples of ES bone metastases. This study indicates that GAP-43 is active in the membrane in HYP-4n cells and drives the formation of microtube networks promoting cell invasion. Further studies are required to prove directly the role of GAP-43 in ES bone metastasis and chemoresistance. Citation Format: Tyler Biermann, Nicholas Cohen, Yasmine Wazni, Stefano Vicini, Jason Tilan, Joanna Kitlinska. Growth-associated protein 43 (GAP-43): the link to Ewing sarcoma bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 106.