Abstract 87: EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma

Abstract

Abstract Background: Ewing Sarcoma (EWS) is the second most common malignant bone tumor of children and adolescents. Patients with metastatic or recurrent disease have very poor outcomes. The receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R) has been implicated in EWS tumorigenesis and development of metastatic disease, with anti-IGF1R antibodies and kinase inhibitors in clinical studies. However, with only ~10% of patients achieving objective responses, delineation of novel pathways that facilitate IGF1R-driven oncogenesis in EWS could provide avenues for more effective therapy. Intracellular trafficking plays a central role in regulating RTK expression and signaling, and this pathway is frequently deregulated in cancer cells. The EPS15 homology domain-containing (EHD) proteins regulate intracellular traffic of cell surface receptors, including RTKs. We observed high frequency (67%) of EHD1 overexpression in 266 primary EWS patient tumor tissues and Kaplan-Meier survival analysis of publicly available mRNA expression data showed that high EHD1 expression was associated with shorter patient survival. Objective: This study aims to comprehend the underlying role of EHD1 in EWS oncogenesis. Study design: Three EHD1-expressing EWS cell lines, TC71, SKES1, and A673, were engineered with doxycycline-inducible EHD1 shRNAs to assess the impact of EHD1 knockdown (KD) on in vitro oncogenic properties. Stable EHD1-Crispr-KO and mouse EHD1-rescue TC71 and A673 cell line models were developed and characterized. Further, non-targeting control or EHD1-KD TC71 cells engineered with RFP-luciferase reporter were implanted in the tibia of nude mice in an orthotopic xenograft model and tumor growth was monitored by IVIS imaging. Results: EHD1-KD led to a significant impairment of EWS cell proliferation, migration, invasion, soft-agar colony formation, and tumor-sphere formation in vitro and reduced tumor growth in nude mice. Using a phospho-RTK profiling antibody array, we found reduced phospho-IGF1R levels upon EHD1-KD, identifying IGF1R as a potential target of regulation by EHD1. Western blotting showed a reduction in total IGF1R levels, and flow cytometric and immunofluorescence analyses revealed a pronounced decrease in the cell surface IGF1R levels upon EHD1 KD/KO in EWS cell lines. EHD1-KO EWS cell lines also exhibited a defect in IGF1-dependent migration and proliferation, and the phenotypes were restored by mouse EHD1 rescue. IGF1R and EHD1 were found to colocalize intracellularly and to co-immunoprecipitate after IGF1 stimulation. Finally, EHD1 depletion was found to impair the IGF-1R-mediated activation of downstream AKT and MAPK pathways. Conclusion: Our studies indicate a novel requirement of the intracellular traffic regulator EHD1 in sustaining IGF1R mediated oncogenesis in EWS. Future studies will aim to evaluate dual targeting of EHD1 and IGF1R in EWS. Citation Format: Sukanya Chakraborty, Bhopal C. Mohapatra, Sameer Mirza, Aaqib M. Bhat, Matthew D. Storck, Isidro Machado, José Antonio López, Antonio Llomart Bosch, Donald W. Coulter, Gargi Ghosal, Vimla Band, Hamid Band. EHD1 is required for IGF1R-mediated oncogenic signaling in Ewing Sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 87.