Characterization of Trafficking and Metabolic Functions of the Endocytic Regulator EHD1 in Ewing Sarcoma

Author

Sukanya Chakraborty, University of Nebraska Medical CenterFollow

ORCID ID

0000-0003-1238-7962

Graduation Date

Fall 12-15-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Interdisciplinary Graduate Program in Biomedical Sciences

First Advisor

Hamid Band

Abstract

Ewing sarcoma (EWS) is the second most common bone malignancy in children and adolescents. Despite improvement in survival due to advances in multimodality treatment strategies, patients with metastatic or recurrent disease have very poor outcomes. Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown.

Studies presented in this dissertation establish that EHD1 overexpression is a feature of nearly 90% EWS patient tumors with high EHD1 expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. Receptor tyrosine kinase (RTK) antibody arrays identified the Insulin-like growth factor 1 receptor (IGF-1R) as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.

Cancer cells display a distinct metabolic phenotype known as the Warburg effect, where even in the presence of sufficient oxygen, they prefer to use glycolysis as their main source of energy with increased glucose uptake and glycolytic flux, a phenomenon that was termed as aerobic glycolysis by Otto Warburg. In this study, we demonstrate that EHD1 regulates glucose uptake in EWS cells in an IGF-1R dependent manner. In addition, by Seahorse Extracellular Flux analysis, we show that EHD1 regulates the glycolytic proton efflux rate of EWS cells. Using LC-MS/MS based metabolomics and PCR profiler array analyses, we establish that loss of EHD1 results in decreased expression of glycolytic genes and corresponding decrease in glycolytic metabolites. Additionally, EHD1-knockout EWS cells also show an increase in tricarboxylic acid (TCA) cycle metabolite levels and expression of TCA cycle genes. Taken together, we present evidence of the crucial role of the EHD1-IGF-1R axis in the acquisition of the Warburg metabolic phenotypes in EWS cells.

The studies above have contributed to unraveling an essential role of the endocytic recycling regulator EHD1 in RTK trafficking and metabolic rewiring of EWS cells, thus opening new avenues to understand the roles of EHD1 in cancer cell biology and for therapeutic targeting in EWS.

Comments

2023 Copyright, the authors

Recommended Citation

Chakraborty, Sukanya, "Characterization of Trafficking and Metabolic Functions of the Endocytic Regulator EHD1 in Ewing Sarcoma" (2023). Theses & Dissertations. 775.
https://digitalcommons.unmc.edu/etd/775