Dissertations, Theses, and Capstone Projects

Date of Degree

9-2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biology

Advisor

Shahana S. Mahajan

Committee Members

Frida Kleiman

Moira Sauane

Daniel Keedy

Maria Contel

Suzie Chen

Subject Categories

Biology | Cancer Biology | Cell Biology | Molecular Biology

Keywords

Patient derived Xenografts, Yes-associated protein, p73, bone cancer, drug repurposing

Abstract

Osteosarcoma is a rare but aggressive bone malignancy most commonly affecting adolescents and young adults (AYA). With 5-year survival rate stagnating over the last four decades despite the advancements in multi-modal therapy, drug repurposing offers a rapid path to new osteosarcoma treatments. Recent studies suggest that Riluzole, a drug approved for amyotrophic lateral sclerosis (ALS), may be repurposed for osteosarcoma treatment due to its ability to slow tumor progression and induce apoptosis. Previously, we have shown that Riluzole increases reactive oxygen species (ROS), activating c-Abl, which phosphorylates YAP in the nucleus. YAP then forms a complex with p73 to enhance Bax expression, triggering apoptosis. EGR1, a zinc-finger transcription factor, is significantly downregulated in osteosarcoma but is known to promote apoptosis via ROS induction in other cancers. However, its role in Riluzole- mediated apoptosis in osteosarcoma remains unexplored. In this study, we examined EGR1 expression across multiple osteosarcoma lines following Riluzole treatment. qPCR and western blot analyses revealed a significant upregulation of EGR1 at both mRNA and protein levels. Selectively, we treated U2OS (WT p53) and LM7 (p53 null) osteosarcoma cell lines, as well as osteosarcoma patient derived xenograft (PDX) models, with Riluzole. EGR1 expression was assessed by qPCR and Western blot, while chromatin immunoprecipitation (ChIP-qPCR) was used to evaluate EGR1 and YAP/p73 binding to pro-apoptotic Bax gene promoter. EGR1 induction and apoptosis, in vivo, were examined via immunohistochemistry.

Our results show that Riluzole induced a rapid, ROS-dependent upregulation of EGR1 within 1 h in U2OS and by 4 h in LM7 cells. Rescue experiments using antioxidants confirmed that Riluzole-induced ROS directly stimulates EGR1 expression. Chromatin- immunoprecipitation coupled with qPCR demonstrated that EGR1 in complex with YAP/p73, binds to the Bax promoter, enhancing its transcription. Furthermore, we assessed EGR1 expression in osteosarcoma PDX lines which reveals significant increase in EGR1 levels post-Riluzole treatment. Immunohistochemical analysis of Riluzole-treated osteosarcoma metastatic mouse tissues revealed increased EGR1 expression in vivo. Collectively, our findings establish EGR1 as a critical mediator of Riluzole-induced apoptosis in osteosarcoma by facilitating Bax activation via the YAP/p73 complex. By positioning EGR1 as a link between oxidative stress and pro-apoptotic transcriptional program, our findings highlight EGR1 to be promising, both, as a mechanistic target and as a therapeutic biomarker in repurposed-drug strategies for AYA with osteosarcoma.

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