Date of Award

3-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Susan A. Miranda, PhD

Committee

Michael Bishop, MD; Kevin Freeman, PhD, Mark Hatley, MD, PhD; Gustavo A. Miranda-Carboni, PhD; Tiffany N. Seagroves, PhD

Keywords

WNT5B; Osteosarcoma; Cancer stem cells; Glycosaminoglycans; ROR1; Chemoresistance; Metastasis

Abstract

Osteosarcoma is a pediatric bone cancer that has no targeted therapy and has had no treatment advances for the last three decades. Osteosarcoma frequently metastasizes to the lungs and is highly chemotherapy resistant, reducing patient survival with metastatic disease to 20% after 5 years. New therapies targeted to metastasis and drug resistance in osteosarcoma are essential to improving patient outcomes.

Osteosarcoma is a pediatric bone cancer that has no targeted therapy and has had no treatment advances for the last three decades. Osteosarcoma frequently metastasizes to the lungs and is highly chemotherapy resistant, reducing patient survival with metastatic disease to 20% after 5 years. New therapies targeted to metastasis and drug resistance in osteosarcoma are essential to improving patient outcomes. We found that WNT5B is the most expressed WNT in osteosarcoma patients and correlates with both metastasis and survival. In osteosarcoma spheroids, both protein and mRNA levels of WNT5B are enhanced in the stem cell population compared to adherent cells. Further, WNT5B upregulates the expression of the stemness gene SOX2 and directs stemness phenotypes, such as sphere size, migration, and sphere forming efficiency.

Additionally, WNT5B enhances osteosarcoma chemoresistance to methotrexate. In vivo, WNT5B significantly increases osteosarcoma metastasis to the lungs, and these metastases are morphologically different between control and WNT5B knockout. We revealed that this morphologic difference is due to enhanced degradation of the glycosaminoglycan hyaluronic acid mediated by WNT5B. Further, ROR1 was identified as the receptor through which WNT5B signals, and we determined the effects of D10, a monoclonal antibody which targets ROR1. Next, we characterized the signaling pathway changes between adherent cells and stem cells, identifying FZD2 as the co-receptor which WNT5B signals through in stem cells. WNT5B signals through the WNT/PCP pathway in adherent cells, but switches to a non-conventional signaling pathway in osteosarcoma stem cells. Finally, RNA-sequencing was performed on 143B osteosarcoma adherent cells versus stem cells, comparing control and WNT5B knockout cells. The RNA-sequencing data provided validation of the results presented in this dissertation, as well as providing future directions for studying WNT5B in osteosarcoma.

Through revealing a novel role for WNT5B in osteosarcoma cancer stem cells, therapy resistance and metastasis, we present the WNT5B pathway as a candidate for therapeutically targeting osteosarcoma stem cells in patients.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0001-8780-3305

DOI

10.21007/aetd.cghs.2024.0002

Additional Files
Perkins 3.12.24. AETD DOA.pdf (299 kB)
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