GSBS Dissertations and Theses

ORCID ID

0000-0001-7527-5533

Publication Date

2020-04-08

Document Type

Doctoral Dissertation

Academic Program

MD/PhD

Department

Program in Molecular Medicine

First Thesis Advisor

Craig J. Ceol

Keywords

zebrafish, melanoma, melanocyte, mitf, mitfa, BMP signaling, monoclonal antibody, therapy

Abstract

Melanoma is one of the most aggressive and deadly forms of skin cancer. Arising from melanocytes, a pigment cell population derived from the neural crest, melanomas often adopt characteristics associated with the neural crest – the ability to rapidly proliferate, migrate and invade throughout the body. Historically, these characteristics along with a baseline resistance to chemotherapy have made melanoma extremely difficult to treat. Improvements in targeted and immunotherapeutic options have improved patient outcomes, but many patients still experience limited durable responses to therapy. In order to improve patient outcomes, new potential avenues of therapy must be identified based on the underlying pathogenesis of the disease. We previously identified and characterized the function of a novel melanoma oncogene, GDF6, uncovering a role in promoting melanoma cell survival and dedifferentiation by activating a neural crest identity. Here, we have a) identified a role for GDF6-activated BMP signaling during melanocyte development that forms a basis for its oncogenic role in melanoma, b) determined BMP signaling may play a role in promoting a neural crest-like state during melanoma initiation, and c) assayed novel monoclonal antibodies targeting GDF6 for use as blocking antibodies to treat advanced melanoma.

Previous work identified GDF6 as a melanoma oncogene that promotes melanoma progression through suppression of apoptosis and differentiation in melanoma cells, by regulating neural crest factor expression and neural crest identity, suggesting a potential role for GDF6 in the embryonic neural crest. Additional studies had previously identified roles for GDF6 and its orthologous genes in specific biological contexts, including embryonic neuronal cell survival, bone and cartilage development, embryonic eye development, and bone and ligament repair in adult tissue. Furthermore, a study had indicated a role for a GDF6 ortholog, gdf6a, during zebrafish neural crest induction, but had not uncovered any specific role for gdf6a in further development of the neural crest or in any neural crest derivatives. We determined blocking gdf6a-activated BMP signaling acts to increase melanocyte development during embryogenesis by increasing the proportion of neural crest cells activating the pigment cell marker, mitfa. Furthermore, we showed the increase in melanocytes is at the expense of the iridophore population. These results indicate GDF6 function in melanoma is a reiteration of the normal physiological function of GDF6 during embryonic melanocyte development from the neural crest.

Given these results and our previous findings of the role of GDF6-activated BMP signaling established melanomas, we hypothesized a potential role for GDF6-activated BMP signaling during melanoma initiation. Previous studies have determined neural crest identity and neural crest-like characteristics to be crucial during multiple phases of melanoma, including initiation, progression, and metastasis. We evaluated melanoma initiating lesions to determine the potential impact of BMP signaling on development and progression of these lesions. We found early lesions in our model to have active BMP signaling and that modulation of BMP signaling could alter the rate of development of these lesions in our animals. Furthermore, BMP modulation ultimately impacted the development of these lesions into melanomas. Together, these results indicate BMP signaling is a potential driving pathway during melanoma initiation and progression.

Finally, we wanted to determine the therapeutic potential of targeting GDF6 in order to treat patients with advanced melanoma. Given our previous findings and mechanism of ligand-activated BMP signaling, we hypothesized a monoclonal antibody targeting GDF6 could block GDF6 activity at its receptor on melanoma cells, thus inhibiting GDF6-activated BMP signaling. Monoclonal antibodies have been widely used as therapy in cancer as well as many other rheumatologic and immunologic conditions. We established a panel of GDF6-targeting antibodies via a hybridoma approach. We then assessed the antibodies ability to identify mammalian GDF6 in vitro and performed functional assays to determine if anti-GDF6 antibody treatment yielded the expected results of inhibiting GDF6-activated BMP signaling. We observed decreased pathway activity, decreased cell viability, and increased cell death in melanoma cells treated with anti-GDF6 antibodies in vitro. We further investigated whether these antibodies could exert anti-melanoma effects in vivo. Together, these results indicate potential therapeutic value for our antibodies in treating GDF6-positive melanomas.

DOI

10.13028/2kjx-k232

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Available for download on Saturday, April 16, 2022

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