Graduation Year

2024

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Conor C. Lynch, Ph.D.

Committee Member

Eric Le-Lau, Ph.D.

Committee Member

Keiran S. Smalley, Ph.D.

Committee Member

Antonio L. Amelio, Ph.D.

Committee Member

Julia A. Rhoades, Ph.D.

Keywords

Histone deacetylase, Lung metastasis, Neuropilin-1, Osteosarcoma, Vesicular glutamate transporter

Abstract

Osteosarcoma (OS) is a bone cancer that commonly metastasizes to the lung. Despite advances in the development of targeted therapies for treating solid malignancies, broad-acting chemotherapies remain the first line of treatment for OS. In assaying the efficacy of approved therapeutics for non-OS malignancies, we identified the histone deacetylase 1 and 2 (HDAC1 and 2) inhibitor, romidepsin, as effective for treating established lung metastatic OS. However, romidepsin has noted toxicities in humans, thus we aimed to define the primary mechanisms through which HDAC1/2 mediates OS progression to identify more selective druggable targets/pathways. Microarray and proteomic analyses of romidepsin-treated OS cells revealed many genes to be differentially regulated including neuropilin-1 (NRP1) and vesicular glutamate transporter (SLC17A7)—genes that we studied in more detail to identify mechanisms underlying the effects of romidepsin.

Our results show that silencing of NRP1 significantly reduced OS proliferation, migration, invasion, and adhesion in vitro. More strikingly, in vivo, reduced NRP1 expression significantly mitigated the lung metastatic potential of OS in two independent models (K7M2 and SAOS-LM7). Mechanistically, our data point to NRP1 mediating this effect via the downregulation expression/activity of migration machinery including SRC, FAK, and ROCK1. For SLC17A7, we noted that transient overexpression of SLC17A7 significantly reduced OS proliferation. We have also shown that glutamate levels were reduced in the conditioned media upon treatment with romidepsin. Based on these data, we anticipate that SLC17A7 is a crucial osteosarcoma tumor suppressor and that altering glutamate levels is a novel strategy to prevent osteosarcoma growth. We have used the FDA-approved drug riluzole to therapeutically target glutamate levels, which inhibits glutamate export from the cells. Indeed, in vivo, riluzole treatment effectively inhibited OS growth and metastasis. Given that our K7M2 in vivo studies were conducted in immunocompetent BALB/c mice, we also leveraged emerging spatial transcriptomic technology to understand more about the osteosarcoma lung metastatic microenvironment.

In conclusion, we have found that HDAC inhibitors potently inhibit lung metastatic osteosarcoma and does so in part by blocking NRP1 expression and enhancing the expression of SLC17A7. Our results suggest two potential new avenues for the treatment and prevention on lung metastatic osteosarcoma.

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