Dissertations, Theses, and Capstone Projects

Date of Degree

9-2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biology

Advisor

Shahana S. Mahajan

Committee Members

Andrew L. Wolfe

Cathy Savage-Dunn

Frida Kleiman

Vinagolu K. Rajasekhar

Subject Categories

Biology | Cancer Biology | Cell Biology

Keywords

Osteosarcoma, Metastasis, COL1A1, COL1A2, Dedifferentiation

Abstract

Metastatic osteosarcoma (OS) is associated with a markedly poor prognosis, with a five-year survival rate below 30%, compared to over 70% for primary osteosarcoma. This disparity suggests that metastatic osteosarcoma possesses enhanced aggressiveness and proliferative ability. However, the mechanisms driving this phenotype remain poorly understood. In this study, we hypothesized that specific molecular factors regulate the transition from primary to metastatic osteosarcoma. To address this, we analyzed RNA-seq datasets from matched primary and metastatic osteosarcoma cell lines obtained from the GEO database. Our analysis identified significant downregulation of collagen type I alpha 1 chain (COL1A1) and COL1A2 collagen type I alpha 2 chain in metastatic osteosarcoma. To assess their functional roles, we generated stable shCOL1A1 and shCOL1A2 knockdown cell lines in primary osteosarcoma cells and transiently overexpressed these genes in metastatic osteosarcoma cells. We then assessed cell proliferation using MTT proliferation assays and metastatic potential using gelatin zymography. Functional studies demonstrated that modulation of COL1A1 and COL1A2 influenced osteosarcoma cell proliferation and metastatic behavior in a cell line-dependent manner, suggesting that their reduced expression is not merely a passive consequence of metastatic progression. Given that COL1A1 and COL1A2 are osteoblast-associated genes, we next investigated whether their reduced expression reflects osteosarcoma differentiation status. Consequently, we ABSTRACT v examined several osteoblast differentiation markers and found that some osteoblast differentiation markers, including OCN, SPARC, SP7, and DMP1, are consistently downregulated in metastatic osteosarcoma cell lines. These results suggest that reduced COL1A1 and COL1A2 expressions might be associated with a loss of osteoblastic identity, consistent with a dedifferentiated state. Lastly, we reanalyzed our previous RNA-seq datasets to investigate the mechanisms underlying dedifferentiation during metastatic transformation. This analysis revealed that epigenetic modification, particularly histone modifications, are involved in this process. Consequently, we assessed COL1A1 and COL1A2 expressions after treatment with histone deacetylases (HDACs) inhibitors or a histone acetyltransferase (HAT) inhibitor to determine whether these gene expressions are regulated by epigenetic modification. Histone deacetylases (HDACs) inhibition, but not histone acetyltransferase (HAT) inhibition, reduced COL1A1 and COL1A2 expressions in primary osteosarcoma cells, suggesting a role for histone deacetylases (HDACs) in regulating their expression. Furthermore, promoter analysis using the PROMO transcription factor prediction tool identified candidate transcription factors that may recruit epigenetic regulators to the COL1A1 and COL1A2 promoters. This analysis identified CCAAT/enhancer-binding protein beta (CEBPβ), Yin Yang 1 (YY1), and Paired Box 5 (PAX5) as potential transcription factors that may recruit epigenetic regulators to the COL1A1 and COL1A2 promoters. Collectively, these findings suggest that epigenetic dysregulation contributes to the loss of osteoblastic features during metastatic osteosarcoma progression. Additionally, COL1A1 and COL1A2 may serve as markers of osteosarcoma dedifferentiation, providing insight into the mechanisms underlying metastatic progression. More broadly, this work highlights the importance of cellular plasticity and epigenetic regulation in cancer progression and metastasis.

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