Date of Degree

2-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Olorunseun Ogunwobi

Committee Members

Jill Bargonetti

Benjamin Ortiz

Xingxing Zang

Moira Sauane

Subject Categories

Cancer Biology | Cell Biology

Keywords

metastasis, CTCs, cell migration, EMT, immune evasion, fibronectin

Abstract

Metastasis is the leading cause of cancer deaths worldwide. However, there exist only limited treatment options and they are often ineffective. An important aspect of metastasis that requires study, but has previously been understudied is circulating tumor cells (CTCs). CTCs are a critical step in the metastatic cascade. They can be analyzed for the identification of key mechanisms in metastasis. To this end, we isolated CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) and a human xenograft mouse model of castration-resistant prostate cancer (CRPC). From these mouse models, primary tumor and CTC lines were established. Functional characterization of cells revealed CTCs to be more migratory than primary tumor cells. Additionally, CTCs underwent epithelial-to-mesenchymal transition (EMT) as observed through E-cadherin decrease and SLUG and fibronectin (FN1) increases. Taking advantage of the intact immune system present in the BALB/c syngeneic mouse model we used, we investigated the major histocompatibility complex class I (MHCI) cell surface molecule present on tumor cells and recognizable by cytotoxic T lymphocytes (CTLs). MHCI expression was decreased in CTCs. To investigate additional immune-evading mechanisms, CTC secretory profiles were examined. Endostatin, C-X-C motif chemokine 5 (CXCL5), and proliferin secretions were decreased in CTCs in comparison to primary tumor cells. Moreover, decreased intracellular endostatin expression was found in CTCs. Taken together, our findings suggest that CTCs exhibit distinct characteristics from primary tumor cells. Given that CTCs were more migratory and expressed higher levels of Fibronectin (FN1), SLUG, and Integrin B1 (ITGB1), we investigated FN1, SLUG, and ITGB1 relationship to CTC migration and one another. Knockdown studies of FN1, SLUG, and ITGB1 confirmed each to be important for CTC migration. Furthermore, FN1 was observed to regulate ITGB1 and SLUG. FN1’s regulation of these two molecules, however, involves two separate and independent pathways by which FN1 increases migration. Finally, we tested whether FN1 required ITGB1 and SLUG to exert its effects on CTC migration. We observed that while FN1 was able to induce migration, it required ITGB1 and SLUG to maximally enhance CTC migration. FN1 was previously mostly known for its role in being an extracellular matrix (ECM) protein, frequently overexpressed in cancers. Very little is known about FN1’s intracellular role and ability to regulate other molecules. This is the first time to our knowledge that FN1 is being reported to regulate ITGB1 and SLUG as a molecular mechanism for CTC migration. Further CTC studies will undoubtedly uncover additional important mechanisms of cancer metastasis.

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