Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Anatomy and Cell Biology

Supervisor

Alison, Allan L.

Abstract

Prostate cancer is the second leading cause of cancer-related deaths in American men. Most of these deaths occur as the result of metastasis, which is associated with an epithelial-to-mesenchymal transition (EMT). EMT leads to greater migratory/invasive capacity and resistance to therapy. During metastasis and associated EMT, cancer cells shed from the primary tumor and disseminate throughout the body as circulating tumor cells (CTCs) in the bloodstream. CTCs have been correlated with increased metastatic disease, reduced survival, and therapy response/resistance. Assessing CTCs presents an opportunity to study cancer progression/treatment effectiveness from a blood test. However, outstanding questions regarding CTCs and EMT has resulted in hesitation in the clinical adoption of CTCs. This thesis aimed to gain a greater understanding of the functional role of EMT in CTC generation, detection, and metastatic behavior in prostate cancer. We converged available CTC isolation technologies to develop protocols for EMT-independent isolation and molecular analysis of CTCs in pre-clinical mouse and human samples. We compared the effectiveness of CellSearch® and Parsortix® for analyzing CTCs in prostate cancer patients and observed that both technologies were equally effective at enumerating CTCs across the clinical spectrum of prostate cancer metastasis. This resulted in identification of 24 genes whose altered expression in patient CTCs may be influencing disease progression, including several related to EMT. We then investigated how EMT affects cell morphology, phenotype, and marker expression by knockdown of the EMT-inducing transcription factor Zeb1 in mesenchymal prostate cancer cells using shRNA (Zeb1KD cells). We observed an aggressive partial (p-EMT) phenotype in Zeb1KD cells compared to controls, which was mitigated by treatment with the demethylating drug 5-azacytidine. Lastly, we identify a unique panel of p-EMT markers for aggressive disease using methylation chip analysis. This research provides the groundwork for increasing future accessibility of CTC liquid biopsies for prostate cancer patients of any disease stage and/or EMT status, thus allowing a greater number of patients to benefit from a personalized medicine approach to combating disease progression and improving outcomes.

Summary for Lay Audience

Prostate cancer is the second leading cause of cancer-related deaths among American men. Most of these deaths occur because of the spread of cancer from the prostate to distant organs in the body such as bone, which is very difficult to treat. Cancer cells spread by escaping from the original prostate tumor and entering into the bloodstream, where they become circulating tumor cells (CTCs). Capturing and studying CTCs in the bloodstream is an opportunity for early detection of disease progression and the use of more aggressive treatments which may ultimately extent patient lives. In this thesis we addressed 3 scientific questions based on the current challenges of capturing CTCs from the blood. Question 1: How can we capture every CTC from the blood in mouse and human samples? Question 2: How can we identify CTCs in prostate cancer patients in order to better identify aggressive cancer? Question 3: What are the biological changes that make cancer cells more likely to enter the blood and spread, and how can these changes be identified and targeted? One of the major challenges with capturing CTCs is that each CTC has different characteristics. Therefore, to answer Question 1 we assessed how current CTC technologies work and developed new ways to capture every CTC in blood samples from mouse models and humans. This formed the basis for answering Question 2, where we assessed how CTCs could be effectively detected at different stages of prostate cancer and identified gene signatures that were altered in patient CTCs. Finally, in Question 3 we mimicked the biological changes that prostate cancer cells undergo as they enter the bloodstream and become more aggressive and demonstrated ways of identifying and targeting these changes that could be applied clinically in the future to benefit patients. Together, this knowledge lays the groundwork for more effective capture of CTCs from the blood in order to help prostate cancer patients use a personalized medicine approach to combat disease progression and improve outcomes.

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