Date of Award

2016-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Igor C. Almeida

Second Advisor

Marc B. Cox

Abstract

Prostate cancer (PCa) is the leading non-cutaneous malignancy and the second deadliest among American men. PCa mortality rates among African American men are much higher than any other ethnic group, and the same is true for men of African ancestry world-wide. There is also a lack of reliable diagnostic markers and effective treatment options. Extracellular vesicles (EVs) have been observed to play an important role in cancer processes such as promotion of tumor growth and metastasis. They are also a promising source of diagnostic markers. This study addresses these problems by studying the proteome of EVs derived from PCa cells LNCaP, 22RV1, E006AA-hT, which was derived from an African American PCa patient, and non-cancerous prostate epithelial cell line RWPE1. The enrichment of EVs was conducted using differential centrifugation, where we cleared cell supernatant of cell debris and apoptotic bodies and centrifuged for 2 hours at 100,000xg to pellet EVs (EV2). The supernatant from this spin was subsequently centrifuged for 16 hours to pellet any remaining EVs (EV16). The EV enrichment was deemed successful through transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA); which showed that EV2 and EV16 differ in overall size. Western blotting confirmed the presence of at least one EV marker (ALIX or TSG101) in EV2 and EV16 from all cell lines. Proteomic analysis revealed existing qualitative differences between EV2 and EV16 from the same cell-line. We also observed 67 proteins common only in PCa cell EV2 fractions and 18 in EV16. Each cell-derived EV fraction also had proteins that were exclusive. A differential proteomic analysis using normalized spectral count values revealed cancer-related proteins present across all cell lines that differed in abundance, some of them significantly different when compared to RWPE1. Among the most notable proteins are Vinculin, Thy-1, and multiple subunits of the 26S protease complex, TCP1 containing ring complex (TRiC), and the COP9 signalosome complex. We also observed proteins that were significantly abundant in E006AA-hT EVs which are known to contribute to migration and metastasis, characteristic of E006AA-hT In vivo. In summary, our analysis of PCa cell-derived EVs identified proteins in two EV fractions which are significantly abundant in comparison to non-cancerous prostate cell-derived EVs, and have been reported to play a role in cancer. This knowledge provides the basis to further understand the prostate tumor microenvironment in African American and Caucasian men, and suggests candidates for validation in bio fluids for the development of diagnostic tools.

Language

en

Provenance

Received from ProQuest

File Size

146 pages

File Format

application/pdf

Rights Holder

Gloria Polanco

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