Prostate cancer is the most commonly diagnosed cancer and the second most leading cause of cancer deaths in American men (www.cancer.org). Most prostate cancer-related deaths are due to the metastatic form of the disease. The 5-year relative survival rate in patient's diagnosed with metastatic prostate cancer is just 28%, as compared to 100% in patient's diagnosed with localized prostate cancer. This clearly indicates the lack of effective treatment available for metastatic prostate cancer. MicroRNAs (miRNAs) are small (18~23 nucleotide long) non-coding RNAs that can influence gene expression by binding to the 3'-untranslated region of coding RNAs at the post-transcriptional level. Some miRNAs has been termed as oncomirs due to their role in promoting tumor growth, invasion and metastasis. One such oncomir is microRNA-21 (miR-21) whose levels are often up-regulated in a number of cancers, including prostate cancer. MiR-21 increases the survival and invasiveness of cancer cells by suppressing its target tumor suppressor genes, namely programmed cell death 4 (PDCD4) and maspin. Thus, drugs which target miR-21 for inhibition could provide novel treatment options for metastatic prostate cancer. Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic phytoalexin found in high quantities in various dietary sources, such as grapes, red wine, berries and peanuts. Various reports have demonstrated a significant role of resveratrol in the management of several old age diseases including cancer. The efficacy of resveratrol as an anti-cancer agent resides in its ability to interfere with cell proliferation and metastasis and enhancement of apoptosis. Resveratrol has been shown to act on several intracellular targets to exert these effects. However, the exact mechanism by which resveratrol mediates its beneficial cancer chemotherapeutic actions are not clear and is the focus of this study. Based on the reported data, we hypothesized that resveratrol mediates its anti-cancer action against metastatic prostate cancer by inhibiting the signaling pathway which involves miR-21 expression and function. To address this hypothesis, we show that resveratrol decreased cell viability, migration and invasiveness of androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. These effects of resveratrol were associated with the inhibition of miR-21, since over-expression of miR- 21 with pre-miR-21 oligonucleotides attenuated resveratrol's effect on these cells. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21 and knockdown of PDCD4 by short interfering (si) RNA reversed the resveratrol's effect on prostate cancer cells. PC-3M-MM2 cells also exhibits high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002, a known PI3-kinase inhibitor. MiR-21 expression in these cells appears to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibits the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Future investigation into the molecular mechanisms revealed that resveratrol suppressed prostate cancer growth by decreasing the levels of insulin-like growth factor-1 (IGF-1) and its receptor (R). Previous studies had associated elevated levels of serum IGF-1 with high risk of prostate cancer. IGF-1, after binding to its receptors, acts as a potent mitogen which stimulates cancer cell growth and proliferation mainly by activating Akt signaling pathway. Interestingly, this effect of resveratrol on IGF-1/IGF-1R was independent of its effect on miR-21. In summary, our data show that resveratrol exerts its anti-cancer effect on metastatic prostate cancer cells, at least in part, by targeting Akt/miR-21 pathway. These data highlight a potential molecular mechanism for resveratrol's anti-cancer action for the treatment of metastatic prostate cancer and suggest that inhibition of the IGF-1/Akt/miR-21 pathway is a rationale approach for the treatment prostate cancer metastasis.

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Molecular profiling of blood for several purposes, 1) To identify prostate cancer biomarkers, 2) to identify commonalities between asthma and mood disorders, and 3) to identify mRNAs that may be involved in psychobiotic changes to behaviour. / Every cell of the body has the opportunity to secrete molecules into the blood. These molecules: proteins, RNAs, and DNAs, can be secreted freely, or within extracellular vesicles (EV). The complement of specific molecules secreted by cells can vary in accordance with changes to their immediate environment, such as disease in a particular organ. Cells of the immune system which circulate in the blood may also change the rates at which they produce these molecules in response to a disease or unusual event occurring somewhere within the body. The full complement of proteins, RNAs, or DNAs from all sources within the blood can therefore be measured to garner information about disease states and communication between every tissue of the body. In this body of work, we leveraged this to address three separate challenges within medical science. First, we utilized blood as a source of biomarkers for disease and disease severity; isolating EVs from the blood of prostate cancer patients and healthy subjects and characterized their proteins with mass spectrometry to identify potential biomarkers for prostate cancer and its stages. Next, we explored the ability of blood to identify commonalities between distinct but often comorbid diseases; here we utilized publicly available datasets to identify transcripts or gene sets potentially facilitating the relationship between PTSD, MDD, and asthma. Finally, we utilized differential gene and gene sets expression to gain mechanistic insight into microbiota-gut-brain axis; investigating the hippocampus and blood of mice fed one of two psychobiotic bacteria: Lactobacillus rhamnosus JB1, Lactobacillus reuteri 6475. The analysis identified several mRNA expression differences potentially responsible for the mood-altering characteristics of these psychobiotic bacteria. This body of work illustrates the utility of blood omics data for addressing many problems within medical science, and highlights the large scale of information stored within the blood. / Thesis / Doctor of Philosophy (Medical Science) / Every cell of the body has the opportunity to secrete molecules into the blood. These molecules: proteins, ribonucleic acids (RNAs), and deoxyribonucleic acids (DNAs), can be secreted freely, or within small membrane compartments called extracellular vesicles (EV). Specific molecules are secreted more or less by cells depending on changes to their immediate environment, such as disease in a particular organ. We leveraged this to the benefit of medical science in three separate scenarios: 1) using the molecular contents of EVs to determine when someone has prostate cancer, and at what stage; 2) examining RNAs of the blood to determine why so many with asthma also have depression or PTSD; 3) measuring RNAs in the blood and hippocampus of mice to better understand how certain bacteria in the gut can alleviate depression. This work illustrates the utility of blood in tackling many challenging problems within medical science.

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Recent advances in whole genome sequencing have led to many discoveries in the mechanisms involved in carcinogenesis. Genomic characterization of premalignant lesions in numerous cancers has led to new prevention strategies, early detection, and treatment options that have saved lives and improved the quality of life for the people suffering from these cancers. Prostate cancer (PCa) is the most common cancer in men in the United States (US) and the second leading cause of death in men from all cancers. However, in African American men (AAM) the mortality rate from PCa is 2.4 times higher than European American men (EAM). In addition, AAM are more likely to get PCa and have a higher PCa burden at diagnosis than their EAM counterparts. This may suggest that there are racial/ethnic differences in the mechanism of carcinogenesis in PCa. PCa and its premalignant lesion, high grade prostatic intraepithelial neoplasia (HGPIN), are one of the most heterogeneous and complex cancers for scientists to determine the mechanisms of carcinogenesis. Due to this complexity, research on HGPIN and PCa has been difficult to carry out and interpret. Projects have been undertaken and progress has been made in the discovery of some genes involved in PCa and potential drivers of initiation, progression and aggressiveness of PCa. However, these studies have mostly been conducted among EAM and have little ethnic diversity. Discovery of new prevention, early detection and treatment methods for PCa will not be possible without advances in the genetic characterization of the pathways of carcinogenesis of PCa among ethnically diverse study populations. To date there are no known genetic characterizations of HGPIN and PCa in AAM. This study aims to characterize chromosomal copy number aberrations (CNA) in paired HGPIN and PCa in AAM. By utilizing advanced microarray techniques, we will determine the degree to which HGPIN and PCa share CNA and identify CNA that may be involved in PCa progression. This study will lay the foundation for future research into CNA that may be used as potential biomarkers for early detection of neoplasms of high-risk for development into PCa in AAM. The discovery of biomarkers and the characterization of the mechanisms involved in PCa progression may lead to treatment options for the prevention of PCa and an overall better outcome for AAM suffering from PCa.

The current standard treatment of prostate cancer by androgen deprivation therapy involves using drugs such as bicalutamide (Casodex) to antagonistically block androgen receptors that are normally present within prostate cells. Usually, the therapy is successful in the short run at limiting the growth of prostate cancer. However, in virtually all cases tumors begin to grow aggressively again after several months of treatment and new therapies must be started. The mechanism by which these prostate cells transform from androgen sensitive to androgen independent and anti-androgen resistant is unclear. In this study, we investigated the role of microRNAs, small 15 to 18 nucleotide regulatory RNAs, in regulating the desensitization of prostate cancer cells to the androgen receptor antagonist drug bicalutamide. In order to identify significant microRNAs, quantitative PCR was used to obtain genome-wide microRNA expression levels of 885 human microRNAs at different timepoints for androgen sensitive LNCaP cancer cells treated with bicalutamide and for untreated control cells in tissue culture. Analysis of microRNA expression by clustering analysis and by statistical comparisons of treatment groups resulted in identification of 28 microRNAs that have altered expression in the progression process. In silico target prediction analysis was performed with the microRNAs shown to have altered expression, and a group of genes predicted to be under microRNA regulatory control during cancer progression to resistance was identified. A microRNA expression profile can be useful in developing more effective prognostic and therapeutic tools for prostate cancer.

Current treatments for prostate cancer (PCa) are marred with high relapse frequency and development of progressively aggressive cancers; developing new treatment options for PCa remains crucial. In this project, a series of synthetic compounds based on natural products will be screened to identify inhibitors for Aurora-A kinase (Aur-A). Aur-A facilitates centrosome separation and bipolar spindle formation during mitosis. Aur-A is overexpressed in metastatic PCa cells, and is a good candidate for targeted therapies. Compound libraries are designed using natural compounds that contain simple structural elements as starting points for developing drug like libraries. High-throughput screening of these libraries will be used to identify potent antimitotic agents that selectively affect cancer cells but not normal cells. A combination of in vitro protein assays – quantifying protein activity – cell-based assays – measuring cell growth and proliferation – and cell-reporter assays – to determine which metabolic pathway the compound affects – were used to identify potential inhibitors. Through these methods, we have identified several compounds, with special consideration to thiazole piperazine compounds, to successfully inhibit proliferation of metastatic PCa cells.