<p>  </p> <p><strong>ABSTRACT</strong></p> <p>The worldwide obesity prevalence has almost tripled since 1973 according to the World Health Organization. In the United States, the disease is especially prevalent, with a recorded prevalence of 41.9 percent in 2017, as reported by the National Health and Nutrition Examination Survey. Obesity is associated with an increased risk of heart disease, type 2 diabetes, stroke, non-alcoholic fatty liver disease (NAFLD), and certain types of cancers, including prostate cancer (PCa). While obesity is preventable and reversible, it is a relapsing disease that requires long-term intervention. Furthermore, accumulating evidence shows obesity is not simply a matter of lack of willpower but the re-wired and altered biology that may need medical treatment. Therefore, researchers have been searching for effective approaches to treat obesity and obesity-related diseases. To this end, my research focuses on exploring the role of the sterol O-acyltransferase (SOAT) enzyme and how the inhibition of the enzyme benefits the treatment of obesity and PCa. In addition, I also studied the molecular signatures of NAFLD, with a special focus on altered lipid metabolism using proteomics and determined the protein oligomerization profiles. The major lines of research are summarized in the following and discussed in greater detail in chapters 2 to 5. </p> <p>SOAT enzyme catalyzes the conversion of free cholesterol into its storage form, cholesteryl ester. Our group previously showed that increased SOAT1 expression is associated with increased adipogenesis <em>in vitro</em> and increased adiposity in adipose tissue. When SOAT1 activity was blocked using the pharmacological inhibitor avasimibe, lipid droplet formation and expansion during adipogenesis were suppressed. We further showed that non-orally administered avasimibe led to significant fat mass loss in diet-induced obese (DIO) mice with concomitant food intake suppression and decreased expression of lipogenic genes in adipose tissue. Based on the promising use of avasimibe as an anti-obesity medication, I sought to answer whether avasimibe can enhance the weight loss effect of glucagon-like peptide-1 receptor agonist (GLP-1RA) by accelerating fat mass loss (chapter 2). We found subcutaneous administration of avasimibe can significantly potentiate the weight-reducing effect of the GLP-1RA in DIO mice.</p> <p>Inspired by the lipid droplet modulatory role of the SOAT1 enzyme, I also expanded my dissertation project to cancer (chapter 3). I found that low SOAT1 expression is associated with favorable patient outcomes among PCa patients who had previously undergone anti-hormone therapy. Since the current treatment option, anti-androgen drug enzalutamide, induces mechanisms of resistance in a short period, I hypothesized that blockage of SOAT1 activity using avasimibe would enhance the enzalutamide action and help overcome the resistance. To test this hypothesis, I characterized lipidomic signatures of PCa cells in response to enzalutamide and avasimibe treatments. Then, I tested the anti-cancer effect of the combined treatment in cell cultures and in xenograft tumors in nude mice. I found the combined treatment was significantly more effective in inhibiting cancer cell proliferation and tumor growth than each drug treatment alone. These findings provide insights into molecular signatures associated with enzalutamide treatment outcomes and can serve as a prelude to developing a therapeutic regimen targeting cholesterol metabolism. </p> <p>Among the comorbidities of obesity, NAFLD is very common in obese adults and the prevalence is close to 50–90%. We launched the third project (chapter 4) where we compared the liver proteome from lean mice and DIO mice. To date, most of the omics studies on DIO have been monolithic and very few have explored the multi-omic aspects of fatty liver tissue. To address this gap, we integrated global proteomics, phosphoproteomics and lipidomics to determine molecular signatures of the fatty liver. We identified a range of biological pathways that were altered, and we showed how the alterations in lipid content and amount were correlated with the alterations in the liver proteome and phosphoproteome. The results shed light on the interrelated nature of these biological processes. This was hypothesis-generating study that provided extensive data that could guide future investigations. </p> <p>We followed up on the third project and employed extensive measures to determine the protein oligomerization profiles of the fatty liver (chapter 5). Understanding the modes of protein oligomerization is important since proteins typically exert their biological functions by interacting with other proteins to form protein complexes. We used size exclusion chromatography (SEC) to fractionate liver proteins into 32 fractions based on their size and conducted label-free quantitation of each fraction using mass spectrometry. We successfully obtained elution profiles of individual proteins for subsequent comparison. Our approach enabled the identification of 1172 proteins found common in four liver samples (two lean livers and two fatty livers) for correlation profiling. We discovered that protein elution profiles were highly conserved in the fatty livers despite the metabolic disease state. At the same time, we identified several proteins with different elution profiles between the lean and the fatty livers, which could potentially mediate the hepatic dysfunctions displayed in NAFLD. This study delivers novel pieces of information about protein complex formation in fatty livers.</p> <p>The four research projects included in this dissertation explored obesity and obesity related diseases. Cholesterol accumulation is manifested as lipid metabolism is altered during the progression of obesity in adipose tissue and in PCa. Pharmacological inhibition of SOAT is responsible for cholesterol accumulation was effective in weight management in DIO and demonstrated anti-cancer effect in PCa models together with enzalutamide. These findings suggest that SOAT could be a therapeutic target for diseases featuring cholesteryl ester accumulation. Subsequent projects explored the liver proteome and revealed. In the subsequent projects, liver proteome showed a clear distinction between lean mice and obese mice. The identified proteins in these studies could facilitate the development of targeted therapies for treating NAFLD.</p>

Targeting molecular alterations as an effective treatment for isocitrate dehydrogenasewildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.

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