Elham Hosseini-Beheshti

Exosomes as Biomarker Enriched Microvesicles: Characterization of Exosomal Proteins Derived from a Panel of Prostate Cell Lines with Distinct AR Phenotypes

Prostate cancer is the leading type of cancer diagnosed in men. In 2010, ∼217,730 new cases of prostate cancer were reported in the United States. Prompt diagnosis of the disease can substantially improve its clinical outcome. Improving capability for early detection, as well as developing new therapeutic targets in advanced disease are research priorities that will ultimately lead to better patient survival. Eukaryotic cells secrete proteins via distinct regulated mechanisms which are either ER/Golgi dependent or microvesicle mediated. The release of microvesicles has been shown to provide a novel mechanism for intercellular communication. Exosomes are nanometer sized cup-shaped membrane vesicles which are secreted from normal and cancerous cells. They are present in various biological fluids and are rich in characteristic proteins. Exosomes may thus have potential both in facilitating early diagnosis via less invasive procedures or be candidates for novel therapeutic approaches for castration resistance prostate cancer. Because exosomes have been shown previously to have a role in cell-cell communication in the local tumor microenvironment, conferring activation of numerous survival mechanisms, we characterized constitutive lipids, cholesterol and proteins from exosomes derived from six prostate cell lines and tracked their uptake in both cancerous and benign prostate cell lines respectively. Our comprehensive proteomic and lipidomic analysis of prostate derived exosomes could provide insight for future work on both biomarker and therapeutic targets for the treatment of prostate cancer.

Reproducibility and efficiency of serum-derived exosome extraction methods.

OBJECTIVES Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. DESIGN AND METHODS We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. RESULTS Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. CONCLUSION ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EVs into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression.

Regulation of local steroidogenesis in the brain and in prostate cancer: Lessons learned from interdisciplinary collaboration

Sex steroids play critical roles in the regulation of the brain and many other organs. Traditionally, researchers have focused on sex steroid signaling that involves travel from the gonads via the circulation to intracellular receptors in target tissues. This classic concept has been challenged, however, by the growing number of cases in which steroids are synthesized locally and act locally within diverse tissues. For example, the brain and prostate carcinoma were previously considered targets of gonadal sex steroids, but under certain circumstances, these tissues can upregulate their steroidogenic potential, particularly when circulating sex steroid concentrations are low. We review some of the similarities and differences between local sex steroid synthesis in the brain and prostate cancer. We also share five lessons that we have learned during the course of our interdisciplinary collaboration, which brought together neuroendocrinologists and cancer biologists. These lessons have important implications for future research in both fields.

Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa). However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR) as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.

Next-generation steroidogenesis inhibitors, dutasteride and abiraterone, attenuate but still do not eliminate androgen biosynthesis in 22RV1 cells in vitro.

Castration resistant prostate cancer (CRPC) is often lethal and inevitably develops after androgen ablation therapy. However, in the majority of cases it remains androgen dependent. CRPC tumors have the ability to synthesize their own androgens from cholesterol by engaging in de novo steroidogenesis. We investigated the potential of 22RV1 prostate cancer cells to convert the supplemented steroid precursors within this pathway under the effects of current clinical steroidogenesis inhibitors such as abiraterone and dutasteride, either alone or in combination. Under steroid starved conditions, enzymes responsible for de novo steroidogenesis were upregulated. Testosterone and dihydrotestosterone (DHT) were formed by using both dehydroepiandrosterone (DHEA) and progesterone as substrates. Formation of testosterone and DHT was higher following incubation with DHEA compared to progesterone. Progesterone decreased the mRNA expression of enzymes responsible for steroidogenesis. Abiraterone treatment decreased testosterone production but increased several precursor steroids in both classical and backdoor pathways in the presence of progesterone. In contrast, the DHT levels were elevated following treatment with abiraterone when progesterone was absent. Dutasteride decreased the formation of testosterone, DHT and precursor steroids in the backdoor pathway but increased steroid precursors in the classical steroidogenesis pathway. The combination of abiraterone and dutasteride decreased testosterone and DHT in the presence of progesterone but increased DHT in the absence of progesterone. Abiraterone inhibited androgen receptor (AR) activation but not to the same extent as MDV3100. However, abiraterone and dutasteride treatment, either alone or in combination, were more effective in decreasing prostate specific antigen secretion into the media than MDV3100. Thus, while interventions with these drugs alone or in combination fail to completely inhibit steroidogenesis in the 22RV1 cells, the combined inhibition of androgen production and blockade of AR can exceed the effect of MDV3100. Further characterization of bypass mechanisms that may develop as a response to these inhibitors is necessary to achieve optimal suppression of testosterone and DHT synthesis as a part of therapeutic regimens for the treatment of CRPC.

Characterization of Antioxidant Capacity from Fruits with Distinct Anthocyanin Biosynthetic Pathways

The antioxidant capacity of individual anthocyanins is well established. Less information is available however, about the relative contribution to which specific anthocyanins in a complex mixture affect total antioxidant capacity in different soft fruit sources; especially those that share a similar pathway for anthocyanin synthesis. The objectives of this work were to compare the antioxidant capacity of two different soft fruits, blackcurrant and grape, which share similarities in anthocyanin biosynthetic pathways but are composed of distinctly different anthocyanin profiles. Anthocyanin composition profiles of grape and blackcurrant were characterized by High Performance Liquid Chromatography/Mass Spectrometry (HPLC). ORAC (Oxygen Radical Absorbance Capacity) and ABTS (2,2’-azinobis 3-ethylbenzothiazoline- 6-sulfonic acid) assays were used for antioxidant activity quantification. An anthocyanin antioxidant capacity index (AACI) was derived from the product of antioxidant (ORAC) activity for each of major anthocyanins present in blackcurrant and grape, and the sum of anthocyanins recovered from purified fruit extracts to determine the extent that the total antioxidant activity derived from different anthocyanin combinations. Blackcurrant contained four predominant anthocyanins, cyanidin3-glucoside (Cy3G), delphinidin3-glucoside (Dp3G), cyanidin3-rutinoside (Cy3R), and delphinidin3- rutinoside (Dp3R). Major anthocyanins found in grape were malvidin3-glucoside (Mv3G), Dp3G, Cy3G, petunidin3-glucoside (Pt3G), and peonidin3-glucoside (Pn3G). A greater (p<0.05) total antioxidant capacity existed for blackcurrant compared to grape when measured by ORAC and ABTS methods. An antioxidant synergy was confirmed for blackcurrant and wind grape thus indicating that this phenomenon is a factor for characterizing total antioxidant activity in both blackcurrant and wine grape.

Role of hepatic Annexin A6 in fatty acid-induced lipid droplet formation

ABSTRACT Annexin A6 (AnxA6) has been implicated in the regulation of endo‐/exocytic pathways, cholesterol transport, and the formation of multifactorial signaling complexes in many different cell types. More recently, AnxA6 has also been linked to triglyceride storage in adipocytes. Here we investigated the potential role of AnxA6 in fatty acid (FA) – induced lipid droplet (LD) formation in hepatocytes. AnxA6 was associated with LD from rat liver and HuH7 hepatocytes. In oleic acid (OA) ‐loaded HuH7 cells, substantial amounts of AnxA6 bound to LD in a Ca2+‐independent manner. Remarkably, stable or transient AnxA6 overexpression in HuH7 cells led to elevated LD numbers/size and neutral lipid staining under control conditions as well as after OA loading compared to controls. In contrast, overexpression of AnxA1, AnxA2 and AnxA8 did not impact on OA‐induced lipid accumulation. On the other hand, incubation of AnxA6‐depleted HuH7 cells or primary hepatocytes from AnxA6 KO‐mice with OA led to reduced FA accumulation and LD numbers. Furthermore, morphological analysis of liver sections from A6‐KO mice revealed significantly lower LD numbers compared to wildtype animals. Interestingly, pharmacological inhibition of cytoplasmic phospholipase A2&agr; (cPLA2&agr;)‐dependent LD formation was ineffective in AnxA6‐depleted HuH7 cells. We conclude that cPLA2&agr;‐dependent pathways contribute to the novel regulatory role of hepatic AnxA6 in LD formation. HIGHLIGHTSAnxA6 binds LD from rat liver and HuH7 hepatocytes in a Ca2+‐independent manner.AnxA6, but not AnxA1, AnxA2 or AnxA8 promote LD formation.AnxA6 knockdown in HuH7 and AnxA6‐KO hepatocytes show reduced FA accumulation.Liver sections from A6‐KO mice revealed significantly lower LD numbers.Inhibiting cPLA2&agr;‐dependent LD formation is ineffective in AnxA6‐depleted cells.

Extracellular vesicles as mediators of immunopathology in infectious diseases

In the last decades, extracellular vesicles have emerged as important elements in cell–cell communication and as key players in disease pathogenesis via transmission of their cargo between different cells. Various works have described different subpopulations of these membrane structures, based on their cell of origin, biogenesis, size, biophysical properties and cargo. In addition to their pathophysiological role in the development and progression of different diseases including infectious diseases, neurodegenerative disorders and cancer, extracellular vesicles are now recognized for their potential as novel therapeutic targets and intelligent drug delivery system. Here, we have reviewed the most recent data on different subtypes of extracellular vesicles, focusing on microvesicles and exosomes and their subpopulations, their involvement in immune‐mediated pathogenesis of various infectious diseases and their role as potential therapeutic targets.

Extracellular vesicles and microvascular pathology: Decoding the active dialogue

Extracellular vesicles (EV) are a heterogeneous collection of membrane‐surrounded structures released from all studied cells, under both physiological and pathological conditions. These nano‐size vesicles carry complex cargoes including different classes of proteins, lipids and nucleic acids and are known to act as a communication and signalling vesicles in various cellular process. In addition to their role in development and progression of pathological disorders which make them potentially great biomarkers, EV have beneficial effects, as they take part in homeostasis. In this review we have analysed the evidence for the role of microvesicles and exosomes secreted from other cells on microvascular endothelium (EV uptake) as well as the role of endothelial‐derived vesicles on their neighbouring and distant cells (EV release).