[Show abstract][Hide abstract] ABSTRACT: Background:
Prostate cancer is the most frequently diagnosed cancer in men and the third leading cause of cancer related deaths among men living in developed countries. Biomarkers that predict disease outcome at the time of initial diagnosis would substantially aid disease management.
Proteins extracted from formalin-fixed paraffin-embedded tissue were identified using nanoflow liquid chromatography-MALDI MS/MS or after separation by one- or two-dimensional electrophoresis. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000963. A list of potential biomarker candidates, based on proposed associations with prostate cancer, was derived from the 320 identified proteins. Candidate biomarkers were then examined by multiplexed Western blotting of archival specimens from men with premetastatic disease and subsequent disease outcome data. Annexin A2 provided the best prediction of risk of metastatic disease (log-rank Chi squared p = 0. 025). A tumor/control tissue >2-fold relative abundance increase predicted early biochemical failure, while <2-fold change predicted late or no biochemical failure.
This study confirms the potential for use of archival FFPE specimens in the search for prognostic biomarkers for prostate cancer and suggests that annexin A2 abundance in diagnostic biopsies is predictive for metastatic potential. Protein profiling each cancer may lead to an overall reduction in mortality from metastatic prostate cancer as well as reduced treatment associated morbidity.
[Show abstract][Hide abstract] ABSTRACT: Introduction & Objectives
There is a large body of evidence showing that prostate cancer and its progression to castrate resistant disease is driven by the action of androgens and the androgen receptor. Exosomes, nanosize vesicles secreted by cells, have been shown to be involved in cell to cell communication, and evidence from studies in other types of cancer suggests that the exosomes and microvesicles are regulators which mediate adaptive cancer processes.
LNCaP cells were grown in androgen deprived condition in charcoal stripped serum (CSS) and treated with androgen dihydrotestosterone (DHT) and androgen antagonist MDV 3100. The exosomes from conditioned media from LNCaP cells were isolated by serial ultracentrifugation, purified exosomes were assessed by electron microscopy, BCA assay and western blot. Exosome fraction consists of currently accepted exosomal marker such as Alix and TSG101 were analysed by mass spectrometry.
Treatment with DHT changed the composition of exosomal protein content. 78/577 total proteins were found in all samples, including CD9, Alix, TSG101, HSP90-beta, and the prostate specific biomarker, Prostate Specific Membrane Antigen (PSMA), which was found in all exosome samples irrespective of growth conditions. Ingenuity pathway analysis showed that DHT treatment increased the number of proteins known to be involved in cell survival in comparison with LNCaPs grown in CSS.
Our data support the hypothesis that androgens through the androgen receptor play a regulatory role in the secretion process, both qualitatively and quantitatively determining exosome size, content, and number in the context of the androgen sensitive prostate cancer cell, LNCaP. Our data indicate that androgens not only mediate prostate cancer progression through regulating gene transcription, but also alter cell to cell communications between prostate cancer cells via secreted nanosize vesicles such as exosomes. The data support that exosome analysis has the potential to detect and differentiate prostate cancer response to androgen targeted therapies. Exosome isolation and characterisation from advanced prostate cancer patients undergoing systemic therapy could provide an indicator of treatment response and treatment resistance.
Urological Society of Australia and New Zealand 67th ASM 2014 Annual Scientific Meeting; 01/2014
[Show abstract][Hide abstract] ABSTRACT: Introduction
Androgen Receptor (AR) stimulation activates androgen response genes to upregulate hormone dependent prostate cancer (PCa) cell proliferation. As androgen deprivation therapy will upregulate androgen down-regulated genes, characterising exosomes from advanced PCa patients on systemic therapy may provide indicators of treatment response. We report here the effects of androgen deprivation, dihydrotestosterone (DHT) and androgen antagonist MDV3100 on exosomes from AR+ LNCaP & DuCaP cells.
Cells in 5% CSS were treated with Ethanol or 10 nM dihydrotestosterone (DHT) +/- 10 mM MDV3100. Exosomes isolated from conditioned media by stepwise ultracentrifugation (Thery et al. 2006) were confirmed by EM before downstream proteomics & transcriptomics analysis.
DHT & MDV3100 treatments changed the composition of LNCaP exosomal proteins and miRNA content. By mass spec, 161/338 total proteins were seen in all samples, including exosome markers CD9, CD81, Alix, TSG101, and prostate specific biomarker, Prostate Specific Membrane Antigen. DHT upregulated CD9, Alix & TSG101 in DuCap while androgen deprivation affected their expression in LNCaP cells. DHT significantly altered the size of exosomes secreted by LNCaP, but not DuCaP cells. Ingenuity pathway analysis on LNCaP exosomes showed that DHT upregulated cell proliferative and survival pathways. Functional assays showed that treating androgen deprived LNCaP cells with exosomes from DHT treated cells increased their proliferative rate of LNCaP cells. SiRNA of Alix inhibited LNCaP cell proliferation confirming that altering exosome biogenesis plays a role in cellular proliferation.
Our data support the idea that androgens, through the AR, regulate exosome secretion in AR+ PCa LNCaP & DuCaP cells. Androgens not only mediate PCa progression by directly regulating AR related gene transcription, but also alter cell-cell communications between PCa cells via secreted nanosized vesicles such as exosomes.
Supported by USA DoD – Postdoctoral Training Award [W81XWH-12-1-0047]; Movember Global Action Plan for Exosome Biomarkers & Australian Government Department of Health and Ageing.
ISEV Congress of the International Society for Extracellular Vesicles; 01/2014
[Show abstract][Hide abstract] ABSTRACT: There is a large body of evidence showing that prostate cancer and its progression to castrate resistant disease is driven by the action of androgens and the androgen receptor. Exosomes, nanosize vesicles secreted by cells, are shown to be involved in cell to cell communication, and evidence from studies in other types of cancer, suggests that the exosomes and microvesicles are regulators which mediate adaptive cancer processes including metastatic progression. We aim to investigate the involvement of androgens in the exosome secretion process in prostate cancer. Our data support the hypothesis that androgens through the androgen receptor play a regulatory role in the secretion process, both qualitatively and quantitatively determining exosome size, content, and number in the context of the androgen sensitive prostate cancer cell, LNCaP. We isolated the exosomes from LNCaP cells conditioned medium by serial ultracentrifugation and confirmed the presence of exosomes by electron microscopy. Treatment with dihydrotestosterone (DHT) changes the composition of protein content, as characterised by mass spectrometry. The presence of androgen receptor antagonist drug, MDV3100, alters the population of exosomes secreted by LNCaP cells. These data indicate that androgens not only mediate prostate cancer progression through regulating gene transcription, but also alter cell to cell communications between prostate cancer cells via exosomes. The data supports that exosome analysis has the potential to detect and differentiate prostate cancer response to androgen targeted therapies. Exosome isolation and characterisation from advanced prostate cancer patients undergoing systemic therapy could provide an indicator of treatment response and treatment resistance.
Prostate Cancer World Congress & 14th Australasian Prostate Cancer Conference, Abstract 24730; 08/2013
[Show abstract][Hide abstract] ABSTRACT: Over 20,000 Australian men are diagnosed with prostate cancer with more than 3,500 deaths/year from this disease. Early prostate cancer (PCa) is treated by surgery or radiation, androgen depletion therapy (ADT) is subsequently used for those who fail treatment, as PCa is androgen regulated. Among these, 25% of cases develop castrate resistant PCa (CRPC) with a rising PSA, an androgen regulated gene, despite low androgen levels in the serum. The underlying mechanisms for progression to CRPC are complex. PCa cells that survive ADT arrest adapt to a low androgen environment through various mechanisms which maintain Androgen Receptor (AR) signalling and continue to proliferate. The chemotherapeutic drug, docetaxel, is commonly used to treat CRPC patients in the clinic, but ~30% of patients who receive docetaxel therapy relapse and suffer from severe side effects.
Biomarkers which could define whether patients will respond to ADT and drug treatments, are needed. Exosomes may provide novel biomarkers that will alter with treatment, potentially providing markers that reflect PCa progression and drug resistant disease. We investigated the effects of DHT on exosome secretion using AR+ LNCaP and 22RV1 cell lines as an in vitro PCa model. DHT changes the protein profile of exosomes isolated from both cell lines, implying that in PCa, the AR plays a role in selecting the content of exosome and confirming that exosome analysis has the potential to provide novel biomarkers for PCa progression and drug resistance.
Translational Research Institute Inaugural Poster Symposium; 01/2013
[Show abstract][Hide abstract] ABSTRACT: The research was aimed at finding which membrane proteins of the rumen bacterium Butyrivibrio proteoclasticus are involved in the uptake of carbohydrates resulting from extracellular enzymatic degradation of hemicellulose and fructan. The proteomic analysis of cells grown with fructose or xylan as the sole substrate identified 13 membrane proteins predicted to function as carbohydrate transporters. One protein detected was the membrane component of a fructose-specific phosphoenolpyruvate:sugar phosphotransferase system believed to be involved in the fructose uptake following extracellular fructan breakdown. The other 12 proteins were all ABC transport system substrate-binding proteins, nine of which belong to functional category COG1653 that includes proteins predicted to transport oligosaccharides. Four of the SBPs were significantly upregulated in xylan grown cells, and three of these were found in polysaccharide utilisation loci where they are clustered with other genes involved in hemicellulose breakdown and metabolism. It is possible that the carbon source available regulates a wider network of genes. The information on the mechanisms used by rumen bacteria to take up carbohydrates from their environment may improve our understanding of the ruminant digestion and facilitate strategies for improved pasture and stored feed utilisation.
Journal of proteomics 12/2011; 75(11):3138-44. DOI:10.1016/j.jprot.2011.12.013 · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Plant polysaccharide-degrading rumen microbes are fundamental to the health and productivity of ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-producing anaerobic bacterium with a key role in hemicellulose degradation in the rumen. Gel-based proteomics was used to examine the growth-phase-dependent abundance patterns of secreted proteins recovered from cells grown in vitro with xylan or xylose provided as the sole supplementary carbon source. Five polysaccharidases and two carbohydrate-binding proteins (CBPs) were among 30 identified secreted proteins. The endo-1,4-β-xylanase Xyn10B was 17.5-fold more abundant in the culture medium of xylan-grown cells, which suggests it plays an important role in hemicellulose degradation. The secretion of three nonxylanolytic enzymes and two CBPs implies they augment hemicellulose degradation by hydrolysis or disruption of associated structural polysaccharides. Sixteen ATP-binding cassette (ABC) transporter substrate-binding proteins were identified, several of which had altered relative abundance levels between growth conditions, which suggests they are important for oligosaccharide uptake. This study demonstrates that B. proteoclasticus modulates the secretion of hemicellulose-degrading enzymes and ATP-dependent sugar uptake systems in response to growth substrate and supports the notion that this organism makes an important contribution to polysaccharide degradation in the rumen.
Journal of Proteome Research 11/2011; 11(1):131-42. DOI:10.1021/pr200864j · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Determining the role of rumen microbes and their enzymes in plant polysaccharide breakdown is fundamental to understanding digestion and maximising productivity in ruminant animals. Butyrivibrio proteoclasticus B316(T) is a gram-positive, butyrate-forming rumen bacterium with a key role in plant polysaccharide degradation. The 4.4 Mb genome consists of 4 replicons; a chromosome, a chromid and two megaplasmids. The chromid is the smallest reported for all bacteria, and the first identified from the phylum Firmicutes. B316 devotes a large proportion of its genome to the breakdown and reassembly of complex polysaccharides and has a highly developed glycobiome when compared to other sequenced bacteria. The secretion of a range of polysaccharide-degrading enzymes which initiate the breakdown of pectin, starch and xylan, a subtilisin family protease active against plant proteins, and diverse intracellular enzymes to break down oligosaccharides constitute the degradative capability of this organism. A prominent feature of the genome is the presence of multiple gene clusters predicted to be involved in polysaccharide biosynthesis. Metabolic reconstruction reveals the absence of an identifiable gene for enolase, a conserved enzyme of the glycolytic pathway. To our knowledge this is the first report of an organism lacking an enolase. Our analysis of the B316 genome shows how one organism can contribute to the multi-organism complex that rapidly breaks down plant material in the rumen. It can be concluded that B316, and similar organisms with broad polysaccharide-degrading capability, are well suited to being early colonizers and degraders of plant polysaccharides in the rumen environment.
PLoS ONE 08/2010; 5(8):e11942. DOI:10.1371/journal.pone.0011942 · 3.23 Impact Factor