Proteomic analysis of microvesicles released by the human prostate cancer cell line PC-3

Cancer biomarkers are invaluable tools for cancer detection, prognosis, and treatment. Recently, microvesicles have appeared as a novel source for cancer biomarkers. We present here the results from a proteomic analysis of microvesicles released to the extracellular environment by the metastatic prostate cancer cell line PC-3. Using nanocapillary liquid chromatography-tandem mass spectrometry 266 proteins were identified with two or more peptide sequences. Further analysis showed that 16% of the proteins were classified as extracellular and that intracellular proteins were annotated in a variety of locations. Concerning biological processes, the proteins found in PC-3 cell-released microvesicles are mainly involved in transport, cell organization and biogenesis, metabolic process, response to stimulus, and regulation of biological processes. Several of the proteins identified (tetraspanins, annexins, Rab proteins, integrins, heat shock proteins, cytoskeletal proteins, 14-3-3 proteins) have previously been found in microvesicles isolated from other sources. However, some of the proteins seem to be more specific to the vesicular population released by the metastatic prostate cancer PC-3 cell line. Among these proteins are the tetraspanin protein CD151 and the glycoprotein CUB domain-containing protein 1. Interestingly, our results show these proteins are promising biomarkers for prostate cancer and therefore candidates for clinical validation studies in biological fluids.     

The proteomics of prostate cancer exosomes

Exosomes and other microvesicles are emerging as rich reservoirs of tumor-specific proteins and biomarkers for cancer detection and progression. For prostate cancer, exosomes secreted by the prostate can be isolated from prostatic secretions, seminal fluid, tissue, urine or blood for further proteomic analysis. Structurally, prostate-derived exosomes are distinct in size, membrane composition and specific prostate protein content, potentially providing a novel and easily isolatable source of biomarkers from clinical biofluids. The key to these isolation strategies will be the targeting of specific prostatic proteins expressed in these exosomes, thus requiring detailed proteomic characterizations. A summary of ongoing efforts to characterize the proteome of these unique prostate cancer-associated exosomes and their potential applications for use in biomarker assays is presented.     

Mining the malignant ascites proteome for pancreatic cancer biomarkers

Pancreatic cancer (PC) is one of the most lethal malignancies and disease‐specific biomarkers are desperately needed for better diagnosis, prognosis, monitoring treatment efficacy and for accelerating the development of novel targeted therapeutics. Being an advanced stage manifestation and a proximal fluid in contact with cancer tissues, the ascitic fluid presents itself as a promising rich source of biomarkers. Herein, we present a comprehensive proteomic analysis of pancreatic ascitic fluid. To fractionate the complex ascites proteome, we adopted a multi‐dimensional chromatographic approach that included size‐exclusion, ion‐exchange and lectin‐affinity chromatographic techniques. Our detailed proteomic analysis with high‐resolution Orbitrap^® mass spectrometer resulted in the identification of 816 proteins. We followed rigorous filtering criteria that consisted of PC‐specific information obtained from three publicly available databases (Oncomine, Protein Atlas and Unigene) to segregate 20 putative biomarker candidates for future validation. Since these proteins are of membranous and extra‐cellular origin, most are glycosylated, and many of them are over‐expressed in cancer tissues, the probability of these proteins entering the peripheral blood circulation is high. Their validation as serological PC biomarkers in the future is highly warranted.     

Application of mass spectrometry to the discovery of biomarkers for detection of prostate cancer

There has been an impressive emergence of mass spectrometry based technologies applied toward the study of proteins. Equally notable is the rapid adaptation of these technologies to biomedical approaches in the realm of clinical proteomics. Concerted efforts toward the elucidation of the proteomes of organ sites or specific disease state are proliferating and from these efforts come the promise of better diagnostics/prognostics and therapeutic intervention. Prostate cancer has been a focus of many such studies with the promise of improved care to patients via biomarkers derived from these proteomic approaches. The newer technologies provide higher analytical capabilities, employ automated liquid handling systems, fractionation techniques and bioinformatics tools for greater sensitivity and resolving power, more robust and higher throughput sample processing, and greater confidence in analytical results. In this prospects, we summarize the proteomic technologies applied to date in prostate cancer, along with their respective advantages and disadvantages. The development of newer proteomic strategies for use in future applications is also discussed.     

Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice

Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, alpha enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.     

Temporal variations of the postnatal rat urinary proteome as a reflection of systemic maturation

The rat kidney matures during the first 2 wk of life, suggesting that temporal variations in the urinary proteome may occur during this period. We describe the urine proteome during postnatal development in the rat and demonstrate specific proteomic changes corresponding to developmental milestones. Urine was collected from 30 rats at five postnatal (P) days of life (P1, P3, P7, P14, and >P30) by bladder aspiration. The proteome was assessed by nano-ESI-LC-MS/MS. For identification, we used stringent criteria to provide a 1% false positive rate at the peptide level. The proteins in common at each time interval decreased during postnatal maturation. When comparing all five developmental times, six proteins were ubiquitously present. We detected 14 proteins involved with cellular adhesion, structure, or proliferation and differentiation only during neonatal development. Additionally, 30 proteins were specific to adults, of which 13 originated from the prostate or seminal vesicle. This is the first MS characterization of the normal urinary proteome in early postnatal rodent development that demonstrates distinct differences correlating with different stages of tissue maturation. Further characterization of the normal urinary proteome may provide the basis for identification of urinary biomarkers of diseases of the urinary tract.     

Evaluation of an in vitro model of androgen ablation and identification of the androgen responsive proteome in LNCaP cells

Proteins responsive to androgen and anti-androgen may be involved in the development and progression of prostate cancer and the ultimate failure of androgen-ablation therapy. These proteins represent potential diagnostic and therapeutic targets for improved management of prostate cancer. We have investigated the effect of androgen (R1881) and anti-androgen (bicalutamide) on the androgen-responsive prostate cancer LNCaP cell line using a quantitative gel-based proteomic approach. Prior to analysis, the in vitro system was evaluated for reproducibility and validated by appropriate molecular responses to treatment. Six replicate samples were independently generated and analysed by 2-D DIGE. According to strict statistical criteria, 197 spots were differentially expressed, of which we have successfully identified 165 spots corresponding to 125 distinct proteins. Following androgen supplementation, 108 spots (68 proteins) were increased and 57 spots (39 proteins) were decreased. Essentially no difference was observed between control and anti-androgen-treated samples, confirming the absence of "off-target" effects of bicalutamide. Identified proteins were involved in diverse processes including the stress response and intracellular signalling. The potential contribution to disease of these processes and identified constituent proteins are discussed. This rigorous, statistically supported study of androgen responses has provided a number of potential candidates for development as diagnostic/prognostic markers and drug targets.     

The human urine mannose 6-phosphate glycoproteome

Glycoproteins containing the mannose 6-phosphate (Man-6-P) modification represent a class of proteins of considerable biomedical importance. They include over sixty different soluble lysosomal hydrolases and accessory proteins, deficiencies of which result in over forty different known human genetic diseases. In addition, there are patients with lysosomal storage diseases of unknown etiology and lysosomal proteins have been implicated in pathophysiological processes associated with Alzheimer disease, arthritis, and cancer. The aim of this study was to explore urine as a source for the proteomic investigation of lysosomal storage disorders as well as for biomarker studies on the role of Man-6-P containing proteins in other human diseases. To this end, urinary proteins were affinity purified on immobilized Man-6-P receptors, digested with trypsin, and analyzed using nanospray LC/MS/MS. This resulted in identification of 67 proteins, including 48 known lysosomal proteins and 9 proteins that may be lysosomal. The identification of a large proportion of the known set of soluble lysosomal proteins with relatively few contaminants suggests that urine represents a promising substrate for the development of comparative proteomic methods for the investigation of lysosomal disorders and other diseases involving Man-6-P glycoproteins.     

Prostate cancer and the genomic revolution: Advances using microarray analyses

The emerging technology of microarray analysis allows the establishment of molecular portraits of prostate cancer and the discovery of novel genes involved in the carcinogenesis process. Many novel genes have already been identified using this technique, and functional analyses of these genes are currently being tested. The combination of microarray analysis with other recently developed high-throughput techniques, such as proteomics, tissue arrays, and gene promoter-methylation, especially using tissue microdissection methods, will provide us with more comprehensive insights into how prostate cancer develops and responds to gene-targeted therapies. Animal models of prostate cancer are being characterized by high throughput techniques to better define the similarities and differences between those models and the human disease, and to determine whether particular models may be useful for specific targeted therapies in pre-clinical studies. Although profiling of mRNA expression provides important information of gene expression, the development of proteomic technologies will allow for an even more precise global insight into cellular signaling and structural alterations during prostate carcinogenesis. Not only will the "omic" revolution change basic science, but it will lead to a new era of molecular medicine.     

The discovery of putative urine markers for the specific detection of prostate tumor by integrative mining of public genomic profiles

Urine has emerged as an attractive biofluid for the noninvasive detection of prostate cancer (PCa). There is a strong imperative to discover candidate urinary markers for the clinical diagnosis and prognosis of PCa. The rising flood of various omics profiles presents immense opportunities for the identification of prospective biomarkers. Here we present a simple and efficient strategy to derive candidate urine markers for prostate tumor by mining cancer genomic profiles from public databases. Prostate, bladder and kidney are three major tissues from which cellular matters could be released into urine. To identify urinary markers specific for PCa, upregulated entities that might be shed in exosomes of bladder cancer and kidney cancer are first excluded. Through the ontology-based filtering and further assessment, a reduced list of 19 entities encoding urinary proteins was derived as putative PCa markers. Among them, we have found 10 entities closely associated with the process of tumor cell growth and development by pathway enrichment analysis. Further, using the 10 entities as seeds, we have constructed a protein-protein interaction (PPI) subnetwork and suggested a few urine markers as preferred prognostic markers to monitor the invasion and progression of PCa. Our approach is amenable to discover and prioritize potential markers present in a variety of body fluids for a spectrum of human diseases.     

Urinary extracellular microvesicles: Isolation methods and prospects for urinary proteome

Extracellular microvesicles (EVs) are membranous vesicles, which are released from diverse cells. These EVs have also been found in a wide range of body fluids. The cargo of EVs, including proteins, lipids, carbohydrates, and nucleic acids, can be stably preserved in EVs. Researchers have found that EVs can mediate intercellular communication by shuttling the cargo components. Therefore, EVs can be used for the identification of disease‐specific biomarkers. As one class of EVs, urinary exosomes can reflect the status of the renal system. Moreover, urinary exosome analysis can minimize the interference of high abundant proteins in the whole urine sample. Therefore, urinary exosomes have gained much attention in recent years. In this review, we present a comprehensive summary of urinary exosome studies in recent years, including collection, storage, and isolation methods. The normal and disease proteomic analyses of urinary exosomes are also presented. Thus, this review may provide a valuable reference for future research.     

Implications for RNase L in prostate cancer biology

Recently, the interferon (IFN) antiviral pathways and prostate cancer genetics and have surprisingly converged on a single-strand specific, regulated endoribonuclease. Genetics studies from several laboratories in the U.S., Finland, and Israel, support the recent identification of the RNase L gene, RNASEL, as a strong candidate for the long sought after hereditary prostate cancer 1 (HPC1) allele. Results from these studies suggest that mutations in RNASEL predispose men to an increased incidence of prostate cancer, which in some cases reflect more aggressive disease and/or decreased age of onset compared with non-RNASEL linked cases. RNase L is a uniquely regulated endoribonuclease that requires 5'-triphosphorylated, 2',5'-linked oligoadenylates (2-5A) for its activity. The presence of both germline mutations in RNASEL segregating with disease within HPC-affected families and loss of heterozygosity (LOH) in tumor tissues suggest a novel role for the regulated endoribonuclease in the pathogenesis of prostate cancer. The association of mutations in RNASEL with prostate cancer cases further suggests a relationship between innate immunity and tumor suppression. It is proposed here that RNase L functions in counteracting prostate cancer by virtue of its ability to degrade RNA, thus initiating a cellular stress response that leads to apoptosis. This monograph reviews the biochemistry and genetics of RNase L as it relates to the pathobiology of prostate cancer and considers implications for future screening and therapy of this disease.     

Challenges and solutions in proteomics

The accelerated growth of proteomics data presents both opportunities and challenges. Large-scale proteomic profiling of biological samples such as cells, organelles or biological fluids has led to discovery of numerous key and novel proteins involved in many biological/disease processes including cancers, as well as to the identification of novel disease biomarkers and potential therapeutic targets. While proteomic data analysis has been greatly assisted by the many bioinformatics tools developed in recent years, a careful analysis of the major steps and flow of data in a typical highthroughput analysis reveals a few gaps that still need to be filled to fully realize the value of the data. To facilitate functional and pathway discovery for large-scale proteomic data, we have developed an integrated proteomic expression analysis system, iProXpress, which facilitates protein identification using a comprehensive sequence library and functional interpretation using integrated data. With its modular design, iProXpress complements and can be integrated with other software in a proteomic data analysis pipeline. This novel approach to complex biological questions involves the interrogation of multiple data sources, thereby facilitating hypothesis generation and knowledge discovery from the genomic-scale studies and fostering disease diagnosis and drug development.     

Molecular insight in cancer treatment and prevention

This article explores the impact of new insights in the biology of cancer on the treatment and the prevention of this disease. There are two types of targeted cancer treatment, afforded by the molecular profile of cancer. One concerns the use of agents targeted on a specific component of the cancer cells (e.g., CD20 in lymphoma) or on a specific survival function of the cancer cell (growth-factor-receptor interaction; transduction cascade). The other concerns the recognition of tumors that are more or less likely to benefit from cytotoxic chemotherapy according to their genomic or proteomic profile. Cancer prevention may benefit from new molecular insight in cancer biology as these processes allow early diagnosis of cancer, identification of patients at risk for cancer, and may provide intermediate markers for chemoprevention studies.     

Tissue proteomics for cancer biomarker development: laser microdissection and 2D-DIGE

Novel cancer biomarkers are required to achieve early diagnosis and optimized therapy for individual patients. Cancer is a disease of the genome, and tumor tissues are a rich source of cancer biomarkers as they contain the functional translation of the genome, namely the proteome. Investigation of the tumor tissue proteome allows the identification of proteomic signatures corresponding to clinico-pathological parameters, and individual proteins in such signatures will be good biomarker candidates. Tumor tissues are also a rich source for plasma biomarkers, because proteins released from tumor tissues may be more cancer specific than those from non-tumor cells. Two-dimensional difference gel electrophoresis (2D-DIGE) with novel ultra high sensitive fluorescent dyes (CyDye DIGE Fluor satulation dye) enables the efficient protein expression profiling of laser-microdissected tissue samples. The combined use of laser microdissection allows accurate proteomic profiling of specific cells in tumor tissues. To develop clinical applications using the identified biomarkers, collaboration between research scientists, clinicians and diagnostic companies is essential, particularly in the early phases of the biomarker development projects. The proteomics modalities currently available have the potential to lead to the development of clinical applications, and channeling the wealth of produced information towards concrete and specific clinical purposes is urgent.     

Proteomics of M-phase entry: 'Omen' vs. 'Omre', the battle for oocyte quality and beyond

The understanding of cell cycle regulation benefited greatly from omic approaches. Because the cell cycle engine relies heavily on proteins, proteomic methods play a key role in identification of cell cycle players. The proteomic approach delivers an enormous volume of data, but it often lacks comprehensiveness. To ensure the comprehensiveness of results the discovery of novel proteins must be followed by functional analysis. Using Xenopus laevis oocytes in two different proteomic screens, we have recently identified a number of proteins whose behavior suggested specific and unexpected roles in M-phase entry. Functional analysis of EP45 identified in one of these screens has shown that M-phase entry is stimulated by Oocyte-Maturation-ENhancer ('Omen') activity. The second screen suggests the presence of an antagonistic activity, which we call 'Omre' (Oocyte- -Maturation-REpressor). The equilibrium between Omen and Omre activities may determine the quality of oocytes and further embryo development via participation in making the decision whether to enter oocyte maturation. It remains an open question whether similar activities operate during mitotic divisions in embryonic and adult cells. Identifying such activities in somatic cells might impact on cancer treatments.     

Proteomics in prostate cancer biomarker discovery

Despite advances in molecular medicine, genomics, proteomics and translational research, prostate cancer remains the second most common cause of cancer-related mortality for men in the Western world. Clearly, early detection, targeted treatment and post-treatment monitoring are vital tools to combat this disease. Tumor markers can be useful for diagnosis and early detection of cancer, assessment of prognosis, prediction of therapeutic effect and treatment monitoring. Such tumor markers include prostate-specific antigen (prostate), cancer antigen (CA)15.3 (breast), CA125 (ovarian), CA19.9 (gastrointestinal) and serum α-fetoprotein (testicular cancer). However, all of these biomarkers lack sensitivity and specificity and, therefore, there is a large drive towards proteomic biomarker discovery. Current research efforts are directed towards discovering biosignatures from biological samples using novel proteomic technologies that provide high-throughput, in-depth analysis and quantification of the proteome. Several of these studies have revealed promising biomarkers for use in diagnosis, assessment of prognosis, and targeting treatment of prostate cancer. This review focuses on prostate cancer proteomic biomarker discovery and its future potential.