Dataset Information

Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker.

ABSTRACT: In prostate cancer and other malignancies sensitive and robust biomarkers are lacking or have relevant limitations. Prostate specific antigen (PSA), the only biomarker widely used in prostate cancer, is suffering from low specificity. Exosomes offer new perspectives in the discovery of blood-based biomarkers. Here we present a proof-of principle study for a proteomics-based identification pipeline, implementing existing data sources, to exemplarily identify exosome-based biomarker candidates in prostate cancer.Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation. Exosome and control samples were analyzed on an LTQ-Orbitrap XL mass spectrometer. Proteomic data is available via ProteomeXchange with identifier PXD003651. We developed a scoring scheme to rank 64 proteins exclusively found in PC3 exosomes, integrating data from four public databases and published mass spectrometry data sets. Among the top candidates, we focused on the tight junction protein claudin 3. Retests under serum-free conditions using immunoblotting and immunogold labeling confirmed the presence of claudin 3 on PC3 exosomes. Claudin 3 levels were determined in the blood plasma of patients with localized (n = 58; 42 with Gleason score 6-7, 16 with Gleason score ?8) and metastatic prostate cancer (n = 11) compared with patients with benign prostatic hyperplasia (n = 15) and healthy individuals (n = 15) using ELISA, without prior laborious exosome isolation. ANOVA showed different CLDN3 plasma levels in these groups (p = 0.004). CLDN3 levels were higher in patients with Gleason ?8 tumors compared with patients with benign prostatic hyperplasia (p = 0.012) and Gleason 6-7 tumors (p = 0.029). In patients with localized tumors CLDN3 levels predicted a Gleason score ? 8 (AUC = 0.705; p = 0.016) and did not correlate with serum PSA.By using the described workflow claudin 3 was identified and validated as a potential blood-based biomarker in prostate cancer. Furthermore this workflow could serve as a template to be used in other cancer entities.

SUBMITTER: Worst TS

PROVIDER: S-EPMC5461549 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker.

Worst Thomas Stefan TS von Hardenberg Jost J Gross Julia Christina JC Erben Philipp P Schnölzer Martina M Hausser Ingrid I Bugert Peter P Michel Maurice Stephan MS Boutros Michael M

Molecular & cellular proteomics : MCP 20170409 6

In prostate cancer and other malignancies sensitive and robust biomarkers are lacking or have relevant limitations. Prostate specific antigen (PSA), the only biomarker widely used in prostate cancer, is suffering from low specificity. Exosomes offer new perspectives in the discovery of blood-based biomarkers. Here we present a proof-of principle study for a proteomics-based identification pipeline, implementing existing data sources, to exemplarily identify exosome-based biomarker candidates in ...[more]

PMID: 28396511

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Project description:In the United States alone one in five newly diagnosed cancers in men are prostate carcinomas (PCa). Androgen receptor (AR) status and the PI3K-AKT-mTOR signal transduction pathway are critical in PCa. After initial response to single drugs targeting these pathways resistance often emerges, indicating the need for combination therapy. Here, we address the question of efficacy of drug combinations and development of resistance mechanisms to targeted therapy by a systems pharmacology approach. We combine targeted perturbation with detailed observation of the molecular response by mass spectrometry. We hypothesize that the molecular short-term (24?h) response reveals details of how PCa cells adapt to counter the anti-proliferative drug effect. With focus on six drugs currently used in PCa treatment or targeting the PI3K-AKT-mTOR signal transduction pathway, we perturbed the LNCaP clone FGC cell line by a total of 21 treatment conditions using single and paired drug combinations. The molecular response was analyzed by the mass spectrometric quantification of 52 proteins. Analysis of the data revealed a pattern of strong responders, i.e., proteins that were consistently downregulated or upregulated across many of the perturbation conditions. The downregulated proteins, HN1, PAK1, and SPAG5, are potential early indicators of drug efficacy and point to previously less well-characterized response pathways in PCa cells. Some of the upregulated proteins such as 14-3-3 proteins and KLK2 may be useful early markers of adaptive response and indicate potential resistance pathways targetable as part of combination therapy to overcome drug resistance. The potential of 14-3-3? (YWHAZ) as a target is underscored by the independent observation, based on cancer genomics of surgical specimens, that its DNA copy number and transcript levels tend to increase with PCa disease progression. The combination of systematic drug perturbation combined with detailed observation of short-term molecular response using mass spectrometry is a potentially powerful tool to discover response markers and anti-resistance targets.

Project description:BackgroundThe high incidence of pre-eclampsia, which affects 2-7% of all pregnancies, remains a major health concern. Detection of pre-eclampsia before the appearance of clinical symptoms is essential to allow early intervention, and would benefit from identification of plasma/serum biomarkers to help guide diagnosis and treatment. Liquid biopsy has emerged as a promising source of protein biomarkers that circumvents some of the inherent challenges of proteome-wide analysis of plasma/serum. In this respect, purified exosomes have the added benefit of being carriers of intercellular communication both in physiological and pathological conditions.MethodsWe compared the protein complement of purified exosomes from three different collections of control and pre-eclamptic serum samples, obtained at the end of the second trimester of pregnancy and at delivery. We employed shotgun label-free proteomics to investigate differential protein expression, which was then validated by targeted proteomics.ResultsWe developed a purification method that yielded highly enriched exosome preparations. The presence of specific pregnancy protein markers suggested that a significant proportion of purified exosomes derived from tissues related to pregnancy. Quantitative proteomic analyses allowed us to identify 10, 114 and 98 differentially-regulated proteins in the three sample collections, with a high degree of concordance. Functional analysis suggested that these proteins participate in biological processes related to pre-eclampsia, including angiogenesis, inflammation and cell migration. The differential abundance of 66 proteins was validated by targeted proteomics. Finally, we studied the impact of the pre-eclampsia-associated exosomes in the proteome using an in vitro cellular model.ConclusionsWe have identified and validated differential exosomal proteins in liquid biopsy of pregnant women that open new possibilities for early detection of pre-eclampsia. Additionally, the functional impact of the proteome composition of purified pre-eclamptic exosomes in target cells provides new information to better understand changes in embryo-maternal interactions and, consequently, the pathogenesis of this disease.

Dataset Information

Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker.

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Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker.

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