Abnormal aggregation of α-synuclein and sustained microglial activation are important contributors to the pathogenic processes in Parkinson's disease. However, the relationship between disease-associated protein aggregation and microglia-mediated neuroinflammation remains unknown. Here, using a combination of in silico, in vitro, and in vivo approaches, we show that extracellular α-synuclein released from neuronal cells is an endogenous agonist for toll-like receptor 2 (TLR2), which activates inflammatory responses in microglia. TLR2 ligand activity of α-synuclein is conformation-sensitive; only specific types of oligomer can interact with and activate TLR2. This paracrine interaction between neuron-released oligomeric α-synuclein and TLR2 in microglia suggests that both of these proteins are novel therapeutic targets for modification of neuroinflammation in Parkinson's disease and related neurological diseases.

Large oncosomes (LO) are atypically large (1-10μm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrepTM) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.

Over the past decade, the identification of cancer-associated factors has been a subject of primary interest not only for understanding the basic mechanisms of tumorigenesis but also for discovering the associated therapeutic targets. However, aminoacyl-tRNA synthetases (ARSs) have been overlooked, mostly because many assumed that they were simply 'housekeepers' that were involved in protein synthesis. Mammalian ARSs have evolved many additional domains that are not necessarily linked to their catalytic activities. With these domains, they interact with diverse regulatory factors. In addition, the expression of some ARSs is dynamically changed depending on various cellular types and stresses. This Analysis article addresses the potential pathophysiological implications of ARSs in tumorigenesis.

Prostate cancer (PC) is a biologically heterogeneous disease with variable molecular alterations underlying cancer initiation and progression. Despite recent advances in understanding PC heterogeneity, better methods for classification of PC are still needed to improve prognostic accuracy and therapeutic outcomes. In this study we computationally assembled a large virtual cohort (n=1,321) of human PC transcriptome profiles from 38 distinct cohorts and, using pathway activation signatures of known relevance to PC, developed a novel classification system consisting of 3 distinct subtypes (named PCS1 to 3). We validated this subtyping scheme in 10 independent patient cohorts and 19 laboratory models of PC, including cell lines and genetically engineered mouse models. Analysis of subtype-specific gene expression patterns in independent datasets derived from luminal and basal cell models provides evidence that PCS1 and PCS2 tumors reflect luminal subtypes, while PCS3 represents a basal subtype. We show that PCS1 tumors progress more rapidly to metastatic disease in comparison to PCS2 or PCS3, including PSC1 tumors of low Gleason grade. To apply this finding clinically, we developed a 37-gene panel that accurately assigns individual tumors to one of the 3 PCS subtypes. This panel was also applied to circulating tumor cells (CTCs) and provided evidence that PCS1 CTCs may reflect enzalutamide resistance. In summary, PCS subtyping may improve accuracy in predicting the likelihood of clinical progression and permit treatment stratification at early and late disease stages.

Treatment of prostate cancer (PC) by androgen suppression promotes the emergence of aggressive variants that are androgen receptor- (AR-) independent. Here we identify the transcription factor ONECUT2 (OC2) as a master regulator of AR networks in metastatic castration-resistant prostate cancer (mCRPC). OC2 acts as a survival factor in mCRPC models, suppresses the AR transcriptional program by direct regulation of AR target genes and the AR licensing factor FOXA1, and activates genes associated with neural differentiation and progression to lethal disease. OC2 appears active in a substantial subset of human prostate adenocarcinoma and neuroendocrine tumors. Inhibition of OC2 by a newly identified small molecule suppresses metastasis in mice. These findings suggest that OC2 displaces AR-dependent growth and survival mechanisms in many cases where AR remains expressed, but where its activity is bypassed. OC2 is also a potential drug target in the metastatic phase of aggressive PC.

To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, α-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level.

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).

Communication between cancer cells and the tumor microenvironment results in the modulation of complex signaling networks that facilitate tumor progression. Here, we describe a new mechanism of intercellular communication originating from large oncosomes (LO), which are cancer cell-derived, atypically large (1-10 mm) extracellular vesicles (EV). We demonstrate that, in the context of prostate cancer, LO harbor sustained AKT1 kinase activity, nominating them as active signaling platforms. Active AKT1 was detected in circulating EV from the plasma of metastatic prostate cancer patients and was LO specific. LO internalization induced reprogramming of human normal prostate fibroblasts as reflected by high levels of a-SMA, IL6, and MMP9. In turn, LO-reprogrammed normal prostate fibroblasts stimulated endothelial tube formation in vitro and promoted tumor growth in mice. Activation of stromal MYC was critical for this reprogramming and for the sustained cellular responses elicited by LO, both in vitro and in vivo in an AKT1-dependent manner. Inhibition of LO internalization prevented activation of MYC and impaired the tumor-supporting properties of fibroblasts. Overall, our data show that prostate cancer-derived LO powerfully promote establishment of a tumor-supportive environment by inducing a novel reprogramming of the stroma. This mechanism offers potential alternative options for patient treatment. Cancer Res; 77(9); 2306-17.Ó2017 AACR.