Sungyong You

Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia

Abnormal aggregation of α-synuclein and sustained microglial activation are important contributors to the pathogenic processes of Parkinsons 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. The 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 Parkinsons disease and related neurological diseases.

Proteomic analysis of urinary exosomes from patients of early IgA nephropathy and thin basement membrane nephropathy

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 MSE 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.

Loss of caveolin‐1 in prostate cancer stroma correlates with reduced relapse‐free survival and is functionally relevant to tumour progression

Levels of caveolin‐1 (Cav‐1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav‐1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav‐1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav‐1 in the stroma correlated with reduced relapse‐free survival (p = 0.009), suggesting a role for stromal Cav‐1 in inhibiting advanced disease. Silencing of Cav‐1 by shRNA in WPMY‐1 prostate fibroblasts resulted in up‐regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5‐fold increase in TGF‐β1 and γ‐synuclein (SNCG) gene expression. Moreover, silencing of Cav‐1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down‐regulation of TGF‐β1 and SNCG, suggesting that loss of Cav‐1 in the stroma can influence Akt‐mediated signalling in the tumour microenvironment. Cav‐1‐depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav‐1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up‐regulation of TGF‐β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma. Copyright

Urinary exosomes and proteomics

A number of highly abundant proteins in urine have been identified through proteomics approaches, and some have been considered as disease-biomarker candidates. These molecules might be clinically useful in diagnosis of various diseases. However, none has proven to be specifically indicative of perturbations of cellular processes in cells associated with urogenital diseases. Exosomes could be released into urine which flows through the kidney, ureter, bladder and urethra, with a process of filtration and reabsorption. Urinary exosomes have been recently suggested as alternative materials that offer new opportunities to identify useful biomarkers, because these exosomes secreted from epithelial cells lining the urinary track might reflect the cellular processes associated with the pathogenesis of diseases in their donor cells. Proteomic analysis of such urinary exosomes assists the search of urinary biomarkers reflecting pathogenesis of various diseases and also helps understanding the function of urinary exosomes in urinary systems. Thus, it has been recently suggested that urinary exosomes are one of the most valuable targets for biomarker development and to understand pathophysiology of relevant diseases.

p21-Activated kinase 4 promotes prostate cancer progression through CREB.

Prostate cancer is initially androgen-dependent but, over time, usually develops hormone- and chemo-resistance. The present study investigated a role for p21-activated kinase 4 (PAK4) in prostate cancer progression. PAK4 activation was markedly inhibited by H89, a specific protein kinase A (PKA) inhibitor, and PAK4 was activated by the elevation of cAMP. The catalytic subunit of PKA interacted with the regulatory domain of PAK4, and directly phosphorylated PAK4 at serine 474 (S474). Catalytically active PAK4 enhanced the transcriptional activity of CREB independent of S133 phosphorylation. Stable knockdown of PAK4 in PC-3 and DU145 prostate cancer cells inhibited tumor formation in nude mice. Decreased tumorigenicity correlated with decreased expression of CREB and its targets, including Bcl-2 and cyclin A1. Additionally, in androgen-dependent LNCap-FGC cells, PAK4 regulated cAMP-induced neuroendocrine differentiation, which is known to promote tumor progression. Finally, PAK4 enhanced survival and decreased apoptosis following chemotherapy. These results suggested that PAK4 regulates progression toward hormone- and chemo-resistance in prostate cancer, and this study identified both a novel activation mechanism and potential downstream effector pathways. Therefore, PAK4 may be a promising therapeutic target in prostate cancer.

NF‐AT5 is a critical regulator of inflammatory arthritis

OBJECTIVE To investigate the role of NF-AT5, an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in patients with rheumatoid arthritis (RA). METHODS The expression of NF-AT5 in synovial tissue and synoviocytes from RA patients was examined by immunohistochemistry and Western blot analysis, respectively. Messenger RNA (mRNA) in RA synoviocytes and human umbilical vein endothelial cells (HUVECs) transfected with dummy small interfering RNA (siRNA) or NF-AT5 siRNA were profiled using microarray technology. Assays to determine synoviocyte apoptosis and proliferation were performed in the presence of NF-AT5 siRNA. VEGF₁₆₅-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wound migration of HUVECs. Experimental arthritis was induced in mice by injection of anti-type II collagen antibody. RESULTS NF-AT5 was highly expressed in rheumatoid synovium, and its activity was increased by proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor α. The mRNA profiling of synoviocytes and HUVECs transfected with NF-AT5-targeted siRNA revealed 3 major changes in cellular processes associated with the pathogenesis of RA: cell cycle and survival, angiogenesis, and cell migration. Consistent with these results, NF-AT5 knockdown in RA synoviocytes and HUVECs inhibited their proliferation/survival and impeded angiogenic processes in HUVECs. Mice with NF-AT5 haploinsufficiency (NF-AT5(+/-)) developed a very limited degree of synovial proliferation, as seen on histologic analysis, and decreased angiogenesis, and they exhibited a nearly complete suppression of experimentally induced arthritis. CONCLUSION NF-AT5 regulates synovial proliferation and angiogenesis in chronic arthritis.

RANK- and c-Met-mediated signal network promotes prostate cancer metastatic colonization

Prostate cancer (PCa) metastasis to bone is lethal and there is no adequate animal model for studying the mechanisms underlying the metastatic process. Here, we report that receptor activator of NF-κB ligand (RANKL) expressed by PCa cells consistently induced colonization or metastasis to bone in animal models. RANK-mediated signaling established a premetastatic niche through a feed-forward loop, involving the induction of RANKL and c-Met, but repression of androgen receptor (AR) expression and AR signaling pathways. Site-directed mutagenesis and transcription factor (TF) deletion/interference assays identified common TF complexes, c-Myc/Max, and AP4 as critical regulatory nodes. RANKL–RANK signaling activated a number of master regulator TFs that control the epithelial-to-mesenchymal transition (Twist1, Slug, Zeb1, and Zeb2), stem cell properties (Sox2, Myc, Oct3/4, and Nanog), neuroendocrine differentiation (Sox9, HIF1α, and FoxA2), and osteomimicry (c-Myc/Max, Sox2, Sox9, HIF1α, and Runx2). Abrogating RANK or its downstream c-Myc/Max or c-Met signaling network minimized or abolished skeletal metastasis in mice. RANKL-expressing LNCaP cells recruited and induced neighboring non metastatic LNCaP cells to express RANKL, c-Met/activated c-Met, while downregulating AR expression. These initially non-metastatic cells, once retrieved from the tumors, acquired the potential to colonize and grow in bone. These findings identify a novel mechanism of tumor growth in bone that involves tumor cell reprogramming via RANK–RANKL signaling, as well as a form of signal amplification that mediates recruitment and stable transformation of non-metastatic bystander dormant cells.

Identification of key regulators for the migration and invasion of rheumatoid synoviocytes through a systems approach

Significance Rheumatoid arthritis (RA) is an autoimmune disorder that affects ∼1% of the population. Macrophage- and fibroblast-like synoviocytes are thought to play central roles in the pathogenesis of RA. They have shared and distinct features defining RA pathology. However, these features have not been systematically explored. Using global gene expression profiling, we identified molecular signature and biological network models underlying the pathologic features of macrophage-like vs. fibroblast-like synoviocytes. Based on the network models, we selected key regulators, including POSTN and TWIST1, responsible for synoviocyte migration/invasion causing joint destruction, and their validity was experimentally confirmed. Our findings provide a comprehensive and systematic basis for mechanisms explaining RA pathogenesis and also for identification of therapeutic targets for RA. Rheumatoid synoviocytes, which consist of fibroblast-like synoviocytes (FLSs) and synovial macrophages (SMs), are crucial for the progression of rheumatoid arthritis (RA). Particularly, FLSs of RA patients (RA-FLSs) exhibit invasive characteristics reminiscent of cancer cells, destroying cartilage and bone. RA-FLSs and SMs originate differently from mesenchymal and myeloid cells, respectively, but share many pathologic functions. However, the molecular signatures and biological networks representing the distinct and shared features of the two cell types are unknown. We performed global transcriptome profiling of FLSs and SMs obtained from RA and osteoarthritis patients. By comparing the transcriptomes, we identified distinct molecular signatures and cellular processes defining invasiveness of RA-FLSs and proinflammatory properties of RA-SMs, respectively. Interestingly, under the interleukin-1β (IL-1β)–stimulated condition, the RA-FLSs newly acquired proinflammatory signature dominant in RA-SMs without losing invasive properties. We next reconstructed a network model that delineates the shared, RA-FLS–dominant (invasive), and RA-SM–dominant (inflammatory) processes. From the network model, we selected 13 genes, including periostin, osteoblast-specific factor (POSTN) and twist basic helix–loop–helix transcription factor 1 (TWIST1), as key regulator candidates responsible for FLS invasiveness. Of note, POSTN and TWIST1 expressions were elevated in independent RA-FLSs and further instigated by IL-1β. Functional assays demonstrated the requirement of POSTN and TWIST1 for migration and invasion of RA-FLSs stimulated with IL-1β. Together, our systems approach to rheumatoid synovitis provides a basis for identifying key regulators responsible for pathological features of RA-FLSs and -SMs, demonstrating how a certain type of cells acquires functional redundancy under chronic inflammatory conditions.

Integrated classification of prostate cancer reveals a novel luminal subtype with poor outcome

Prostate cancer is a biologically heterogeneous disease with variable molecular alterations underlying cancer initiation and progression. Despite recent advances in understanding prostate cancer heterogeneity, better methods for classification of prostate cancer 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 prostate cancer transcriptome profiles from 38 distinct cohorts and, using pathway activation signatures of known relevance to prostate cancer, developed a novel classification system consisting of three distinct subtypes (named PCS1-3). We validated this subtyping scheme in 10 independent patient cohorts and 19 laboratory models of prostate cancer, 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 with 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 three PCS subtypes. This panel was also applied to circulating tumor cells (CTC) 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. Cancer Res; 76(17); 4948-58. ©2016 AACR.

Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancer regulate the tumor microenvironment.

Epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitors (TKIs), including gefitinib, are the first‐line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR‐TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib‐resistant nonsmall cell lung cancer cells using proteomics analysis. Nano‐LC–MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib‐induced apoptosis. Dose‐ and time‐dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.