Landscape of MicroRNA Regulatory Network Architecture and Functional Rerouting in Cancer.

Somatic mutations are a major source of cancer development, and many driver mutations have been identified in protein coding regions. However, the function of mutations located in miRNA and their target binding sites throughout the human genome remains largely unknown. Here, we built detailed cancer-specific miRNA regulatory networks across 30 cancer types to systematically analyze the effect of mutations in miRNAs and their target sites in 3' untranslated region (3' UTR), coding sequence (CDS), and 5' UTR regions. A total of 3,518,261 mutations from 9,819 samples were mapped to miRNA-gene interactions (mGI). Mutations in miRNAs showed a mutually exclusive pattern with mutations in their target genes in almost all cancer types. A linear regression method identified 148 candidate driver mutations that can significantly perturb miRNA regulatory networks. Driver mutations in 3'UTRs played their roles by altering RNA binding energy and the expression of target genes. Finally, mutated driver gene targets in 3' UTRs were significantly downregulated in cancer and functioned as tumor suppressors during cancer progression, suggesting potential miRNA candidates with significant clinical implications. A user-friendly, open-access web portal (mGI-map) was developed to facilitate further use of this data resource. Together, these results will facilitate novel noncoding biomarker identification and therapeutic drug design targeting the miRNA regulatory networks. SIGNIFICANCE: A detailed miRNA-gene interaction map reveals extensive miRNA-mediated gene regulatory networks with mutation-induced perturbations across multiple cancers, serving as a resource for noncoding biomarker discovery and drug development.

Comprehensive Network Analysis Reveals Alternative Splicing-Related lncRNAs in Hepatocellular Carcinoma.

It is increasingly appreciated that long non-coding RNAs (lncRNAs) associated with alternative splicing (AS) could be involved in aggressive hepatocellular carcinoma. Although many recent studies show the alteration of RNA alternative splicing by deregulated lncRNAs in cancer, the extent to which and how lncRNAs impact alternative splicing at the genome scale remains largely elusive. We analyzed RNA-seq data obtained from 369 hepatocellular carcinomas (HCCs) and 160 normal liver tissues, quantified 198,619 isoform transcripts, and identified a total of 1,375 significant AS events in liver cancer. In order to predict novel AS-associated lncRNAs, we performed an integration of co-expression, protein-protein interaction (PPI) and epigenetic interaction networks that links lncRNA modulators (such as splicing factors, transcript factors, and miRNAs) along with their targeted AS genes in HCC. We developed a random walk-based multi-graphic (RWMG) model algorithm that prioritizes functional lncRNAs with their associated AS targets to computationally model the heterogeneous networks in HCC. RWMG shows a good performance evaluated by the ROC curve based on cross-validation and bootstrapping strategies. As a conclusion, our robust network-based framework has derived 31 AS-related lncRNAs that not only validates known cancer-associated cases MALAT1 and HOXA11-AS, but also reveals new players such as DNM1P35 and DLX6-AS1with potential functional implications. Survival analysis further provides insights into the clinical significance of identified lncRNAs.

Functional interplay between YY1 and CARM1 promotes oral carcinogenesis.

Coactivator associated arginine methyltransferase 1 (CARM1) has been functionally implicated in maintenance of pluripotency, cellular differentiation and tumorigenesis; where it plays regulatory roles by virtue of its ability to coactivate transcription as well as to modulate protein function as an arginine methyltransferase. Previous studies establish an oncogenic function of CARM1 in the context of colorectal and breast cancer, which correlate to its overexpressed condition. However, the mechanism behind its deregulated expression in the context of cancer has not been addressed before. In the present study we uncover an oncogenic function of CARM1 in the context of oral cancer, where it was found to be overexpressed. We also identify YY1 to be a positive regulator of CARM1 gene promoter, where silencing of YY1 in oral cancer cell line could lead to reduction in expression of CARM1. In this context, YY1 showed concomitant overexpression in oral cancer patient samples compared to adjacent normal tissue. Cell line based experiments as well as xenograft study revealed pro-neoplastic functions of YY1 in oral cancer. Transcriptomics analysis as well as qRT-PCR validation clearly indicated pro-proliferative, pro-angiogenic and pro-metastatic role of YY1 in oral cancer. We also show that YY1 is a substrate of CARM1 mediated arginine methylation, where the latter could coactivate YY1 mediated reporter gene activation in vivo. Taken together, CARM1 and YY1 were found to regulate each other in a positive feedback loop to facilitate oral cancer progression.

Epigenetic Regulation of the PTEN-AKT-RAC1 Axis by G9a Is Critical for Tumor Growth in Alveolar Rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. As transient and stable modifications to chromatin have emerged as critical mechanisms in oncogenic signaling, efforts to target epigenetic modifiers as a therapeutic strategy have accelerated in recent years. To identify chromatin modifiers that sustain tumor growth, we performed an epigenetic screen and found that inhibition of lysine methyltransferase G9a significantly affected the viability of ARMS cell lines. Targeting expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo. Transcriptome and chromatin immunoprecipitation-sequencing analysis provided mechanistic evidence that the tumor-suppressor PTEN was a direct target gene of G9a. G9a repressed PTEN expression in a methyltransferase activity-dependent manner, resulting in increased AKT and RAC1 activity. Re-expression of constitutively active RAC1 in G9a-deficient tumor cells restored oncogenic phenotypes, demonstrating its critical functions downstream of G9a. Collectively, our study provides evidence for a G9a-dependent epigenetic program that regulates tumor growth and suggests targeting G9a as a therapeutic strategy in ARMS. SIGNIFICANCE: These findings demonstrate that RAC1 is an effector of G9a oncogenic functions and highlight the potential of G9a inhibitors in the treatment of ARMS.

Unravelling disparate roles of NOTCH in bladder cancer.

The Notch pathway has been implicated in both oncogenic and tumour-suppressive roles in cancer depending on the tissue type and cellular context. However, until recently, little was known about the pathway in bladder cancer. Studies have revealed that NOTCH1 copy number and expression are decreased in bladder cancer and NOTCH1 activation in bladder cancer cell lines reduces proliferation, suggesting that NOTCH1 acts as a tumour suppressor. Furthermore, in transgenic models, bladder cancer is promoted by bladder-specific inactivation of a component of the γ-secretase complex, which liberates the intracellular domain of neurogenic locus Notch homologue protein (NOTCH) and starts the signalling cascade. By contrast, further work has demonstrated that NOTCH2 acts as an oncogene that promotes cell proliferation and metastasis through epithelial-to-mesenchymal transition, cell cycle progression, and maintenance of stemness. Studies indicating that NOTCH1 and NOTCH2 have opposite effects on the progression of bladder cancer could give rise to potential therapeutic approaches aimed at blocking or restoring the Notch pathway.

Stressing the (Epi)Genome: Dealing with Reactive Oxygen Species in Cancer.

SIGNIFICANCE: Growing evidence indicates cross-talk between reactive oxygen species (ROS) and several key epigenetic processes such as DNA methylation, histone modifications, and miRNAs in normal physiology and human pathologies including cancer. This review focuses on how ROS-induced oxidative stress, metabolic intermediates, and epigenetic processes influence each other in various cancers. Recent Advances: ROS alter chromatin structure and metabolism that impact the epigenetic landscape in cancer cells. Several site-specific DNA methylation changes have been identified in different cancers and are discussed in the review. We also discuss the interplay of epigenetic enzymes and miRNAs in influencing malignant transformation in an ROS-dependent manner. CRITICAL ISSUES: Loss of ROS-mediated signaling mostly by epigenetic regulation may promote tumorigenesis. In contrast, augmented oxidative stress because of high ROS levels may precipitate epigenetic alterations to effect various phases of carcinogenesis. We address both aspects in the review. FUTURE DIRECTIONS: Several drugs targeting ROS are under various stages of clinical development. Recent analysis of human cancers has revealed pervasive deregulation of the epigenetic machinery. Thus, a better understanding of the cross-talk between ROS and epigenetic alterations in cancer could lead to the identification of new drug targets and more effective treatment modalities.

Conversion-to-open in laparoscopic appendectomy: A cohort analysis of risk factors and outcomes.

BACKGROUND: Identifying risk factors for conversion from laparoscopic to open appendectomy could select patients who may benefit from primary open appendectomy. We aimed to develop a predictive scoring model for conversion from laparoscopic to open based on pre-operative patient characteristics. METHODS: A retrospective review of the State Inpatient Database (2007-2011) was performed using derivation (N = 71,617) and validation (N = 143,235) cohorts of adults ≥ 18 years with acute appendicitis treated by laparoscopic-only (LA), conversion from laparoscopic to open (CA), or primary open (OA) appendectomy. Pre-operative variables independently associated with CA were identified and reported as odds ratios (OR) with 95% confidence intervals (CI). A weighted integer-based scoring model to predict CA was designed based on pre-operative variable ORs, and complications between operative subgroups were compared. RESULTS: Independent predictors of CA in the derivation cohort were age ≥40 (OR 1.67; CI 1.55-1.80), male sex (OR 1.25; CI 1.17-1.34), black race (OR 1.46; CI 1.28-1.66), diabetes (OR 1.47; CI 1.31-1.65), obesity (OR 1.56; CI 1.40-1.74), and acute appendicitis with abscess or peritonitis (OR 7.00; CI 6.51-7.53). In the validation cohort, the CA predictive scoring model had an optimal cutoff score of 4 (range 0-9). The risk of conversion-to-open was ≤5% for a score <4, compared to 10-25% for a score ≥4. On composite outcomes analysis controlling for all pre-operative variables, CA had a higher likelihood of infectious/inflammatory (OR 1.44; CI 1.31-1.58), hematologic (OR 1.31; CI 1.17-1.46), and renal (OR 1.22; CI 1.06-1.39) complications compared to OA. Additionally, CA had a higher likelihood of infectious/inflammatory, respiratory, cardiovascular, hematologic, and renal complications compared to LA. CONCLUSIONS: CA patients have an unfavorable complication profile compared to OA. The predictors identified in this scoring model could help select for patients who may benefit from primary open appendectomy.

BcCluster: A Bladder Cancer Database at the Molecular Level.

BACKGROUND: Bladder Cancer (BC) has two clearly distinct phenotypes. Non-muscle invasive BC has good prognosis and is treated with tumor resection and intravesical therapy whereas muscle invasive BC has poor prognosis and requires usually systemic cisplatin based chemotherapy either prior to or after radical cystectomy. Neoadjuvant chemotherapy is not often used for patients undergoing cystectomy. High-throughput analytical omics techniques are now available that allow the identification of individual molecular signatures to characterize the invasive phenotype. However, a large amount of data produced by omics experiments is not easily accessible since it is often scattered over many publications or stored in supplementary files. OBJECTIVE: To develop a novel open-source database, BcCluster (http://www.bccluster.org/), dedicated to the comprehensive molecular characterization of muscle invasive bladder carcinoma. MATERIALS: A database was created containing all reported molecular features significant in invasive BC. The query interface was developed in Ruby programming language (version 1.9.3) using the web-framework Rails (version 4.1.5) (http://rubyonrails.org/). RESULTS: BcCluster contains the data from 112 published references, providing 1,559 statistically significant features relative to BC invasion. The database also holds 435 protein-protein interaction data and 92 molecular pathways significant in BC invasion. The database can be used to retrieve binding partners and pathways for any protein of interest. We illustrate this possibility using survivin, a known BC biomarker. CONCLUSIONS: BcCluster is an online database for retrieving molecular signatures relative to BC invasion. This application offers a comprehensive view of BC invasiveness at the molecular level and allows formulation of research hypotheses relevant to this phenotype.

Variability in length of stay after uncomplicated pulmonary lobectomy: is length of stay a quality metric or a patient metric?†.

OBJECTIVES: Previous studies have identified predictors of prolonged length of stay (LOS) following pulmonary lobectomy. LOS is typically described to have a direct relationship to postoperative complications. We sought to determine the LOS and factors associated with variability after uncomplicated pulmonary lobectomy. METHODS: Analysing the State Inpatient Databases, Healthcare Cost and Utilization Project, Agency for Healthcare Research and Quality database, we reviewed lobectomies performed (2009-11) on patients in California, Florida and New York. LOS and comorbidities were identified. Multivariable regression analysis (MVA) was used to determine factors associated with LOS greater than the median. Patients with postoperative complications or death were excluded. RESULTS: Among 22 647 lobectomies performed, we identified 13 099 patients (58%) with uncomplicated postoperative courses (mean age = 66 years; 56% female; 76% white, 57% Medicare; median DEYO comorbidity score = 3, 55% thoracotomy, 45% thoracoscopy/robotic). There was a wide distribution in LOS [median LOS = 5 days; interquartile range (IQR) 4-7]. By MVA, predictors of prolonged LOS included, age ≥ 75 years [odds ratio (OR) 1.7, 95% confidence interval (CI) 1.4-2.0], male gender (OR 1.2, 95% CI 1.1-1.2), chronic obstructive pulmonary disease (OR 1.6, 95% CI 1.5-1.7) and other comorbidities, Medicaid payer (OR 1.7, 95% CI 1.4-2.1) versus private insurance, thoracotomy (OR 3.0, 95% CI 2.8-3.3) versus video-assisted thoracoscopic surgery/robotic approach and low hospital volume (OR 2.4, 95% CI 2.1-2.6). CONCLUSIONS: Variability exists in LOS following even uncomplicated pulmonary lobectomy. Variability is driven by clinical factors such as age, gender, payer and comorbidities, but also by surgical approach and volume. All of these factors should be taken into account when designing clinical care pathways or when allocating payment resources. Attempts to define an optimal LOS depend heavily upon the patient population studied.

Incidence and Factors Associated With Hospital Readmission After Pulmonary Lobectomy.

BACKGROUND: Readmission rates after major procedures are used to benchmark quality of care. We sought to identify readmission diagnoses and factors associated with readmission in patients undergoing pulmonary lobectomy. METHODS: Analyzing the State Inpatient Databases (Healthcare Cost and Utilization Project), we reviewed all lobectomies performed from 2009 to 2011 in California, Florida, and New York. The group was subdivided into open (OL) versus minimally invasive lobectomy (MIL; thoracoscopic/robotic). We used unique identifiers to determine 30- and 90-day readmission rates and diagnoses and performed regression analysis to determine factors associated with readmission. RESULTS: A total of 22,647 lobectomies were identified (58.8% OL vs 41.2% MIL; median age, 68 years; median length of stay, 6 days). Most patients (59.8%) had routine discharge home (home health care, 29.4%; transfer to other facility, 8.8%; mortality, 1.9%). The 30-day readmission rate was 11.5% (OL 12.0% vs MIL 10.8%, p = 0.01), while the 90-day readmission rate was 19.8% (OL 21.1% vs MIL 17.9%, p < 0.001). The most common readmission diagnoses were pulmonary (24.1%), cardiovascular (16.3%), and complications related to surgical/medical procedures (15.1%). Preoperative factors associated with readmission included male gender (odds ratio, 1.19), Medicaid payer (odds ratio, 1.29), and several individual comorbidities. Surgical approach and postoperative complications were not independently associated with readmission. CONCLUSIONS: Readmission is a frequent event after pulmonary lobectomy and is strongly associated with preoperative demographic factors and comorbidities. Resources and services should be directed to patients at risk for readmission and multicomponent care pathways developed that may circumvent the need for repeat hospitalization.

Incidence and implications of postoperative supraventricular tachycardia after pulmonary lobectomy.

OBJECTIVE: We sought to determine the rate of postoperative supraventricular tachycardia (POSVT) in patients undergoing pulmonary lobectomy, and its association with adverse outcomes. METHODS: Using the State Inpatient Database, from the Healthcare Cost and Utilization Project, we reviewed lobectomies performed (2009-2011) in California, Florida, and New York, to determine POSVT incidence. Patients were grouped by presence or absence of POSVT, with or without other complications. Stroke rates were analyzed independently from other complications. Multivariable regression analysis was used to determine factors associated with POSVT. RESULTS: Among 20,695 lobectomies performed, 2449 (11.8%) patients had POSVT, including 1116 (5.4%) with isolated POSVT and 1333 (6.4%) with POSVT with other complications. Clinical predictors of POSVT included age ≥75 years, male gender, white race, chronic obstructive pulmonary disease, congestive heart failure, thoracotomy surgical approach, and pulmonary complications. POSVT was associated with an increase of: stroke (odds ratio [OR] 1.74; 95% confidence interval [CI] 1.03-2.94); in-hospital death (OR 1.85; 95% CI 1.45-2.35); LOS (OR 1.33; 95% CI 1.29-1.37); and readmission (OR 1.29; 95% CI 1.04-1.60). The stroke rate was <1% in patients who had isolated POSVT, and 1.5% in patients with POSVT with other complications. Patients with isolated POSVT had increased readmission and LOS, and a marginal increase in stroke rate, compared with patients with an uncomplicated course. CONCLUSIONS: POSVT is common in patients undergoing pulmonary lobectomy and is associated with adverse outcomes. Comparative studies are needed to determine whether strict adherence to recently published guidelines will decrease the rate of stroke, readmission, and death after POSVT in thoracic surgical patients.

Inhibition of p300 lysine acetyltransferase activity by luteolin reduces tumor growth in head and neck squamous cell carcinoma (HNSCC) xenograft mouse model.

Chromatin acetylation is attributed with distinct functional relevance with respect to gene expression in normal and diseased conditions thereby leading to a topical interest in the concept of epigenetic modulators and therapy. We report here the identification and characterization of the acetylation inhibitory potential of an important dietary flavonoid, luteolin. Luteolin was found to inhibit p300 acetyltransferase with competitive binding to the acetyl CoA binding site. Luteolin treatment in a xenografted tumor model of head and neck squamous cell carcinoma (HNSCC), led to a dramatic reduction in tumor growth within 4 weeks corresponding to a decrease in histone acetylation. Cells treated with luteolin exhibit cell cycle arrest and decreased cell migration. Luteolin treatment led to an alteration in gene expression and miRNA profile including up-regulation of p53 induced miR-195/215, let7C; potentially translating into a tumor suppressor function. It also led to down-regulation of oncomiRNAs such as miR-135a, thereby reflecting global changes in the microRNA network. Furthermore, a direct correlation between the inhibition of histone acetylation and gene expression was established using chromatin immunoprecipitation on promoters of differentially expressed genes. A network of dysregulated genes and miRNAs was mapped along with the gene ontology categories, and the effects of luteolin were observed to be potentially at multiple levels: at the level of gene expression, miRNA expression and miRNA processing.

Identification of ageing-associated naturally occurring peptides in human urine.

To assess normal and pathological peptidomic changes that may lead to an improved understanding of molecular mechanisms underlying ageing, urinarypeptidomes of 1227 healthy and 10333 diseased individuals between 20 and 86 years of age were investigated. The diseases thereby comprised diabetes mellitus, renal and cardiovascular diseases. Using age as a continuous variable, 116 peptides were identified that significantly (p < 0.05; |ρ|≥0.2) correlated with age in the healthy cohort. The same approach was applied to the diseased cohort. Upon comparison of the peptide patterns of the two cohorts 112 common age-correlated peptides were identified. These 112 peptides predominantly originated from collagen, uromodulin and fibrinogen. While most fibrillar and basement membrane collagen fragments showed a decreased age-related excretion, uromodulin, beta-2-microglobulin and fibrinogen fragments showed an increase. Peptide-based in silico protease analysis was performed and 32 proteases, including matrix metalloproteinases and cathepsins, were predicted to be involved in ageing. Identified peptides, predicted proteases and patient information were combined in a systems biology pathway analysis to identify molecular pathways associated with normal and/or pathological ageing. While perturbations in collagen homeostasis, trafficking of toll-like receptors and endosomal pathways were commonly identified, degradation of insulin-like growth factor-binding proteins was uniquely identified in pathological ageing.

Protein interactome of muscle invasive bladder cancer.

Muscle invasive bladder carcinoma is a complex, multifactorial disease caused by disruptions and alterations of several molecular pathways that result in heterogeneous phenotypes and variable disease outcome. Combining this disparate knowledge may offer insights for deciphering relevant molecular processes regarding targeted therapeutic approaches guided by molecular signatures allowing improved phenotype profiling. The aim of the study is to characterize muscle invasive bladder carcinoma on a molecular level by incorporating scientific literature screening and signatures from omics profiling. Public domain omics signatures together with molecular features associated with muscle invasive bladder cancer were derived from literature mining to provide 286 unique protein-coding genes. These were integrated in a protein-interaction network to obtain a molecular functional map of the phenotype. This feature map educated on three novel disease-associated pathways with plausible involvement in bladder cancer, namely Regulation of actin cytoskeleton, Neurotrophin signalling pathway and Endocytosis. Systematic integration approaches allow to study the molecular context of individual features reported as associated with a clinical phenotype and could potentially help to improve the molecular mechanistic description of the disorder.

Integrating proteomics profiling data sets: a network perspective.

Understanding disease mechanisms often requires complex and accurate integration of cellular pathways and molecular networks. Systems biology offers the possibility to provide a comprehensive map of the cell's intricate wiring network, which can ultimately lead to decipher the disease phenotype. Here, we describe what biological pathways are, how they function in normal and abnormal cellular systems, limitations faced by databases for integrating data, and highlight how network models are emerging as a powerful integrative framework to understand and interpret the roles of proteins and peptides in diseases.

Epigenetic response in mice mastitis: Role of histone H3 acetylation and microRNA(s) in the regulation of host inflammatory gene expression during Staphylococcus aureus infection.

BACKGROUND: There is renewed interest towards understanding the host-pathogen interaction in the light of epigenetic modifications. Although epithelial tissue is the major site for host-pathogen interactions, there is handful of studies to show how epithelial cells respond to pathogens. Bacterial infection in the mammary gland parenchyma induces local and subsequently systemic inflammation that results in a complex disease called mastitis. Globally Staphylococcus aureus is the single largest mastitis pathogen and the infection can ultimately result in either subclinical or chronic and sometimes lifelong infection. RESULTS: In the present report we have addressed the differential inflammatory response in mice mammary tissue during intramammary infection and the altered epigenetic context induced by two closely related strains of S. aureus, isolated from field samples. Immunohistochemical and immunoblotting analysis showed strain specific hyperacetylation at histone H3K9 and H3K14 residues. Global gene expression analysis in S. aureus infected mice mammary tissue revealed a selective set of upregulated genes that significantly correlated with the promoter specific, histone H3K14 acetylation. Furthermore, we have identified several differentially expressed known miRNAs and 3 novel miRNAs in S. aureus infected mice mammary tissue by small RNA sequencing. By employing these gene expression data, an attempt has been made to delineate the gene regulatory networks in the strain specific inflammatory response. Apparently, one of the isolates of S. aureus activated the NF-κB signaling leading to drastic inflammatory response and induction of immune surveillance, which could possibly lead to rapid clearance of the pathogen. The other strain repressed most of the inflammatory response, which might help in its sustenance in the host tissue. CONCLUSION: Taken together, our studies shed substantial lights to understand the mechanisms of strain specific differential inflammatory response to S. aureus infection during mastitis. In a broader perspective this study also paves the way to understand how certain bacteria can evade the immune surveillance and cause sustained infection while others are rapidly cleared from the host body.

SERS and MD simulation studies of a kinase inhibitor demonstrate the emergence of a potential drug discovery tool.

We demonstrate the use of surface-enhanced Raman spectroscopy (SERS) as an excellent tool for identifying the binding site of small molecules on a therapeutically important protein. As an example, we show the specific binding of the common antihypertension drug felodipine to the oncogenic Aurora A kinase protein via hydrogen bonding interactions with Tyr-212 residue to specifically inhibit its activity. Based on SERS studies, molecular docking, molecular dynamics simulation, biochemical assays, and point mutation-based validation, we demonstrate the surface-binding mode of this molecule in two similar hydrophobic pockets in the Aurora A kinase. These binding pockets comprise the same unique hydrophobic patches that may aid in distinguishing human Aurora A versus human Aurora B kinase in vivo. The application of SERS to identify the specific interactions between small molecules and therapeutically important proteins by differentiating competitive and noncompetitive inhibition demonstrates its ability as a complementary technique. We also present felodipine as a specific inhibitor for oncogenic Aurora A kinase. Felodipine retards the rate of tumor progression in a xenografted nude mice model. This study reveals a potential surface pocket that may be useful for developing small molecules by selectively targeting the Aurora family kinases.

Phosphorylation of multifunctional nucleolar protein nucleophosmin (NPM1) by aurora kinase B is critical for mitotic progression.

The functional association of NPM1 with Aurora kinases is well documented. Surprisingly, although NPM1 is a well characterized phosphoprotein, it is unknown whether it is a substrate of Aurora kinases. We have found that Aurora kinases A and B can phosphorylate NPM1 at a single serine residue, Ser125, in vitro and in vivo. Phosphorylated-S125-NPM1 (pS125-NPM1) localizes to the midbody region during late cytokinesis where it colocalizes with Aurora B. The overexpression of mutant (S125A) NPM1 resulted in the deregulation of centrosome duplication and mitotic defects possibly due to cytokinesis failure. These data suggest that Aurora kinase B-mediated phosphorylation of NPM1 plays a critical role during mitosis, which could have wider implications in oncogenesis.

Clinical proteomic biomarkers: relevant issues on study design & technical considerations in biomarker development.

Biomarker research is continuously expanding in the field of clinical proteomics. A combination of different proteomic-based methodologies can be applied depending on the specific clinical context of use. Moreover, current advancements in proteomic analytical platforms are leading to an expansion of biomarker candidates that can be identified. Specifically, mass spectrometric techniques could provide highly valuable tools for biomarker research. Ideally, these advances could provide with biomarkers that are clinically applicable for disease diagnosis and/ or prognosis. Unfortunately, in general the biomarker candidates fail to be implemented in clinical decision making. To improve on this current situation, a well-defined study design has to be established driven by a clear clinical need, while several checkpoints between the different phases of discovery, verification and validation have to be passed in order to increase the probability of establishing valid biomarkers. In this review, we summarize the technical proteomic platforms that are available along the different stages in the biomarker discovery pipeline, exemplified by clinical applications in the field of bladder cancer biomarker research.

Probing p300/CBP associated factor (PCAF)-dependent pathways with a small molecule inhibitor.

PCAF (KAT2B) belongs to the GNAT family of lysine acetyltransferases (KAT) and specifically acetylates the histone H3K9 residue and several nonhistone proteins. PCAF is also a transcriptional coactivator. Due to the lack of a PCAF KAT-specific small molecule inhibitor, the exclusive role of the acetyltransferase activity of PCAF is not well understood. Here, we report that a natural compound of the hydroxybenzoquinone class, embelin, specifically inhibits H3Lys9 acetylation in mice and inhibits recombinant PCAF-mediated acetylation with near complete specificity in vitro. Furthermore, using embelin, we have identified the gene networks that are regulated by PCAF during muscle differentiation, further highlighting the broader regulatory functions of PCAF in muscle differentiation in addition to the regulation via MyoD acetylation.