EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Leydig cell ovarian tumor - clinical case description and literature review.

Leydig cell ovarian tumors constitute not only a medical problem for clinicians but also a social problem - which is why women with symptoms of hirsutism relatively quickly contact physicians for medical consultation. Leydig cell ovarian tumor is a rare sex cord-gonadal stromal tumor which constitutes less than 0.5% of ovarian tumors. These cancers appear at all ages but the majority of the cases concern women in the perimenopause. In the majority of cases (70-85%), the growth is accompanied by androgen secretion, together with virilization and hirsutism. The presence of hormonally active ovarian cancers should be suspected in cases of rapidly growing symptoms of masculinization, especially when the level of free testosterone in the blood exceeds the upper limit for the given age more than three times. In diagnosing postmenopausal hyperandrogenism, it is necessary to take into account hormonally active ovarian tumors, as well as adrenal cancers. It is important to exclude other causes of hyperandrogenism, e.g. endocrinopathies (acromegaly or hypothyroidism), or iatrogenic and idiopathic factors. In order to make the diagnosis and implement the proper treatment method faster, an interdisciplinary team of physicians specializing in endocrinology, gynecology and oncology is crucial. This paper contains a study of two cases concerning Leydig cell ovarian tumors in women of postmenopausal age with symptoms of masculinization and hirsutism.

Genome-wide association study yields variants at 20p12.2 that associate with urinary bladder cancer.

Genome-wide association studies (GWAS) of urinary bladder cancer (UBC) have yielded common variants at 12 loci that associate with risk of the disease. We report here the results of a GWAS of UBC including 1670 UBC cases and 90 180 controls, followed by replication analysis in additional 5266 UBC cases and 10 456 controls. We tested a dataset containing 34.2 million variants, generated by imputation based on whole-genome sequencing of 2230 Icelanders. Several correlated variants at 20p12, represented by rs62185668, show genome-wide significant association with UBC after combining discovery and replication results (OR = 1.19, P = 1.5 × 10(-11) for rs62185668-A, minor allele frequency = 23.6%). The variants are located in a non-coding region approximately 300 kb upstream from the JAG1 gene, an important component of the Notch signaling pathways that may be oncogenic or tumor suppressive in several forms of cancer. Our results add to the growing number of UBC risk variants discovered through GWAS.

A novel hypoxia-associated subset of FN1 high MITF low melanoma cells: identification, characterization, and prognostic value.

In many human cancers, the epithelial-to-mesenchymal transition has an important role in the induction of cancer stem-like cells, and hence, in the causation of intratumoral heterogeneity. This process, also referred to as mesenchymal mimicry, is, however, only poorly understood in melanoma and histological correlation is still lacking. In an immunohistochemical analysis of a large prospective series of 220 primary and metastatic melanomas for the well-known epithelial-to-mesenchymal transition marker FN1, we observed melanoma cells with high FN1 expression in metastases with ischemic necrosis, but rarely or not at all in samples lacking evidence of hypoxia. In a blinded, retrospective series of 82 melanoma metastases with 10-year follow-up, the presence of clusters of these FN1(high) melanoma cells correlated significantly with shortened melanoma-specific survival, highlighting the prognostic value of their presence. We describe in detail the unique light- and electron-microscopic features of these FN1(high) melanoma cells, enabling their identification in routinely hematoxylin-and-eosin-stained sections. In addition, by laser microdissection and subsequent gene expression analysis and immunohistochemistry, we highlight their distinctive, molecular phenotype that includes expression of various markers of the epithelial-to-mesenchymal transition (eg, ZEB1) and of melanoma stem-like cells (eg, NGFR), and lack of immunoreactivity for the melanocytic marker MITF. This phenotype could be reproduced in vitro by culturing melanoma cells under hypoxic conditions. Functionally, the hypoxic microenvironment was shown to induce a more migratory and invasive cell type. In conclusion, we identified a novel clinically relevant FN1(high)MITF(low) cell type in melanoma associated with ischemic necrosis, and propose that these cells reside at the crossroad of the epithelial-to-mesenchymal transition and stem-like cell induction, plausibly triggered by the hypoxic environment.

Urinary bladder cancer susceptibility markers. What do we know about functional mechanisms?

Genome-wide association studies (GWAS) have been successful in the identification of the several urinary bladder cancer (UBC) susceptibility loci, pointing towards novel genes involved in tumor development. Despite that, functional characterization of the identified variants remains challenging, as they mostly map to poorly understood, non-coding regions. Recently, two of the UBC risk variants (PSCA and UGT1A) were confirmed to have functional consequences. They were shown to modify bladder cancer risk by influencing gene expression in an allele-specific manner. Although the role of the other UBC risk variants is unknown, it can be hypothesized-based on studies from different cancer types-that they influence cancer susceptibility by alterations in regulatory networks. The insight into UBC heritability gained through GWAS and further functional studies can impact on cancer prevention and screening, as well as on the development of new biomarkers and future personalized therapies.

Uncovering the role of hypoxia inducible factor-1α in skin carcinogenesis.

The hypoxia inducible factor-1α (HIF-1α) is a pleiotropic transcription factor typically activated in response to low oxygen tension as well as other stress factors in normoxic conditions. Upon activation HIF-1α mediates the transcriptional activation of target genes involved in a variety of processes comprising stress adaptation, metabolism, growth and invasion, but also apoptotic cell death. The molecular mechanisms, signaling pathways and downstream targets evoked by the activation of HIF-1α in epidermal cells are becoming increasingly understood and underscore the participation of HIF-1α in crucial processes including malignant transformation and cancer progression. Recent studies have implicated HIF-1α as an integral part of the multifaceted signal transduction initiated by the exposure of keratinocytes to ultraviolet radiation B (UVB), which represents the most ubiquitous hazard for human skin and the principal risk factor for skin cancer. HIF-1α activation by UVB exposure contributes to either repair or the removal of UVB-damaged keratinocytes by inducing apoptosis, thus revealing a tumor suppressor role for HIF-1α in these cells. On the other hand, the constitutive expression of HIF-1α evoked by the mild hypoxic state of the skin has been implicated as a positive factor in the transformation of normal melanocytes into malignant melanoma, one of the most aggressive types of human cancers. Here we review the uncovered and complex role of HIF-1α in skin carcinogenesis.

Molecular Phenotyping of AR Signaling for Predicting Targeted Therapy in Castration Resistant Prostate Cancer.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

Highly fluorescent copper nanoclusters for sensing and bioimaging.

Metal nanoclusters (NCs), typically consisting of a few to tens of metal atoms, bridge the gap between organometallic compounds and crystalline metal nanoparticles. As their size approaches the Fermi wavelength of electrons, metal NCs exhibit discrete energy levels, which in turn result in the emergence of intriguing physical and chemical (or physicochemical) properties, especially strong fluorescence. Compared with noble metals, copper is a relatively earth-abundant and cost-effective metal. Theoretical and experimental studies have shown that copper NCs (CuNCs) possess unique photoluminescent properties. To highlight these achievements, this review begins by providing an overview of a multitude of factors that play central roles in the fluorescence of CuNCs. Additionally, a critical perspective of how the aggregation of CuNCs can efficiently improve the florescent stability, tunability and intensity is also discussed. Following, we present representative applications of CuNCs in detection and in-vivo/in-vitro imaging and point out that in-situ generation of CuNCs for sensing and bioimaging may be an entry point for the in-depth studies of CuNCs as an intriguing probe. Finally, we outline current challenges and our perspective on the development of CuNCs.

LINC00857 expression predicts and mediates the response to platinum-based chemotherapy in muscle-invasive bladder cancer.

Approximately 20% of patients with bladder cancer are diagnosed with muscle-invasive disease (MIBC). The treatment involves radical cystectomy, but almost 50% of patients with MIBC eventually relapse and develop metastasis. The use of platinum-based chemotherapy in the neoadjuvant setting or for metastatic patients has been shown to improve the overall survival in a subset of patients. Unfortunately, no biomarkers are available to select patients with MIBC who will benefit from chemotherapy or to monitor the efficacy of the treatment. Recently, long noncoding RNAs (lncRNAs) were shown to regulate a variety of processes involved in the development and progression of cancer, including bladder cancer. Moreover, several lncRNAs have been shown to play a role in chemotherapy resistance. Here, we analyzed lncRNA expression associated with response to platinum-based chemotherapy in metastatic MIBC using data from the MiTranscriptome lncRNA expression database. Expression of the lncRNA, LINC00857, was found to be upregulated in tumors from patients that did not respond to platinum-based chemotherapy. Moreover, high expression of LINC00857 is correlated with shorter recurrence-free and overall survival of patients with MIBC. Knockdown of LINC00857 significantly decreased cell viability of bladder cancer cell lines through the induction of apoptosis. Furthermore, LINC00857 knockdown sensitized UM-UC-3 and T24 bladder cancer cells to cisplatin, via the negative regulation of the LMAN1 gene. Our data indicate that LINC00857 plays an important role in the regulation of response to platinum-based chemotherapy. LINC00857 potentially could serve as a novel prognostic and predictive biomarker and might be a therapeutic target to overcome cisplatin resistance in patients with MIBC.

Identification of an enhancer region within the TP63/LEPREL1 locus containing genetic variants associated with bladder cancer risk.

PURPOSE: Genome-wide association studies (GWAS) have led to the identification of a bladder cancer susceptibility variant (rs710521) in a non-coding intergenic region between the TP63 and LEPREL1 genes on chromosome 3q28, suggesting a role in the transcriptional regulation of these genes. In this study, we aimed to functionally characterize the 3q28 bladder cancer risk locus. METHODS: Fine-mapping was performed by focusing on the region surrounding rs710521, and variants were prioritized for further experiments using ENCODE regulatory data. The enhancer activity of the identified region was evaluated using dual-luciferase assays. CRISPR/Cas9-mediated deletion of the enhancer region was performed and the effect of this deletion on cell proliferation and gene expression levels was evaluated using CellTiter-Glo and RT-qPCR, respectively. RESULTS: Fine-mapping of the GWAS signal region led to the identification of twenty SNPs that showed a stronger association with bladder cancer risk than rs710521. Using publicly available data on regulatory elements and sequences, an enhancer region containing the bladder cancer risk variants was identified. Through reporter assays, we found that the presence of the enhancer region significantly increased ΔNTP63 promoter activity in bladder cancer-derived cell lines. CRISPR/Cas9-mediated deletion of the enhancer region reduced the viability of bladder cancer cells by decreasing the expression of ΔNTP63 and p63 target genes. CONCLUSIONS: Taken together, our data show that bladder cancer risk-associated variants on chromosome 3q28 are located in an active enhancer region. Further characterization of the allele-specific activity of the identified enhancer and its target genes may lead to the identification of novel signaling pathways involved in bladder carcinogenesis.

Identification of long non-coding RNAs that stimulate cell survival in bladder cancer.

For many years, research on the biology underlying bladder cancer focused on protein-coding genes which cover only about 3% of the human genome. Recently, it was discovered that a large part of the human genome is actively transcribed as long non-coding RNAs (lncRNAs). LncRNAs are master regulators of gene expression and several lncRNAs were shown to play a role in bladder cancer development and progression. Here, we analyzed lncRNA expression in muscle-invasive bladder cancer (MIBC) using the MiTranscriptome database of cancer lncRNA expression profiles, and we studied their function in bladder cancer-derived tumor cells. Analysis of the MiTranscriptome lncRNA expression data revealed four MIBC subgroups, which partially overlapped with the four mRNA clusters identified by The Cancer Genome Atlas consortium. Up-regulation of three lncRNAs CAT266, CAT1297, and CAT1647 in bladder cancer, in comparison to normal urothelium, was confirmed in an independent series of normal, non-muscle invasive (NMIBC) and MIBC tissue samples. Furthermore, expression levels of CAT1297 were found to be correlated with disease-free and overall survival in MIBC. Knockdown of CAT266, CAT1297, and CAT1647 decreased cell viability and colony formation, due to the induction of apoptosis. In conclusion, our data show that lncRNAs expression is de-regulated in MIBC and three aberrantly expressed transcripts regulate proliferation and apoptosis. Our data indicate that lncRNAs play an important role in MIBC development and progression and are a treasure chest for the discovery of new biomarkers.

Independent Replication of Published Germline Polymorphisms Associated with Urinary Bladder Cancer Prognosis and Treatment Response.

BACKGROUND: Many studies investigated the prognostic or predictive relevance of single nucleotide polymorphisms (SNPs) in biologically plausible genes in urinary bladder cancer (UBC) patients. Most published SNP associations have never been replicated in independent patient series. OBJECTIVE: To independently replicate all previously reported associations between germline SNPs and disease prognosis or treatment response in UBC. METHODS: A Pubmed search was performed to identify studies published by July 1, 2014 reporting on germline SNP associations with UBC prognosis or treatment response. For the replication series, consisting of 1,284 non-muscle-invasive bladder cancer (NMIBC) and 275 muscle-invasive or metastatic bladder cancer (MIBC) patients recruited through the Netherlands Cancer Registry, detailed clinical data were retrieved from medical charts. Patients were genotyped using a genome-wide SNP array. SNP association with recurrence-free, progression-free, and overall survival (OS) within specific patient and treatment strata was tested using Cox regression analyses. RESULTS: For only six of the 114 evaluated SNPs, the association with either UBC prognosis or treatment response was replicated at the p <  0.05 level: rs1799793 (ERCC2) and rs187238 (IL18) for BCG recurrence; rs6678136 (RGS4) and rs11585883 (RGS5) for NMIBC progression; rs12035879 (RGS5) and rs2075786 (TERT) for MIBC OS. CONCLUSIONS: Non-replicated genetic associations in the literature require cautious interpretation. This single replication does not provide definitive proof of association for the six SNPs, and non-replication of other SNPs may result from population-specific effects or the retrospective patient enrollment.

Prognostic relevance of urinary bladder cancer susceptibility loci.

In the last few years, susceptibility loci have been identified for urinary bladder cancer (UBC) through candidate-gene and genome-wide association studies. Prognostic relevance of most of these loci is yet unknown. In this study, we used data of the Nijmegen Bladder Cancer Study (NBCS) to perform a comprehensive evaluation of the prognostic relevance of all confirmed UBC susceptibility loci. Detailed clinical data concerning diagnosis, stage, treatment, and disease course of a population-based series of 1,602 UBC patients were collected retrospectively based on a medical file survey. Kaplan-Meier survival analyses and Cox proportional hazard regression were performed, and log-rank tests calculated, to evaluate the association between 12 confirmed UBC susceptibility variants and recurrence and progression in non-muscle invasive bladder cancer (NMIBC) patients. Among muscle-invasive or metastatic bladder cancer (MIBC) patients, association of these variants with overall survival was tested. Subgroup analyses by tumor aggressiveness and smoking status were performed in NMIBC patients. In the overall NMIBC group (n = 1,269), a statistically significant association between rs9642880 at 8q24 and risk of progression was observed (GT vs. TT: HR = 1.08 (95% CI: 0.76-1.54), GG vs. TT: HR = 1.81 (95% CI: 1.23-2.66), P for trend = 2.6 × 10(-3)). In subgroup analyses, several other variants showed suggestive, though non-significant, prognostic relevance for recurrence and progression in NMIBC and survival in MIBC. This study provides suggestive evidence that genetic loci involved in UBC etiology may influence disease prognosis. Elucidation of the causal variant(s) could further our understanding of the mechanism of disease, could point to new therapeutic targets, and might aid in improvement of prognostic tools.

Autophagy-dependent suppression of cancer immunogenicity and effector mechanisms of innate and adaptive immunity.

The inspection of the mechanisms through which autophagy modulates immunogenic cell death revealed that the autophagic response of cancer cells to reactive oxygen species-dependent endoplasmic reticulum stress suppresses the exposure of calreticulin on the cell surface, the phenotypic maturation of dendritic cells (DCs) as well as their ability to release interleukin-6 and to support the proliferative expansion of (interferon γ-producing) CD4+ and CD8+ T lymphocytes. These findings unveil an unprecedented role for therapy-induced autophagy in suppressing key mechanisms that underlie anticancer immune responses as elicited by immunogenic cell death.

Immature, Semi-Mature, and Fully Mature Dendritic Cells: Toward a DC-Cancer Cells Interface That Augments Anticancer Immunity.

Dendritic cells (DCs) are the sentinel antigen-presenting cells of the immune system; such that their productive interface with the dying cancer cells is crucial for proper communication of the "non-self" status of cancer cells to the adaptive immune system. Efficiency and the ultimate success of such a communication hinges upon the maturation status of the DCs, attained following their interaction with cancer cells. Immature DCs facilitate tolerance toward cancer cells (observed for many apoptotic inducers) while fully mature DCs can strongly promote anticancer immunity if they secrete the correct combinations of cytokines [observed when DCs interact with cancer cells undergoing immunogenic cell death (ICD)]. However, an intermediate population of DC maturation, called semi-mature DCs exists, which can potentiate either tolerogenicity or pro-tumorigenic responses (as happens in the case of certain chemotherapeutics and agents exerting ambivalent immune reactions). Specific combinations of DC phenotypic markers, DC-derived cytokines/chemokines, dying cancer cell-derived danger signals, and other less characterized entities (e.g., exosomes) can define the nature and evolution of the DC maturation state. In the present review, we discuss these different maturation states of DCs, how they might be attained and which anticancer agents or cell death modalities (e.g., tolerogenic cell death vs. ICD) may regulate these states.

Cancer immunogenicity, danger signals, and DAMPs: what, when, and how?

Cancer immunosurvelliance usually leads to formation of cancer cells that have been "immunoedited" to resist anti-tumor immunity. One of the consequences of immunoediting that is, reduced immunogenicity, is an important roadblock in revival of stable and long-lasting anti-tumor immune responses. Research done during the last decade has shown that emission by the dying cancer cells of immunomodulatory factors or damage-associated molecular patterns (DAMPs), which can act as danger signals, is a critical event in accentuating the immunogenicity of cancer cells, in response to a subset of anticancer treatments. Recent evidence has defined that an apoptotic cell death subroutine and its underlying biochemistry, which has been termed as "immunogenic cell death (ICD)" or "immunogenic apoptosis," is required for the efficient emission of DAMPs and inciting anti-tumor immunity. Here, we review the basic concepts of ICD, like cancer immunogenicity, danger signals, and DAMPs. Moreover, we discuss the emerging molecular links between endoplasmic reticulum (ER) stress, induction of a viral response-like gene expression, danger signals, and anti-tumor immunity. We envisage that along with ER stress-based trafficking of DAMPs (which is a "short-range communicator" of danger), the accompanying induction of a viral response-like gene expression and the secretion of anti-tumorigenic cytokines may become a crucial signature of ICD induction by anticancer therapy.

Inducers of immunogenic cancer cell death.

Recently, cytokine-based pro-tumourigenic signalling has been found to play a major role in the immune system's pro-tumourigenic activity. On the other hand, other recent findings have shown that immunogenic cancer cell death triggered by certain anticancer modalities might reset the dysfunctional immune system towards the activation of a long-lasting protective anti-tumour response. Therefore, using inducers of immunogenic cell death (ICD) that can prevent or impede tumour-promoting cytokine signalling is one of the best ways of instigating or restoring efficient anti-tumour immunity. In this review it is discussed, how the different ICD inducers interact with the immune system and influence cytokine-based pro-tumourigenic signalling. We believe that it is crucial to discover or develop new anti-cancer therapeutic modalities that can induce ICD and impede tumour-promoting cytokine signalling.

ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death.

Calreticulin surface exposure (ecto-CALR), ATP secretion, maturation of dendritic cells (DCs) and stimulation of T cells are prerequisites for anticancer therapy-induced immunogenic cell death (ICD). Recent evidence suggests that chemotherapy-induced autophagy may positively regulate ICD by favoring ATP secretion. We have recently shown that reactive oxygen species (ROS)-based endoplasmic reticulum (ER) stress triggered by hypericin-mediated photodynamic therapy (Hyp-PDT) induces bona fide ICD. However, whether Hyp-PDT-induced autophagy regulates ICD was not explored. Here we showed that, in contrast to expectations, reducing autophagy (by ATG5 knockdown) in cancer cells did not alter ATP secretion after Hyp-PDT. Autophagy-attenuated cancer cells displayed enhanced ecto-CALR induction following Hyp-PDT, which strongly correlated with their inability to clear oxidatively damaged proteins. Furthermore, autophagy-attenuation in Hyp-PDT-treated cancer cells increased their ability to induce DC maturation, IL6 production and proliferation of CD4(+) or CD8(+) T cells, which was accompanied by IFNG production. Thus, our study unravels a role for ROS-induced autophagy in weakening functional interaction between dying cancer cells and the immune system thereby helping in evasion from ICD prerequisites or determinants.