Gene-Transcript Expression in Urine Supernatant and Urine Cell-Sediment Are Different but Equally Useful for Detecting Prostate Cancer.

There is considerable interest in urine as a non-invasive liquid biopsy to detect prostate cancer (PCa). PCa-specific transcripts such as the TMPRSS2:ERG fusion gene can be found in both urine extracellular vesicles (EVs) and urine cell-sediment (Cell) but the relative usefulness of these and other genes in each fraction in PCa detection has not been fully elucidated. Urine samples from 76 men (PCa n = 40, non-cancer n = 36) were analysed by NanoString for 154 PCa-associated genes-probes, 11 tissue-specific, and six housekeeping. Comparison to qRT-PCR data for four genes (PCA3, OR51E2, FOLH1, and RPLP2) was strong (r = 0.51-0.95, Spearman p < 0.00001). Comparing EV to Cells, differential gene expression analysis found 57 gene-probes significantly more highly expressed in 100 ng of amplified cDNA products from the EV fraction, and 26 in Cells (p < 0.05; edgeR). Expression levels of prostate-specific genes (KLK2, KLK3) measured were ~20× higher in EVs, while PTPRC (white-blood Cells) was ~1000× higher in Cells. Boruta analysis identified 11 gene-probes as useful in detecting PCa: two were useful in both fractions (PCA3, HOXC6), five in EVs alone (GJB1, RPS10, TMPRSS2:ERG, ERG_Exons_4-5, HPN) and four from Cell (ERG_Exons_6-7, OR51E2, SPINK1, IMPDH2), suggesting that it is beneficial to fractionate whole urine prior to analysis. The five housekeeping genes were not significantly differentially expressed between PCa and non-cancer samples. Expression signatures from Cell, EV and combined data did not show evidence for one fraction providing superior information over the other.

A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG.

The H19X-encoded miR-424(322)/503 cluster regulates multiple cellular functions. Here, it is reported for the first time that it is also a critical linchpin of fat mass expansion. Deletion of this miRNA cluster in mice results in obesity, while increasing the pool of early adipocyte progenitors and hypertrophied adipocytes. Complementary loss and gain of function experiments and RNA sequencing demonstrate that miR-424(322)/503 regulates a conserved genetic program involved in the differentiation and commitment of white adipocytes. Mechanistically, it is demonstrated that miR-424(322)/503 targets γ-Synuclein (SNCG), a factor that mediates this program rearrangement by controlling metabolic functions in fat cells, allowing adipocyte differentiation and adipose tissue enlargement. Accordingly, diminished miR-424(322) in mice and obese humans co-segregate with increased SNCG in fat and peripheral blood as mutually exclusive features of obesity, being normalized upon weight loss. The data unveil a previously unknown regulatory mechanism of fat mass expansion tightly controlled by the miR-424(322)/503 through SNCG.

Loss of microRNA-135b Enhances Bone Metastasis in Prostate Cancer and Predicts Aggressiveness in Human Prostate Samples.

About 70% of advanced-stage prostate cancer (PCa) patients will experience bone metastasis, which severely affects patients' quality of life and progresses to lethal PCa in most cases. Hence, understanding the molecular heterogeneity of PCa cell populations and the signaling pathways associated with bone tropism is crucial. For this purpose, we generated an animal model with high penetrance to metastasize to bone using an intracardiac percutaneous injection of PC3 cells to identify PCa metastasis-promoting factors. Using genomic high-throughput analysis we identified a miRNA signature involved in bone metastasis that also presents potential as a biomarker of PCa progression in human samples. In particular, the downregulation of miR-135b favored the incidence of bone metastases by significantly increasing PCa cells' migratory capacity. Moreover, the PLAG1, JAKMIP2, PDGFA, and VTI1b target genes were identified as potential mediators of miR-135b's role in the dissemination to bone. In this study, we provide a genomic signature involved in PCa bone growth, contributing to a better understanding of the mechanisms responsible for this process. In the future, our results could ultimately translate into promising new therapeutic targets for the treatment of lethal PCa.

Dural Venous Sinus Variations in Idiopathic Subarachnoid Hemorrhage: A New Indicator of the Venous Origin with Diagnostic Usefulness?

OBJECTIVE: Venous hypertension associated with a primitive basal vein of Rosenthal (BVR) has been noted as the most likely cause of idiopathic subarachnoid hemorrhage (iSAH). Other types of venous drainage variations have been scarcely studied but may further explain the cases not associated with a BVR anomaly. Our aim was to investigate if dural venous sinus (DVS) anomalies are related with iSAH. METHODS: A total of 76 patients diagnosed with iSAH were identified from a prospectively maintained database and their angiographic findings compared with 76 patients diagnosed with aneurysmal subarachnoid hemorrhage. RESULTS: On top of the BVR variations, our data showed a higher prevalence of transverse sinus hypoplasia (47.4% vs. 28.9%; P = 0.019), superior petrosal sinus hypoplasia (32.9% vs. 13.2%; P = 0.003), and clival plexus hyperplasia (65.8% vs. 43.4%; P = 0.005) in patients with iSAH. Analyzing by total number of angiograms, the iSAH group showed also a higher prevalence of inferior petrosal sinus hyperplasia (36.2% vs. 25%; P = 0.003). Of the patients with iSAH without a primitive BVR, 84% harbored ≥1 perimesencephalic DVS variation and the overall number of venous drainage variations was significantly higher in patients with iSAH. CONCLUSIONS: In addition to the well-documented BVR anomalies, there seems to be a significant relationship of other DVS variations in patients with iSAH. Transverse sinus hypoplasia, superior petrosal sinus hypoplasia, inferior petrosal sinus hyperplasia, and clival plexus hyperplasia were significantly more frequent in patients with iSAH. The presence of ≥3 of those variations would increase the suspicion of a nonaneurysmatic subarachnoid hemorrhage and could help avoid a second angiogram.

Methodology for the at-home collection of urine samples for prostate cancer detection.

Urine from patients with prostate cancer (PCa) contains gene transcripts that have been used for PCa diagnosis and prognosis. Historically, patient urine samples have been collected after a digital rectal examination of the prostate, which was thought necessary to boost the levels of prostatic secretions in the urine. We herein describe methodology that allows urine to be collected by patients at home and then posted to a laboratory for analysis. RNA yields and quality were comparable to those for post digital rectal examination urine, and there was improved sensitivity for the detection of TMPRSS2:ERG transcripts by RT-PCR. The At-Home collection protocol has opened up the potential to perform large-scale PCa studies without the inconvenience, cost, discomfort and expense of patients having to visit the clinic.

Multiple immunofluorescence assay identifies upregulation of Active ß-catenin in prostate cancer.

OBJECTIVES: To apply a systems pathology-based approach to the quantification of nuclear Active ß-catenin and human leukocyte antigen class I, and assess the biomarker involvement in a cohort of prostate tumor patients. RESULTS: The systems pathology approach applied allows a precise quantification of the marker expression in the different cell compartments as well as the determination of the areas that coexpress two markers. Our data shows that the accumulation of ß-catenin in the nuclear compartment is significantly decreased in the adjacent normal areas when compared to tumor of the same patients (p < 0.001). In conclusion, the application of this novel multiple immunofluorescence assay demonstrates that the upregulation of Active ß-catenin is a relatively common feature of prostate tumor development, and further supports the activation of the Wnt/ß-catenin pathway in prostate cancer progression.

Low-dose statin treatment increases prostate cancer aggressiveness.

Prostate cancer is diagnosed late in life, when co-morbidities are frequent. Among them, hypertension, hypercholesterolemia, diabetes or metabolic syndrome exhibit an elevated incidence. In turn, prostate cancer patients frequently undergo chronic pharmacological treatments that could alter disease initiation, progression and therapy response. Here we show that treatment with anti-cholesterolemic drugs, statins, at doses achieved in patients, enhance the pro-tumorigenic activity of obesogenic diets. In addition, the use of a mouse model of prostate cancer and human prostate cancer xenografts revealed that in vivo simvastatin administration alone increases prostate cancer aggressiveness. In vitro cell line systems supported the notion that this phenomenon occurs, at least in part, through the direct action on cancer cells of low doses of statins, in range of what is observed in human plasma. In sum, our results reveal a prostate cancer experimental system where statins exhibit an undesirable effect, and warrant further research to address the relevance and implications of this observation in human prostate cancer.

Quantitative RNA Analysis from Urine Using Real Time PCR.

Urine is emerging as a biological fluid suitable to perform liquid biopsy in a minimally invasive manner, a fundamental attribute for prevention and early detection of cancer. Urine biomarkers can be analyzed in voided urine, in urine sediment, and urine supernatant. In the case of urothelial carcinoma, in which tumor cells are in direct contact with urine, the assessment of the levels of biomarkers in the urinary cell fraction appears to be the most promising approach to identify diagnostic and prognostic biomarkers in a noninvasive way. Here, we describe a protocol to collect and process urine samples to obtain urinary exfoliated cells. Furthermore, we describe the methodology to isolate RNA from urinary cells and to quantify gene expression levels from these urinary cells.

Prostate Tumor Overexpressed-1 (PTOV1) promotes docetaxel-resistance and survival of castration resistant prostate cancer cells.

Metastatic prostate cancer is presently incurable. The oncogenic protein PTOV1, first described in prostate cancer, was reported as overexpressed and significantly correlated with poor survival in numerous tumors. Here, we investigated the role of PTOV1 in prostate cancer survival to docetaxel and self-renewal ability. Transduction of PTOV1 in docetaxel-sensitive Du145 and PC3 cells significantly increased cell survival after docetaxel exposure and induced docetaxel-resistance genes expression (ABCB1, CCNG2 and TUBB2B). In addition, PTOV1 induced prostatospheres formation and self-renewal genes expression (ALDH1A1, LIN28A, MYC and NANOG). In contrast, Du145 and PC3 cells knockdown for PTOV1 significantly accumulated in the G2/M phase, presented a concomitant increased subG1 peak, and cell death by apoptosis. These effects were enhanced in docetaxel-resistant cells. Analyses of tumor datasets show that PTOV1 expression significantly correlated with prostate tumor grade, drug resistance (CCNG2) and self-renewal (ALDH1A1, MYC) markers. These genes are concurrently overexpressed in most metastatic lesions. Metastases also show PTOV1 genomic amplification in significant co-occurrence with docetaxel-resistance and self-renewal genes. Our findings identify PTOV1 as a promoter of docetaxel-resistance and self-renewal characteristics for castration resistant prostate cancer. The concomitant increased expression of PTOV1, ALDH1A1 and CCNG2 in primary tumors, may predict metastasis and bad prognosis.

Targeted proteomics in urinary extracellular vesicles identifies biomarkers for diagnosis and prognosis of prostate cancer.

Rapid and reliable diagnosis of prostate cancer (PCa) is highly desirable as current used methods lack specificity. In addition, identification of PCa biomarkers that can classify patients into high- and low-risk groups for disease progression at early stage will improve treatment decision-making. Here, we describe a set of protein-combination panels in urinary extracellular vesicles (EVs), defined by targeted proteomics and immunoblotting techniques that improve early non-invasive detection and stratification of PCa patients.We report a two-protein combination in urinary EVs that classifies benign and PCa patients (ADSV-TGM4), and a combination of five proteins able to significantly distinguish between high- and low-grade PCa patients (CD63-GLPK5-SPHM-PSA-PAPP). Proteins composing the panels were validated by immunohistochemistry assays in tissue microarrays (TMAs) confirming a strong link between the urinary EVs proteome and alterations in PCa tissues. Moreover, ADSV and TGM4 abundance yielded a high diagnostic potential in tissue and promising TGM4 prognostic power. These results suggest that the proteins identified in urinary EVs distinguishing high- and low grade PCa are a reflection of histological changes that may be a consequence of their functional involvement in PCa development. In conclusion, our study resulted in the identification of protein-combination panels present in urinary EVs that exhibit high sensitivity and specificity for PCa detection and patient stratification. Moreover, our study highlights the potential of targeted proteomic approaches-such as selected reaction monitoring (SRM)-as diagnostic assay for liquid biopsies via urinary EVs to improve diagnosis and prognosis of suspected PCa patients.

Cachexia-associated adipose tissue morphological rearrangement in gastrointestinal cancer patients.

BACKGROUND AND AIMS: Cachexia is a syndrome characterized by marked involuntary loss of body weight. Recently, adipose tissue (AT) wasting has been shown to occur before the appearance of other classical cachexia markers. We investigated the composition and rearrangement of the extracellular matrix, adipocyte morphology and inflammation in the subcutaneous AT (scAT) pad of gastrointestinal cancer patients. METHODS: Surgical biopsies for scAT were obtained from gastrointestinal cancer patients, who were signed up into the following groups: cancer cachexia (CC, n = 11), weight-stable cancer (WSC, n = 9) and weight-stable control (non-cancer) (control, n = 7). The stable weight groups were considered as those with no important weight change during the last year and body mass index <25 kg/m(2). Subcutaneous AT fibrosis was quantified and characterized by quantitative PCR, histological analysis and immunohistochemistry. RESULTS: The degree of fibrosis and the distribution and collagen types (I and III) were different in WSC and CC patients. CC patients showed more pronounced fibrosis in comparison with WSC. Infiltrating macrophages surrounding adipocytes and CD3 Ly were found in the fibrotic areas of scAT. Subcutaneous AT fibrotic areas demonstrated increased monocyte chemotactic protein 1 (MCP-1) and Cluster of Differentiation (CD)68 gene expression in cancer patients. CONCLUSIONS: Our data indicate architectural modification consisting of fibrosis and inflammatory cell infiltration in scAT as induced by cachexia in gastrointestinal cancer patients. The latter was characterized by the presence of macrophages and lymphocytes, more evident in the fibrotic areas. In addition, increased MCP-1 and CD68 gene expression in scAT from cancer patients may indicate an important role of these markers in the early phases of cancer.

Inhibition of the autocrine IL-6-JAK2-STAT3-calprotectin axis as targeted therapy for HR-/HER2+ breast cancers.

HER2-positive (HER2(+)) breast adenocarcinomas are a heterogeneous group in which hormone receptor (HR) status influences therapeutic decisions and patient outcome. By combining genome-wide RNAi screens with regulatory network analysis, we identified STAT3 as a critically activated master regulator of HR(-)/HER2(+) tumors, eliciting tumor dependency in these cells. Mechanistically, HR(-)/HER2(+) cells secrete high levels of the interleukin-6 (IL-6) cytokine, inducing the activation of STAT3, which in turn promotes a second autocrine stimulus to increase S100A8/9 complex (calprotectin) production and secretion. Increased calprotectin levels activate signaling pathways involved in proliferation and resistance. Importantly, we demonstrated that inhibition of the IL-6-Janus kinase 2 (JAK2)-STAT3-calprotectin axis with FDA-approved drugs, alone and in combination with HER2 inhibitors, reduced the tumorigenicity of HR(-)/HER2(+) breast cancers, opening novel targeted therapeutic opportunities.

Clinical significance of proliferative inflammatory atrophy finding in prostatic biopsies.

BACKGROUND: Proliferative inflammatory atrophy (PIA) has been involved in prostatic carcinogenesis. However, little is known about the clinical significance of a PIA finding in prostatic biopsies (PBs). The aim of this study is to determine the incidence of prostate inflammatory atrophy (PIA) in prostate biopsies (PBs), its association to high-grade prostatic intraepithelial neoplasia (HGPIN), prostate cancer (PCa), and tumor aggressiveness. METHODS: Prospective and observational study of PIA lesion in 528 extended PBs and 200 radical prostatectomy specimens (RPS). OUTCOME MEASUREMENTS: PIA, HGPIN, PCa incidence, Gleason score, clinical and pathologic tumor stage and insignificant tumor rate. Univariate and multivariate analysis. RESULTS: Overall incidence of PIA and HGPIN was 30.3% and 54%. In RPS, the incidence was 30.5% and 72%, respectively. No significant association was found between PIA and HGPIN. Overall PCa detection rate in PBs was 38.1%. PCa was found in 27.5% PBs with PIA and 42.7% of those without PIA, P < 0.001. In contrast, PCa was detected in 50.9% of PBs with HGPIN and 23% of those without HGPIN, P = 0.001. Multivariate analysis revealed that PIA decreased the risk of PCa, OR:0.59 (95%CI:0.37-0.95), P = 0.029, while HGPIN increased OR:3.16 (95%CI:2.04-4.90), P = 0.001. PIA was not related to Gleason grade and clinical stage, however it was associated to an insignificant tumors increase, OR:3.08 (95%CI:1.09-8.7), P = 0.033. The information in RPS suggests that PIA is associated with less aggressive tumors and a higher probability of insignificant tumors. CONCLUSIONS: PIA is present in one third of PBs, HGPIN in one half of them, and no association exists between both lesions. Contrary to HGPIN, PIA finding is associated to lower risk of PCa detection. Tumors accompanying PIA seem to be less aggressive and have a greater probability of being insignificant.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Urinary biomarkers for the detection of prostate cancer in patients with high-grade prostatic intraepithelial neoplasia.

INTRODUCTION: High-grade prostatic intraepithelial neoplasia (HGPIN) is a recognized precursor stage of PCa. Men who present HGPIN in a first prostate biopsy face years of active surveillance including repeat biopsies. This study aimed to identify non-invasive prognostic biomarkers that differentiate early on between indolent HGPIN cases and those that will transform into actual PCa. METHODS: We measured the expression of 21 candidate mRNA biomarkers using quantitative PCR in urine sediment samples from a cohort of 90 patients with initial diagnosis of HGPIN and a posterior follow up of at least two years. Uni- and multivariate statistical analyses were applied to analyze the candidate biomarkers and multiplex models using combinations of these biomarkers. RESULTS: PSMA, PCA3, PSGR, GOLM, KLK3, CDH1, and SPINK1 behaved as predictors for PCa presence in repeat biopsies. Multiplex models outperformed (AUC = 0.81-0.86) the predictive power of single genes, including the FDA-approved PCA3 (AUC = 0.70). With a fixed sensitivity of 95%, the specificity of our multiplex models was of 41-58%, compared to the 30% of PCA3. The PPV of our models (30-38%) was also higher than the PPV of PCA3 (27%), suggesting that benign cases could be more accurately identified. Applying statistical models, we estimated that 33% to 47% of repeat biopsies could be prevented with a multiplex PCR model, representing an easy applicable and significant advantage over the current gold standard in urine sediment. DISCUSSION: Using multiplex RTqPCR-based models in urine sediment it is possible to improve the current diagnostic method of choice (PCA3) to differentiate between benign HGPIN and PCa cases.

The microRNA 424/503 cluster reduces CDC25A expression during cell cycle arrest imposed by transforming growth factor ß in mammary epithelial cells.

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor ß (TGF-ß) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-ß. Mechanistically, we showed that after TGF-ß exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-ß/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR(+)) mammary epithelial cells in vivo.

Role of serum cholesterol and statin use in the risk of prostate cancer detection and tumor aggressiveness.

The aim of this study was to analyze the relationship between statin use along with serum cholesterol levels and prostate cancer (PCa) detection and aggressiveness. Statin users of three years or more and serum cholesterol levels (SC) were assessed in 2408 men scheduled for prostate biopsy. SC was classified as normal (NSC: <200 mg/dL) or high (HSC: >200 mg/dL). High-grade PCa (HGPCa) was considered if the Gleason score was greater than 7. Statin users comprised 30.9% of those studied. The PCa detection rate was 31.2% of men on statins and 37% of non-statin users (p<0.006). The PCa detection rate was 26.3% in men with NSC and 40.6% in those with HSC (p<0.001). In the subset of NSC men, the PCa rate was 26.5% for statin users and 26.2% for non-users (p=0.939), while in men with HSC, the PCa rate was 36.4% for statin users and 42.0% for non-statin users (p=0.063). The HGPCa rate was 41.8% for statin users and 32.5% for non-users (p=0.012). NSC men had a 53.8% rate of HGPCa, while the rate was only 27.6% in HSC men (p<0.001). NSC men on statins had an HGPCa rate of 70.2%, while non-statin users had a rate of 41.2% (p<0.001). The HGPCa rate for HSC men on statins was 18.8%, while the rate was 30.0% (p=0.011) for non-users. Logistic regression analysis suggested that serum cholesterol levels could serve as an independent predictor of PCa risk, OR 1.87 (95% CI 1.56-2.24) and HGPCa risk, OR 0.31 (95% CI 0.23-0.44), while statin usage could not. Statin treatment may prevent PCa detection through serum cholesterol-mediated mechanisms. A disturbing increase in the HGPCa rate was observed in statin users who normalized their serum cholesterol.

MicroRNAs as prognostic markers in ovarian cancer.

Ovarian cancer (OC) is the most lethal gynecological malignancy among women. Over 70% of women with OC are diagnosed in advanced stages and most of these cases are incurable. Although most patients respond well to primary chemotherapy, tumors become resistant to treatment. Mechanisms of chemoresistance in cancer cells may be associated with mutational events and/or alterations of gene expression through epigenetic events. Although focusing on known genes has already yielded new information, previously unknown non-coding RNAs, such as microRNAs (miRNAs), also lead insight into the biology of chemoresistance. In this review we summarize the current evidence examining the role of miRNAs as biomarkers of response and survival to therapy in OC. Beside their clinical implications, we also discuss important differences between studies that may have limited their use as clinical biomarkers and suggest new approaches.

A differential pattern of gene expression in skeletal muscle of tumor-bearing rats reveals dysregulation of excitation­contraction coupling together with additional muscle alterations.

INTRODUCTION: Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass. METHODS: By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia-inducing tumor, the AH-130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor-bearing animals, as compared with their healthy counterparts. RESULTS: The results show important differences in gene expression in EDL skeletal muscle between tumor-bearing animals with cachexia and control animals. CONCLUSIONS: The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome.