La desregulación de la maquinaria de splicing está directamente asociada con la agresividad del cáncer de próstata

Premio extraordinario de Trabajo Fin de Máster curso 2018/2019. Máster en Investigación Biomédica TraslacionalLa desregulación de la expresión de variantes de splicing se ha postulado recientemente como una nueva huella molecular del cáncer. Así, aunque la aparición de variantes de splicing aberrantes (p.ej.: AR-v7, sst5TMD4, etc.) se ha asociado a un incremento de la agresividad del cáncer de próstata (CaP) y/o al desarrollo del cáncer de próstata resistente a la castración (CPRC), hasta la fecha no se ha explorado si el mecanismo molecular subyacente a este fenómeno está relacionado con una desregulación de la maquinaria celular responsable del proceso de splicing (componentes del spliceosoma y factores de splicing). Por ello, en este proyecto se analizaron los niveles de expresión de un conjunto representativo de componentes del spliceosoma (n=15) y factores de splicing (n=27) en dos cohortes de muestras de CaP: 1) muestras de CaP clínicamente localizadas (n=84) y su respectivo tejido adyacente no tumoral (n=84); y, 2) muestras de biopsias de CaP agresivo (n=42). Nuestros resultados reflejaron una profunda desregulación en el nivel de expresión de multitud de componentes de la maquinaria de splicing (i.e. 7 componentes del spliceosoma y 19 factores de splicing) al comparar muestras de CaP frente a las correspondientes regiones adyacentes no tumorales. Además, la sobreexpresión de SNRNP200, SRRM1 y SRSF3 (a nivel de ARNm y proteína) se asoció con relevantes parámetros clínicos (p.ej.: grado Gleason, estadio tumoral, presencia de metástasis, recidiva bioquímica) y moleculares (p.ej.: expresión de AR-v7) de agresividad tumoral en muestras de CaP. A su vez, se llevaron a cabo ensayos funcionales (proliferación/migración celular) y mecanísticos [análisis de la expresión génica (PCR cuantitativa) y proteica (Western-blot)] en líneas celulares derivadas de próstata normal (PNT2) y tumoral (22Rv1, LNCaP, DU145 y PC-3) en respuesta al silenciamiento de SNRNP200, SRRM1 y SRSF3. Estos análisis revelaron un descenso en la tasa de proliferación/migración de células de CaP a través de la modulación de los niveles de expresión de variantes de splicing oncogénicas (p.ej.: AR-v7, PKM2, XBP1-S), así como de una alteración de rutas de señalización oncogénicas (p.ej.: p-AKT, p-JNK) claves en el desarrollo y/o agresividad del CaP. En conjunto, nuestros resultados demuestran que la maquinaria de splicing se encuentra drásticamente alterada en CaP, donde SNRNP200, SRRM1 y SRSF3 podrían representar nuevas dianas para el diagnóstico/pronóstico y tratamiento del CaP y CPRC

Gold Nanosystems Covered with Doxorubicin/DNA Complexes: A Therapeutic Target for Prostate and Liver Cancer

Different gold nanosystems covered with DNA and doxorubicin (Doxo) were designed and synthesized for cancer therapy, starting from Au@16-Ph-16 cationic nanoparticles and DNA–Doxo complexes prepared under saturation conditions. For the preparation of stable, biocompatible, and small-sized compacted Au@16-Ph-16/DNA–Doxo nanotransporters, the conditions for the DNA–Doxo compaction process induced by gold nanoparticles were first explored using fluorescence spectroscopy, circular dichroism and atomic force microscopy techniques. The reverse process, which is fundamental for Doxo liberation at the site of action, was found to occur at higher CAu@16-Ph-16 concentrations using these techniques. Zeta potential, dynamic light scattering and UV–visible spectroscopy reveal that the prepared compacted nanosystems are stable, highly charged and of adequate size for the effective delivery of Doxo to the cell. This fact is verified by in vitro biocompatibility and internalization studies using two prostate cancer-derived cell lines (LNCaP and DU145) and one hepatocellular carcinoma-derived cell line (SNU-387), as well as a non-tumor prostate (PNT2) cell line and a non-hepatocarcinoma hepatoblastoma cell line (Hep-G2) model used as a control in liver cells. However, the most outstanding results of this work are derived from the use of the CI+NI combined treatments which present strong action in cancer-derived cell lines, while a protective effect is observed in non-tumor cell lines. Hence, novel therapeutic targets based on gold nanoparticles denote high selectivity compared to conventional treatment based on free Doxo at the same concentration. The results obtained show the viability of both the proposed methodology for internalization of compacted nanocomplexes inside the cell and the effectiveness of the possible treatment and minimization of side effects in prostate and liver cancer

Comparative Cytotoxic Activity of Hydroxytyrosol and Its Semisynthetic Lipophilic Derivatives in Prostate Cancer Cells

A high adherence to a Mediterranean diet has been related to numerous beneficial effects in human health, including a lower incidence and mortality of prostate cancer (PCa). Olive oil is an important source of phenolic bioactive compounds, mainly hydroxytyrosol (HT), of this diet. Because of the growing interest of this compound and its derivatives as a cancer chemopreventive agent, we aimed to compare the in vitro effect of HT isolated from olive mill wastewaters and five semisynthetic alkyl ether, ester, and nitro-derivatives against prostate cancer (PCa) cell lines. The effect in cell proliferation was determined in RWPE-1, LNCaP, 22Rv1, and PC-3 cells by resazurin assay, the effect in cell migration by wound healing assay, and tumorsphere and colony formation were evaluated. The changes in key signaling pathways involved in carcinogenesis were assessed by using a phosphorylation pathway profiling array and by Western blotting. Antiproliferative effects of HT and two lipophilic derivatives [hydroxytyrosyl acetate (HT-Ac)/ethyl hydroxytyrosyl ether (HT-Et)] were significantly higher in cancerous PC-3 and 22Rv1 cells than in non-malignant RWPE-1 cells. HT/HT-Ac/HT-Et significantly reduced migration capacity in RWPE-1 and PC-3 and prostatosphere size and colony formation in 22Rv1, whereas only HT-Ac and HT-Et reduced these functional parameters in PC-3. The cytotoxic effect in 22Rv1 cells was correlated with modifications in the phosphorylation pattern of key proteins, including ERK1/2 and AKT. Consistently, HT-Ac and HT-Et decreased p-AKT levels in PC-3. In sum, our results suggest that HT and its lipophilic derivatives could be considered as potential therapeutic tools in PCa

Unleashing the Diagnostic, Prognostic and Therapeutic Potential of the Neuronostatin/GPR107 System in Prostate Cancer

Certain components of the somatostatin-system play relevant roles in Prostate Cancer (PCa), whose most aggressive phenotype (Castration-Resistant-PCa (CRPC)) remains lethal nowadays. However, neuronostatin and the G protein-coupled receptor 107 (GPR107), two novel members of the somatostatin-system, have not been explored yet in PCa. Consequently, we investigated the pathophysiological role of NST/GPR107-system in PCa. GPR107 expression was analyzed in well-characterized PCa patient′s cohorts, and functional/mechanistic assays were performed in response to GPR107-silencing and NST-treatment in PCa cells (androgen-dependent (AD: LNCaP) and androgen-independent (AI: 22Rv1/PC-3), which are cell models of hormone-sensitive and CRPC, respectively), and normal prostate cells (RWPE-1 cell-line). GPR107 was overexpressed in PCa and associated with key clinical parameters (e.g., advance stage of PCa, presence of vascular invasion and metastasis). Furthermore, GPR107-silencing inhibited proliferation/migration rates in AI-PCa-cells and altered key genes and oncogenic signaling-pathways involved in PCa aggressiveness (i.e., KI67/CDKN2D/MMP9/PRPF40A, SST5TMD4/AR-v7/In1-ghrelin/EZH2 splicing-variants and AKT-signaling). Interestingly, NST treatment inhibited proliferation/migration only in AI-PCa cells and evoked an identical molecular response than GPR107-silencing. Finally, NST decreased GPR107 expression exclusively in AI-PCa-cells, suggesting that part of the specific antitumor effects of NST could be mediated through a GPR107-downregulation. Altogether, NST/GPR107-system could represent a valuable diagnostic and prognostic tool and a promising novel therapeutic target for PCa and CRPC

LRP10, PGK1 and RPLP0: best reference genes in periprostatic adipose tissue under obesity and prostate cancer conditions

Obesity (OB) is a metabolic disorder characterized by adipose tissue dysfunction that has emerged as a health problem of epidemic proportions in recent decades. OB is associated with multiple comorbidities, including some types of cancers. Specifically, prostate cancer (PCa) has been postulated as one of the tumors that could have a causal relationship with OB. Particularly, a specialized adipose tissue (AT) depot known as periprostatic adipose tissue (PPAT) has gained increasing attention over the last few years as it could be a key player in the pathophysiological interaction between PCa and OB. However, to date, no studies have defined the most appropriate internal reference genes (IRGs) to be used in gene expression studies in this AT depot. In this work, two independent cohorts of PPAT samples (n = 20/n = 48) were used to assess the validity of a battery of 15 literature-selected IRGs using two widely used techniques (reverse transcription quantitative PCR [RT-qPCR] and microfluidic-based qPCR array). For this purpose, ΔCt method, GeNorm (v3.5), BestKeeper (v1.0), NormFinder (v.20.0), and RefFinder software were employed to assess the overall trends of our analyses. LRP10, PGK1, and RPLP0 were identified as the best IRGs to be used for gene expression studies in human PPATs, specifically when considering PCa and OB conditions

Clinical Utility of Ghrelin-O-Acyltransferase (GOAT) Enzyme as a Diagnostic Tool and Potential Therapeutic Target in Prostate Cancer

Recent data suggested that plasma Ghrelin O-Acyl Transferase enzyme (GOAT) levels could represent a new diagnostic biomarker for prostate cancer (PCa). In this study, we aimed to explore the diagnostic and prognostic/aggressiveness capacity of GOAT in urine, as well as to interrogate its putative pathophysiological role in PCa. We analysed urine/plasma levels of GOAT in a cohort of 993 patients. In vitro (i.e., cell-proliferation) and in vivo (tumor-growth in a xenograft-model) approaches were performed in response to the modulation of GOAT expression/activity in PCa cells. Our results demonstrate that plasma and urine GOAT levels were significantly elevated in PCa patients compared to controls. Remarkably, GOAT significantly outperformed PSA in the diagnosis of PCa and significant PCa in patients with PSA levels ranging from 3 to 10 ng/mL (the so-called PSA grey-zone). Additionally, urine GOAT levels were associated to clinical (e.g., Gleason-score, PSA levels) and molecular (e.g., CDK2/CDK6/CDKN2A expression) aggressiveness parameters. Indeed, GOAT overexpression increased, while its silencing/blockade decreased cell-proliferation in PCa cells. Moreover, xenograft tumors derived from GOAT-overexpressing PCa (DU145) cells were significantly higher than those derived from the mock-overexpressing cells. Altogether, our results demonstrate that GOAT could be used as a diagnostic and aggressiveness marker in urine and a therapeutic target in PCa

Analysis of the common genetic component of large-vessel vasculitides through a meta- Immunochip strategy

Giant cell arteritis (GCA) and Takayasu's arteritis (TAK) are major forms of large-vessel vasculitis (LVV) that share clinical features. To evaluate their genetic similarities, we analysed Immunochip genotyping data from 1,434 LVV patients and 3,814 unaffected controls. Genetic pleiotropy was also estimated. The HLA region harboured the main disease-specific associations. GCA was mostly associated with class II genes (HLA-DRB1/HLA-DQA1) whereas TAK was mostly associated with class I genes (HLA-B/MICA). Both the statistical significance and effect size of the HLA signals were considerably reduced in the cross-disease meta-analysis in comparison with the analysis of GCA and TAK separately. Consequently, no significant genetic correlation between these two diseases was observed when HLA variants were tested. Outside the HLA region, only one polymorphism located nearby the IL12B gene surpassed the study-wide significance threshold in the meta-analysis of the discovery datasets (rs755374, P?=?7.54E-07; ORGCA?=?1.19, ORTAK?=?1.50). This marker was confirmed as novel GCA risk factor using four additional cohorts (PGCA?=?5.52E-04, ORGCA?=?1.16). Taken together, our results provide evidence of strong genetic differences between GCA and TAK in the HLA. Outside this region, common susceptibility factors were suggested, especially within the IL12B locus

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

A Large-Scale Genetic Analysis Reveals a Strong Contribution of the HLA Class II Region to Giant Cell Arteritis Susceptibility

We conducted a large-scale genetic analysis on giant cell arteritis (GCA), a polygenic immune-mediated vasculitis. A case-control cohort, comprising 1,651 case subjects with GCA and 15,306 unrelated control subjects from six different countries of European ancestry, was genotyped by the Immunochip array. We also imputed HLA data with a previously validated imputation method to perform a more comprehensive analysis of this genomic region. The strongest association signals were observed in the HLA region, with rs477515 representing the highest peak (p = 4.05 × 10−40, OR = 1.73). A multivariate model including class II amino acids of HLA-DRβ1 and HLA-DQα1 and one class I amino acid of HLA-B explained most of the HLA association with GCA, consistent with previously reported associations of classical HLA alleles like HLA-DRB1∗04. An omnibus test on polymorphic amino acid positions highlighted DRβ1 13 (p = 4.08 × 10−43) and HLA-DQα1 47 (p = 4.02 × 10−46), 56, and 76 (both p = 1.84 × 10−45) as relevant positions for disease susceptibility. Outside the HLA region, the most significant loci included PTPN22 (rs2476601, p = 1.73 × 10−6, OR = 1.38), LRRC32 (rs10160518, p = 4.39 × 10−6, OR = 1.20), and REL (rs115674477, p = 1.10 × 10−5, OR = 1.63). Our study provides evidence of a strong contribution of HLA class I and II molecules to susceptibility to GCA. In the non-HLA region, we confirmed a key role for the functional PTPN22 rs2476601 variant and proposed other putative risk loci for GCA involved in Th1, Th17, and Treg cell function