Identificación de nuevos biomarcadores y dianas terapéuticas en la interacción patológica entre la desregulación del sistema endocrino-metabólico y el cáncer de próstata

Obesity (OB) is a metabolic chronic disease with a growing incidence and elevated morbidities and mortality [1, 2]. OB is also associated with elevated economic costs for the health systems representing a worldwide public health problem [3, 4]. Specifically, in 2016, 650 million adults were obese [5]; among them, 10% to 30% presented with metabolic disorders [6] including type 2 diabetes (T2DM), hypertension (HT), heart disease, high total cholesterol and triglycerides, stroke, obstructive sleep apnea, nonalcoholic fatty liver disease, and certain types of cancer [7-9]. In this sense, bariatric surgery (BS) is a well-recognized treatment for OB that has superior outcomes for patients who have been unable to keep weight loss by nonsurgical methods [10]. Worldwide, the most commonly performed bariatric procedures are laparoscopic Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG), wherein both procedures encompass nearly 80% of all bariatric operations worldwide [11]. BS induces and maintains substantial weight loss through a variety of mechanisms, including caloric restriction (due to anatomical surgical-induced changes of the gastrointestinal tract), increased meal-induced thermogenesis, modulation of hypothalamic neuronal circuits (that regulate energy balance and/or appetite regulation), modifications in gut-brain signaling pathways and changes in taste, food preferences, and eating behavior patterns [12-15]. All these changes are accompanied by reduction in metabolic complications, especially T2DM [16], and a rapid alteration of the inflammatory status, due in part to the modulation of cytokine production after BS, which may be also accompanied by improvement in other metabolic complications [17]. In this context, inflammation is a protective immune response, wherein innate immune function depends upon recognition of pathogen-associated molecular patterns, which are derived from invading pathogens, and danger-associated molecular patterns, being induced endogenously by germline-encoded pattern recognition receptors (PRRs). The activation of PRRs induces production of interferon-α, interferon-β, and proinflammatory cytokines [18]. These responses are mediated by the inflammasome machinery, an intracellular multiprotein complex that plays a central role in innate immunity and is responsible for the activation of inflammatory responses. This complex is composed of some families of PRRs, including the nucleotide-binding domain, leucine-rich repeat containing proteins [nucleotide binding oligomerization (NOD) domain-like receptors (NLRs)], and by the absent in melanoma 2-like (AIM2- like) receptors [19]. NLRs and AIM-like receptors can oligomerize and act as caspase-1– activating scaffolds—for example, activating the proinflammatory interleukin (IL)-1 family through the production of IL-18 and IL-1β [20]. Remarkably, the activity of these inflammasome components is regulated by different regulatory proteins, metabolic pathways, and a regulatory mitochondrial hub [21]. Consequently, the activation of these inflammasome components leads to the secretion of diverse inflammatory cytokines and the activation of key receptors in immune cells, thus inducing the activation of inflammatory cascades, which can lead in some cases in cellcycle alterations and DNA damage [20, 21]. Different components of the inflammasome machinery have been linked to a variety of autoinflammatory, autoimmune, and metabolic disorders including atherosclerosis, T2DM, and OB [22]. Some studies have found that inflammasome components are dysregulated in several tissues/cells including peripheral blood mononuclear cells (PBMCs), and that these changes may be associated with the development of atherosclerosis, autoimmune (e.g., multiple sclerosis), and neurodegenerative (e.g., Alzheimer´s or Parkinson´s) diseases [20]. In the same line, alterations in some specific inflammasome components have been described in OB, but their specific roles are still not well understood [23]. However, to the best of our knowledge, changes in inflammasome components induced by acute weight loss related to BS have not been fully described yet. Interestingly, it has been reported that inflammasome-induced cytokines promote OB-related inflammatory diseases following stimulation by high-fat diet metabolites and adipose tissue changes that occur during weight gain [24], which suggests that inflammasome modulation due to weight loss may be associated to an improvement in OB-related comorbidities. As mentioned above, OB is the most common cause of insulin resistance; however, overt diabetes does not develop in most obese individuals, as they can preserve normoglycemia through an enhanced insulin secretion, resulting in compensatory hyperinsulinemia [25]. Conversely, in susceptible individuals, compensatory hyperinsulinemia cannot be maintained, consequently developing prediabetes and T2DM [6]. In this sense, it is critical to understand the molecular mechanisms involved in the onset of OB and its associated comorbidities. In fact, it has been demonstrated that the development and progression of OB and other associated comorbidities, including insulin resistance [26], is closely linked to the dysregulation of key metabolic factors, especially those implicated in the cellular control of the energy balance (e.g. insulin, leptin, glucose transporters, etc.), which are, in turn, controlled by specific cellular regulatory mechanisms, such as microRNAs. MicroRNAs (or miRNAs) are short non-coding RNAs (20-22 nucleotides) that act at post-transcriptional level to regulate gene expression [27]. Evidence is accumulating that circulating miRNAs can act as endocrine factors in that they are released to the circulation by many tissues [28]. Indeed, miRNAs seem to serve as endocrine and paracrine messengers that facilitate communication between donor and target cells, thereby potentially exerting important roles in metabolic organs crosstalk [28]. For these reasons, it has been postulated that circulating miRNAs could exert crucial roles in the development and progression of OB and its related comorbidities. Previous, scattered studies have reported that some miRNAs are dysregulated in plasma samples of obese subjects, including miR-142-3p, miR-221 or miR-222 [29-34]. However, these results are inconsistent among studies and cannot be compared due to several factors, including different cohort characteristics (obese, morbid obese, diabetic), study designs (cross-sectional, transversal) or detection methods (TaqMan, SYBR Green, micro or macro arrays, small RNA or miRNA sequencing). Hence, the whole pattern of dysregulations of miRNAs in obese condition is still to be reliably defined. Indeed, it has been shown by previous studies that high-throughput technologies may represent one of the most precise and reliable tools for the quantification of miRNAs [35]. Unfortunately, although several of the previously mentioned studies have reported the dysregulation of different miRNAs in OB condition, to the best of our knowledge there are not studies to date describing the whole miRNome using microRNAs specific arrays. Finally, as previously mentioned, OB promotes the appearance of multiple pathological problems/defects, which cause serious alterations of the homeostasis of the organism (e.g. dysregulation of hormones, growth and inflammatory factors, etc.) that frequently promote or influence the development of the severe pathologies including different endocrine-related tumors/cancers [36]. In this context, it is important to mention that Prostate Cancer (PCa) has emerged as the most frequent tumor type among men and represents a severe health problem worldwide [49]. A key limitation in PCa management is that the gold-standard screen test is based on the plasma levels of prostate-specific antigen (PSA), a biomarker that exhibits profound drawbacks, especially in the so-called “grey zone” (defined as a PSA range of 3-10 ng/mL) [37]. In fact, PSA test displays low specificity in that multiple factors can increase PSA levels without necessarily indicating the presence of a tumor, such as benign prostatic hyperplasia or inflammatory conditions. In addition, PSA test is not able to accurately distinguish clinically relevant tumors from indolent cases [38]. For these reasons, the anatomo-pathological analysis of prostate biopsies, which represent a highly invasive technique, is still necessary to appropriately diagnose PCa nowadays. Therefore, there is an important unmet clinical need for the identification and validation of new, reliable and specific non-invasive PCa diagnostic biomarkers, ideally from a personalized point of view and showing prognostic and/or therapeutic potential. In this sense, miRNAs are attractive biomarker candidates as they can be reproducibly extracted from a wide range of biologic samples and are generally stable and resistant to various storage conditions [28, 39]. Indeed, recent studies have suggested a relationship between circulating miRNAs and PCa presence and outcome [40-42]. Specifically, recent studies have identified some miRNAs differentially present in plasma samples from PCa patients; however, only some of them seem to be specifically derived from PCa tissues [43-45]. Unfortunately, although various studies have identified some putative PCa-specific miRNAs (e.g. miR-141 [46], miR-375 [47], and miR-21 [48]), there is not a consensus in the utility of plasma miRNAs as circulating non-invasive biomarkers for PCa. In fact, there are no studies describing the dysregulation of the whole miRNome in PCa vs. healthy patients. For these reasons, this field requires further investigation in order to ascertain whether a specific miRNA or a plasma/serum miRNA signature could be associated with PCa risk and provide diagnostic and prognostic value through a fast, easy, and non-invasive test. Based on all the information mentioned above, in this Doctoral Thesis we deploy a set of innovative clinical and experimental approaches commonly used in our team, including numerous clinical samples and cellular models, to pursue a triple OBJECTIVE with the ultimate goal of identifying new and more personalized diagnostic, prognostic, and therapeutic tools for the management of OB and PCa. To that end, we explored: 1) Changes in components of the inflammasome machinery (i.e., inflammasome components and inflammatoryassociated factors) in obese patients after BS and their relation with clinical/biochemical parameters at baseline and after bariatric surgery; 2) The differential expression pattern of miRNAs in OB and their putative role in OB -related comorbidities such as insulin resistance; and, 3) The dysregulation of the human miRNome in PCa patients, considering the OB condition of the patients (normoweight, overweight, or OB), and the potential pathophysiological role and the molecular mechanisms underlying the role of miR-107 in PCa…La obesidad (OB) es una enfermedad crónica metabólica con una incidencia creciente y una elevada morbilidad y mortalidad [1, 2]. La obesidad también se asocia a elevados costes económicos para los sistemas de salud y representa un problema de salud pública a nivel mundial [3, 4]. En concreto, en 2016, 650 millones de adultos eran obesos [5]; entre ellos, entre el 10% y el 30% presentaban trastornos metabólicos [6], entre los que se encuentran la diabetes tipo 2 (T2DM), la hipertensión (HT), las cardiopatías, el colesterol total y los triglicéridos elevados, los accidentes cerebrovasculares, la apnea obstructiva del sueño, la enfermedad del hígado graso no alcohólico y ciertos tipos de cáncer [7-9]. En este sentido, la cirugía bariátrica (BS) es un tratamiento bien reconocido para la OB que tiene resultados superiores para los pacientes que no han podido mantener la pérdida de peso por métodos no quirúrgicos [10]. En todo el mundo, los procedimientos bariátricos más realizados son el bypass gástrico en Y de Roux (RYGB) y la gastrectomía en manga (SG) por vía laparoscópica, donde ambos procedimientos abarcan casi el 80% de todas las operaciones bariátricas del mundo [11]. La BS induce y mantiene una pérdida de peso sustancial a través de diversos mecanismos, como la restricción calórica (debido a los cambios anatómicos inducidos por la cirugía en el tracto gastrointestinal), el aumento de la termogénesis inducida por las comidas, la modulación de los circuitos neuronales hipotalámicos (que regulan el equilibrio energético y/o la regulación del apetito), las modificaciones en las vías de señalización intestino-cerebro y los cambios en el gusto, las preferencias alimentarias y los patrones de conducta alimentaria [12-15]. Todos estos cambios se acompañan de una reducción de las complicaciones metabólicas, especialmente de la T2DM [16], y de una rápida alteración del estado inflamatorio, debida en parte a la modulación de la producción de citoquinas tras la BS, que puede ir acompañada también de una mejora de otras complicaciones metabólicas [17]. En este contexto, la inflamación es una respuesta inmunitaria protectora, en la que la función inmunitaria innata depende del reconocimiento de patrones moleculares asociados a patógenos, que proceden de patógenos invasores, y de patrones moleculares asociados al peligro, siendo inducidos de forma endógena por receptores de reconocimiento de patrones (PRRs) codificados en la línea germinal. La activación de los PRRs induce la producción de interferón-α, interferón-β y citoquinas proinflamatorias [18]. Estas respuestas están mediadas por la maquinaria del inflamasoma, un complejo multiproteico intracelular que desempeña un papel central en la inmunidad innata y es responsable de la activación de las respuestas inflamatorias. Este complejo está compuesto por algunas familias de PRRs, incluyendo el dominio de unión a nucleótidos, proteínas que contienen repeticiones ricas en leucina [receptores similares al dominio de unión a nucleótidos (NOD)], y por los receptores ausentes en el melanoma 2-like (AIM2- like) [19]. Los receptores tipo NOD (NLRs) y los receptores similares a AIM pueden oligomerizarse y actuar como andamios activadores de la caspasa-1, por ejemplo, activando la familia proinflamatoria de la interleucina (IL)-1 mediante la producción de IL-18 e IL-1β [20]. Sorprendentemente, la actividad de estos componentes del inflamasoma está regulada por diferentes proteínas reguladoras, vías metabólicas y un eje regulador mitocondrial [21]. En consecuencia, la activación de estos componentes del inflamasoma conduce a la secreción de diversas citoquinas inflamatorias y a la activación de receptores clave en las células inmunitarias, induciendo así la activación de cascadas inflamatorias, que pueden conducir en algunos casos a alteraciones del ciclo celular y a daños en el DNA [20, 21]. Diferentes componentes de la maquinaria del inflamasoma se han relacionado con diversos trastornos autoinflamatorios, autoinmunes y metabólicos, como la aterosclerosis, la T2DM y la OB [22]. Algunos estudios han descubierto que los componentes del inflamasoma están desregulados en varios tejidos/células, incluidas las células mononucleares de sangre periférica (PBMC), y que estos cambios pueden estar asociados al desarrollo de la aterosclerosis, las enfermedades autoinmunes (por ejemplo, la esclerosis múltiple) y las neurodegenerativas (por ejemplo, el Alzheimer o el Parkinson) [20]. En la misma línea, se han descrito alteraciones en algunos componentes específicos del inflamasoma en la OB, pero sus funciones específicas aún no se conocen bien [23]. Sin embargo, hasta donde sabemos, aún no se han descrito completamente los cambios en los componentes del inflamasoma inducidos por la pérdida de peso aguda relacionada con el BS. Curiosamente, se ha informado de que las citocinas inducidas por el inflamasoma promueven las enfermedades inflamatorias relacionadas con la OB tras la estimulación de los metabolitos de la dieta alta en grasas y los cambios en el tejido adiposo que se producen durante el aumento de peso [24], lo que sugiere que la modulación del inflamasoma debido a la pérdida de peso puede estar asociada a una mejora de las comorbilidades relacionadas con la OB. Como se ha mencionado anteriormente, la OB es la causa más común de resistencia a la insulina; sin embargo, la diabetes manifiesta no se desarrolla en la mayoría de los individuos obesos, ya que pueden preservar la normoglucemia mediante una mayor secreción de insulina, lo que da lugar a una hiperinsulinemia compensatoria [25]. Por el contrario, en los individuos susceptibles, la hiperinsulinemia compensatoria no se puede mantener, por lo que se desarrolla prediabetes y T2DM [6]. En este sentido, es fundamental comprender los mecanismos moleculares implicados en la aparición de la OB y sus comorbilidades asociadas. De hecho, se ha demostrado que el desarrollo y la progresión de la OB y otras comorbilidades asociadas, incluida la resistencia a la insulina [26], están estrechamente relacionados con la desregulación de factores metabólicos clave, especialmente los implicados en el control celular del balance energético (por ejemplo, la insulina, la leptina, los transportadores de glucosa, etc.), que a su vez están controlados por mecanismos reguladores celulares específicos, como los microARN. Los microRNAs (o miRNAs) son RNA cortos no codificantes (20-22 nucleótidos) que actúan a nivel post-transcripcional para regular la expresión génica [27]. Se están acumulando pruebas de que los miRNAs circulantes pueden actuar como factores endocrinos, ya que son liberados a la circulación por muchos tejidos [28]. De hecho, los miRNAs parecen servir como mensajeros endocrinos y paracrinos que facilitan la comunicación entre las células donantes y las dianas, ejerciendo así potencialmente importantes funciones en la integración de los órganos metabólicos [28]. Por estas razones, se ha postulado que los miRNAs circulantes podrían ejercer papeles cruciales en el desarrollo y la progresión de la OB y sus comorbilidades relacionadas. Estudios anteriores y dispersos han informado de que algunos miRNAs están desregulados en muestras de plasma de sujetos obesos, incluyendo miR-142-3p, miR- 221 o miR-222 [29-34]. Sin embargo, estos resultados son inconsistentes entre los estudios y no pueden ser comparados debido a varios factores, incluyendo las diferentes características de la cohorte (obesos, obesos mórbidos, diabéticos), los diseños de los estudios (paralelo, transversal) o los métodos de detección (TaqMan, SYBR Green, micro o macro arrays, secuenciación de ARN pequeños o miRNA). Por lo tanto, el patrón completo de desregulación de los miRNAs en la condición de obesidad está aún por definir de forma fiable. De hecho, estudios anteriores han demostrado que las tecnologías de alto rendimiento pueden representar una de las herramientas más precisas y fiables para la cuantificación de miRNAs [35]. Lamentablemente, aunque varios de los estudios mencionados anteriormente han informado de la desregulación de diferentes miRNAs en la condición de OB, hasta donde sabemos no hay estudios hasta la fecha que describan todo el miRNome utilizando arrays específicos de microRNAs. Por último, como se ha mencionado anteriormente, la OB promueve la aparición de múltiples problemas/defectos patológicos, que provocan graves alteraciones de la homeostasis del organismo (por ejemplo, desregulación de las hormonas, factores de crecimiento e inflamatorios, etc.) que con frecuencia promueven o influyen en el desarrollo de las patologías graves, incluyendo diferentes tumores/cánceres relacionados con el sistema endocrino [36]. En este contexto, es importante mencionar que el Cáncer de Próstata (PCa) se ha convertido en el tipo de tumor más frecuente entre los hombres y representa un grave problema de salud en todo el mundo [36]. Una limitación clave en el manejo del PCa es que la prueba de cribado de referencia se basa en los niveles plasmáticos del antígeno prostático específico (PSA), un biomarcador que presenta profundos inconvenientes, especialmente en la llamada "zona gris" (definida como un rango de PSA de 3-10 ng/mL) [37]. De hecho, la prueba del PSA muestra una baja especificidad, ya que hay múltiples factores que pueden aumentar los niveles de PSA sin indicar necesariamente la presencia de un tumor, como la hiperplasia prostática benigna o las afecciones inflamatorias. Además, la prueba del PSA no es capaz de distinguir con precisión los tumores clínicamente relevantes de los casos indolentes [38]. Por estas razones, el análisis anatomopatológico de las biopsias de próstata, que representan una técnica altamente invasiva, sigue siendo necesario para diagnosticar adecuadamente el PCa en la actualidad. Por lo tanto, existe una importante necesidad clínica insatisfecha de identificar y validar nuevos biomarcadores diagnósticos de PCa no invasivos, fiables y específicos, idealmente desde un punto de vista personalizado y que muestren un potencial pronóstico y/o terapéutico. En este sentido, los miRNAs son atractivos candidatos a biomarcadores ya que pueden ser extraídos de forma reproducible de una amplia gama de muestras biológica y son generalmente estables y resistentes a diversas condiciones de almacenamiento [28, 39]. De hecho, estudios recientes han sugerido una relación entre los miRNAs circulantes y la presencia y el resultado del PCa [40-42]. En concreto, estudios recientes han identificado algunos miRNAs presentes de forma diferencial en muestras de plasma de pacientes con PCa; sin embargo, sólo algunos de ellos parecen derivar específicamente de los tejidos del PCa [43-45]. Lamentablemente, aunque varios estudios han identificado algunos miRNAs putativos específicos del PCa (por ejemplo, miR-141 [46], miR

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

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

Oncogenic Role of Secreted Engrailed Homeobox 2 (EN2) in Prostate Cancer

Engrailed variant-2 (EN2) has been suggested as a potential diagnostic biomarker; however, its presence and functional role in prostate cancer (PCa) cells is still controversial or unknown. Here, we analyzed 1) the expression/secretion profile of EN2 in five independent samples cohorts from PCa patients and controls (prostate tissues and/or urine) to determine its utility as a PCa biomarker; and 2) the functional role of EN2 in normal (RWPE1) and tumor (LNCaP/22Rv1/PC3) prostate cells to explore its potential value as therapeutic target. EN2 was overexpressed in our two cohorts of PCa tissues compared to control and in tumor cell lines compared with normal-like prostate cells. This profile was corroborated in silico in three independent data sets [The Cancer Genome Atlas(TCGA)/Memorial Sloan Kettering Cancer Center (MSKCC)/Grasso]. Consistently, urine EN2 levels were elevated and enabled discrimination between PCa and control patients. EN2 treatment increased cell proliferation in LNCaP/22Rv1/PC3 cells, migration in RWPE1/PC3 cells, and PSA secretion in LNCaP cells. These effects were associated, at least in the androgen-sensitive LNCaP cells, with increased AKT and androgen-receptor phosphorylation levels and with modulation of key cancer-associated genes. Consistently, EN2 treatment also regulated androgen-receptor activity (full-length and splicing variants) in androgen-sensitive 22Rv1 cells. Altogether, this study demonstrates the potential utility of EN2 as a non-invasive diagnostic biomarker for PCa and provides novel and valuable information to further investigate its putative utility to develop new therapeutic tools in PCa

Global overview of the management of acute cholecystitis during the COVID-19 pandemic (CHOLECOVID study)

Background: This study provides a global overview of the management of patients with acute cholecystitis during the initial phase of the COVID-19 pandemic. Methods: CHOLECOVID is an international, multicentre, observational comparative study of patients admitted to hospital with acute cholecystitis during the COVID-19 pandemic. Data on management were collected for a 2-month study interval coincident with the WHO declaration of the SARS-CoV-2 pandemic and compared with an equivalent pre-pandemic time interval. Mediation analysis examined the influence of SARS-COV-2 infection on 30-day mortality. Results: This study collected data on 9783 patients with acute cholecystitis admitted to 247 hospitals across the world. The pandemic was associated with reduced availability of surgical workforce and operating facilities globally, a significant shift to worse severity of disease, and increased use of conservative management. There was a reduction (both absolute and proportionate) in the number of patients undergoing cholecystectomy from 3095 patients (56.2 per cent) pre-pandemic to 1998 patients (46.2 per cent) during the pandemic but there was no difference in 30-day all-cause mortality after cholecystectomy comparing the pre-pandemic interval with the pandemic (13 patients (0.4 per cent) pre-pandemic to 13 patients (0.6 per cent) pandemic; P = 0.355). In mediation analysis, an admission with acute cholecystitis during the pandemic was associated with a non-significant increased risk of death (OR 1.29, 95 per cent c.i. 0.93 to 1.79, P = 0.121). Conclusion: CHOLECOVID provides a unique overview of the treatment of patients with cholecystitis across the globe during the first months of the SARS-CoV-2 pandemic. The study highlights the need for system resilience in retention of elective surgical activity. Cholecystectomy was associated with a low risk of mortality and deferral of treatment results in an increase in avoidable morbidity that represents the non-COVID cost of this pandemic

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Global overview of the management of acute cholecystitis during the COVID-19 pandemic (CHOLECOVID study)

Background: This study provides a global overview of the management of patients with acute cholecystitis during the initial phase of the COVID-19 pandemic. Methods: CHOLECOVID is an international, multicentre, observational comparative study of patients admitted to hospital with acute cholecystitis during the COVID-19 pandemic. Data on management were collected for a 2-month study interval coincident with the WHO declaration of the SARS-CoV-2 pandemic and compared with an equivalent pre-pandemic time interval. Mediation analysis examined the influence of SARS-COV-2 infection on 30-day mortality. Results: This study collected data on 9783 patients with acute cholecystitis admitted to 247 hospitals across the world. The pandemic was associated with reduced availability of surgical workforce and operating facilities globally, a significant shift to worse severity of disease, and increased use of conservative management. There was a reduction (both absolute and proportionate) in the number of patients undergoing cholecystectomy from 3095 patients (56.2 per cent) pre-pandemic to 1998 patients (46.2 per cent) during the pandemic but there was no difference in 30-day all-cause mortality after cholecystectomy comparing the pre-pandemic interval with the pandemic (13 patients (0.4 per cent) pre-pandemic to 13 patients (0.6 per cent) pandemic; P = 0.355). In mediation analysis, an admission with acute cholecystitis during the pandemic was associated with a non-significant increased risk of death (OR 1.29, 95 per cent c.i. 0.93 to 1.79, P = 0.121). Conclusion: CHOLECOVID provides a unique overview of the treatment of patients with cholecystitis across the globe during the first months of the SARS-CoV-2 pandemic. The study highlights the need for system resilience in retention of elective surgical activity. Cholecystectomy was associated with a low risk of mortality and deferral of treatment results in an increase in avoidable morbidity that represents the non-COVID cost of this pandemic