Papel de la fosfatasa SHP-1 en el cáncer de próstata resistente a la castración

El cáncer de próstata (CP) es la segunda causa de muerte por cáncer en hombres en el mundo occidental. Inicialmente, el desarrollo de este tipo de cáncer es dependiente de andrógenos, por ello, el tratamiento convencional consiste en la deprivación androgénica. Aunque se consigue la regresión del tumor, en un alto porcentaje de pacientes progresa hacia formas más agresivas de la enfermedad denominadas cáncer de próstata resistente a la castración (CRPC). Entre los mecanismos por los cuales se desarrolla la resistencia se ha descrito la activación del receptor de andrógenos (AR) a través de vías independientes de andrógenos, el cual se activará, translocará al núcleo y aumentará la expresión de genes relacionados con la supervivencia y proliferación de las células cancerosas. Uno de los principales reguladores negativos de estas vías es la proteína SHP-1, una fosfatasa encargada de desfosforilar los residuos de tirosina de multitud de proteínas de estas vías. En este trabajo nos planteamos conocer mejor el papel de esta fosfatasa en el desarrollo del CRPC y su función sobre el AR. Para ello, silenciaremos SHP-1 mediante la técnica CRISPR/Cas9, una técnica reciente de edición genética, y determinaremos su papel en la fosforilación y actividad del AR, y como afecta a la supervivencia y proliferación de dos líneas celulares del cáncer de próstata con diferente sensibilidad a los tratamientos hormonales. Este estudio tendrá la finalidad de obtener nueva información sobre el papel de esta fosfatasa en fases más agresivas del cáncer con el fin de encontrar nuevas dianas terapéuticas que mejoren los tratamientos actuales de esta enfermedad y, por tanto, la supervivencia de estos paciente

Estudio de la regulación de la histona metilasa EZH2 por la proteína tirosina fosfatasa SHP-1 en cáncer de próstata

El cáncer de próstata (CP) es uno de los tumores malignos más frecuentes en varones de países desarrollados. La deprivación androgénica es uno de los tratamientos más efectivos para el cáncer de próstata metastásico; sin embargo, con el tiempo, el tumor progresa a una fase independiente de éstos para la que no existe tratamiento. El estudio de los mecanismos moleculares implicados en esta fase de la enfermedad permitirá el desarrollo de nuevas terapias encaminadas a solventar este problema. En este trabajo estudiamos el papel de SHP-1, una tirosina fosfatasa que actúa como regulador negativo de vías de señalización relacionadas con la progresión tumoral, como posible regulador indirecto de EZH2 y su interacción con el receptor de andrógenos (AR) a través de la vía PI3K/AKT para la activación transcripcional de genes diana de este. En primer lugar, se determinará si existe un complejo proteico formado por SHP-1, AKT, EZH2 Y AR mediante ensayos de captura y unión o “pull down” y co-inmunoprecipitación. Posteriormente, el estudio de esta fosfatasa se realizará mediante su silenciamiento por RNA de interferencia con el objetivo de analizar posibles cambios en la actividad de AKT, EZH2 y AR. Además, se realizarán ensayos de proliferación celular con un inhibidor de EZH2 (GSK126) con el fin de determinar si SHP-1 modula su efecto. La existencia de cambios en la actividad de las proteínas o de la efectividad del inhibidor constituyen un paso previo para investigar las consecuencias funcionales de SHP-1 en la progresión del cáncer de próstat

Caracterización de clones estables de SHP-1 en líneas celulares de cáncer de próstata

El cáncer de próstata (CP) es uno de los problemas sanitarios más relevantes de nuestra sociedad actual. A pesar de disponer de diversos tratamientos para abordarla, el avance de la enfermedad hacia un estadio refractario conocido como cáncer de próstata resistente a la castración (CRPC) hace que hoy en día siga teniendo altos índices de mortalidad. Muchos de los mecanismos moleculares involucrados en su desarrollo están relacionados con cambios en el receptor de andrógenos (AR), que gobierna el desarrollo normal y patológico de la próstata. Entre estos mecanismos encontramos cambios en la expresión y función de coreguladores de AR, así como la activación del receptor por vías independientes de andrógenos. En estos mecanismos participan una serie de proteínas, entre las que encontramos un complejo de señalización conformado por AKT-EZH2-AR. La evidencia científica respalda el papel de EZH2 como activador independiente de AR, función promovida al ser fosforilada por AKT. Debido a la función reguladora que la fosfatasa SHP-1 podría tener sobre AKT, hipotetizamos sobre el papel que esta podría tener sobre el complejo y, por tanto, sobre el avance de la enfermedad hacia CRPC. Para estudiarlo, obtuvimos mediante CRISPR/Cas9 clones estables de distintas líneas celulares de CP con el gen de SHP-1 silenciado para así poder determinar si existían o no diferencias en una serie de parámetros celulares. El silenciamiento promovió diversas modificaciones en el modelo sensible a andrógenos que no se observaron en los modelos de CRPC, entre las que encontramos una menor sensibilidad a GSK126, una disminución del gen regulado por AR, FKBP5, y la adquisición de un fenotipo neuroendocrino. Los datos apoyan la hipótesis de que SHP-1 podría ser un posible regulador de este complejo de señalización, aunque futuros estudios son necesarios para determinar con exactitud de qué manera

Modulation of guanosine triphosphatase activity of G proteins by arachidonic acid in rat Leydig cell membranes

Previous results from our group have indicated that arachidonic acid decrease cAMP production through a modification of heterotrimericGproteins.\ud In the present study, we have characterized the high affinity GTPase activity present in Leydig cell membranes and its regulation by fatty acids. The high-affinity GTPase activity, measured as [g32P] GTP hydrolysis rate, was both time and protein concentration dependent. Arachidonic acid elicited a dose-dependent inhibition of enzyme activity with an IC50 5 26.7 6 1.1 mM. The existence of only two double bonds in linoleic acid is reflected by a decrease in its inhibitory activity (IC5053462.3mM). Saturated fatty\ud acids showed no effect at this level. The kinetic analysis as interpreted by Lineweaver-Burk plots, indicated that 50 mM arachidonic acid had no effect on the apparent affinity for GTP, but resulted in a 40% decreases in the maximal velocity of the reaction. Arachidonic acid modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 59-lipoxygenase, cyclooxygenase, or epoxygenase P-450. The addition of arachidonic acid to pertussis toxin-treated membranes had no effect on the enzyme activity, indicating that arachidonic acid does not modify the GTPase activity present in Gas protein. However, ADP-ribosylation with cholera toxin followed by arachidonic acid treatment led to a further 40% inhibition\ud when compared with cholera toxin treatment alone. These results allowed us to postulate that arachidonic acid inhibits the GTPase activity of Gi protein family. To further analyze the mechanism of\ud arachidonic acid inhibition of GTPase activity, the effect of arachidonic acid on the [35S]GTPgS binding was studied. No effect of this fatty acid on GTP binding was found. Combining our previous results\ud with those found here, we can conclude that arachidonic acid maintains Gi proteins in their active state, which in turn inhibit adenylate cyclase and results in decrease cAMP levels

The Role of Epigenetics in the Progression of Clear Cell Renal Cell Carcinoma and the Basis for Future Epigenetic Treatments

Clear cell renal cell carcinoma (ccRCC) is curable when diagnosed at an early stage, but when disease is non-confined it is the urologic cancer with worst prognosis. Antiangiogenic treatment and immune checkpoint inhibition therapy constitute a very promising combined therapy for advanced and metastatic disease. Many exploratory studies have identified epigenetic markers based on DNA methylation, histone modification, and ncRNA expression that epigenetically regulate gene expression in ccRCC. Additionally, epigenetic modifiers genes have been proposed as promising biomarkers for ccRCC. We review and discuss the current understanding of how epigenetic changes determine the main molecular pathways of ccRCC initiation and progression, and also its clinical implications. Despite the extensive research performed, candidate epigenetic biomarkers are not used in clinical practice for several reasons. However, the accumulated body of evidence of developing epigenetically-based biomarkers will likely allow the identification of ccRCC at a higher risk of progression. That will facilitate the establishment of firmer therapeutic decisions in a changing landscape and also monitor active surveillance in the aging population. What is more, a better knowledge of the activities of chromatin modifiers may serve to develop new therapeutic opportunities. Interesting clinical trials on epigenetic treatments for ccRCC associated with well established antiangiogenic treatments and immune checkpoint inhibitors are revisited.Instituto de Salud Carlos IIIComisión Europe

Claudin-3 Loss of Expression Is a Prognostic Marker in Castration-Resistant Prostate Cancer

Castration-resistant prostate cancer (CRPC) development is the foremost concern after treatment of patients with high risk with locally advanced or metastatic prostate cancer. Androgen receptor (AR) is the main driver of CRPC development, through its interaction with epigenetic modifier genes, placing epigenetics modifications in the forefront of CRPC development. Comparing the DNA methylation and expression profile of androgen-sensitive and -refractory prostate cancer cells, we describe the epigenetic silencing of claudin-3 (CLDN3) in AR positive cells resistant to androgen deprivation (LNCaP-abl). CLDN3 silencing was associated with DNA methylation, loss of histone acetylation and H3K27 methylation, and was re-expressed by the combined treatment with the epigenetic modulators Aza and SAHA. From a functional point of view, CLDN3 loss was associated with increased cellular invasion. Immunohistochemical analysis showed decreased CLDN3 expression in samples from CRPC patients. Interestingly, CLDN3 expression was significantly decreased in samples from patients with high total Gleason score (>= 8) and locally advanced tumors. Finally, CLDN3 loss of expression was associated with worse disease-free survival and time to clinical progression. In conclusion, our findings strongly indicate that epigenetic silencing of CLDN3 is a common event in CRPC that could be useful as a molecular marker for the prognosis of prostate cancer patients and to discriminate aggressive from indolent prostate tumors

A DNA hypermethylation profile reveals new potential biomarkers for the evaluation of prognosis in urothelial bladder cancer

DNA hypermethylation has emerged as a molecular biomarker for the evaluation of cancer diagnosis and prognosis. We define a methylation signature of bladder cancer and evaluate whether this profile assesses prognosis of patients. Genome-wide methylation analysis was performed on 70 tumor and 10 normal bladder samples. Hypermethylation status of 1505 CpGs present in the promoter region of 807 genes was studied. Thirty-three genes were significantly hypermethylated in >= 10% of the tumors. Three clusters of patients were characterized by their DNA methylation profile, one at higher risk of dead of disease (p = 0.0012). Association between cluster distribution and stage (p = 0.02) or grade (p = 0.02) was demonstrated. Hypermethylation of JAK3 and absence of hypermethylation of EYA4, GAT6, and SOX1 were associated with low-grade non-invasive disease. On the other hand, in high-grade invasive disease hypermethylation of CSPG2, HOXA11, HOXA9, HS3ST2, SOX1, and TWIST1 was associated with muscle invasiveness. A panel of hypermethylated genes including APC, CSPG2, EPHA5, EYA4, HOXA9, IPF1, ISL1, JAK3, PITX2, SOX1, and TWIST1 predicted cancer-specific survival and SOX1 (HR = 3.46), PITX2 (HR = 4.17), CSPG2 (HR = 5.35), and JAK3 hypermethylation (HR = 0.19) did so independently. Silencing of genes by hypermethylation is a common event in bladder cancer and could be used to develop diagnostic and prognostic markers. Combined hypermethylation of SOX1, PITX2, or CSPG2 signals patients at higher risk of death from bladder cancer

Epigenetic Regulation of Gfi1 in Endocrine-Related Cancers: A Role Regulating Tumor Growth

Prostate and breast cancer constitute the most common cancers among men and women worldwide. The aging population is one of the main risk factors for prostate and breast cancer development and accumulating studies link aging with epigenetic changes. Growth factor independence-1 (Gfi1) is a transcriptional repressor with an important role in human malignancies, including leukemia, colorectal carcinoma, and lung cancer, but its role in prostate and breast cancer is unknown. We have found that Gfi1 epigenetic silencing is a common event in prostate and breast cancer. Gfi1 re-expression in prostate and breast cancer cell lines displaying Gfi1 epigenetic silencing decreases cell proliferation, reduced colony formation density, and tumor growth in nude mice xenografts. In addition, we found that Gfi1 repress alpha 1-anti-trypsin (AAT) and alpha 1-anti-chymotrypsin (ACT) expression, two genes with important functions in cancer development, suggesting that Gfi1 silencing promotes tumor growth by increasing AAT and ACT expression in our system. Finally, Gfi1 epigenetic silencing could be a promising biomarker for prostate cancer progression because it is associated with shorter disease-free survival. In conclusion, our findings strongly indicate that Gfi1 epigenetic silencing in prostate and breast cancer could be a crucial step in the development of these two-well characterized endocrine related tumors.Instituto de Salud Carlos II

Autocrine regulation of human prostate carcinoma cell proliferation by somatostatin through the modulation of the SH2 domain containing protein tyrosine phosphatase (SHP)-1

The present study was intended to gain additional information on the growth regulation of prostate by somatostatin\ud (SRIF) and the intracellular events involved. The humanprostate adenocarcinoma cell lines PC-3 and LNCaP produce SRIF and express subtypes 2 and 5 of SRIF receptors. The secretion\ud of SRIF is related to the proliferative status of these cells; an inverse relationship exists between cell proliferation and the amount of secreted SRIF. Moreover, the growth of PC-3 cells\ud is inhibited by SRIF overexpression and increased by blockage of endogenous SRIF. Coincident with the increase in SRIF\ud secretion, the activity and levels of theSH2domain containing protein tyrosine phosphatase (SHP)-1, present in PC-3 cells are augmented, but the effect can be partially prevented by neutralization of secreted endogenously SRIF. The activity of SHP-1 is also stimulated by the SRIF analog RC160. Overexpression of SHP-1 induces inhibition of PC-3 cell growth.\ud SHP-1 is also present in normal prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and well differentiated adenocarcinoma. In contrast, no signal is detected in poorly differentiated prostate cancer. These findings demonstrate that SRIF inhibits PC-3 and LNCaP cell proliferation through an autocrine/paracrine SRIF loop. This effect could be mediated by activation of the tyrosine phosphatase SHP-1 detected in these cells as well as in human prostate and prostate cancer.Fundación para la Investigación en UrologíaMinisterio de Asuntos Exteriore

Effects of acute nicotine and mecamylamine administration on somatostatin concentration and binding in the rat brain

Since nicotine and somatostin have regulatory effects on locomotor activity it was of interest to determine whether the receptors for somatostin are modulated by the cholinergic nicotine-like effects. An i.v. dose of 0.3 mg/kg nicotine induced an increase in the concentrations of somatostatin-like immunoreactivity at 4 min in the parietal cortex and at 15 min in the hippocampus. These changes were associated with a significant increase in the total number of specific somatostatin receptors in the parietal cortex at 15 min and in the hippocampus at 30 min following injection. To determine if the above mentioned changes are related to the nicotine activation of central nicotine-like acetylcholine receptors, a cholinergic nicotinic blocking agent, mecamylamine, was administered before the nicotine injection. Pretreatment with mecamylamine (5.0 mg/kg i.v.) prevented the nicotine-induced changes in somatostatin level and binding in both brain areas. Mecamylamine alone had no observable effect on the somatostatinergic system. These results suggest that the somatostatinergic system can be regulated by nicotine-like acetylcholine receptors and may be involved in some of the behavioral central effects of nicotine