Diseño y caracterización de inhibidores de la fascina para el bloqueo de la invasión y la metástasis en cáncer colorrectal.

El adenocarcinoma serrado (ACS) es un subtipo histológico de cáncer colorrectal reconocido por la Organización Mundial de la Salud (OMS) y que representa alrededor del 9% de los carcinomas colorrectales. En comparación con el carcinoma convencional (CC), el ACS tiene peor pronóstico y supervivencia. Estas características son debidas a que el ACS presenta un débil desarrollo de la respuesta inmune, un frente invasor muy activo y no responde de manera eficiente a terapias dirigidas anti-EGFR debido a la alta frecuencia de mutaciones en KRAS/BRAF. No obstante, recientemente, se han identificado posibles dianas moleculares que podrían favorecer la búsqueda de nuevos tratamientos específicos para el ACS. En esta línea se ha identificado la fascina, una proteína que se encuentra sobre-expresada en este subtipo histológico de cáncer colorrectal y que es una proteína clave en el reordenamiento del citoesqueleto de actina ya que participa en la formación de filopodios y lamelipodios implicados en el movimiento celular. Debido al papel fundamental de la fascina en el empaquetamiento de haces de actina, esta proteína se convierte en una diana para el bloqueo de la invasión tumoral. Objetivos: El principal objetivo de de la presente Tesis ha sido buscar nuevos inhibidores contra la fascina en cáncer colorrectal mediante cribado farmacológico. Posteriormente, hemos evaluado, mediante técnicas in vitro, el efecto de estos inhibidores en la proliferación, movilidad e invasión en líneas celulares de cáncer colorrectal con distintos niveles de expresión basal de la fascina. Igualmente, se analizó la capacidad anti-invasora y anti-metastásica de los inhibidores en modelos in vivo de pez cebra y murinos. Resultados: Se realizó una búsqueda, mediante modelado molecular, de potenciales compuestos anti-fascina de una librería de 9591 compuestos (que incluían 2037 aprobados por la Food and Drug Administration (FDA). Fruto de estudios in silico, identificamos varios compuestos como potenciales inhibidores de la fascina entre los que caben destacar: la imipramina (IMIP), el raltegravir (RAL) y el monastrol (MON). Previamente, el grupo del Dr. HY Huang descubrió el compuesto G2 mediante screening farmacológico y lo caracterizó como un inhibidor de la fascina capaz de bloquear la migración celular, la invasión y la metástasis en cáncer de mama. . Así pues, en esta Tesis, hemos testado las propiedades anti-fascina del compuesto G2, la IMIP, el RAL y el MON mediante ensayos in vitro e in vivo en los que se ha empleado la migrastatina (MGS) como fármaco control anti-fascina. En todos los casos, se observó un efecto antimigratorio y anti-invasor al tratar líneas celulares de cáncer de colon como HCT-116 y DLD-1 con los compuestos anti-fascina en los ensayos in vitro. También se ha observado la capacidad anti-metastásica de los inhibidores in vivo (pez cebra) y hemos estudiado el efecto que tienen la IMIP y el RAL en un modelo murino de tumor primario. Conclusiones: El cribado de fármacos juega un papel fundamental en la investigación traslacional. Hemos encontrado nuevas propiedades antimigratorias y anti-invasoras de los compuestos G2, IMIP, RAL y MON en células de cáncer colorrectal. Además, existe un efecto dosis dependiente de estos compuestos que ha sido demostrado mediante un modelo in vivo de invasión y metástasis tumoral en pez cebra. Los resultados del modelo murino obtenidos en la presente Tesis son aún preliminares y no fueron significativos en comparación con los grupos control. Ello apunta a la necesidad de evaluar en el futuro, el efecto sinérgico de quimioterapéuticos junto con los inhibidores de la fascina descritos en este trabajo. Los presentes resultados abalan el reposicionamiento de fármacos en la investigación clínica, así como la utilidad terapéutica de estos fármacos para el tratamiento de las células tumorales metastásicas más agresivas, como las que se desarrollan en el ACS.Medicin

The FDA-Approved Antiviral Raltegravir Inhibits Fascin1-Dependent Invasion of Colorectal Tumor Cells In Vitro and In Vivo

Background: Fascin1 is the key actin-bundling protein involved in cancer invasion and metastasis whose expression is associated with bad prognosis in tumor from different origins. Methods: In the present study, virtual screening (VS) was performed for the search of Fascin1 inhibitors and RAL, an FDA-approved inhibitor of human immunodeficiency virus-1 (HIV-1) integrase, was identified as a potential Fascin1 inhibitor. Biophysical techniques including nuclear magnetic resonance (NMR) and differential scanning fluorimetry (DSF) were carried out in order to confirm RAL as a Fascin1 blocker. The effect of RAL on actin-bundling activity Fascin1 was assessed by transmission electron microscopy (TEM), immunofluorescence, migration, and invasion assays on two human colorectal adenocarcinoma cell lines: HCT-116 and DLD-1. In addition, the anti-metastatic potential of RAL was in vivo evaluated by using the zebrafish animal model. Results: NMR and DSF confirmed in silico predictions and TEM demonstrated the RAL-induced disorganization of the actin structure compared to control conditions. The protrusion of lamellipodia in cancer cell line overexpressing Fascin1 (HCT-116) was abolished in the presence of this drug. By following the addition of RAL, migration of HCT-116 and DLD-1 cell lines was significantly inhibited. Finally, using endogenous and exogenous models of Fascin1 expression, the invasive capacity of colorectal tumor cells was notably impaired in the presence of RAL in vivo assays; without undesirable cytotoxic effects. Conclusion: The current data show the in vitro and in vivo efficacy of the antiretroviral drug RAL in inhibiting human colorectal cancer cells invasion and metastasis in a Fascin1-dependent manner

The FDA-Approved Antiviral Raltegravir Inhibits Fascin1-Dependent Invasion of Colorectal Tumor Cells In Vitro and In Vivo

Simple Summary: Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Serrated adenocarcinoma (SAC) has been recently recognized by the WHO as a histological CRC with bad prognosis. Consistent with previous evidence, our group identified Fascin1 as a protein directly related to the invasiveness of tumor cells, overexpressed and positively correlated with worse survival in various carcinomas, including SAC. Therefore, Fascin1 has emerged as an ideal target for cancer treatment. In the present study, virtual screening has been carried out from a library of 9591 compounds, thus identifying the FDA-approved anti-retroviral raltegravir (RAL) as a potential Fascin1 blocker. In vitro and in vivo results show that RAL exhibits Fascin1-binding activity and Fascin1-dependent anti-invasive and anti-metastatic properties against CRC cells both in vitro and in vivo. Abstract: Background: Fascin1 is the key actin-bundling protein involved in cancer invasion and metastasis whose expression is associated with bad prognosis in tumor from different origins. Methods: In the present study, virtual screening (VS) was performed for the search of Fascin1 inhibitors and RAL, an FDA-approved inhibitor of human immunodeficiency virus-1 (HIV-1) integrase, was identified as a potential Fascin1 inhibitor. Biophysical techniques including nuclear magnetic resonance (NMR) and differential scanning fluorimetry (DSF) were carried out in order to confirm RAL as a Fascin1 blocker. The effect of RAL on actin-bundling activity Fascin1 was assessed by transmission electron microscopy (TEM), immunofluorescence, migration, and invasion assays on two human colorectal adenocarcinoma cell lines: HCT-116 and DLD-1. In addition, the anti-metastatic potential of RAL was in vivo evaluated by using the zebrafish animal model. Results: NMR and DSF confirmed in silico predictions and TEM demonstrated the RAL-induced disorganization of the actin structure compared to control conditions. The protrusion of lamellipodia in cancer cell line overexpressing Fascin1 (HCT-116) was abolished in the presence of this drug. By following the addition of RAL, migration of HCT-116 and DLD-1 cell lines was significantly inhibited. Finally, using endogenous and exogenous models of Fascin1 expression, the invasive capacity of colorectal tumor cells was notably impaired in the presence of RAL in vivo assays; without undesirable cytotoxic effects. Conclusion: The current data show the in vitro and in vivo efficacy of the antiretroviral drug RAL in inhibiting human colorectal cancer cells invasion and metastasis in a Fascin1-dependent manner.Instituto de Salud Carlos III Spanish GovernmentEuropean Commission PI15/00626 PI18/00144European Commission's Corbel Program PID-3630 2334 2428Spanish Ministry of Economy and Competitiveness MINECO CTQ2017-87974-RFundación Seneca 20988/PI/18 20646/JLI/18UCAM FPI05-UCAM/17Junta Provincial Murcia Predoctoral Asociación Española contra el Cáncer (AECC) PRDMU1900

Biology and Therapeutic Targets of Colorectal Serrated Adenocarcinoma; Clues for a Histologically Based Treatment against an Aggressive Tumor

Serrated adenocarcinoma (SAC) is a tumor recognized by the WHO as a histological subtype accounting for around 9% of colorectal carcinomas. Compared to conventional carcinomas, SACs are characterized by a worse prognosis, weak development of the immune response, an active invasive front and a frequent resistance to targeted therapy due to a high occurrence of KRAS or BRAF mutation. Nonetheless, several high-throughput studies have recently been carried out unveiling the biology of this cancer and identifying potential molecular targets, favoring a future histologically based treatment. This review revises the current evidence, aiming to propose potential molecular targets and specific treatments for this aggressive tumor

Low Performance of a Clinical-Genetic Model in the Estimation of Time in Therapeutic Range in Acenocoumarol-Adherent Patients with Nonvalvular Atrial Fibrillation: The Quality of Anticoagulation Challenge

Background. Anticoagulation with vitamin K antagonists continues to be a challenging task given the difficulty of achieving a correct time in therapeutic range (TTR). The SAMeTT2R2 score has been proposed to identify patients that will be good responders. In this study we aimed to analyse clinical and genetic factors involved in a correct level of anticoagulation in patients with atrial fibrillation and thereby potentially improve the diagnostic performance of SAMeTT2R2 score. Methods. We prospectively included 212 consecutive patients with nonvalvular atrial fibrillation under treatment with acenocoumarol for at least 6 months that were attended in a cardiology outpatient clinic and were categorized as adherent to medication. We carried out a multivariate regression analysis to detect the independent predictive factors of good control. In all patients VKORC1, CYP2C9⁎2, CYP2C9⁎3, and MIR133A2 genotyping was performed. Results. A total of 128 (60.4%) patients presented TTR <70% (average TTR = 63.2). We identified body mass index (OR 0.94, 95%CI 0.89-0.99, p=0.032) and regular vitamin K intake (OR 0.53, 95%CI 0.28-0.99, p= 0.046) as independent predictors of poor anticoagulation control. The discriminatory power of a clinical-genetic model derived from our cohort was significantly better compared to the SAMeTT2R2 score (C-statistic 0.658 versus 0.524, p<0.001). Conclusions. In our study the SAMeTT2R2 score revealed a poor ability in the prediction of TTR. Besides SAMeTT2R2, body mass index and possibly vitamin K intake should be taken into account when deciding the optimal anticoagulation strategy. The information provided by the identified genotypes was marginal

Low Performance of a Clinical-Genetic Model in the Estimation of Time in Therapeutic Range in Acenocoumarol-Adherent Patients with Nonvalvular Atrial Fibrillation: The Quality of Anticoagulation Challenge

Background. Anticoagulation with vitamin K antagonists continues to be a challenging task given the difficulty of achieving a correct time in therapeutic range (TTR). The SAMeTT2R2 score has been proposed to identify patients that will be good responders. In this study we aimed to analyse clinical and genetic factors involved in a correct level of anticoagulation in patients with atrial fibrillation and thereby potentially improve the diagnostic performance of SAMeTT2R2 score. Methods. We prospectively included 212 consecutive patients with nonvalvular atrial fibrillation under treatment with acenocoumarol for at least 6 months that were attended in a cardiology outpatient clinic and were categorized as adherent to medication. We carried out a multivariate regression analysis to detect the independent predictive factors of good control. In all patients VKORC1, CYP2C9⁎2, CYP2C9⁎3, and MIR133A2 genotyping was performed. Results. A total of 128 (60.4%) patients presented TTR <70% (average TTR = 63.2). We identified body mass index (OR 0.94, 95%CI 0.89-0.99, p=0.032) and regular vitamin K intake (OR 0.53, 95%CI 0.28-0.99, p= 0.046) as independent predictors of poor anticoagulation control. The discriminatory power of a clinical-genetic model derived from our cohort was significantly better compared to the SAMeTT2R2 score (C-statistic 0.658 versus 0.524, p<0.001). Conclusions. In our study the SAMeTT2R2 score revealed a poor ability in the prediction of TTR. Besides SAMeTT2R2, body mass index and possibly vitamin K intake should be taken into account when deciding the optimal anticoagulation strategy. The information provided by the identified genotypes was marginal

ColPortal, an integrative multiomic platform for analysing epigenetic interactions in colorectal cancer

Abstract Colorectal cancer (CRC) is the third leading cause of cancer mortality worldwide. Different pathological pathways and molecular drivers have been described and some of the associated markers are used to select effective anti-neoplastic therapy. More recent evidence points to a causal role of microbiota and altered microRNA expression in CRC carcinogenesis, but their relationship with pathological drivers or molecular phenotypes is not clearly established. Joint analysis of clinical and omics data can help clarify such relations. We present ColPortal, a platform that integrates transcriptomic, microtranscriptomic, methylomic and microbiota data of patients with colorectal cancer. ColPortal also includes detailed information of histological features and digital histological slides from the study cases, since histology is a morphological manifestation of a complex molecular change. The current cohort consists of Caucasian patients from Europe. For each patient, demographic information, location, histology, tumor staging, tissue prognostic factors, molecular biomarker status and clinical outcomes are integrated with omics data. ColPortal allows one to perform multiomics analyses for groups of patients selected by their clinical data