Papel del sistema ubiquitina proteasoma en la respuesta a terapias antimitóticas y su significado clínico en cáncer

Taxanes are established in the treatment of metastatic breast cancer and early breast cancer as potent chemotherapy agents. However, their therapeutic usefulness s limited by de-novo refractoriness or acquired resistance. Despite all efforts, the mechanisms of resistance have not yet been fully understood. The aim of this thesis was to find new predictive markers of recurrence in breast cancer and new predictive markers of response to taxanes treatments We investigated the role of FBXW7 and their substrates MCL1 and PLK1 in regulating the apoptotic response to paclitaxel treatment in breast cancer cells and their expression in breast cancer tissues. FBXW7 is a component of SCF (complex of SKP1, CUL1 and F-box-protein)-type ubiquitin ligases that targets several oncoproteins for ubiquitination and degradation by the proteasome. FBXW7 regulates cellular apoptosis by targetingMCL1 for ubiquitination. Recently, we identified PLK1 as a new substrate of FBXW7 modulating the intra-S-phase DNA-damage checkpoint. Paclitaxel-sensitive MDA-MB- 468 and a paclitaxel-resistant MDA-MB-468R subclone were used to study the role of FBXW7 and substrates in paclitaxel-induced apoptosis. Our results showed that forced expression of FBXW7 or downregulation of MCL1 or PLK1 restored sensitivity to paclitaxel in MDA-MB-468R cells. By contrary, FBXW7-silenced MDA-MB-468 cells became resistant to paclitaxel. The expression of FBXW7 and substrates were studied in 296 invasive carcinomas by immunohistochemistry and disease-free survival was analyzed in a subset of patients treated with paclitaxel. In breast cancer tissues, loss of FBXW7 correlated with adverse prognosis markers and loss of FBXW7 and MCL1 or PLK1 accumulation were associated with diminished disease-free survival in paclitaxeltreated patients. According to our results FBXW7, MCL1 and PLK1 may be relevant predictive markers of tumor progression and response to paclitaxel treatment. Also we demonstrated that FBXW7 regulates the response to paclitaxel by targeting MCL1 and PLK1 in breast cancer cells and thus targeting these substrates may be a valuable adjunct for paclitaxel treatment. Herein, due the importance of MCL1 in mitotic cell death we used obatoclax in combination with paclitaxel as a mechanism of sensitization to antimitotic drugs in breast cancer MDA-MB-231 cells. We also highlight the importance of the order of the administration of both drugs and its effect on the response to these treatments. Our results showed that the administration of obatoclax enables to restore the sensitivity to paclitaxel in MDA-MB-231 cells. Finally, we analyzed the epithelial–mesenchymal transition (EMT) as a mechanism which is involved in the development of acquired resistance to paclitaxel in breast cancer. EMT is a a complex process by which epithelial cells transit to mesenchymal phenotype in order to adquire enhanced migratory capacity, invasiveness, increased resitance to apoptosis and production of extracellular matrix components. Herein, we studied the role of EDIL3, a αvβ3 integrin ligand, as a novel regulator of EMT which contributes to paclitaxel resistance in breast cancer cells. The analysis of the whole genome expression profiles of breast cancer cells allowed us to identify that EDIL3 and mesenquimal makers were hightly expressed in paclitaxel resistant MDA-MB-468R. Silencing EDIL3 gene discreased mesenchymal markers and restored sensitivity to paclitaxel in MDA-MB-231 cells. Disrupting the ligation of EDIL3 to integrins via RGD-blocking by cilengitide showed that EDIL3 is abundantly secreted by paclitaxel resistant MDA-MB-468R and MDA-MB-231. The administration of cilengitide in combination with paclitaxel resulted in the increased of sensitivity to paclitaxel in MDA-MB-231 breast cancer cells. Our findings demonstrated that EDIL3 may be contribute to the development of paclitaxel resistance and the administration of paclitaxel combinated with cilengitide as a new and effective therapeutic strategy for the treatment of breast cancer.Los taxanos se utilizan en el tratamiento del cáncer de mama metastásico y en las etapas tempranas de la enfermedad como potentes agentes quimioterápicos. Sin embargo, su utilidad terapéutica está limitada debido al desarrollo de resistencias de novo o adquiridas. A pesar de todos los esfuerzos, los mecanismos de resistencia no se conocen completamente. El objetivo de esta tesis es encontrar nuevos marcadores predictivos de recurrencia en cáncer de mama y de respuesta al tratamiento con taxanos. Hemos investigado el papel de FBXW7 y de sus sustratos MCL1 y PLK1 en la regulación de la respuesta apoptótica en el tratamiento con paclitaxel en células tumorales de mama así como su expresión en carcinomas de mama. FBXW7 es un componente del complejo de ubiquitina ligasa SCF (SKP1, CUL1 y F-box) que se encarga de ubiquitinar a numerosas oncoproteínas para su degradación por el proteasoma. FBXW7 regula la apoptosis celular mediante la ubiquitinación de MCL1. Recientemente, hemos identificado que PLK1 es un nuevo sustrato de FBXW7 que modula el punto de control del daño al ADN en la fase S. Hemos utilizado las células sensibles a paclitaxel MDA-MB-468 y resistentes a paclitaxel MDA-MB-468R para estudiar el papel de FBXW7 y sus sustratos en la apoptosis inducida por paclitaxel. Nuestros resultados demuestran que la sobreexpresión de FBXW7 o el silenciamiento de MCL1 o PLK1 restablecen la sensibilidad a paclitaxel en las células MDA-MB- 468R. Por el contrario, las células MDA-MB-468 silenciadas para FBXW7 se convierten en resistentes a paclitaxel. Asimismo, estudiamos la expresión de FBXW7 y sus sustratos mediante inmunohistoquímica en 296 carcinomas invasivos y analizamos el tiempo libre de enfermedad en un subconjunto de pacientes tratados con paclitaxel. En los tejidos de cáncer de mama, la pérdida de FBXW7 correlacionó con marcadores de pronóstico adverso y la pérdida de FBXW7 y la acumulación de MCL1 o PLK1 se asoció con disminución del tiempo libre de enfermedad en los pacientes tratados con paclitaxel. De acuerdo con nuestros resultados, FBXW7, MCL1 y PLK1 podrían ser marcadores predictivos relevantes de la progresión tumoral y la respuesta al tratamiento con paclitaxel. Además demostramos que FBXW7 regula la respuesta a paclitaxel a través del control de MCL1 y PLK1 en células de cáncer de mama y por lo tanto estos sustratos podrían tener un valor añadido como dinas farmacológicas en el tratamiento con paclitaxel. En este sentido, dada la importancia de MCL1 en la muerte celular en mitosis hemos analizado el tratamiento de obatoclax en combinación con paclitaxel como mecanismo de sensibilización a fármacos antimitóticos en las células MDA-MB- 231. Además resaltamos la importancia del orden en la administración de ambos fármacos y su efecto en la respuesta al tratamiento. Nuestros resultados demuestran que la administración de obatoclax permite restablecer la sensibilidad al tratamiento con paclitaxel en las células MDA-MB-231. Finalmente, hemos analizado el fenómeno de transición epitelio-mesénquima (EMT) como un mecanismo involucrado en el desarrollo de la resistencia adquirida a paclitaxel en cáncer de mama. EMT es un proceso complejo en el que las células epiteliales cambian a un fenotipo mesenquimal para adquirir mayor capacidad migratoria, invasividad, incremento de la resistencia a apoptosis y producción de componentes de la matriz extracelular. En este contexto, hemos estudiado el papel de EDIL3, un ligando de integrina αvβ3, como un novedoso regulador de EMT que contribuye a la resistencia a paclitaxel en células tumorales de mama. El análisis de la expresión génica en células de cáncer de mama nos ha permitido identificar que EDIL3 y marcadores mesenquimales se encontraban sobreexpresados en las células resistentes a paclitaxel MDA-MB-468R. El silenciamiento de EDIL3 disminuyó la expresión de marcadores esenquimales y restableció la sensibilidad a paclitaxel en las células MDA-MB-231. La perturbación de la unión de EDIL3 a la integrina mediante el bloqueo del motivo de unión a integrina RGD a través de la administración de cilengitide mostró que EDIL3 se secretaba de manera más abundante en las células resistentes a paclitaxel. La administración de cilengitide en combinación con paclitaxel resultó en un incremento de la sensibilidad a paclitaxel en las células MDA-MB-231. Nuestros hallazgos demostraron que EDIL3 podría contribuir al desarrollo de resistencia a paclitaxel y que la administración de paclitaxel en combinación con cilengitide podría suponer una estrategia terapéutica efectiva en el tratamiento de cáncer de mama

Obatoclax and Paclitaxel Synergistically Induce Apoptosis and Overcome Paclitaxel Resistance in Urothelial Cancer Cells

Paclitaxel is a treatment option for advanced or metastatic bladder cancer after the failure of first-line cisplatin and gemcitabine, although resistance limits its clinical benefits. Mcl-1 is an anti-apoptotic protein that promotes resistance to paclitaxel in different tumors. Obatoclax, a BH3 mimetic of the Bcl-2 family of proteins, antagonizes Mcl-1 and hence may reverse paclitaxel resistance in Mcl-1-overexpressing tumors. In this study, paclitaxel-sensitive 5637 and -resistant HT1197 bladder cancer cells were treated with paclitaxel, obatoclax, or combinations of both. Apoptosis, cell cycle, and autophagy were measured by Western blot, flow cytometry, and fluorescence microscopy. Moreover, Mcl-1 expression was analyzed by immunohistochemistry in bladder carcinoma tissues. Our results confirmed that paclitaxel alone induced Mcl-1 downregulation and apoptosis in 5637, but not in HT1197 cells; however, combinations of obatoclax and paclitaxel sensitized HT1197 cells to the treatment. In obatoclax-treated 5637 and obatoclax + paclitaxel-treated HT1197 cells, the blockade of the autophagic flux correlated with apoptosis and was associated with caspase-dependent cleavage of beclin-1. Obatoclax alone delayed the cell cycle in 5637, but not in HT1197 cells, whereas combinations of both retarded the cell cycle and reduced mitotic slippage. In conclusion, obatoclax sensitizes HT1197 cells to paclitaxel-induced apoptosis through the blockade of the autophagic flux and effects on the cell cycle. Furthermore, Mcl-1 is overexpressed in many invasive bladder carcinomas, and it is related to tumor progression, so Mcl-1 expression may be of predictive value in bladder cancer.España, Sistema Público Andaluz Biobanco y ISCIII-Red de Biobancos PT17/0015/004

Loss of FBXW7 and accumulation of MCL1 and PLK1 promote paclitaxel resistance in breast cancer

FBXW7 is a component of SCF (complex of SKP1, CUL1 and F-box-protein)-type ubiquitin ligases that targets several oncoproteins for ubiquitination and degradation by the proteasome. FBXW7 regulates cellular apoptosis by targeting MCL1 for ubiquitination. Recently, we identified PLK1 as a new substrate of FBXW7 modulating the intra-S-phase DNA-damage checkpoint. Taxanes are frequently used in breast cancer treatments, but the acquisition of resistance makes these treatments ineffective. We investigated the role of FBXW7 and their substrates MCL1 and PLK1 in regulating the apoptotic response to paclitaxel treatment in breast cancer cells and their expression in breast cancer tissues. Paclitaxel-sensitive MDA-MB-468 and a paclitaxel-resistant MDA-MB-468R subclone were used to study the role of FBXW7 and substrates in paclitaxel-induced apoptosis. Forced expression of FBXW7 or downregulation of MCL1 or PLK1 restored sensitivity to paclitaxel in MDA-MB-468R cells. By contrary, FBXW7-silenced MDA-MB-468 cells became resistant to paclitaxel. The expression of FBXW7 and substrates were studied in 296 invasive carcinomas by immunohistochemistry and disease-free survival was analyzed in a subset of patients treated with paclitaxel. In breast cancer tissues, loss of FBXW7 correlated with adverse prognosis markers and loss of FBXW7 and MCL1 or PLK1 accumulation were associated with diminished disease-free survival in paclitaxel-treated patients. We conclude that FBXW7 regulates the response to paclitaxel by targeting MCL1 and PLK1 in breast cancer cells and thus targeting these substrates may be a valuable adjunct for paclitaxel treatment. Also, FBXW7, MCL1 and PLK1 may be relevant predictive markers of tumor progression and response to paclitaxel treatment.España, Ministerio de Economía y Competitividad SAF2014- 53799-C2-1/2-REspaña, Consejería de Salud AI-0025-2015España, Consejería de Innovación Ciencia y Empresa P10- CTS-624

La dispensación y uso seguro de medicamentos

No aplicaLa Organización mundial de la salud OMS define a la farmacovigilancia como la ciencia y las actividades relativas a la detección, evaluación, comprensión y prevención de los efectos adversos de los medicamentos o cualquier otro problema de salud relacionado con ellos, actualmente esto permite que su venta y uso sean controlados en las instituciones prestadoras de servicios de salud (IPS) al igual que en los establecimientos independientes como farmacias y droguerías de esta manera se garantiza la seguridad de los usuarios que adquieren los medicamentos y dispositivos médicos y además se reduce el riesgo de que se presente una reacción adversa a un medicamento (RAM). El objetivo de este trabajo es diseñar e implementar el programa de farmacovigilancia para el establecimiento farmacéutico minorista GRUPAL-SALUD, el cual ha venido presentando problemas en el proceso de dispensación de medicamentos, por lo cual se vio la necesidad de implementa una estrategia de capacitación para todo el personal farmacéutico que está actualmente laborando en pro de que se pueda realizar este proceso minimizando y eliminado los errores que se puedan cometer en el momento de hacer la entrega al usuario además de los problemas relacionados con medicamentos (PRM) que se puedan presentar. Para complementar con la capacitación se elabora e implementa el manual para el manejo adecuado de medicamentos Look-Alike Sound-Alike LASA en donde se involucran aquellos medicamentos que cuentan con una similitud visual o fonética con otro. Palabras clave: farmacovigilancia, dispensación, antibióticos, medicamentos LASA, reacción adversa a un medicamento.The World Health Organization WHO defines pharmacovigilance as the science and activities related to the detection, evaluation, understanding and prevention of adverse effects of drugs or any other health problem related to them, currently this allows their sale and use to be controlled in the institutions providing health services (IPS) as well as in independent establishments such as pharmacies and drugstores, thus ensuring the safety of users who purchase drugs and medical devices and also reducing the risk of an adverse drug reaction (ADR). The objective of this work is to design and implement the pharmacovigilance program for the retail pharmaceutical establishment FARMASALUD, which has been presenting problems in the process of dispensing drugs, so it was necessary to implement a training strategy for all the pharmaceutical staff that is currently working in order to be able to perform this process minimizing and eliminating errors that can be made at the time of delivery to the user in addition to drug-related problems (DRP) that may occur. To complement the training, a manual for the proper handling of Look-Alike Sound-Alike LASA drugs is developed and implemented, which involves those drugs that have a visual or phonetic similarity with another drug. Key words: pharmacovigilance, dispensing, antibiotics, LASA drugs, adverse drug reaction

Addressing Cervical Cancer Disparities in Texas: Expansion of a Community-Based Prevention initiative For Medically Underserved Populations

Although cervical cancer is preventable, significant disparities exist in access to screening and prevention services. In medically underserved areas (MUAs) of Texas, these rates are 55% higher compared to the remainder of the US. In 2019, we expanded a multicomponent, comprehensive program to improve cervical cancer prevention in partnership with 13 clinics and mobile vans in MUAs of Texas. Our multicomponent intervention program consists of community education and patient navigation coupled with a training/mentoring program for local medical providers to perform diagnostic procedures and treatment for patients with abnormal screening results. Hands-on training courses to learn these skills are coupled with biweekly telementoring conferences using Project ECHO® (Extension for Community Healthcare Outcomes). This program was implemented in 2015 and expanded to other MUAs in Texas in 2019. From March 2019 to August 2022, 75,842 individuals were educated about cervical cancer screening and HPV vaccination. A total of 44,781 women underwent screening for cervical cancer, and 2,216 underwent colposcopy and 264 underwent LEEP. High-grade cervical dysplasia was diagnosed in 658 individuals and invasive cervical cancer in 33 individuals. We trained 22 providers to perform colposcopy and/or LEEP. In addition, 78 Project ECHO telementoring sessions were held with an average of 42 attendees per session, with 72 individual patient cases discussed. Our comprehensive community-based prevention initiative for medically underserved populations has led to a significant number of individuals undergoing cervical cancer screening in MUAs, as well as improved access to colposcopy and LEEP services

Utilization of mechanical power and associations with clinical outcomes in brain injured patients: a secondary analysis of the extubation strategies in neuro-intensive care unit patients and associations with outcome (ENIO) trial

Background: There is insufficient evidence to guide ventilatory targets in acute brain injury (ABI). Recent studies have shown associations between mechanical power (MP) and mortality in critical care populations. We aimed to describe MP in ventilated patients with ABI, and evaluate associations between MP and clinical outcomes. Methods: In this preplanned, secondary analysis of a prospective, multi-center, observational cohort study (ENIO, NCT03400904), we included adult patients with ABI (Glasgow Coma Scale ≤ 12 before intubation) who required mechanical ventilation (MV) ≥ 24 h. Using multivariable log binomial regressions, we separately assessed associations between MP on hospital day (HD)1, HD3, HD7 and clinical outcomes: hospital mortality, need for reintubation, tracheostomy placement, and development of acute respiratory distress syndrome (ARDS). Results: We included 1217 patients (mean age 51.2 years [SD 18.1], 66% male, mean body mass index [BMI] 26.3 [SD 5.18]) hospitalized at 62 intensive care units in 18 countries. Hospital mortality was 11% (n = 139), 44% (n = 536) were extubated by HD7 of which 20% (107/536) required reintubation, 28% (n = 340) underwent tracheostomy placement, and 9% (n = 114) developed ARDS. The median MP on HD1, HD3, and HD7 was 11.9 J/min [IQR 9.2-15.1], 13 J/min [IQR 10-17], and 14 J/min [IQR 11-20], respectively. MP was overall higher in patients with ARDS, especially those with higher ARDS severity. After controlling for same-day pressure of arterial oxygen/fraction of inspired oxygen (P/F ratio), BMI, and neurological severity, MP at HD1, HD3, and HD7 was independently associated with hospital mortality, reintubation and tracheostomy placement. The adjusted relative risk (aRR) was greater at higher MP, and strongest for: mortality on HD1 (compared to the HD1 median MP 11.9 J/min, aRR at 17 J/min was 1.22, 95% CI 1.14-1.30) and HD3 (1.38, 95% CI 1.23-1.53), reintubation on HD1 (1.64; 95% CI 1.57-1.72), and tracheostomy on HD7 (1.53; 95%CI 1.18-1.99). MP was associated with the development of moderate-severe ARDS on HD1 (2.07; 95% CI 1.56-2.78) and HD3 (1.76; 95% CI 1.41-2.22). Conclusions: Exposure to high MP during the first week of MV is associated with poor clinical outcomes in ABI, independent of P/F ratio and neurological severity. Potential benefits of optimizing ventilator settings to limit MP warrant further investigation

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke — the second leading cause of death worldwide — were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries

Pagos provisionales del impuesto sobre la renta y la aplicación de la reducción

La generación de efectivo es uno de los principales objetivos de las empresas. La mayoría de sus actividades van encaminadas a provocar, un flujo adecuado de dinero que permita, entre otra cosas, financiar la operación y cumplir con sus obligaciones ante la autoridad

Pagos provisionales del impuesto sobre la renta y la aplicación de la reducción

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