Implication of VHL, ERK5, and HIF-1alpha in clear cell renal cell carcinoma: Molecular basis

Objectives: To determine the expression status of several proteins related to VHL gene function and its relationship with common clinicopathological parameters. Material and methods: Observational, analytical, cross-sectional study with 50 patients diagnosed with clear cell renal cell carcinoma. The study analyzed VHL mutations and hypermethylation as well as protein expression of VHL, CA-IX, HIF-1alpha, VEGF, ERK1/2, and ERK5, relating them to clinical variables. A bivariate and multivariate descriptive logistical regression analysis was performed, using the presence of metastasis at diagnosis as dependent variable. Results: The study identified 13 (26%) VHL mutations related to nuclear grade (P = 0.036). VHL hypermethylation was found in 20% of cases. VHL expression was associated with the presence of mutations (P = 0.013), and the absence of expression was associated with nuclear grade and the presence of metastasis (P<0.05). HIF-1alpha was negative in only 5 cases. Vascular endothelial growth factor (VEGF) was positive in 31 of 47 cases and was associated with Fuhrman nuclear grade, presence of metastasis, and stage (P<0.05). ERK5 expression was increased in 58% of cases and associated with the presence of metastasis and more advanced stages (P<0.05). In the logistic regression analysis, the only variable remaining in the model was VEGF expression (P = 0.014). Conclusions: VEGF has prognostic value in clear cell renal cell carcinoma, and ERK5 may be a new prognostic marker in this type of tumor owing to its relationship with metastasis and more advanced stages

MKP1 mediates chemosensitizer effects of E1a in response to cisplatin in non-small cell lung carcinoma cells

The adenoviral gene E1a is known to enhance the antitumor effect of cisplatin, one of the cornerstones of the current cancer chemotherapy. Here we study the molecular basis of E1a mediated sensitivity to cisplatin in an experimental model of Non-small cell lung cancer. Our data show how E1a blocks the induction of autophagy triggered by cisplatin and promotes the apoptotic response in resistant cells. Interestingly, at the molecular level, we present evidences showing how the phosphatase MKP1 is a major determinant of cisplatin sensitivity and its upregulation is strictly required for the induction of chemosensitivity mediated by E1a. Indeed, E1a is almost unable to promote sensitivity in H460, in which the high expression of MKP1 remains unaffected by E1a. However, in resistant cell as H1299, H23 or H661, which display low levels of MKP1, E1a expression promotes a dramatic increase in the amount of MKP1 correlating with cisplatin sensitivity. Furthermore, effective knock down of MKP1 in H1299 E1a expressing cells restores resistance to a similar extent than parental cells. stores resistance to a similar extent than parental cells. In summary, the present work reinforce the critical role of MKP1 in the cellular response to cisplatin highlighting the importance of this phosphatase in future gene therapy approach based on E1a gene

Exploiting the potential of autophagy in cisplatin therapy: a new strategy to overcome resistance

Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach

Exploiting the potential of autophagy in cisplatin therapy: A new strategy to overcome resistance

Resistance to cisplatin is a major challenge in the current cancer therapy. In order to explore new therapeutic strategies to cisplatin resistance, we evaluated, in a model of lung cancer (H1299 and H460 cell lines), the nature of the pathways leading to cell death. We observed that H1299 displayed a natural resistance to cisplatin due to an inability to trigger an apoptotic response that correlates with the induction of autophagy. However, pharmacological and genetic approaches showed how autophagy was a mechanism associated to cell death rather than to resistance. Indeed, pro-autophagic stimuli such as mTOR or Akt inhibition mediate cell death in both cell lines to a similar extent. We next evaluated the response to a novel platinum compound, monoplatin, able to promote cell death in an exclusive autophagy-dependent manner. In this case, no differences were observed between both cell lines. Furthermore, in response to monoplatin, two molecular hallmarks of cisplatin response (p53 and MAPKs) were not implicated, indicating the ability of this pro-autophagic compound to overcome cisplatin resistance. In summary, our data highlight how induction of autophagy could be used in cisplatin resistant tumours and an alternative treatment for p53 mutated patient in a synthetic lethally approach.This work was supported by grants from Fundación Leticia Castillejo Castillo and Ministerio de Economía y Competitividad (grant SAF2012-30862 to RSP and grant CTQ2011-24434 to FAJ). RSP Research Institute, and the work carried out in his laboratory receive support from the European Community through the regional development funding program (FEDER). JGC received funding from the Regional Ministry of Education and Science of Castilla–La Mancha (FPI-JCCM) and from Fundación Leticia Castillejo Castillo. MCC and RSP have a contract from the INCRECYT progra

ERK5/BMK1 is a novel target of the tumor suppressor VHL: implication in clear cell renal carcinoma

Hi ha quatre pàgines de material suplementari sense numeracióExtracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Estudio de la vía de señalización mediada por VHL y MAPKs en el carcinoma renal de células claras esporádico. Implicaciones clínicas pronósticas

El carcinoma de células renales (CCR) representa aproximadamente el 90% de las patologías renales malignas en adultos, siendo la variedad de células claras la más frecuente. Dentro de los factores genéticos que pueden afectar a este tipo de tumores destacan las alteraciones en el gen supresor tumoral VHL. Este gen codifica una proteína denominada VHL la cual desarrolla su función como supresor tumporal provocando la degradación de HIF-1a, factor de transcripción cuya misión es la transactivación de ciertos genes cuyo producto proteico aumenta la disponibilidad de oxígeno (VGEF, PDGF, EPO, etc.), o que están relacionados con la regulación del pH (CA-IX) cuando el oxígeno ambiental es limitado. En presencia de una actividad normal de VHL y con condiciones ambientales de oxígeno normales, HIF-1a se degrada y no ejerce su función. Sin embargo, en condiciones de hipoxia, VHL no ejerce su función ubiquitín ligasa, HIF-1a no es degradado y se activa la transcripción de sus genes diana. A su vez se ha descrito el hecho de que la proteína VHL controle tanto a HIF1-a como a ERK5, proteína perteneciente al grupo de las MAPK que se ha relacionado con vascularización y, a igual que las proteínas ERK 1,2, con proliferación celular. Todo ello hace interesante evaluar la implicación de la ruta de señalización mediada por VHL y MAPK en el CCR. -Determinar la existencia de mutaciones y alteraciones epigenéticas (metilación del promotor) en el gen VHL en tejido renal tumoral de pacientes con carcinoma renal esporádico de célula clara y caracterizar, por western blot y/o inmunohistoquímica, el estado de expresión de las distintas proteínas que forman parte de la vía metabólica de señalización desencadenada por VHL, como CA-IX, HIF-1a y VEGF y de las MAPK implicadas en proliferación como ERK5 y ERK1/2. -Valorar la utilidad de la determinación de la expresión de la proteína CA-IX como biomarcador en el carcinoma de células renales y cuál de los métodos para la detección de su expresión es más útil. -Analizar si la expresión de VHL se asocia con la expresión de las otras proteínas implicadas en su vía de señalización. -Determinar el valor pronóstico y diagnóstico de la existencia de mutaciones y metilación en el gen VHL, así como de la expresión de VHL y de las proteínas de las vías de señalización analizadas. Estudio descriptivo observacional, analítico y transversal sobre 76 pacientes diagnosticados de CCR y tratados quirúrgicamente en el Servicio de Urología del Complejo Hospitalario Universitario de Albacete entre los años 2006 y 2011. Se obtuvieron muestras de tejido tumoral y parénquima renal de los especímenes quirúrgicos. A dichas muestras se les realizó un análisis de expresión de la proteína CA-IX por inmunohistoquímica, además se les extrajo proteína para realizar el análisis de expresión de la misma proteína mediante la técnica de western blot. De esos 76 pacientes se seleccionaron los 50 pacientes diagnosticados de CCRCC a los que se les extrajo ADN para realizar el estudio genético y epigenético de VHL. Para el estudio de las mutaciones del gen se amplificaron los 3 exones que conforman el gen VHL mediante la técnica de PCR y se sometieron secuenciación automática. Para el estudio epigenético se analizó el análisis de hipermetilación de tres amplicones que componen el mayor número de sitios CpG del promotor de VHL. También se extrajo proteína a las muestras tumorales y de parénquima renal sano de esos 50 pacientes, para realizar el análisis de expresión de las proteínas ERK5 y ERK1,2 por western blot; y se realizó un análisis de expresión de las proteínas VHL, HIF-1a y VEGF por inmunohistoquímica. La edad media de los pacientes fue de 64,1 años, afectando un 68,4% a hombres y un 31,6% mujeres. La cirugía realizada mayoritariamente consistió en nefrectomía radical (84,2%). Respecto a la clínica, el hallazgo incidental de los tumores fue la causa más frecuente (55,9%). El tipo histopatológico más frecuente fue el de células claras encontrado en 50 casos (65,8%), seguido del cromófobo (17,1%). En un 40,3% de los casos el grado nuclear de Fuhrman fue el 2. La mayoría de los tumores se situaron por debajo del estadio pT2 siendo el pT1b el más frecuente encontrado en un 34,2% de los casos. El 27,6% de los casos presentaron metástasis a distancia en el momento del diagnóstico. Con respecto al estadio TNM, el estadio I fue el mas frecuente con 39 casos de los 76 (51,3%). En cuanto a la expresión de CA-IX, todos los CCRCC fueron positivos para CA-IX. Podemos considerar ambas técnicas como válidas para el diagnóstico de CCRCC. No se encontró relación entre la expresión de CA-IX y el grado de Fuhrman ni con el estadio pT. Sí se comprobó que en estadios más altos así como en presencia de metástasis había un mayor porcentaje de casos que expresaban la proteína CA-IX. Con respecto al estado mutacional del gen VHL, se detectaron 13 mutaciones (26%) en los 50 pacientes de CCRCC distribuidas en los 3 exones pero con mayor porcentaje en el exón 1 (53,8%) y siendo la deleción la más frecuente (38,5%). Se encontró un mayor número de mutaciones en el riñón derecho. Con respecto al grado de Fuhrman, las mutaciones se dieron con mayor frecuencia en los grados 1 y 2 (p= 0,036). No se observó que la existencia de mutación se correlacionara con estadio pT, pN, pM ni TNM. De 46 pacientes analizados con CCRCC se encontraron 9 pacientes con hipermetilación en el promotor del gen VHL. Apareció hipermetilación en mayor porcentaje en los tumores que presentaron síntomas. Se observó una tendencia lineal entre la presencia de metástasis a distancia y una mayor proporción de casos conhipermetilación del gen VHL. Más del 50% de los casos expresaron la proteína VHL por inmunohistoquímica, los casos que no expresaron VHL correspondieron a tumores con Fuhrman más altos (14,30% de grado 3 y 54,50% de grado 4). La expresión de HIF-1a fue positiva en la gran mayoría de los casos (89,4%), así como la expresión de ERK1,2 (94%). En cuanto a VEGF y ERK5, su expresión correlacionó con estadios TNM avanzados y con la presencia de metástasis. Además la expresión de VEGF se relacionó también con grados de Fuhrman altos. CONCLUSIONES CA-IX es un marcador específico del carcinoma renal de células claras, técnicas como la inmunohistoquímica y de western blot son válidas para su detección pudiendo servir de apoyo a las técnicas diagnósticas de CCRCC basadas en la morfología tumoral. El CCRCC presenta alteraciones en el gen VHL, por mutación y/o metilación, en porcentajes de 26% y 19,6%. Dichas alteraciones no correlacionan con la expresión de la proteína por inmunohistoquímica. La expresión de VHL no se correlacionó con la expresión de las otras proteínas implicadas en la vía de señalización, pero su ausencia se asocia a grados de Fuhrman altos, indicando peor pronóstico. En cuanto a HIF-1a, su diana directa, se expresa en un alto porcentaje en el CCRCC, lo que al igual que CA-IX lo haría buen biomarcador. Las proteínas VEGF y ERK5, pueden establecerse como marcador pronóstico. La expresión de ambas proteínas está correlacionada entre sí y se expresan, en mayor porcentaje, en grados de Furhman y estadios TNM más altos. Además, su expresión está directamente ligada a la presencia de metástasis a distancia

Spanish version of the short European Health Literacy Survey Questionnaire HLS-Q12 : Transcultural adaptation and psychometric properties

Health literacy has a direct impact on the health of populations. It is related to education, capacity for self-care, and management of health resources. The Health Literacy Survey Questionnaire HLS-Q12 is one of the reference instruments but has not yet been adapted to Spanish. The aims of the study were to cross-culturally adapt and evaluate the psychometric properties of the Spanish version of the HLS-Q12. Data was collected from June 2020 to March 2022. The sample consisted of 60 patients who initiated cancer treatment for the first time within a clinical trial. Double direct translation, back-translation, cognitive debriefing with a 10-patient sample, and an expert committee were used for cross-cultural adaptation. For validation of the HLS-Q12, a psychometric analysis was performed to assess feasibility, reliability, sensitivity to change and construct validity with other measures such as health-related quality of life, empowerment, and health needs. The HLS-Q12 is equivalent at the semantic, conceptual, and content level to the original version and its psychometric properties demonstrated good internal consistency with a Cronbach's alpha of 0.88 and a McDonald's omega of 0.91, a high degree of fit for the confirmatory factor analysis, and a statistically significant sensitivity to change (p = 0.025). Based on robust psychometric values, the Spanish version of HLS-Q12 was found to be a good cross-culturally adapted tool for collecting correct information on health literacy in cancer patients regardless of tumour type or stage. Although more studies are needed, this version of HLS-Q12 could be used in research for collecting data on the health literacy needs of Spanish-speaking patients

Prognostic Value of the VHL, HIF-1α, and VEGF Signaling Pathway and Associated MAPK (ERK1/2 and ERK5) Pathways in Clear-Cell Renal Cell Carcinoma. A Long-Term Study

Background: The prognostic value of molecular markers in renal cell carcinoma has been investigated in several studies. Although their value is still not confirmed, various proteins are important. We describe the effect on long-term survival of the status of the von Hippel-Lindau (VHL) hypoxia-inducible factor 1-α (HIF1-α) signaling pathway as well as associated mitogen-activated protein kinase (extracellular signal-regulated kinase [ERK]1/2 and ERK5). Patients and methods: A prospective, longitudinal cohort study was conducted with 50 patients diagnosed with clear-cell renal cell carcinoma to analyze VHL mutations and hypermethylation as well as VHL, HIF1-α, vascular endothelial growth factor (VEGF), ERK1/2, and ERK5 protein expression. Overall survival (OS), disease-specific survival (DSS), and progression- or recurrence-free survival (PFS) were analyzed using the Kaplan-Meier method. Mantel-Haenszel was used for comparisons, and Cox proportional risk models were also constructed. Results: Follow-up was 66.9 months. There were 23 (46.0%) deaths, of which 17 (73.9%) were caused by the tumor. Mean periods were 85.6 months for OS and 94.3 months for DSS. A total of 22 (44.0%) patients showed progression (PFS, 78.1 months). VHL expression (P = .045) and > 10% of HIF1-α expression (P = .034) were associated with greater OS. DSS was greater in patients without VHL methylation (P = .012), with > 10% HIF1-α expression (P = .037), or with ERK5 protein underexpression. Greater PFS was associated with absence of VHL methylation (P = .045), presence of VHL expression (P 10% (P = .04), and ERK5 protein underexpression (P = .011). The presence of VHL mutation and/or methylation and VEGF expression had no prognostic value. Fuhrman nuclear grade and Tumor, Node, Metastases (TNM) stage were the only variables that remained in the Cox model. Conclusion: The HIF1-α and ERK5 pathway has prognostic value. Patients with no VHL or HIF1-α expression and ERK5 overexpression had a worse course of disease. VHL or VEGF status had no prognostic value. Only TNM stage and Fuhrman nuclear grade remained in the Cox model and, therefore, are still essential in prognostic biomarker panels

Targeting basal-like breast tumors with bromodomain and extraterminal domain (BET) and polo-like kinase inhibitors

Metastatic triple negative breast cancer (TNBC) is an incurable disease with limited therapeutic options, and no targeted therapies available. Triple negative tumors and the basal-like genomic subtype, are both characterized by a high proliferation rate and an increase in cell division. In this context, protein kinases involved in the mitotic formation have a relevant role in this tumor subtype. Recently, Bromodomain and extraterminal domain (BET) inhibitors have shown to be active in this disease by modulating the expression of several transcription factors. In this article, by using an >in silico> approach, we identified genomic functions that can be inhibited pharmacologically in basal-like tumors. Functional annotation analyses identified >cell division> and >regulation of transcription> as upregulated functions. When focus on cell division, we identified the polo-like kinase 1 (PLK) as an upregulated kinase. The PLK inhibitor Volasertib had the strongest anti-proliferative effect compared with other inhibitors against mitotic kinases. Gene expression analyses demonstrated that the BET inhibitor JQ1 reduced the expression of kinases involved in cell division, and synergized with Volasertib in a panel of triple negative cell lines. Combination of both agents augmented cell death. Similarly, combination of both compounds reduced the expression of stem cell markers. Globally, this data demonstrates the synergistic interaction between BET and PLK inhibitors, paving the way for their future clinical development.Instituto de Salud Carlos III (PI13/01444), ACEPAIN; Diputación de Albacete and CRIS Cancer Foundation (to AO). BAE (Beca Ampliación de Estudios) to AO for his stay at Yale University. Ministry of Economy and Competitiveness of Spain (BFU2012–39151), the Instituto de Salud Carlos III through the Spanish Cancer Centers Network Program (RD12/0036/0003), the scientific foundation of the AECC and the CRIS Foundation (to AP). The work carried out in the EU laboratories receive support from the European Community through the regional development funding program (FEDER).Peer Reviewe