miR-200c-3p, miR-222-5p, and miR-512-3p Constitute a Biomarker Signature of Sorafenib Effectiveness in Advanced Hepatocellular Carcinoma

Background: Sorafenib constitutes a suitable treatment alternative for patients with advanced hepatocellular carcinoma (HCC) in whom atezolizumab + bevacizumab therapy is contraindicated. The aim of the study was the identification of a miRNA signature in liquid biopsy related to sorafenib response. Methods: miRNAs were profiled in hepatoblastoma HepG2 cells and tested in animal models, extracellular vesicles (EVs), and plasma from HCC patients. Results: Sorafenib altered the expression of 11 miRNAs in HepG2 cells. miR-200c-3p and miR-27a-3p exerted an anti-tumoral activity by decreasing cell migration and invasion, whereas miR-122-5p, miR-148b-3p, miR-194-5p, miR-222-5p, and miR-512-3p exerted pro-tumoral properties by increasing cell proliferation, migration, or invasion, or decreasing apoptosis. Sorafenib induced a change in EVs population with an increased number of larger EVs, and promoted an accumulation of miR-27a-3p, miR-122-5p, miR-148b-3p, miR-193b-3p, miR-194-5p, miR-200c-3p, and miR-375 into exosomes. In HCC patients, circulating miR-200c-3p baseline levels were associated with increased survival, whereas high levels of miR-222-5p and miR-512-3p after 1 month of sorafenib treatment were related to poor prognosis. The RNA sequencing revealed that miR-200c-3p was related to the regulation of cell growth and death, whereas miR-222-5p and miR-512-3p were related to metabolic control. Conclusions: The study showed that Sorafenib regulates a specific miRNA signature in which miR-200c-3p, miR-222-5p, and miR-512-3p bear prognostic value and contribute to treatment response.Instituto de Salud Carlos IIIEuropean Commission PI16/00090 PI19/01266Consejeria de Igualdad, Salud y Politicas Sociales PI-0198-2016Spanish Government FPU17/00026GEIVEX Mobility Fellowships 2020German Research Foundation (DFG)National Health and Medical Research Council (NHMRC) of Australia EST19/01091European CommissionInstituto de Salud Carlos IIIEuropean Commission PI15/00145 PI18/0358 PI18/00768AECC PI044031Instituto de Salud Carlos II

Extracellular vesicles secretion by Lenvatinib and Sorafenib in HepG2 cells and their effect on cell death and proliferation

Motivation: Sorafenib, which acts on the RAF / MEK / ERK pathways through the inhibition of Raf kinase and different tyrosine kinases (VEGFR2, PDGFR, c-Kit receptors), is the drug currently used as a first-line treatment in hepatocellular carcinomas of advanced stage. It has recently been shown that Lenvatinib, another multi-kinase inhibitor, also improves mean progression-free survival and mean time to cancer progression. This finding motivated us to study the possible antiproliferative effects of Lenvatinib compared to Sorafenib, in addition to the secretion profile of extracellular vesicles in HepG2 cultures due to its recognized role in tumor progression and metastasis.Methods: To determine the percentage of proliferating cells in culture, the incorporation of bromodeoxyuridine (BrdU) was used as a marker, while the analysis of the apoptotic activity was done through a colorimetric test that allows detecting the amount of caspase 3/7 existing in culture. It is well known that there is a connection between apoptosis and autophagy, so we decided to study the changes that occurred in the latter process after treatment. For this, the level of expression of LC3-II was determined through an SDS-PAGE coupled to a Western-Blot analysis. The changes produced in the expression of VEGFR-2 and EGFR were also monitored and, finally, the secretion profile of extracellular vesicles was studied through the analysis of the expression of different markers (Lamp1, E-Selectin, CD63, TSG101, Grp78, GM130, Annexin V and Prohibitin) in fractions enriched in exosomes, extracellular vesicles and apoptotic bodies.Results and Conclusions: The results for the group treated with Sorafenib reproduced what has been described so far in the literature referring to hepatocellular carcinoma: decrease in cell proliferation caused by the downregulation of the expression of different growth factors (EGFR and VEGFR-2) and increase of cell death by apoptosis. However, Lenvatinib did not reproduce the pattern we expected for an antineoplastic drug, since it increased cell proliferation. With respect to the secretion profile of extracellular vesicles, no convincing results were obtained. We think that this could be due to the capacity of separation of the different fractions of the protocol used or to the difficulty of obtaining, from them, high amounts of proteins to proceed to its analysis by WB

MicroRNA-200c Attenuates the Tumor-Infiltrating Capacity of Macrophages

Macrophages constitute a major part of the tumor-infiltrating immune cells. Within the tumor microenvironment, they acquire an alternatively activated, tumor-supporting phenotype. Factors released by tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of hsa-miR-200c-3p (miR-200c) in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed the transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and identified numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. miR-200c-mediated reduction in infiltration further correlated with a miR-200c migration signature comprised of the four miR-200c-repressed, predicted targets PPM1F, RAB11FIB2, RDX, and MSN

Assessing Autophagy in Archived Tissue or How to Capture Autophagic Flux from a Tissue Snapshot

Este artículo pertenece a un número especial: Autophagy in CancerAutophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samplesInstitute de Salud Carlos III (ISCIII) y Fondos FEDER de la UE PI14/01085 y PI17/00093Ministerio de Ciencia, Innovación y Universidades RTI2018-096748-B-100 to N.A.Ministerio de Ciencia, Innovación y Universidades FPU17/00026Consejería de Igualdad, Salud y Políticas Sociales PI-0198-2016Fondos FEDER de la UE NORTE-01-0145-FEDER-000013 y los proyectos POCI-01-0145-FEDER-028159 y POCI-01-0145-FEDER-03078

Cytoskeleton Rearrangements during the Execution Phase of Apoptosis

Apoptosis is a regulated energy‐dependent process for the elimination of unnecessary or damaged cells during embryonic development, tissue homeostasis and many pathological conditions. Apoptosis is characterized by specific morphological and biochemical features in which caspase activation has a pivotal role. During apoptosis, cells undergo characteristic morphological reorganizations in which the cytoskeleton participates actively. Traditionally, this cytoskeleton rearrangement has been assigned mainly to actinomyosin ring contraction, with microtubule and intermediate filaments both reported to be depolymerized at early stages of apoptosis. However, recent results have shown that microtubules are reformed during the execution phase of apoptosis forming an apoptotic microtubule network (AMN). Current hypothesis proposes that AMN is required to maintain plasma membrane integrity and cell morphology during the execution phase of apoptosis. AMN disruption provokes apoptotic cell collapse, secondary necrosis and the subsequent release of toxic molecules which can damage surrounding cells and promote inflammation. Therefore, AMN formation in physiological or pathological apoptosis is essential for tissue homeostasis

Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells

Background/Aims: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. Methods: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. Results: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15 P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172 P-Cdk4/ Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors increased the protein expression of FKBP12 and FKBP51 that appeared to play pro-survival role. Interestingly, the administration of immunosuppressants yields a specific pattern of miRNAs. Tacrolimus and mTOR inhibitors decreased miR-92a-1-5p, miR-197-3p, miR-483-3p and miR- 720, and increased miR-22-3p, miR-376a-3p, miR-663b, miR-886-5p, miR-1300 and miR-1303 expressions in HepG2 cells. Conclusion: The more potent pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with an increased activation of PERK and p53 signaling, and p21 protein expression. FKBP12 and FKBP51 appeared to be the most relevant partners of Tacrolimus and mTOR inhibitors exerting a pro-survival effect in HepG2 cells. The observed effects of immunosuppressants were related to a specific miRNA signature in liver cancer cellsEspaña Ministry of Economy and Competitiveness (MINECO) cofinanced by the ERDF (BFU2016-75352-P AEI/FEDER, EU

Four weeks versus six weeks of ampicillin plus ceftriaxone in Enterococcus faecalis native valve endocarditis: A prospective cohort study

Enterococcus faecalis infective endocarditis (EFIE) is a severe disease of increasing incidence. The objective was to analyze whether the outcome of patients with native valve EFIE (NVEFIE) treated with a short course of ampicillin plus ceftriaxone (4wAC) was similar to patients treated according to international guidelines (6wAC). Between January 2008 and June 2018, 1,978 consecutive patients with definite native valve IE were prospectively included in a national registry. Outcomes of patients with NVEFIE treated with 4wAC were compared to those of patients who received 6wAC. Three hundred and twenty-two patients (16.3%) had NVEFIE. One hundred and eighty-three (56.8%) received AC. Thirty-nine patients (21.3%) were treated with 4wAC for four weeks and 70 patients (38.3%) with 6wAC. There were no differences in age or comorbidity. Patients treated 6wAC presented a longer duration of symptoms before diagnosis (21 days, IQR 7-60 days vs. 7 days, IQR 1-22 days; p = 0.002). Six patients presented perivalvular abscess and all of these received 6wAC. Surgery was performed on 14 patients (35.9%) 4wAC and 34 patients (48.6%) 6wAC (p = 0.201). In-hospital mortality, one-year mortality and relapses among 4wAC and 6wAC patients were 10.3% vs. 11.4% (p = 0.851); 17.9% vs. 21.4% (p = 0.682) and 5.1% vs. 4.3% (p = 0.833), respectively. In conclusion, a four-week course of AC may be considered as an alternative regimen in NVEFIE, notably in patients with shorter duration of symptoms and those without perivalvular abscess. These results support the performance of a randomized clinical trial to evaluate the efficacy of this short regimen.This work was supported in part by the “Fondo de Investigaciones Sanitarias” (FIS) grant 17/01251 from the “Instituto de Salud Carlos III”, Madrid, Spain awarded to JMM. JMM received a personal 80:20 research grant from the Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, during 2017–19. JMP was member of the Endocarditis Team of the Hospital Clinic of Barcelona, Spain when this project was approved by the GAMES Steering Committee.

Assessing autophagy in archived tissue or how to capture autophagic flux from a tissue snapshot

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.This work was supported by grants from the Bernese Cancer League, “Stiftung für klinisch-experimentelle Tumorforschung”, and the Werner and Hedy Berger-Janser Foundation for Cancer Research (to M.H.); by Institute of Health Carlos III (ISCIII) and FEDER funds from the EU (PI14/01085 and PI17/00093) and supported by Miguel Servet contract by ISCIII and FSE funds (CPII16/00023) (to M.M.); from the Spanish Ministry of Science, Innovation and Universities (RTI2018-096748-B-100 to N.A.); from the University Professor Training Fellowship, Ministry of Science, Innovation and University, Government of Spain (FPU17/00026) (to P.C.O); from the ISCIII (PI16/00090 and PI19/01266) and the Andalusian Government (Consejería de Igualdad, Salud y Políticas Sociales, PI-0198-2016) for their financial support, and from the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCIII and co-financed by European Development Regional Fund (EDRF) “A way to achieve Europe” for their financial support (to J.M.), from Breakthrough Cancer Research, Ireland funding (to S.L.M); from the PI18/00442 grant integrated into the State Plan for R & D + I2013-2016 and funded by the ISCIII and the ERDF, a way to make Europe (to G.V.); from the Luxembourg National Research Fund (C18/BM/12670304/COMBATIC to B.J.); from the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, by the European Regional Development Fund (FEDER), through the Competitiveness Factors Operational Programme (COMPETE) (NORTE-01-0145-FEDER-000013) and from the projects POCI-01-0145-FEDER-028159 and POCI-01-0145-FEDER-030782 by FEDER, through the COMPETE (to P.L.); from National funds, through the Foundation for Science and Technology (FCT) (to P.L.); from ARRS—the Slovenian research agency, programme P1-0140: Proteolysis and its regulation (led by B. Turk) (to E.Ž.); from the Swiss Cancer Research (KFS-3360-02-2014) (to A.P, and M.P.T.) (KFS-3409-02-2014), and the Swiss National Science Foundation (31003A_173219) (to M.P.T.)

Mural Endocarditis: The GAMES Registry Series and Review of the Literature

Introduction: Mural infective endocarditis (MIE) is a rare type of endovascular infection. We present a comprehensive series of patients with mural endocarditis. Methods: Patients with infectious endocarditis (IE) from 35 Spanish hospitals were prospectively included in the GAMES registry between 2008 and 2017. MIEs were compared to non-MIEs. We also performed a literature search for cases of MIE published between 1979 and 2019 and compared them to the GAMEs series. Results: Twenty-seven MIEs out of 3676 IEs were included. When compared to valvular IE (VIE) or device-associated IE (DIE), patients with MIE were younger (median age 59 years, p < 0.01). Transplantation (18.5% versus 1.6% VIE and 2% DIE, p < 0.01), hemodialysis (18.5% versus 4.3% VIE and 4.4% DIE, p = 0.006), catheter source (59.3% versus 9.7% VIE and 8.8% DIE, p < 0.01) and Candida etiology (22.2% versus 2% DIE and 1.2% VIE, p < 0.01) were more common in MIE, whereas the Charlson Index was lower (4 versus 5 in non-MIE, p = 0.006). Mortality was similar. MIE from the literature shared many characteristics with MIE from GAMES, although patients were younger (45 years vs. 56 years, p < 0.001), the Charlson Index was lower (1.3 vs. 4.3, p = 0.0001), catheter source was less common (13.9% vs. 59.3%) and there were more IVDUs (25% vs. 3.7%). S. aureus was the most frequent microorganism (50%, p = 0.035). Systemic complications were more common but mortality was similar. Conclusion: MIE is a rare entity. It is often a complication of catheter use, particularly in immunocompromised and hemodialysis patients. Fungal etiology is common. Mortality is similar to other IEs

Role of age and comorbidities in mortality of patients with infective endocarditis

[Purpose]: The aim of this study was to analyse the characteristics of patients with IE in three groups of age and to assess the ability of age and the Charlson Comorbidity Index (CCI) to predict mortality. [Methods]: Prospective cohort study of all patients with IE included in the GAMES Spanish database between 2008 and 2015.Patients were stratified into three age groups:<65 years,65 to 80 years,and ≥ 80 years.The area under the receiver-operating characteristic (AUROC) curve was calculated to quantify the diagnostic accuracy of the CCI to predict mortality risk. [Results]: A total of 3120 patients with IE (1327 < 65 years;1291 65-80 years;502 ≥ 80 years) were enrolled.Fever and heart failure were the most common presentations of IE, with no differences among age groups.Patients ≥80 years who underwent surgery were significantly lower compared with other age groups (14.3%,65 years; 20.5%,65-79 years; 31.3%,≥80 years). In-hospital mortality was lower in the <65-year group (20.3%,<65 years;30.1%,65-79 years;34.7%,≥80 years;p < 0.001) as well as 1-year mortality (3.2%, <65 years; 5.5%, 65-80 years;7.6%,≥80 years; p = 0.003).Independent predictors of mortality were age ≥ 80 years (hazard ratio [HR]:2.78;95% confidence interval [CI]:2.32–3.34), CCI ≥ 3 (HR:1.62; 95% CI:1.39–1.88),and non-performed surgery (HR:1.64;95% CI:11.16–1.58).When the three age groups were compared,the AUROC curve for CCI was significantly larger for patients aged <65 years(p < 0.001) for both in-hospital and 1-year mortality. [Conclusion]: There were no differences in the clinical presentation of IE between the groups. Age ≥ 80 years, high comorbidity (measured by CCI),and non-performance of surgery were independent predictors of mortality in patients with IE.CCI could help to identify those patients with IE and surgical indication who present a lower risk of in-hospital and 1-year mortality after surgery, especially in the <65-year group