The miR-17-92 cluster counteracts quiescence and chemoresistance in a distinct subpopulation of pancreatic cancer stem cells

Objective Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes. Design Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs. Results We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-beta 1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer. Conclusions Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No 256974 (EPC-TM-NET) and No 602783 (CAM-PaC), the Subdireccion General de Evaluacion y Fomento de la Investigacion, Fondo de Investigacion Sanitaria (PS09/02129 \& PI12/02643), and the Programa Nacional de Internacionalizacion de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; Ministerio de Economia y Competitividad, Spain). MC: La Caixa Predoctoral Fellowship.S

Comparison of the Efficiency of Complexes Based on S4(13)-PV Cell-Penetrating Peptides in Plasmid DNA and siRNA Delivery

The successful application of gene therapy approaches is highly dependent on the efficient delivery of nucleic acids into target cells. In the present study, new peptide-based nonviral systems were developed to enhance plasmid DNA and siRNA delivery, aiming at generating appropriate gene delivery and gene silencing tools for preclinical and clinical application. For this purpose, 0 cell-penetrating peptide derived from the wild-type S4(13)-PV peptide was synthesized through the addition of a five-histidine tail to its N-terminus (H-5-S4(13)-PV), and its ability to mediate gene expression and gene silencing was evaluated and compared to that of the wild-type peptide. The histidine-enriched peptide, H-5-S4(13)-PV, proved to be generally more efficient and less toxic than the wild-type peptide in the delivery of plasmid DNA. In addition, complexes of H-5-S4(13)-PV with siRNAs, but not of S4(13)-PV, were efficiently internalized by cells and presented high knockdown activity (63%). Interestingly, systems containing the S4(13)-PV or the H-5-S4(13)-PV peptide exhibited superior biological activity when compared to those containing the reverse NLS or scrambled peptides, suggesting that both the cell-penetrating; sequence and the NLS of the S4(13)-PV peptide influence the competence Of binary and ternary complexes to accomplish nucleic acid delivery. In order to unravel the cancer therapeutic potential of formulations with the histidine-enriched peptide, their efficiency to mediate silencing of the oncogenic protein survivin was evaluated. As opposed to complexes with the wild-type peptide, H-5-S4(13)-PV complexes showed the ability to promote a high survivin knockdown at the level of both protein (44%) and mRNA (73%), in HT1080 cells

Multimodal Treatment Eliminates Cancer Stem Cells and Leads to Long-Term Survival in Primary Human Pancreatic Cancer Tissue Xenografts.

Copyright: 2013 Hermann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.PURPOSE: In spite of intense research efforts, pancreatic ductal adenocarcinoma remains one of the most deadly malignancies in the world. We and others have previously identified a subpopulation of pancreatic cancer stem cells within the tumor as a critical therapeutic target and additionally shown that the tumor stroma represents not only a restrictive barrier for successful drug delivery, but also serves as a paracrine niche for cancer stem cells. Therefore, we embarked on a large-scale investigation on the effects of combining chemotherapy, hedgehog pathway inhibition, and mTOR inhibition in a preclinical mouse model of pancreatic cancer. EXPERIMENTAL DESIGN: Prospective and randomized testing in a set of almost 200 subcutaneous and orthotopic implanted whole-tissue primary human tumor xenografts. RESULTS: The combined targeting of highly chemoresistant cancer stem cells as well as their more differentiated progenies, together with abrogation of the tumor microenvironment by targeting the stroma and enhancing tissue penetration of the chemotherapeutic agent translated into significantly prolonged survival in preclinical models of human pancreatic cancer. Most pronounced therapeutic effects were observed in gemcitabine-resistant patient-derived tumors. Intriguingly, the proposed triple therapy approach could be further enhanced by using a PEGylated formulation of gemcitabine, which significantly increased its bioavailability and tissue penetration, resulting in a further improved overall outcome. CONCLUSIONS: This multimodal therapeutic strategy should be further explored in the clinical setting as its success may eventually improve the poor prognosis of patients with pancreatic ductal adenocarcinoma

Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma

[Objective]: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models.[Design]: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation.[Results]: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment.[Conclusion]: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.JCL-G received support from a 'la Caixa' Foundation (ID 100010434) fellowship (LCF/BQ/DR21/11880011). This study was supported by ISCIII FIS grants PI18/00757 and PI21/01110 (BSJ) and PI18/00267 (LG-B), and grants from the Spanish Ministry of Economy and Innovation SAF2016-76504-R (ACan and FP), PID2019-111052RB-I00 (FP), PID2019-104644RB-I00 (GM-B), a Ramón y Cajal Merit Award RYC-2012–12104 (BSJ) and ISCIII, CIBERONC, CB16/12/00446 (ACar) and CB16/12/00295 (ACan and GM-B), all of them co-financed through Fondo Europeo de Desarrollo Regional (FEDER) 'Una manera de hacer Europa'; a Fero Foundation Grant (BSJ); a Coordinated grant (GC16173694BARB) from the Fundación Científica Asociación Española Contra el Cáncer (FC-AECC) (BSJ); a Miguel Servet award (CP16/00121) (PS); a DFG, German Research Foundation Grant—Project no: 492 436 553 (KG); and a Max Eder Fellowship of the German Cancer Aid (111746) (PCH

S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: Implications for cell internalization

The present work aims to gain insights into the role of peptide–lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide–lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems

Peak left atrial longitudinal strain is associated with all-cause mortality in patients with ventricular functional mitral regurgitation

Abstract Purpose Chronic mitral regurgitation promotes left atrial (LA) remodeling. However, the significance of LA dysfunction in the setting of ventricular functional mitral regurgitation (FMR) has not been fully investigated. Our aim was to assess the prognostic impact of peak atrial longitudinal strain (PALS), a surrogate of LA function, in patients with FMR and reduced left ventricular ejection fraction (LVEF). Methods Patients with at least mild ventricular FMR and LVEF < 50% under optimized medical therapy who underwent transthoracic echocardiography at a single center were retrospectively identified in the laboratory database. PALS was assessed by 2D speckle tracking in the apical 4-chamber view and the study population was divided in two groups according to the best cut-off value of PALS, using receiver operating characteristics (ROC) curve analysis. The primary endpoint-point was all-cause mortality. Results A total of 307 patients (median age 70 years, 77% male) were included. Median LVEF was 35% (IQR: 27 – 40%) and median effective regurgitant orifice area (EROA) was 15mm2 (IQR: 9 – 22mm2). According to current European guidelines, 32 patients had severe FMR (10%). During a median follow-up of 3.5 years (IQR 1.4 – 6.6), 148 patients died. The unadjusted mortality incidence per 100 persons-years increased with progressively lower values of PALS. On multivariable analysis, PALS remained independently associated with all-cause mortality (adjusted hazard ratio 1.052 per % decrease; 95% CI: 1.010 – 1.095; P = 0.016), even after adjustment for several (n = 14) clinical and echocardiographic confounders. Conclusion PALS is independently associated with all-cause mortality in patients with reduced LVEF and ventricular FMR. Graphical Abstrac

Impact of anthropogenic noise on the survival and development of meagre (Argyrosomus regius) early life stages

We are grateful to IPMA-EPPO for the test subjects. We also thank Joaquim Tapisso for the assistance in the bioterium and Marta Bolgan for helping with the preliminary work. This study had the support of FCT through the strategic project UIDB/04292/2020 awarded to MARE, and through project LA/P/0069/2020 granted to the Associate Laboratory ARNET, through the strategic project UIDB/00006/2020 awarded to CE3C; and through project PTDC/BIA-BMA/29662/2017. FCT also attributed individual PhD grant to M.V (SFRH/BD/115562/2016).The growth of human populations has been driving an unprecedent and widespread increase in marine traffic, posing a real threat to marine biodiversity. Even though we are now aware of the negative effects of shipping noise exposure on fish, information about the impact on their early life stages continues to lack. Meagre (Argyrosomus regius) is a vocal fish that uses estuaries with high levels of anthropogenic noise pollution as both breeding areas and nurseries. Here, the effects of boat noise exposure on the development and survival of meagre larvae were studied. Embryos and larvae were exposed to either noise (boat noise playback) or control treatments (coils producing a similar electric field to the speakers) and hatching rate, survival rate, morphometric traits and stress-related biomarkers, at hatching and at 2 days-post-hatching (dph) were analyzed. Results showed no conclusive effects of the impact of boat noise playback, even though there was an increased lipid droplet consumption and a decrease in body depth at 2dph larvae under this stressor. The assessment of oxidative stress and energy metabolism-related biomarkers at hatching showed a marginal decrease in superoxide dismutase (SOD) activity and no changes in DNA damage or electron transport system activity (ETS), although it cannot be disregarded that those effects could only be visible at later stages of larval development. Whether these morphological and developmental results have implications in later stages remains to be investigated. Further studies with longer exposure and wild meagre could help deepen this knowledge and provide a better understanding of how anthropogenic noise can impact meagre early stages.info:eu-repo/semantics/publishedVersio

Targeting of sonic hedgehog and mTOR in pancreatic ductal adenocarcinoma.

<p>(<b>A</b>) Fold increase mRNA expression levels of SHH, GLI-1, and GLI-2 of sphere-derived vs. adherent cells. (<b>B</b>) Western blot analysis of mTOR pathway activity via the assessment of S6 kinase expression (upper panel) and phosphorylation (lower panel) in adherent primary cells versus stem cell-enriched sphere-derived cells. (<b>C</b>) Illustration of experimental setup. Duration of triple therapy is marked by a dark grey box (day 21 to 48), Gem monotherapy with a light grey box (day 48 to 81).</p

Comparison of the <i>in vitro</i> and <i>in vivo</i> effects of Pegylated Gemcitabine.

<p>(<b>A</b>) <i>In vitro</i> effects of Gem and PEG-Gem on apoptosis and cell death as well as CD133 expression (cumulative results of cells obtained from different xenografts). (<b>B</b>) Kaplan-Meier Curve depicting cumulative survival time of all mice pooled by treatment group. For illustrative purposes, selected survival curves of Fig. 2D are depicted again. (<b>C</b>) Tumor growth curves for primary whole-tissue xenografts implanted subcutaneously and orthotopically, respectively. Continuous line depicts Gem+vehicle, dashed line depicts Gem+ SIBI, dotted line depicts Gem+SIBI+Rapa. (<b>D</b>) Kaplan-Meier Curve depicting cumulative survival time of all mice pooled by treatment group. For illustrative purposes, selected survival curves of Fig. 2D are depicted again.</p