Impact of Wnt/β-Catenin Inhibition on Cell Proliferation through CDC25A Downregulation in Soft Tissue Sarcomas.

The Wnt signaling pathway is an important cellular mechanism for regulating differentiation processes as well as cell cycle events, and different inhibitors of this pathway, for example, PRI-724, are showing promising results in clinical trials for treatment of advanced pancreatic adenocarcinoma or ovarian cancer. Growing evidence suggests that Wnt signaling may also be crucial for tumorigenesis and progression of soft tissue sarcomas (STS), a malignant neoplasm with few therapeutic options at an advanced state. Our study with several STS cell lines and primary cultures shows that inhibition of Wnt/β-catenin signaling with PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates. TCF/β-catenin-mediated transcriptional activity is decreased in treated cells, leading to downregulation of its target genes CCND1 and CDC25A. The latter was critical because its downregulation via siRNA was able to mimic the effect of PRI-724 on cell cycle arrest and cell death induction. An evaluation of NCBI/GenBank data confirmed that CDC25A mRNA is elevated in STS patients. Importantly, PRI-724 in combination with standard STS chemotherapeutics doxorubicin or trabectedin enhanced their antitumoral effect in a synergistic manner according to isobolographic analysis, suggesting that Wnt inhibition through PRI-724 could be a beneficial combination regime in patients with advanced STS.This study was financed by Grupo Español de Investigación en Sarcomas (GEIS) and Fundación Mari PazJiménez Casado. MPC is supported by Programa Estrategia de Emprendimiento y Empleo Joven,Garantía Juvenil(Ministerio de Trabajo, Migraciones y Seguridad Social-SOIB.S

Analysis of vaccine responses after anti-CD20 maintenance in B-cell lymphoma in the Balearic Islands. A single reference center experience

IntroductionThe use of maintenance approaches with anti-CD20 monoclonal antibodies has improved the outcomes of B-cell indolent lymphomas but may lead to significant peripheral B-cell depletion. This depletion can potentially hinder the serological response to neoantigens.MethodsOur objective was to analyze the effect of anti-CD20 maintenance therapy in a reliable model of response to neoantigens: SARS-CoV-2 vaccine responses and the incidence/severity ofCOVID-19 in a reference hospital.ResultsIn our series (n=118), the rate of vaccination failures was 31%. Through ROC curve analysis, we determined a cutoff for SARS-CoV-2 vaccine serologic response at 24 months from the last anti-CD20 dose. The risk of severe COVID-19 was notably higher within the first 24months following the last anti-CD20 dose (52%) compared to after this period (just 18%) (p=0.007). In our survival analysis, neither vaccine response nor hypogammaglobulinemia significantly affected OS. While COVID-19 led to a modest mortality rate of 2.5%, this figure was comparable to the OS reported in the general immunocompetent population. However, most patients with hypogammaglobulinemia received intravenous immunoglobulin therapy and all were vaccinated. In conclusion, anti-CD20 maintenance therapy impairs serological responses to SARS-CoV-2 vaccines.DiscussionWe report for the first time that patients during maintenance therapy and up to 24 months after the last anti-CD20 dose are at a higher risk of vaccine failure and more severe cases of COVID-19. Nevertheless, with close monitoring, intravenous immunoglobulin supplementation or proper vaccination, the impact on survival due to the lack of serological response in this high-risk population can be mitigated, allowing for the benefits of anti-CD20 maintenance therapy, even in the presence of hypogammaglobulinemia

Screening for Prognostic microRNAs Associated with Treatment Failure in Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) treatment with R-CHOP regimen produces 5-year progression-free survival and overall survival of around 60-70%. Our objective was to discover prognostic biomarkers allowing early detection of the remaining 30-40% with poor long-term outcome. For this purpose, we applied a novel strategy: from a cohort of DLBCL patients, treated with standard therapy, a discovery group of 12 patients with poor prognosis (advanced stage III-IV, R-IPI > 2) was formed, consisting of six chemoresistant (refractory/early relapse 3 years) subjects. By using microarray assays, the most differentially expressed miRNAs were defined as an initial set of prognostic miRNA candidates. Their expression was then analyzed in a validation cohort of 68 patients and the three miRNAs with the most significant impact on event-free and overall survival were selected. In the DLBCL cell line U-2932 the transfection with miR-1244 and miR-193b-5p, but not miR-1231, blocked the effect of CHOP on cell viability. A subsequent gene set enrichment analysis in patients revealed the implication of the first two miRNAs in cell cycle control and chemoresistance-related pathways, whereas the last one was involved in immunological processes. In conclusion, this novel strategy identified three promising prognostic markers for DLBCL patients at high risk of failure with standard therapy

Regulation of muscarinic acetylcholine receptor sequestration and function by β-arrestin

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic β- arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the β2-adrenergic receptor have demonstrated that β-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of β-arrestin in mAChR sequestration, we determined the effect of overexpressing β-arrestin-1 and the dominant- negative inhibitor of β-arrestin-mediated receptor sequestration, β- arrestin-1 V53D, on mAChR sequestration and function. Sequestration of ml, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing β- arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of β-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen- activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a β-arrestin- and dynamin- dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.</p

WNT/β-Catenin Pathway in Soft Tissue Sarcomas: New Therapeutic Opportunities?

Soft tissue sarcomas (STS) are a very heterogeneous group of rare tumors, comprising more than 50 different histological subtypes that originate from mesenchymal tissue. Despite their heterogeneity, chemotherapy based on doxorubicin (DXR) has been in use for forty years now and remains the standard first-line treatment for locally advanced unresectable or metastatic STS, although overall survival could not be improved by combination with other chemotherapeutics. In this sense, the development of new therapeutic approaches continues to be a largely unmatched goal. The WNT/β-catenin signaling pathway is involved in various fundamental processes for embryogenic development, including the proliferation and differentiation of mesenchymal stem cells. Although the role of this pathway has been widely researched in neoplasms of epithelial origin, little is known about its relevance for mesenchymal neoplasms. This review covers the most important molecular alterations of the WNT signaling pathway in STS. The detection of these alterations and the understanding of their functional consequences for those pathways controlling sarcomagenesis development and progression are crucial to broaden the current knowledge about STS as well as to identify novel drug targets. In this regard, the current therapeutic options and drug candidates to modulate WNT signaling, which are usually classified by their interaction site upstream or downstream of β-catenin, and their presumable clinical impact on STS are also discussed

Down-Regulation of AKT Signalling by Ursolic Acid Induces Intrinsic Apoptosis and Sensitization to Doxorubicin in Soft Tissue Sarcoma.

Several important biological activities have been attributed to the pentacyclic triterpene ursolic acid (UA), being its antitumoral effect extensively studied in human adenocarcinomas. In this work, we focused on the efficacy and molecular mechanisms involved in the antitumoral effects of UA, as single agent or combined with doxorubicin (DXR), in human soft tissue sarcoma cells. UA (5-50 μM) strongly inhibited (up to 80%) the viability of STS cells at 24 h and its proliferation in soft agar, with higher concentrations increasing apoptotic death up to 30%. UA treatment (6-9 h) strongly blocked the survival AKT/GSK3β/β-catenin signalling pathway, which led to a concomitant reduction of the anti-apoptotic proteins c-Myc and p21, altogether resulting in the activation of intrinsic apoptosis. Interestingly, UA at low concentrations (10-15 μM) enhanced the antitumoral effects of DXR by up to 2-fold, while in parallel inhibiting DXR-induced AKT activation and p21 expression, two proteins implicated in antitumoral drug resistance and cell survival. In conclusion, UA is able to induce intrinsic apoptosis in human STS cells and also to sensitize these cells to DXR by blocking the AKT signalling pathway. Therefore, UA may have beneficial effects, if used as nutraceutical adjuvant during standard chemotherapy treatment of STS

Consumption of Virgin Olive Oil Influences Membrane Lipid Composition and Regulates Intracellular Signaling in Elderly Adults With Type 2 Diabetes Mellitus

We aimed to define changes in membrane fatty acids and signaling proteins induced by virgin olive oil (VOO) consumption in elderly persons with type 2 diabetes (n = 16) compared to a control group (n = 28). The fatty acid composition was determined by gas chromatography and G-protein subunits and protein kinase C alpha (PKCα) by immunoblotting. VOO consumption increased the monounsaturated fatty acid content in phospholipids and cholesterol esters in both groups. In contrast, saturated fatty acids were decreased only in phospholipids. The levels of Gαo, Gβ, and PKCα were significantly lower in diabetics than in controls. However, whereas VOO consumption reduced Gαs, Gβ, and PKCα in both groups, reduction in Gαi was observed only in diabetics. These results indicate that long-term VOO consumption modifies the fatty acid composition of plasma membrane, which influences the association of G proteins and PKCα with the lipid bilayer. These combined effects probably account for the positive effects of VOO on glycemic homeostasis.This work was supported by grants AGL2002-00195, AGL2005-00572, SAF2003-00232, and SAF2004-05249 (Ministerio de Educación y Ciencia, Spain), by the Fondo de Investigaciones Sanitarias (FIS; Red Corporativa ISCIII G03/140-2002), and by funds from the Marathon Foundation. J. S. P was a holder of a Juan de la Cierva contract, and O. V. is a Ramón y Cajal Fellow.Peer reviewe

Down-regulation of the AKT/GSK3 / -catenin signalling pathway by the triterpene ursolic acid induced intrinsic apoptosis in human soft tissue sarcomas

Trabajo presentado en la 51th International Conference on the Bioscience of Lipids, celebrada en Bilbao (España) del 07 al 11 de septiembre de 2010

Plant pentacyclic triterpenes perturbe lipid membrane properties

Trabajo presentado en la 51th International Conference on the Bioscience of Lipids, celebrada en Bilbao (España) del 07 al 11 de septiembre de 2010

Natural pentacyclic triterpenes enhance antitumoral effects of doxorubicin through augmenting its intracellular concentration in human sarcoma cells

Trabajo presentado en la 51th International Conference on the Bioscience of Lipids, celebrada en Bilbao (España) del 07 al 11 de septiembre de 2010.Doxorubicin (DXR), an anthracycline antibiotic, is the actual standard chemotherapy for soft tissue sarcomas (STSs), a heterogeneous group of neoplasmas. However, its severe toxicity and the development of drug resistance call for clinically important adjuvants which are able to improve DXR efficacy without producing additional side effects. Oleanolic acid (OLA) and maslinic acid (MLA) are non-toxic pentacyclic triterpenes present in a wide range of plants and human dietary products (e.g., orujo olive oil), which exhibit certain beneficial biological activities, such as antioxidant, anti-inflammatory and antitumoral properties. Here, we examined the anti-proliferative effects of OLA and MLA, which were isolated from pressed olive (O. europaea) fruits, as single agents or in combination with DXR in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells, both prone to DXR resistance. MLA, but not OLA, inhibited cell viability with an IC50 around 50 ¿M in both sarcoma cell lines. Combination of DXR (0.1-10 ¿M) with MLA (30 ¿M) or OLA (80 ¿M) increased the antiproliferative effect of DXR by 1.5-2.5-fold. On the molecular level, OLA and MLA increased the uptake of DXR in a time-dependent manner, leading to intracellular DXR accumulation. Our results show that natural pentacyclic triterpenes enhance the antitumoral effects of DXR, most probably by increasing its intracellular concentration