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

Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients

MET alterations may provide a potential biomarker to evaluate patients who will benefit from treatment with MET inhibitors. Therefore, the purpose of the present study is to investigate the utility of a liquid biopsy-based strategy to assess MET alterations in cancer patients. We analyzed MET amplification in circulating free DNA (cfDNA) from 174 patients with cancer and 49 healthy controls and demonstrated the accuracy of the analysis to detect its alteration in patients. Importantly, a significant correlation between cfDNA concentration and MET copy number (CN) in cancer patients (r = 0.57, p <10−10) was determined. Furthermore, we evaluated two approaches to detect the presence of MET on circulating tumor cells (CTCs), using the CellSearch® and Parsortix systems and monitored patients under anti-EGFR treatment (n = 30) combining both cfDNA and CTCs analyses. This follow-up provides evidence for the potential of MET CN assessment when patients develop resistance to anti-EGFR therapy and a significant association between the presence of CTCs MET+ and the Overall Survival (OS) in head and neck cancer patients (P = 0.05; HR = 6.66). In conclusion, we develop specific and noninvasive assays to monitor MET status in cfDNA/CTCs and demonstrate the utility of plasma MET CN determination as a biomarker for monitoring the appearance of resistance to anti-EGFR therapyThis study was financed by all the donors who participated in the Liquid Biopsy Crowdfunding campaign in 2017. LMR is supported by Asociación Española Contra el Cancer (AECC). ADL is funded by a “Juan Rodés” contract (JR17/00016) from ISCIIIS

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Poliposi adenomatosa familiar i càncer colorectal. Estudi genòmic i anàlisi d'alteracions de la via Wnt.

[cat] Les mutacions germinals en el gen APC són les responsables de la majoria de casos de poliposi adenomatosa familiar. APC participa en diferents processos cel·lulars que inclouen la regulació del cicle cel·lular, l'apoptosi, l'adhesió cel·lular, la migració cel·lular, la transducció de senyals, l'acoblament de microtúbuls i la segregació dels cromosomes. Encara que tots aquests processos estan potencialment lligats amb el càncer, sembla que la funció supressora tumoral del gen resideix en la seva capacitat per regular de manera acurada els nivells intracel·lulars de la β-catenina, component central de la via de Wnt. Aquesta via és important en el desenvolupament embrionari per determinar llinatges cel·lulars específics i en l'homeòstasi cel·lular de determinats teixits com la mucosa intestinal. A part del seu paper clau en la via de Wnt, APC participa en la inestabilitat cromosòmica del càncer colorectal, encara que els mecanismes concrets a través dels quals actua són encara desconeguts en bona part. Els resultats descrits en els tres capítols d'aquesta tesi donen lloc al concepte global que és en les lesions benignes del còlon, els adenomes, ja siguin de pacients de FAP com de càncer colorectal esporàdic, on s'acumulen gran nombre d'alteracions genètiques que afecten a mecanismes molt diversos del funcionament cel·lular, essent especialment importants aquelles alteracions que afecten a la via de Wnt. Totes les alteracions detectades permeten la progressió tumoral ja que afecten a la proliferació, mitosi i moltes altres funcions importants per la cèl·lula. El coneixement de les alteracions moleculars que afecten als adenomes és important per proporcionar més evidències dels mecanismes moleculars i cel·lulars implicats en els primers estadis de la progressió tumoral i donar lloc a noves estratègies de diagnòstic precoç i teràpies preventives, tan importants en la lluita contra el càncer.[eng] Classical Familial Adenomatous Polyposis (FAP) syndrome is caused by APC germline mutations. APC plays important roles in different cellular processes such as cell cycle regulation, apoptosis, cellular adhesion, cellular migration, signal transduction, microtubule binding and chromosome segregation. Although all these processes are potentially related to cancer, it seems that the main tumoral suppressor activity is mediated by its capacity to accurately regulate the β-catenin intracellular levels, which is a central member of the Wnt signalling pathway. This pathway is important in the embryonic development and in cellular homeostasis in certain tissues such as intestinal mucosa. In colorectal cancer, APC is not only believed to be playing a key role in Wnt signalling, but also it is important in chromosomal instability (CIN), although the mechanisms in which APC is participating are still largely unknown. The results presented in this thesis and divided in 3 chapters give us the general idea that in the benign lesions of the colon, called adenomas, in FAP and in sporadic colorectal cancer patients, is where a great number of genetic alterations are accumulated affecting different functional mechanisms in the cell. Those aberrations that alter the Wnt signalling pathway are especially important in the process of tumorigenesis. All the reported alterations enable the tumoral progression because they target proliferation, mitosis and other important functions of the cell. A better understanding of the molecular defects in adenomas is crucial to discover all the molecular and cellular mechanisms operating in the early stages of colorectal cancer and give clues for early diagnosis and prevention

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

TLR/WNT: A Novel Relationship in Immunomodulation of Lung Cancer

The most frequent cause of death by cancer worldwide is lung cancer, and the 5-year survival rate is still very poor for patients with advanced stage. Understanding the crosstalk between the signaling pathways that are involved in disease, especially in metastasis, is crucial to developing new targeted therapies. Toll-like receptors (TLRs) are master regulators of the immune responses, and their dysregulation in lung cancer is linked to immune escape and promotes tumor malignancy by facilitating angiogenesis and proliferation. On the other hand, over-activation of the WNT signaling pathway has been reported in lung cancer and is also associated with tumor metastasis via induction of Epithelial-to-mesenchymal-transition (EMT)-like processes. An interaction between both TLRs and the WNT pathway was discovered recently as it was found that the TLR pathway can be activated by WNT ligands in the tumor microenvironment; however, the implications of such interactions in the context of lung cancer have not been discussed yet. Here, we offer an overview of the interaction of TLR-WNT in the lung and its potential implications and role in the oncogenic process

Disruption of TCF/β-Catenin Binding Impairs Wnt Signaling and Induces Apoptosis in Soft Tissue Sarcoma Cells.

Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin and represent around 1% of adult cancers, being a very heterogeneous group of tumors with more than 50 different subtypes. The Wnt signaling pathway is involved in the development and in the regulation, self-renewal, and differentiation of mesenchymal stem cells, and plays a role in sarcomagenesis. In this study, we have tested pharmacologic inhibition of Wnt signaling mediated by disruption of TCF/β-catenin binding and AXIN stabilization, being the first strategy more efficient in reducing cell viability and downstream effects. We have shown that disruption of TCF/β-catenin binding with PKF118-310 produces in vitro antitumor activity in a panel of prevalent representative STS cell lines and primary cultures. At the molecular level, PKF118-310 treatment reduced β-catenin nuclear localization, reporter activity, and target genes, resulting in an increase in apoptosis. Importantly, combination of PKF118-310 with doxorubicin resulted in enhanced reduction of cell viability, suggesting that Wnt inhibition could be a new combination regime in these patients. Our findings support the usefulness of Wnt inhibitors as new therapeutic strategies for the prevalent STS. Mol Cancer Ther; 16(6); 1166-76. ©2017 AACR

Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10-100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3 ± 1.11% and 68.8 ± 1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5-10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment.</p

Oleanolic and maslinic acid sensitize soft tissue sarcoma cells to doxorubicin by inhibiting the multidrug resistance protein MRP-1, but not P-glycoprotein

The pentacyclic triterpenes oleanolic acid (OLA) and maslinic acid (MLA) are natural compounds present in many plants and dietary products consumed in the Mediterranean diet (e.g., pomace and virgin olive oils). Several nutraceutical activities have been attributed to OLA and MLA, whose antitumoral effects have been extensively evaluated in human adenocarcinomas, but little is known regarding their effectiveness in soft tissue sarcomas (STS). We assessed efficacy and molecular mechanisms involved in the antiproliferative effects of OLA and MLA as single agents or in combination with doxorubicin (DXR) in human synovial sarcoma SW982 and leiomyosarcoma SK-UT-1 cells. As single compound, MLA (10-100 μM) was more potent than OLA, inhibiting the growth of SW982 and SK-UT-1 cells by 70.3±1.11% and 68.8±1.52% at 80 μM, respectively. Importantly, OLA (80 μM) or MLA (30 μM) enhanced the antitumoral effect of DXR (0.5-10 μM) by up to 2.3-fold. On the molecular level, efflux activity of the multidrug resistance protein MRP-1, but not of the P-glycoprotein, was inhibited. Most probably as a consequence, DXR accumulated in these cells. Kinetic studies showed that OLA behaved as a competitive inhibitor of substrate-mediated MRP-1 transport, whereas MLA acted as a non-competitive one. Moreover, none of both triterpenes induced a compensatory increase in MRP-1 expression. In summary, OLA or MLA sensitized cellular models of STS to DXR and selectively inhibited MRP-1 activity, but not its expression, leading to a higher antitumoral effect possibly relevant for clinical treatment. © 2014 Elsevier Inc.Peer Reviewe

Detection of the EGFR G719S mutation in non-small cell lung cancer using Droplet Digital PCR (under review)

[eng] Objectives: The main objectives of the study were 1) to set-up a droplet digital PCR (ddPCR) assay 25 for the non-invasive detection of G719S EGFR mutation in NSCLC patients; 2) to determine the 26 limits of detection of the ddPCR assay for G719S mutation and 3) to compare COBAS® and ddPCR 27 System for G719S quantification in plasma. Materials and methods: Blood samples were collected from 19 patients diagnosed with clinical 29 stage IVA or IVB NSCLC according to the TNM Classification of Malignant Tumors. Then, plasma 30 ctDNA was extracted with the Qiagen Circulating Nucleic Acids kit and quantified by QuantiFluor® 31 dsDNA System. The mutational study of EGFR was carried out by digital droplet PCR (ddPCR) with 32 the QX200 Droplet Digital PCR System with specific probes and primers. Results: We observed the lowest percentage of G719S mutant allele could be detected in a wildtype 34 background was 0,058%. In the specificity analysis, low levels of G719S mutation were detected in 35 healthy volunteers with a peak of 21.65 mutant copies per millilitre of plasma and 6.35 MAFs. In 36 those patients whose tissue biopsy was positive for G719S mutation, mutant alleles could also be 37 detected in plasma using both ddPCR and COBAS® System. Finally, when mutational status was 38 studied using both genotyping techniques, higher mutant copies/ml and higher mutant allele fraction 39 (MAF) correlated with higher Semiquantitative Index obtained by COBAS®. Conclusions: Although tissue biopsies cannot be replaced due to the large amount of information 41 they provide regarding tumor type and structure, liquid biopsy and ddPCR represents a new 42 promising strategy for genetic analysis of tumors from plasma samples. In the present study, G719S 43 mutation was detected in a highly sensitive manner, allowing its monitorization with a non-invasive 44 technique