Publications by authors named "Raquel Buj"

17 Publications

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DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A.

J Cell Biol 2021 Aug 26;220(8). Epub 2021 May 26.

Department of Pharmacology and Chemical Biology, University of Pittsburgh Medical Center Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.
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http://dx.doi.org/10.1083/jcb.202008101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8160577PMC
August 2021

Overexpression of oncogenic H-Ras in hTERT-immortalized and SV40-transformed human cells targets replicative and specialized DNA polymerases for depletion.

PLoS One 2021 7;16(5):e0251188. Epub 2021 May 7.

Department of Pathology, The Jake Gittlen Laboratories for Cancer Research, Penn State University College of Medicine, Hershey, Pennsylvania, United States of America.

DNA polymerases play essential functions in replication fork progression and genome maintenance. DNA lesions and drug-induced replication stress result in up-regulation and re-localization of specialized DNA polymerases η and κ. Although oncogene activation significantly alters DNA replication dynamics, causing replication stress and genome instability, little is known about DNA polymerase expression and regulation in response to oncogene activation. Here, we investigated the consequences of mutant H-RAS G12V overexpression on the regulation of DNA polymerases in h-TERT immortalized and SV40-transformed human cells. Focusing on DNA polymerases associated with the replication fork, we demonstrate that DNA polymerases are depleted in a temporal manner in response to H-RAS G12V overexpression. The polymerases targeted for depletion, as cells display markers of senescence, include the Pol α catalytic subunit (POLA1), Pol δ catalytic and p68 subunits (POLD1 and POLD3), Pol η, and Pol κ. Both transcriptional and post-transcriptional mechanisms mediate this response. Pol η (POLH) depletion is sufficient to induce a senescence-like growth arrest in human foreskin fibroblast BJ5a cells, and is associated with decreased Pol α expression. Using an SV-40 transformed cell model, we observed cell cycle checkpoint signaling differences in cells with H-RasG12V-induced polymerase depletion, as compared to Pol η-deficient cells. Our findings contribute to our understanding of cellular events following oncogene activation and cellular transformation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0251188PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104423PMC
May 2021

Loss of p16: A Bouncer of the Immunological Surveillance?

Life (Basel) 2021 Apr 2;11(4). Epub 2021 Apr 2.

UPMC Hillman Cancer Center, Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

p16 (hereafter called p16) is an important tumor suppressor protein frequently suppressed in human cancer and highly upregulated in many types of senescence. Although its role as a cell cycle regulator is very well delineated, little is known about its other non-cell cycle-related roles. Importantly, recent correlative studies suggest that p16 may be a regulator of tissue immunological surveillance through the transcriptional regulation of different chemokines, interleukins and other factors secreted as part of the senescence-associated secretory phenotype (SASP). Here, we summarize the current evidence supporting the hypothesis that p16 is a regulator of tumor immunity.
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http://dx.doi.org/10.3390/life11040309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065641PMC
April 2021

Suppression of p16 alleviates the senescence-associated secretory phenotype.

Aging (Albany NY) 2021 02 6;13(3):3290-3312. Epub 2021 Feb 6.

Department of Pharmacology and Chemical Biology, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

Oncogene-induced senescence (OIS) is characterized by increased expression of the cell cycle inhibitor p16, leading to a hallmark cell cycle arrest. Suppression of p16 in this context drives proliferation, senescence bypass, and contributes to tumorigenesis. OIS cells are also characterized by the expression and secretion of a widely variable group of factors collectively termed the senescence-associated secretory phenotype (SASP). The SASP can be both beneficial and detrimental and affects the microenvironment in a highly context-dependent manner. The relationship between p16 suppression and the SASP remains unclear. Here, we show that knockdown of p16 decreases expression of the SASP factors and pro-inflammatory cytokines and in multiple models, including OIS and DNA damage-induced senescence. Notably, this is uncoupled from the senescence-associated cell cycle arrest. Moreover, low p16 expression in both cancer cell lines and patient samples correspond to decreased SASP gene expression, suggesting this is a universal effect of loss of p16 expression. Together, our data suggest that p16 regulates SASP gene expression, which has implications for understanding how p16 modulates both the senescent and tumor microenvironment.
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http://dx.doi.org/10.18632/aging.202640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906185PMC
February 2021

ATM inhibition synergizes with fenofibrate in high grade serous ovarian cancer cells.

Heliyon 2020 Sep 29;6(9):e05097. Epub 2020 Sep 29.

Department of Cellular & Molecular Physiology, Penn State College of Medicine, Hershey, PA, USA.

While therapies targeting deficiencies in the homologous recombination (HR) pathway are emerging as the standard treatment for high grade serous ovarian cancer (HGSOC) patients, this strategy is limited to the ~50% of patients with a deficiency in this pathway. Therefore, patients with HR-proficient tumors are likely to be resistant to these therapies and require alternative strategies. We found that the HR gene Ataxia Telangiectasia Mutated (ATM) is wildtype and its activity is upregulated in HGSOC compared to normal fallopian tube tissue. Interestingly, multiple pathways related to metabolism are inversely correlated with expression in HGSOC specimens, suggesting that combining ATM inhibition with metabolic drugs would be effective. Analysis of FDA-approved drugs from the Dependency Map demonstrated that ATM-low cells are more sensitive to fenofibrate, a PPARα agonist that affects multiple cellular metabolic pathways. Consistently, PPARα signaling is associated with expression. We validated that combined inhibition of ATM and treatment with fenofibrate is synergistic in multiple HGSOC cell lines by inducing senescence. Together, our results suggest that metabolic changes induced by ATM inhibitors are a potential target for the treatment of HGSOC.
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http://dx.doi.org/10.1016/j.heliyon.2020.e05097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527645PMC
September 2020

Molecular profiling for acromegaly treatment: a validation study.

Endocr Relat Cancer 2020 06;27(6):375-389

Biomedical Research Networking Center in Rare Diseases (CIBERER), Institute of Health Carlos III (ISCIII), Madrid, Spain.

Pharmacologic treatment of acromegaly is currently based upon assay-error strategy, the first-generation somatostatin receptor ligands (SRL) being the first-line treatment. However, about 50% of patients do not respond adequately to SRL. Our objective was to evaluate the potential usefulness of different molecular markers as predictors of response to SRL. We used somatotropinoma tissue obtained after surgery from a national cohort of 100 acromegalic patients. Seventy-one patients were treated with SRL during at least 6 months under maximal therapeutic doses according to IGF1 values. We analyzed the expression of SSTR2, SSTR5, AIP, CDH1 (E-cadherin), MKI67 (Ki-67), KLK10, DRD2, ARRB1, GHRL, In1-Ghrelin, PLAGL1 and PEBP1 (RKIP) by RT-qPCR and mutations in GNAS gene by Sanger sequencing. The response to SRL was categorized as complete response (CR), partial (PR) or non-response (NR) if IGF1 was normal, between >2<3 SDS or >3 SDS IGF1 at 6 months of follow-up, respectively. From the 71 patients treated, there were 27 CR (38%), 18 PR (25%) and 26 NR (37%). SSTR2, Ki-67 and E-cadherin were associated with SRL response (P < 0.03, P < 0.01 and P < 0.003, respectively). E-cadherin was the best discriminator for response prediction (AUC = 0.74, P < 0.02, PPV of 83.7%, NPV of 72.6%), which was validated at protein level. SSTR5 expression was higher in patients pre-treated with SRL before surgery. We conclude that somatotropinomas showed heterogeneity in the expression of genes associated with SRL response. E-cadherin was the best molecular predictor of response to SRL. Thus, the inclusion of E-cadherin in subsequent treatment-decision after surgical failure may be useful in acromegaly.
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http://dx.doi.org/10.1530/ERC-18-0565DOI Listing
June 2020

p16: cycling off the beaten path.

Mol Cell Oncol 2019 15;6(6):e1677140. Epub 2019 Oct 15.

Department of Cellular & Molecular Physiology, Penn State College of Medicine, Hershey, PA, USA.

p16 (hereafter called p16) is a faithful cellular ally in the fight against tumorigenesis. Although its canonical pathway through retinoblastoma (RB) is well delineated, RB-independent functions for p16 are beginning to emerge. Here we summarize non-canonical roles of p16, including our recent finding on its role in nucleotide metabolism.
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http://dx.doi.org/10.1080/23723556.2019.1677140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816386PMC
October 2019

Suppression of p16 Induces mTORC1-Mediated Nucleotide Metabolic Reprogramming.

Cell Rep 2019 08;28(8):1971-1980.e8

Department of Cellular & Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, USA. Electronic address:

Reprogrammed metabolism and cell cycle dysregulation are two cancer hallmarks. p16 is a cell cycle inhibitor and tumor suppressor that is upregulated during oncogene-induced senescence (OIS). Loss of p16 allows for uninhibited cell cycle progression, bypass of OIS, and tumorigenesis. Whether p16 loss affects pro-tumorigenic metabolism is unclear. We report that suppression of p16 plays a central role in reprogramming metabolism by increasing nucleotide synthesis. This occurs by activation of mTORC1 signaling, which directly mediates increased translation of the mRNA encoding ribose-5-phosphate isomerase A (RPIA), a pentose phosphate pathway enzyme. p16 loss correlates with activation of the mTORC1-RPIA axis in multiple cancer types. Suppression of RPIA inhibits proliferation only in p16-low cells by inducing senescence both in vitro and in vivo. These data reveal the molecular basis whereby p16 loss modulates pro-tumorigenic metabolism through mTORC1-mediated upregulation of nucleotide synthesis and reveals a metabolic vulnerability of p16-null cancer cells.
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http://dx.doi.org/10.1016/j.celrep.2019.07.084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716532PMC
August 2019

Targeting IDH1 as a Prosenescent Therapy in High-grade Serous Ovarian Cancer.

Mol Cancer Res 2019 08 20;17(8):1710-1720. Epub 2019 May 20.

Department of Cellular & Molecular Physiology, Penn State College of Medicine, Hershey, Pennsylvania.

Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer. High-grade serous carcinoma (HGSC) is the most frequently diagnosed and lethal histosubtype of EOC. A significant proportion of patients with HGSC relapse with chemoresistant disease. Therefore, there is an urgent need for novel therapeutic strategies for HGSC. Metabolic reprogramming is a hallmark of cancer cells, and targeting metabolism for cancer therapy may be beneficial. Here, we found that in comparison with normal fallopian tube epithelial cells, HGSC cells preferentially utilize glucose in the TCA cycle and not for aerobic glycolysis. This correlated with universally increased TCA cycle enzyme expression in HGSC cells under adherent conditions. HGSC disseminates as tumor cell spheroids within the peritoneal cavity. We found that wild-type isocitrate dehydrogenase I () is the only TCA cycle enzyme upregulated in both adherent and spheroid conditions and is associated with reduced progression-free survival. IDH1 protein expression is also increased in patients with primary HGSC tumors. Pharmacologic inhibition or knockdown of IDH1 decreased proliferation of multiple HGSC cell lines by inducing senescence. Mechanistically, suppression of IDH1 increased the repressive histone mark H3K9me2 at multiple E2F target gene loci, which led to decreased expression of these genes. Altogether, these data suggest that increased IDH1 activity is an important metabolic adaptation in HGSC and that targeting wild-type IDH1 in HGSC alters the repressive histone epigenetic landscape to induce senescence. IMPLICATIONS: Inhibition of IDH1 may act as a novel therapeutic approach to alter both the metabolism and epigenetics of HGSC as a prosenescent therapy.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-1233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679739PMC
August 2019

Simultaneous isotope dilution quantification and metabolic tracing of deoxyribonucleotides by liquid chromatography high resolution mass spectrometry.

Anal Biochem 2019 03 31;568:65-72. Epub 2018 Dec 31.

AJ Drexel Autism Institute, Drexel University, 3020 Market St Suite 560, Philadelphia, PA, 19104, USA. Electronic address:

Quantification of cellular deoxyribonucleoside mono- (dNMP), di- (dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available CN-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and C isotope tracing from major substrates including C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.
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http://dx.doi.org/10.1016/j.ab.2018.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359880PMC
March 2019

Deoxyribonucleotide Triphosphate Metabolism in Cancer and Metabolic Disease.

Front Endocrinol (Lausanne) 2018 18;9:177. Epub 2018 Apr 18.

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA, United States.

The maintenance of a healthy deoxyribonucleotide triphosphate (dNTP) pool is critical for the proper replication and repair of both nuclear and mitochondrial DNA. Temporal, spatial, and ratio imbalances of the four dNTPs have been shown to have a mutagenic and cytotoxic effect. It is, therefore, essential for cell homeostasis to maintain the balance between the processes of dNTP biosynthesis and degradation. Multiple oncogenic signaling pathways, such as c-Myc, p53, and mTORC1 feed into dNTP metabolism, and there is a clear role for dNTP imbalances in cancer initiation and progression. Additionally, multiple chemotherapeutics target these pathways to inhibit nucleotide synthesis. Less is understood about the role for dNTP levels in metabolic disorders and syndromes and whether alterations in dNTP levels change cancer incidence in these patients. For instance, while deficiencies in some metabolic pathways known to play a role in nucleotide synthesis are pro-tumorigenic (e.g., p53 mutations), others confer an advantage against the onset of cancer (G6PD). More recent evidence indicates that there are changes in nucleotide metabolism in diabetes, obesity, and insulin resistance; however, whether these changes play a mechanistic role is unclear. In this review, we will address the complex network of metabolic pathways, whereby cells can fuel dNTP biosynthesis and catabolism in cancer, and we will discuss the potential role for this pathway in metabolic disease.
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http://dx.doi.org/10.3389/fendo.2018.00177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915462PMC
April 2018

Kallikreins Stepwise Scoring Reveals Three Subtypes of Papillary Thyroid Cancer with Prognostic Implications.

Thyroid 2018 05;28(5):601-612

1 Program for Predictive and Personalized Medicine of Cancer, Germans Trias i Pujol Research Institute (PMPPC-IGTP) , Badalona, Spain .

Background: Papillary thyroid cancer (PTC) is the most common type of thyroid cancer. Unlike most cancers, its incidence has dramatically increased in the last decades mainly due to increased diagnosis of indolent PTCs. Adequate risk stratification is crucial to avoid the over-treatment of low-risk patients, as well as the under-treatment of high-risk patients, but the currently available markers are still insufficient. Kallikreins (KLKs) are emergent biomarkers in cancer, but their involvement in PTC is unknown.

Methods: This study analyzed DNA methylation (HumanMethylation arrays) and gene expression (RNA-Seq) of KLKs, BRAF and RAS mutations, and clinical data from four published thyroid cancer data sets including normal and tumor tissues (n = 73, n = 475, n = 20, and n = 82) as discovery, training, and validation series. The C4.5 classification algorithm was used to generate a decision tree. Disease-free survival was estimated using Kaplan-Meier and Cox approaches. Specific analyses were performed using real-time polymerase chain reaction and immunohistochemistry.

Results: The entire KLK family was deregulated in PTC, displaying a specific epigenetic and transcriptional profile strongly associated with BRAF or RAS mutations. Thus, a decision-tree algorithm was developed based on three KLKs with >80% sensitivity and >95% specificity, identifying BRAF- and RAS-mutated tumors. Notably, tumors lacking these mutations were classified as BRAF- or RAS-like. Most importantly, the KLK algorithm uncovered a novel PTC subtype showing favorable prognostic features.

Conclusions: The KLK algorithm could lead to a new clinically applicable strategy with important implications for the risk stratification of PTC and the management of patients.
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http://dx.doi.org/10.1089/thy.2017.0501DOI Listing
May 2018

Increased Global DNA Hypomethylation in Distant Metastatic and Dedifferentiated Thyroid Cancer.

J Clin Endocrinol Metab 2018 02;103(2):397-406

Consortium for the Study of Thyroid Cancer, CECaT, Barcelona, Spain.

Context: Global DNA hypomethylation is a major event for the development and progression of cancer, although the significance in thyroid cancer remains unclear. Therefore, we aimed to investigate its role in thyroid cancer progression and its potential as a prognostic marker.

Methods: Global hypomethylation of Alu repeats was used as a surrogate marker for DNA global hypomethylation, and was assessed using the Quantification of Unmethylated Alu technique. Mutations in BRAF and RAS were determined by Sanger sequencing.

Results: Ninety primary thyroid tumors were included [28 low-risk differentiated thyroid cancer (DTC), 13 pediatric DTC, 33 distant metastatic DTC, 7 poorly differentiated thyroid cancer (PDTC), and 9 anaplastic thyroid cancer (ATC)], as well as 24 distant metastases and 20 normal thyroid tissues. An increasing hypomethylation was found for distant metastatic DTC [median, 4.0; interquartile range (IQR), 3.1 to 6.2] and PDTC/ATC (median, 9.3; IQR, 7.0 to 12.1) as compared with normal thyroid tissue (median, 2.75; IQR, 2.30 to 3.15), whereas low-risk and pediatric DTC were not affected by hypomethylation. Alu hypomethylation was similar between distant metastases and matched primary tumors. Within distant metastatic DTC, Alu hypomethylation was increased in BRAF vs RAS mutated tumors. Kaplan-Meier and Cox regression analyses showed that thyroid cancer-related and all-cause mortality were associated with tumor hypomethylation, but this association was lost after adjustment for thyroid cancer risk category.

Conclusion: Distant metastatic DTC, PDTC, and ATC were increasingly affected by global Alu hypomethylation, suggesting that this epigenetic entity may be involved in thyroid cancer progression and dedifferentiation.
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http://dx.doi.org/10.1210/jc.2017-01613DOI Listing
February 2018

DNA methylation profiling identifies PTRF/Cavin-1 as a novel tumor suppressor in Ewing sarcoma when co-expressed with caveolin-1.

Cancer Lett 2017 02 25;386:196-207. Epub 2016 Nov 25.

Sarcoma Research Group, Institut d'Investigació Biomèdica de Bellvitge-IDIBELL, L'Hospitalet de Llobregat, Barcelona, 08908, Spain. Electronic address:

Epigenetic modifications have been shown to be important in developmental tumors as Ewing sarcoma. We profiled the DNA methylation status of 15 primary tumors, 7 cell lines, 10 healthy tissues and 4 human mesenchymal stem cells lines samples using the Infinium Human Methylation 450K. Differential methylation analysis between Ewing sarcoma and reference samples revealed 1166 hypermethylated and 864 hypomethylated CpG sites (Bonferroni p < 0.05, δ-β-value with absolute difference of >0.20) corresponding to 392 and 470 genes respectively. Gene Ontology analysis of genes differentially methylated in Ewing sarcoma samples showed a significant enrichment of developmental genes. Membrane and cell signal genes were also enriched, among those, 11 were related to caveola formation. We identified differential hypermethylation of CpGs located in the body and S-Shore of the PTRF gene in Ewing sarcoma that correlated with its repressed transcriptional state. Reintroduction of PTRF/Cavin-1 in Ewing sarcoma cells revealed a role of this protein as a tumor suppressor. Restoration of caveolae in the membrane of Ewing sarcoma cells, by exogenously reintroducing PTRF, disrupts the MDM2/p53 complex, which consequently results in the activation of p53 and the induction of apoptosis.
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http://dx.doi.org/10.1016/j.canlet.2016.11.020DOI Listing
February 2017

Quantification of unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples.

Oncotarget 2016 Mar;7(9):10536-46

Institute of Predictive and Personalized Medicine of Cancer (IMPPC), Badalona, Barcelona, Spain.

Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings.
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http://dx.doi.org/10.18632/oncotarget.7233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891138PMC
March 2016

DNA methylation profiling of well-differentiated thyroid cancer uncovers markers of recurrence free survival.

Int J Cancer 2014 Aug 13;135(3):598-610. Epub 2014 Jan 13.

Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

Thyroid cancer is a heterogeneous disease with several subtypes characterized by cytological, histological and genetic alterations, but the involvement of epigenetics is not well understood. Here, we investigated the role of aberrant DNA methylation in the development of well-differentiated thyroid tumors. We performed genome-wide DNA methylation profiling in the largest well-differentiated thyroid tumor series reported to date, comprising 83 primary tumors as well as 8 samples of adjacent normal tissue. The epigenetic profiles were closely related to not only tumor histology but also the underlying driver mutation; we found that follicular tumors had higher levels of methylation, which seemed to accumulate in a progressive manner along the tumorigenic process from adenomas to carcinomas. Furthermore, tumors harboring a BRAF or RAS mutation had a larger number of hypo- or hypermethylation events, respectively. The aberrant methylation of several candidate genes potentially related to thyroid carcinogenesis was validated in an independent series of 52 samples. Furthermore, through the integration of methylation and transcriptional expression data, we identified genes whose expression is associated with the methylation status of their promoters. Finally, by integrating clinical follow-up information with methylation levels we propose etoposide-induced 2.4 and Wilms tumor 1 as novel prognostic markers related to recurrence-free survival. This comprehensive study provides insights into the role of DNA methylation in well-differentiated thyroid cancer development and identifies novel markers associated with recurrence-free survival.
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http://dx.doi.org/10.1002/ijc.28703DOI Listing
August 2014

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

BMC Mol Biol 2013 Aug 20;14(1):18. Epub 2013 Aug 20.

Department of Brain Ischemia and Neurodegeneration, Institut d'Investigacions Biomèdiques de Barcelona (IIBB)-Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.

Background: Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous.

Results: We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells.

Conclusions: We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.
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http://dx.doi.org/10.1186/1471-2199-14-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765358PMC
August 2013
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