cancer epigenetics and metabolism: connecting the dots€¦ · cancer epigenetics and metabolism:...

CANCER EPIGENETICSCANCER EPIGENETICS

AND METABOLISM:AND METABOLISM:

Connecting the DotsConnecting the Dots

UC BiotechUC BiotechCantanhede, PortugalCantanhede, Portugal

2-3 February 20152-3 February 2015

CAN CER EP I GEN ET ICS AN D METAB OL ISM: Connec t ing the Do ts Ca n tanhede , Po r tuga l 2 - 3 Feb r ua ry 2015

Scientific Commission:Scientific Commission:

Paulo J. Oliveira, Ph.D. (CNC, Coimbra, Portugal)

Frederick Domann, Ph.D. (University of Iowa, Iowa City, IA, U.S.A.)

Maria T. Cunha-Oliveira, Ph.D. (CNC, Coimbra, Portugal)

Vilma A. Sardão, Ph.D. (CNC, Coimbra, Portugal)

Teresa L. Serafim, Ph.D. (CNC, Coimbra, Portugal)

Ignacio Vega-Naredo, Ph.D. (CNC, Coimbra, Portugal)

Organization:Organization:

Paulo J. Oliveira, Ph.D. (CNC, Coimbra, Portugal)

Maria T. Cunha-Oliveira (CNC, Coimbra, Portugal)

Luciana Ferreira (CNC, Coimbra, Portugal)

André Ferreira (CNC, Coimbra, Portugal)

Sandra Tomé (Biocant Park, Cantanhede, Portugal)

Leonor Jesus (CNC, Coimbra, Portugal)

Teresa L. Serafim (CNC, Coimbra, Portugal)

Vilma A. Sardão (CNC, Coimbra, Portugal)

Ignacio Vega-Naredo (CNC, Coimbra, Portugal)

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TABLE OF CONTENTSTABLE OF CONTENTS

Program.................................................................................................3Abstracts from invited speakers Day 1................................................5Abstracts from invited speakers Day 2..............................................11Abstracts from selected oral communications Day 1.......................16Abstracts from selected oral communications Day 2.......................21List of Posters......................................................................................26Abstracts from Poster Communications............................................27Sponsors..............................................................................................37

2


PROGRAMPROGRAM

Day 1, February 2

8:30 – 9:15 Registration

9:15 – 9:30 Opening of the meeting

9:30 – 10:30Opening Plenary Lecture, Paolo Sassone-Corsi, University of California – Irvine, USA “Metabolism and epigenetics: the Clock link”

10:30 – 11:15 Valdemar Maximo, IPATIMUP, University of Porto, Portugal “Mitochondrial regulation of epigenetics and its role in human diseases”

11:15 – 11:45 Coffee break, meet the sponsors

11:45 – 12:30 Gabriella Ficz, Barts Cancer Institute, University of London, UK “Role of epimutations in cellular transformation”

12:30 - 13.00 Michael Rhodes, Nanostring Technologies, USA "Translational Research: fromResearch to Diagnostics. The NanoString Digital Technology"

13:00 – 13:20Selected short oral talk S1: Ana S. Rodrigues, CNC, Portugal,“Pyruvate dehydrogenase and pluripotency: can we affect pluripotency metabolically?”

13:20 - 15:00 Lunch and poster session

15:00 – 16.00Plenary lecture, Carmen Jerónimo, Portuguese Oncology Institute-Porto (IPO-Porto) & ICBAS-UP, Portugal “Epigenetic deregulation in urological tumors: from biology to clinics”

16:00 – 16:45

Florian Pauler, Research Center for Molecular Medicine of the Austrian Academy of Sciences, Austria “Imprinted long non coding (lnc) RNAs as a paradigm for functional lncRNAs in development and cancer”


17:15 – 17:35

Selected short oral talk S2: Ricardo P. Neves, CNC, Portugal and MRC Molecular Haematology Unit, University of Oxford, UK “Connecting the dots between cellular mitochondrial content and global variability in gene expression and alternative splicing”

17:35 – 17:55Selected short oral talk S3: Teresa L. Serafim, CNC, Portugal, “Mitochondrial characterization of human breast cancer cell lines for in vitro cancer research”

17:55 – 18:15

Selected short oral talk S4: Maria I. Almeida, INEB and Instituto de Investigação e Inovação em Saúde, Portugal “Extracellular vesicles derived from mesenchymal stem/stromal cells contain microRNAs involved in osteoblast differentiation”

20:00 Meeting dinner

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CAN CE R EP IGEN ET IC S AN D META BOL IS M: Connec t ing the Do ts Can tanhede , Po r tuga l 2 - 3 F ebr ua r y 2015

DAY 2, February 3

9:30 – 10:30

Plenary Lecture, Frederick Domann, University of Iowa, USA “Mitochondrial redox metabolism and its influence on the epigenetic control of gene expression”

10:30 – 11:15John G. Jones, CNC, University of Coimbra, Portugal “Metabolic flux profiling of cancer cells using deuterated water and 13C-tracers”


11:45 – 12:30

Maarten Van Lohuizen, Netherlands Cancer Institute, Amsterdam, The Netherlands “Unexpected results of prolonged Ezh2 inhibition in an in vivo model for Glioblastoma”

12:30 – 12:50

Selected short oral talk S5: Fátima Martel, Faculty of Medicine, University of Porto, Portugal “The anticarcinogenic effect of the dietary compound kaempferol in a human breast cancer cell line is dependent on inhibition of glucose cellular uptake”

12:50 – 15:00 Lunch and poster session

15:00 – 15.45João T. Barata, Institute of Molecular Medicine, Lisbon, Portugal “Signaling, Oncogenes and T-cell Leukemia”

15:45 – 16:45Roundtable “Cancer Epigenetics and Metabolism: Connecting the Dots?”Moderated by: Frederick Domann, University of Iowa, USA


17:15 – 17:35Selected short oral talk S6: Luciana Ferreira, CNC, Portugal, “Persistent effects inmitochondrial genes transcription after incubation of H9c2 cells with the anticancer drug Doxorubicin: an epigenetic origin?”

17:35 – 17:55

Selected short oral talk S7: Sofia Gomes, Faculty of Pharmacy, University of Lisbon, Portugal “miR-143 and miR-145 overexpression in HCT116 colon cancer cells enhances cetuximab-mediated antibody-dependent cellular cytotoxicity leading to increased apoptosis”

17:55 – 18:15 Selected short oral talk S8: Marcelo Correia, CNC, Portugal, “Histone deacetylaseproteins: a possible glycolytic metabolic shifter in embryonic stem cells”

18:15 – 18:30 Closing session

44


ABSTRACTS FROM ABSTRACTS FROM

INVITED SPEAKERS INVITED SPEAKERS

DAYDAY

11

5


OPENING PLENARY LECTURE

Paolo Sassone-Corsi

Center for Epigenetics and Metabolism,

University of California, Irvine, California 92697,

USA; [email protected]

“COMMON THREADS: EPIGENETICS, METABOLISM AND THE CLOCK”

Circadian rhythms govern a number of fundamental physiological functions in almost allorganisms, from prokaryotes to humans. The circadian clocks are intrinsic time-trackingsystems with which organisms can anticipate environmental changes and adapt to theappropriate time of day. Disruption of these rhythms can have a profound influence to humanhealth and has been linked to depression, insomnia, jet lag, coronary heart disease,neurodegenerative disorders and cancer. At the heart of circadian regulatory pathways is theclock machinery, a remarkably coordinated transcription-translation system that utilizes alsodynamic changes in chromatin transitions and epigenetic control. Recent findings indicate thatregulation goes also the other way, since specific elements of the clock are able to sensechanges in the cellular metabolism. We have uncovered the role of the deacetylases SIRT1 andSIRT6 in partitioning of the circadian epigenome, as well as the contribution of MLL1 as H3K4methyltransferase. Notably, chromosome capture 4C studies have revealed that circadian genesare organized in nuclear interactomes that dictate their coordinated expression,Understanding in full detail the intimate links between cellular metabolism and the circadianclock machinery will provide not only critical insights into system physiology andendocrinology, but also novel avenues for pharmacological intervention towards metabolicdisorders.

66


Valdemar Maximo

Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465 Porto, Portugal; 2Department of Pathology and Oncology, Medical Faculty of the University

of Porto, 4200-319 Porto.

“MITOCHONDRIAL REGULATION OF EPIGENETICS AND ITS ROLE IN HUMANDISEASES”

Mitochondria have a central role in energy uptake and energy production, and as consequencethey play a key role in a wide variety of pathological conditions, such as cancer,neurodegenerative diseases, diabetes and aging. Indeed, mitochondrial dysfunction, andespecially mitochondrial dysfunction caused by mutations in mtDNA have been implicated in awide range of age related pathologies. The mitochondrial activity is largely dependent on theenvironmental availability of nutrients, namely carbohydrates, proteins and fats, and theirconversion, mainly by glycolysis and oxidative phosphorylation (OXPHOS), in energy richcompounds, such as adenosine triphosphate (ATP), acetyl- Coenzyme A (acetyl-CoA), S-adenosyl-methionine (SAM), and nicotinamide adenine dinucleotide (NADH). ATP, acetyl-CoA,SAM, and NAD+/NADH, in turn, are the high-energy substrates for protein (including histones)phosphorylation, acetylation and deacetylation reactions, and DNA and protein methylation,thus contributing to the regulation of several cell signalling pathways and cell epigeneticlandscape. Thus, the interrelationship between mtDNA, mitochondrial activity and cellmetabolism have an essential role in the maintenance of the levels of essential co-factors forcell epigenetic mechanisms regulation, such as DNA methylation and protein acetylation.

The main goal of this study was to unveil how pathogenic mutations in mtDNA and/ormitochondrial variants may lead to epigenetic changes, namely alterations in the pattern ofnuclear DNA methylation and/or acetylation of histones and other proteins.

Our data, showed that mitochondrial dysfunction, caused either by mtDNA mutations orabsence of mtDNA, as well as the presence of specific mitochondrial variants (different mtDNAhaplogroups) induce changes in cell metabolic profile, complex I activity, DNA methylation andprotein acetylation. Furthermore, we observed that mitochondrial dysfunction caused bydepletion of mtDNA, leads to the readjustment of several cellular processes through theacetylation of several proteins, allowing the cell to survive and maintain its homeostasis.

Acknowledgements: This work was partially supported by the Portuguese Science and Technology Foundation(FCT) through the project (PIC/IC/83037/2007). Further funding was obtained from the project ‘Microenvironment,metabolism and cancer’ partially supported by Programa Operacional Regional do Norte (ON.2—O Novo Norte),

under the Quadro de Referência Estratégico Nacional (QREN), and through the Fundo Europeu deDesenvolvimento Regional (FEDER). IPATIMUP is an associate laboratory of the Portuguese Ministry of Science,

Technology and Higher Education and is partially supported by the FCT.

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Gabriella Ficz

Centre for Haemato-Oncology, Barts Cancer Institute, EC1M 6BQ, London, UK

“EPIGENETICS OF GROUND STATE PLURIPOTENCY”

DNA methylation is the last bottleneck in the reprogramming process and incomplete erasurealters the differentiation potential of induced pluripotent cells. We have recently described themechanism of DNA methylation erasure in mouse embryonic stem cells (ESCs) through FGFsignalling inhibition (Erk1/2 and Gsk3b, called 2i) to achieve the blastocyst-like epigeneticground state pluripotency. We showed by whole-genome bisulphite sequencing that 2i inducesrapid and genome-wide demethylation on a scale and pattern similar to that in migratoryprimordial germ cells (PGCs) and early embryos. Major satellites, intracisternal A particles(IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involvesoxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impairedmaintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a andDnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory regionin Dnmt3b that is highly responsive to signaling. These insights provide a framework forunderstanding how signaling pathways regulate reprogramming to an epigenetic ground stateof pluripotency.

88


PLENARY LECTURE

Carmen Jerónimo

Cancer Biology & Epigenetics Group - Research Center; Portuguese Oncology Institute-Porto(IPO-Porto), Porto, Portugal & & Abel Salazar Biomedical Sciences Institute (ICBAS), University

of Porto, Portugal

“EPIGENETIC DEREGULATION IN UROLOGICAL TUMORS: FROM BIOLOGYTO CLINICS”

The most common genitourinary (GU) neoplasms (i.e., prostate, bladder, and kidney cancers),represent the second most prevalent group of tumors only surpassed by lung cancer. All threeare generally clinically silent at their earliest stages, when curative treatment is most likelysuccessful. However, there are no consensual guidelines for GU cancer screening and availablemethods are characterized by suboptimal sensitivity and specificity. Moreover, standardclinical and pathological parameters meet with important limitations in the assessment ofprognosis in an individual basis. An increasing body of evidence is implicating severalepigenetic mechanisms as driving forces of neoplastic transformation. Among these, aberrantDNA methylation is, by far, the most widely studied, followed by deregulated expression ofchromatin machinery proteins and microRNAs. Importantly, cancer-related epigeneticalterations may provide novel cancer biomarkers, intended for early detection and diagnosis,assessment of prognosis, and prediction of response to therapy. The importance of earlydiagnosis in Oncology has been emphasized and although current methodologies (such ascytology, histopathology, immunohistochemistry, etc) play a critical role, molecular markersmay complement or, eventually, replace them if more effective. Because epigenetic alterationsoften precede the emergence of the malignant phenotype, they are advantageous for earlycancer detection. They might also be detected in clinical samples, including those obtained byexfoliative cytology, endoscopic brush techniques, and biopsy, as well as urine, saliva, stool,plasma and sputum samples, allowing for non- or minimally invasive molecular detection oftumors. Moreover, early detection of cancer relapse before it manifests clinically (or onimaging during routine patient follow-up) might be accomplished through epigenetic-basedbiomarkers. Some epigenetic alterations have also been associated with tumor behavior and,thus, may serve as biomarkers for prognosis.

In my presentation I will focus on the major findings obtained by our research team regardingepigenetic mechanisms implicated in urological tumorigenesis as well as the usefulness ofepigenetic-based biomarkers for detection and prognosis in these cancers.

99


Florian Pauler

CeMM Research Center for Molecular Medicineof the Austrian Academy of Sciences,Lazarettgasse 14, AKH BT 25.31090 Vienna, Austria

“IMPRINTED LONG NON CODING (LNC) RNAS AS A PARADIGM FORFUNCTIONAL LNCRNAS IN DEVELOPMENT AND CANCER”

Genomic imprinting is a developmentally important, epigenetic phenomenon that restrictsgene expression to one of the two parental alleles in mammals. Protein coding genes withimprinted expression are therefore active on one parental allele and are silenced on the other.Long non-protein coding (lnc) RNAs are long known to play a key role in this silencing process.Therefore imprinted lncRNAs have been discovery models for the lncRNA mediated silencing.For example the study of the imprinted lncRNA Airn in our lab has identified a novel silencingmechanism that is independent of the lncRNA product and is only dependent on lncRNAtranscription. Recent technological advances revealed that lncRNAs are abundant transcriptsin mouse and human. Importantly lncRNA mapping studies found many novel lncRNAs, someof which are promising tumor markers that might be directly involved in cancer relatedprocesses like metastasis formation. However our work revealed that widely usedbioinformatics pipelines to identify lncRNAs might miss important transcripts like imprintedlncRNAs that have an unusual RNA biology. After the association of lncRNAs with cancer anddisease it is now the next big challenge to identify the mode of action of these lncRNAs toultimately interfere with this function for therapeutic purposes. The findings and toolsestablished for and from the study of imprinted lncRNAs will help in this endeavor.

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INVITED SPEAKERS INVITED SPEAKERS

DAY DAY

22

11


PLENARY LECTURE

Frederick Domann

Department of Radiation Oncology, University of Iowa, Iowa City, IA 52246 USA

“MITOCHONDRIAL REDOX METABOLISM AND ITS INFLUENCE ON THEEPIGENETIC CONTROL OF GENE EXPRESSION”

The earliest eukaryotic life forms simultaneously adopted mitochondria and chromatin, thoughthe molecular explanations for this and the mechanistic link between these apparentlysimultaneous evolutionary events remains unknown. Cells respond to physiological andenvironmental triggers through several key pathways including response to reactive oxygenspecies, nutrient and ATP sensing, DNA damage response, and epigenetic alterations.Mitochondria play a central role in these pathways not only through energetics and ATPproduction but also through metabolites generated in the tricarboxylic acid cycle, alterations inthe balance of redox couples such as NAD+/NADH, and in mitochondria-nuclear signalingrelated to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, andepigenetic regulation. This presentation examines bidirectional interactions betweenmitochondria and cellular pathways in response to physiological and environmental cues witha focus on epigenetic regulation of biological processes that respond to endogenous andexogenous signals through changes in homeostasis and altered mitochondrial function. Whenthe discovery that the enzymes responsible for initiating and perpetuating epigenetic events islinked to metabolism by their cofactors a new paradigm for cellular retrograde signaling canbe created. Here, we summarize the foundation of such a paradigm on the origins of cancer, inwhich metabolic alterations create an epigenetic progenitor that clonally expands to becomecancer. We suggest that metabolic alterations disrupt the production and availability ofcofactors such as S-adenosylmethionine, α-ketoglutarate, NAD(+), and acetyl-CoA to modify theepigenotype of cells. We further speculate that redox biology can change epigenetic eventsthrough oxidation of enzymes and alterations in metabolic cofactors that affect epigeneticevents such as DNA methylation. Combined, these metabolic and redox changes serve as thefoundation for altering the epigenotype of normal cells and creating the epigenetic progenitorof cancer. A more comprehensive understanding of the retrograde signaling mechanismsbetween the mitochondria and nucleus is central to elucidating the integration ofmitochondrial functions with other cellular responses with respect to disease. Recent studieshave highlighted the importance of mitochondrial functions in epigenetic regulation. Furtherdevelopment and application of novel technologies, enhanced experimental models, and asystems-type research approach will help to discern how mitochondrial dysfunction affects keyepigenetic pathways.

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John G. Jones

CNC, University of Coimbra, Portugal

“METABOLIC FLUX PROFILING OF CANCER CELLS USING DEUTERATEDWATER AND 13C-TRACERS”

Cancer cells are programmed to be highly anabolic such that biosynthesis of lipid andnucleotides are able to sustain high rates of proliferation. At the same time, they show amarked dependence on glycolytic over mitochondrial ATP generation for energy needs. Abetter understanding of the relationship between the biosynthetic carbon and energy demandsversus carbon and energy supply of cancer cells may identify critical metabolic bottlenecksthat may represent novel therapeutic targets for their selective demise. To help identify suchtargets, it is important to develop metabolic tracer assays with high coverage of catabolic andanabolic fluxes. To this end, the presentation will focus on how this may be achieved by theintegration of deuterated water (2H2O) - an universal tracer of biosynthetic pathways - with 13C-tracers, which monitor the fate of specific substrates into catabolic and anabolic pathways.This will include methodological advances in NMR and MS measurements of metabolite 2H and13C-enrichment assays and novel metabolic activities of cancer cells that have been revealed bysuch approaches.

1313


Maarten Van Lohuizen

Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands

“UNEXPECTED RESULTS OF PROLONGED EZH2 INHIBITION IN AN IN VIVOMODEL FOR GLIOBLASTOMA”

Repressive Polycomb-group (Pc-G) protein complexes and the counteracting Trithorax-group(Trx-G) of nucleosome remodeling factors are involved in the dynamic maintenance of propergene expression patterns during development, acting at the level of chromatin structure. Assuch, they are important controllers of cell fate and differentiation. When deregulated, thesemaster switches of gene expression are strongly implicated in formation of a diverse set ofcancers. Examples are the Pc-G gene Bmi1 and Ezh2 which are overexpressed in many cancersincluding Glioblastoma. However, recently Ezh2 has also been found to be mutated/inactivatedin other forms of cancer, suggesting a highly context-dependent role as oncogene or tumorsuppressor. An outstanding question is how to identify among the many Pc-G bound genes thecancer-relevant ones. hereto we have recently combined stringent ChIP-seq with customshRNAi library screening in in vivo models for glioblastoma. As Ezh2 is associated with poorprognosis and metastasis in many cancer settings there is an increasing interest fordevelopment of selective Ezh2 inhibitors as potential new ways for epigenetic cancer therapy.We have developed conditional shRNAi inhibition in mouse models for aggressive Glioma tostudy the effects of Ezh2 inhibition on tumor initiation and maintenance. Whereas initialtumor regression upon Ezh2 inhibition was observed in vivo, prolonged Ezh2 inhibition causedunexpected profound changes in tumor plasticity, differentiation status with importantconsequences for tumor progression and treatment options, which will be discussed.

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João T. Barata

Instituto de Medicina Molecular, Lisbon University Medical School, Portugal l

“SIGNALING, ONCOGENES AND T-CELL LEUKEMIA”

Interleukin 7 (IL-7) and its receptor (IL-7R), formed by IL-7Rα (encoded by IL7R) and γc, areessential for normal T-cell development and homeostasis. However, IL-7/IL-7R-mediatedsignaling can also have a “dark side”, promoting T-cell leukemia proliferation in vitro andexpansion in vivo. In addition to the evidence that IL-7 present in the tumor microenvironmenthas a role in accelerating leukemia progression, we and others found that around 10% of T-cellacute lymphoblastic leukemia (T-ALL) patients display oncogenic IL7R gain-of-functionmutations. The IL-7/IL-7R axis activates phosphoinositide 3-kinase/Akt/mammalian target ofrapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, cell cycle progression andgrowth of T-ALL cells. IL-7-mediated PI3K/Akt/mTOR-dependent survival of leukemia cellsrelies, at least in part, on the upregulation of GLUT1 and increased glucose uptake. GLUTactivity appears to associate with IL-7-tiggered upregulation of reactive oxygen species, which,surprisingly, are required for T-ALL cell viability. Interestingly, our RNA sequencing studiesindicate that the impact of IL-7 on the regulation of metabolism-related genes in T-ALL extendsbeyond GLUT1 and suggest that IL-7/IL-7R-mediated signaling may promote a broadtranscriptional program with impact on metabolism. Moreover, we found that IL-7 regulates T-ALL cell viability not only by regulating apoptosis but also autophagy. Notably, IL-7 does so in acomplex manner that involves the activation of both anti-autophagic (PI3K/Akt/mTOR) and pro-autophagic (MEK/Erk) pathways, which ultimately allows IL-7 to promote leukemia cellviability in opposite scenarios (e.g. nutrient-rich versus nutrient-poor conditions) by eitherpreventing or inducing autophagy.

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SELECTED ORAL COMMUNICATIONSSELECTED ORAL COMMUNICATIONS

DAYDAY

11

16


SELECTED SHORT ORAL TALK S1

Ana S. Rodrigues1,2, Marcelo Correia1,2,3, Tânia Perestrelo1,2,3, Sandro L.Pereira2, Maria I. Sousa2,3, Marcelo F. Ribeiro2,4, João Ramalho-Santos2,3,4

1PhD Programme in Experimental Biology and Biomedicine (PDBEB), CNC - Center forNeuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 2Biology of

Reproduction and Stem Cell Group, CNC - Center for Neuroscience and Cell Biology, Universityof Coimbra, Portugal, 3Institute for Interdisciplinary Research (IIIUC), University of Coimbra,

Portugal, 4Department of Life Sciences, Faculty of Sciences and Technology, University ofCoimbra, Coimbra, Portugal

“PYRUVATE DEHYDROGENASE AND PLURIPOTENCY: CAN WE AFFECTPLURIPOTENCY METABOLICALLY?”

Similarly to Inner Cell Mass cells and to some types of tumours, embryonic stem cells (ESC) relymostly on glycolysis for energy supply. ESC present “silent” mitochondria with a low oxidativephosphorylation activity; a high activity of hexokinase II (HK) to favour glucose use, and aphosphorylated (inactive) pyruvate dehydrogenase (PDH) complex to steer pyruvate away fromthe Krebs Cycle. The regulation of PDH activity thus plays an important role in the metabolicflexibility. In order to better understand the metabolic profile of these cells we looked to theregulation of the PDH complex and HK in pluripotent vs. differentiated mouse ESC, with theultimate goal of manipulating metabolism and possibly pluripotency. We set an experimentaldesign using PDHK inhibitor Dichloroacetate (DCA) and the HK inhibitor 3-Bromopyruvate(3BP). Overall viability was not altered but proliferation was affected. Pluripotency wasnegatively affected with both compounds elevating mitochondrial membrane potential andROS production causing alterations in the energetic status of these cells. Also DCA is promotinga PDH more active, which is translated in a more oxidative state, the same was observed for3BP though the effect was not as relevant. Interestingly we also observe a negative effect ofboth compounds on glycolytic enzymes such as HK, Gapdh and PKM. To better understand thepossible metabolic shift we evaluated protein and mRNA levels for Hif 1alfa and p53. Bothcompounds lead to a decrease in both Hif1 alfa and p53 protein levels.

Manipulating metabolism will cause a shift in pluripotency accompanied with mitochondrialalterations and altered metabolic pathways. More importantly we established for the first timethat loss of pluripotency is accompanied by alterations on the PDH cycle independently werewe inhibit metabolism.

Acknowledgements: FCT for funding (grants PTDC/EBB-EBI/101114/2008, PTDC/EBB-EBI/120634/2010 andPTDC/QUI-BIQ/120652/2010. QREN for funding (CENTRO-01-0762-FEDER-00204. CNC funding is supported by FCT(Pest-C/SAU/LA000172011). PhD scholarships attributed to M.C. (SFRH/BD/51681/2011), T.P. (SFRH/BD/51684/2011)

and M.S. (SFRH/BD/86260/2012).

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Ricardo Pires das Neves1,2, Alberto Rastrojo3, Ana Lima1, Begoña Aguado3,Raul Guantes4, Francisco J Iborra2,5

1UC Biotech – Centre for Neurosciences and Cell Biology, Biocant Technology Park, 3060-197Cantanhede, Portugal; 2MRC Molecular Haematology Unit, Weatherall Institute of MolecularMedicine, John Radcliffe Hospital, University of Oxford Headington, Oxford, OX3 9DS, UK;

3Centro Biología Molecular “Severo Ochoa”, CSIC-UAM, C/ Nicolás Cabrera 1; Campus deCantoblanco, 28049 Madrid, Spain; 4Department of Condensed Matter Physics, MaterialsScience Institute ‘Nicolás Cabrera’ and Institute of Condensed Matter Physics (IFIMAC)

Universidad Autónoma de Madrid; Campus de Cantoblanco, 28049 Madrid, Spain; 5CentroNacional de Biotecnología, CSIC, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain.

[email protected]

“CONNECTING THE DOTS BETWEEN CELLULAR MITOCHONDRIAL CONTENTAND GLOBAL VARIABILITY IN GENE EXPRESSION AND ALTERNATIVE

SPLICING.”

Noise in gene expression is a main determinant of phenotypic variability. Increasingexperimental evidence suggests that genome-wide cellular constraints largely contribute to theheterogeneity observed in gene products. It is still unclear, however, which global factorsmostly affect gene expression noise, and to which extent. We have previously shown that thenumber of mitochondria transmitted to daughter cells at mitosis presents broad fluctuationsand this constitutes an important source of variability in transcription rates. The speed oftranscription elongation is directly linked to ATP concentration and to the levels ofmitochondria inside the cell. Using high content image analysis we have simultaneouslymonitored mitochondrial content and the levels of different proteins or markers of geneexpression activity to quantify the contribution of mitochondria to the global variability ingene expression. We found that changes in mitochondria content can account for ~50% of thevariability observed in protein levels. This is the combined result of the effect of mitochondriadosage on transcription and translation apparatus content and activities. Moreover, we foundthat mitochondrial levels have a large impact on alternative splicing, thus modulating both theabundance and type of mRNAs. A simple mathematical model where mitochondrial contentsimultaneously affects transcription rate and splicing site choice can explain the alternativesplicing data. The results of this study suggest that cells use mitochondrial content (and/orprobably function) to control mRNA abundance, translation and alternative splicing, whichmay ultimately impact on cellular phenotype.

Acknowledgments: part of this work was co-funded by FEDER (QREN), through Programa Mais Centro underprojects CENTRO-07-ST24-FEDER-002008, and through Programa Operacional Factores de Competitividade -

COMPETE and National funds via FCT – Fundação para a Ciência e a Tecnologia under project(s) Pest-C/SAU/LA0001/2013-2014 and PTDC/SAU-ENB/113696/2009. Ana Lima has a FCT scholarship SFRH/BD/51942/2012.

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Teresa L. Serafim1, Teresa Cunha-Oliveira1, Vilma A. Sardão1, Cláudia Deus1,Amy Greene2, Edward Perkins2 and Paulo J. Oliveira1.

1Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2Department ofBiomedical Sciences, Mercer University School of Medicine, Savannah Campus, Georgia, USA

“MITOCHONDRIAL CHARACTERIZATION OF HUMAN BREAST CANCER CELLLINES FOR IN VITRO CANCER RESEARCH.”

Mitochondria are organelles present in all nucleated cells, being critical for many cellularfunctions, such as ATP production by oxidative phosphorylation (OXPHOS), cell deathregulation, reactive oxygen species (ROS) generation and ionic homeostasis. Therefore,mitochondria have been implicated in several diseases, including cancer. In fact,comparatively to normal cells, it is suggested that cancer cells present differences in terms ofmitochondrial physiology that can contribute to the general metabolic remodeling observed incancer cells and which can contribute to different resistance to chemotherapeutics. In thepresent work a set of commercial human breast cancer cell lines (MCF-7, HS578T and MDA-MB-231) grown in high-glucose medium, were characterized regarding mitochondrial physiologywith the goal of detecting differences that can explain specific alterations in terms of behaviorand resistance to classic chemotherapeutics. Additionally, the transition to a glucose-free,galactose/glutamine media, which is known to remodel metabolism to an OXPHOS-based, wasalso evaluated. Specific alterations and cell behavior were compared with human normalbreast MCF-12A cell line. The results show that the mitochondrial membrane potential isgenerally higher in tumor cell lines, especially in the MCF-7 cell line, which present condensedmitochondria network. In galactose/glutamine, a remodeling of the mitochondrial networkoccurred, with decreased membrane potential, elongated mitochondrial network of tumor cellsand increased mobility of HS578T cells. The tumor cell lines had also higher content ofmitochondrial complex subunits, confirmed by the increase of specific mitochondrialtranscripts for OXPHOS components and mitochondrial DNA copy number. When investigatingthe effects of classic mitochondrial poisons, the MCF-7 cell line was more susceptible to thecompounds tested.

The data shows that the three commercial breast cancer cell lines tested presented alterationsin mitochondrial physiology, which may impact overall metabolism and resistance tochemotherapeutic-induced cell death. The work presents a simple framework for the validityof personal anti-cancer treatments based on distinct tumor physiology.

Acknowledgments: the present work was support by grants from the Portuguese Foundation for Science andTechnology (PTDC/QUI-QUI/101409/2008 to PJO and SFRH/BPD/75959/2011 to TLS).

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Maria I. Almeida1,2, Andreia M. Silva1,2,3, Susana G. Santos1,2, Mário A.Barbosa1,2,3

1INEB - Instituto de Engenharia Biomédica, Portugal, 2Faculdade de Engenharia, Universidadedo Porto, Portugal; 2Instituto de Investigação e Inovação em Saúde, Universidade do Porto,

Portugal; 3ICBAS – Instituto de Ciências Biomédica Abel Salazar, Universidade do Porto,Portugal; [email protected]

“EXTRACELLULAR VESICLES DERIVED FROM MESENCHYMALSTEM/STROMAL CELLS CONTAIN MICRORNAS INVOLVED IN OSTEOBLAST

DIFFERENTIATION.”

MicroRNAs are small non-coding RNAs involved in several biological processes important forcancer pathogenesis and tissue regeneration, including cell differentiation. Mesenchymalstem/stromal cells (MSC) have great therapeutic potential since they can differentiate intoosteoblasts, chondroblasts and adipocytes. MSC secrete abundant extracellular vesicles (EV),which are known to contain and/or be associated with microRNAs and are able to protect themfrom degradation.

In this study, we investigated the content of MSC-derived EV for microRNAs involved inosteoblast differentiation, and consequently in bone repair. We also studied thecommunication between MSC and others cell types present in the context of an injury, namelymacrophages, via EV.

We used primary MSC isolated from human bone marrow and a mouse preosteoblastic cellline. EV produced by these cells were isolated by differential (ultra)-centrifugation. EV werevisualized by transmission electron microscopy (TEM) and their size was estimated by dynamiclight scattering (DLS). EV RNA was isolated and microRNAs expression was determined byquantitative real-time PCR. For MSC-macrophages cross-talk, EV derived from MSC werestained with PKH26 dye and added to macrophages.

MSC-derived EV displayed a cup-like morphology, as observed by TEM, and an average sizebellow 200nm, as detected by DLS. Several microRNAs involved in MSC osteogenicdifferentiation were detected in EV derived from MSC, namely miR-29b, miR-29c and miR-20a.Additionally, EV derived from MSC were found to be internalized by macrophages, as assessedby confocal microcopy.

Taken together, these results suggest that MSC-derived EV might have a dual role in bone tissuerepair/regeneration, acting both in osteogenic pathways regulation and inflammatoryprocesses.

Authors thank Centro Hospitalar São João for donating buffy coats and bone marrow samples. Work funded byPrograma Operacional Factores de Competitividade–COMPETE, FCT–Fundação para a Ciência e a Tecnologia (PEst-

C/SAU/LA0002/2013; SFRH/BD/85968/2012; SFRH/BPD/91011/2012) and North Region Operational Program-ON.2(‘‘Project on Biomedical Engineering for Regenerative Therapies; Cancer-NORTE-07-0124-FEDER-000005’’), through

QREN. M.I.Almeida would also like to thank to Boehringer Ingelheim Fonds.

2020



SELECTED ORAL COMMUNICATIONSSELECTED ORAL COMMUNICATIONS

DAY DAY

22

21



Cláudia Azevedo1, Ana Correia-Branco1, João R. Araújo1, João T.Guimarães1,2, Elisa Keating1,3, Fátima Martel1

1Department of Biochemistry (U-38 FCT), Faculty of Medicine, University of Porto, Portugal;2Department of Clinical Pathology, São João Hospital Center, Porto, Portugal; 3Center for

Biotechnology and Fine Chemistry, School of Biotechnology, Portuguese Catholic University,Porto, Portugal

“THE ANTICARCINOGENIC EFFECT OF THE DIETARY COMPOUNDKAEMPFEROL IN A HUMAN BREAST CANCER CELL LINE IS DEPENDENT ON

INHIBITION OF GLUCOSE CELLULAR UPTAKE.”

Cancer cells present an altered metabolism, with an increased rate of glucose uptake andlactate production instead of oxidative metabolism (the Warburg effect) [1]. Dietarypolyphenols are known to possess cancer preventive and anticancer effects [2]. Recently, ourgroup verified that the polyphenols quercetin and epigallocatechin-3-gallate inhibit glucoseuptake by the MCF-7 breast cancer cell line [3]. So, we decided to investigate if otherpolyphenols could also interfere with glucose uptake by these cells.

Uptake of 3H-deoxy-D-glucose (3H-DG) by MCF-7 cells was time-dependent, saturable andinhibited by cytochalasin B plus phloridzin. In the short-term (26 min), myricetin, chrysin,genistein, resveratrol, kaempferol and xanthohumol (10-100 µM) inhibited 3H-DG uptake.Kaempferol was found to be the most potent inhibitor of 3H-DG uptake (IC50 of 4 µM (1.6-9.8)),behaving as a mixed-type inhibitor. In the long-term (24h), kaempferol was also able to inhibit3H-DG uptake, associated with a 40% decrease in GLUT1 mRNA levels. Interestingly enough,kaempferol (100 µM) revealed antiproliferative (sulforhodamine B and 3H-thymidineincorporation assays) and cytotoxic (extracellular lactate dehydrogenase activitydetermination) properties, which were mimicked by low extracellular (1 mM) glucoseconditions and reversed by high extracellular (20 mM) glucose conditions. Finally, exposure ofcells to kaempferol induced an increase in extracellular lactate levels over time (to 731±32% ofcontrol after a 24-h exposure), most probably related to inhibition of MCT1-mediated lactatecellular uptake.

In conclusion, kaempferol potently inhibits glucose uptake by MCF-7 cells, apparently bydecreasing GLUT1-mediated glucose uptake. The antiproliferative and cytotoxic effect ofkaempferol in these cells appears to be dependent on this effect.

References:[1] Cairns et al. (2011) Nature Rev Cancer 11:85-95

[2] Gupta and Prakash (2014) J Complement Integr Med 11:151-69[3] Moreira et al. (2013) Exp Cell Res 319:1784-179

2222


SELECTED SHORT ORAL TALK S6:

Luciana L. Ferreira1,2, Teresa Cunha-Oliveira1, Paulo J. Oliveira1

1CNC, Center for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech-BiocantPark, 3060-197 Cantanhede, Portugal; 2Doctoral Programme in Medical Biochemistry and

Biophysics, University of Coimbra, Portugal

“PERSISTENT EFFECTS IN MITOCHONDRIAL GENES TRANSCRIPTION AFTERINCUBATION OF H9C2 CELLS WITH THE ANTICANCER DRUG DOXORUBICIN:

AN EPIGENETIC ORIGIN?”

Doxorubicin (DOX) is a widely used and efficient chemotherapeutic agent prescribed for thetreatment of several types of tumors. However, despite its clinical potential, DOX treatment isassociated with a persistent, cumulative and dose-dependent cardiac toxicity. In fact, thechronic cardiotoxicity can appear years or even decades after the end of the treatment, whichis particularly common in childhood tumor survivors. The exact mechanisms for this persistentcardiotoxic memory remain under-explored and more studies are needed to elucidate thecauses of this delayed toxicity. In this context, the present work aims to investigate in vitropersistent gene expression alterations, with special relevance on mitochondrial related-genes.H9c2 cardiomyoblast cells were incubated with sub-lethal DOX concentrations (0.01 and 0.025micromolar) during 24h, sub-cultured 72h after the end of the treatment and further sub-cultured for 72h after the first cell passage. RNA and DNA were isolated at four different time-points and gene expression and mitochondrial DNA copy number were determined by real-time qPCR analysis. The affected genes included the mitochondrial mt-nd1, mt-cytb, mt-co1 andmt-atp8 genes, whose expression significantly increased nine days after the end of thetreatment, confirming a DOX-dependent persistent effect. However, no statistically significantalteration was observed in the expression of the mitochondrial transcription factor gene tfamor in mitochondrial DNA copy number. In turn, dnmt1 expression consistently decreased in alltime-points, suggesting that alterations in DNA methylation may also occur and play a role inthe regulation of mtDNA transcription. In conclusion, the persistent alterations inmitochondrial genes expression observed in the absence of the drug, confirm that DOX haspersistent effects in cardiac cells. More interestingly, despite complementary analysis is stilllacking, it is also possible that DOX might interfere with epigenetic regulation.

ACKNOWLEDGMENTS: This work was supported by the FCT (Fundação para a Ciência e a Tecnologia) grantsPTDC/DTP-FTO/1180/2012, PEst-C/SAU/LA0001/2013-2014 and co-funded by FEDER/Compete and National Budget,

CENTRO-07-ST24-FEDER-002008, as well as personal fellowships (SFRH/BD/52429/2013L.F.).

2323



Sofia E. Gomes1, André E. S. Simões1, Diane M. Pereira1, Rui E. Castro1,Alexandra Fernandes2, Catarina Rodrigues2, Pedro M Borralho1, Cecília M.

P. Rodrigues1

1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,Lisbon, Portugal; 2Faculty of Sciences and Technology, New University of Lisbon, Lisbon,

Portugal

“MIR-143 AND MIR-145 OVEREXPRESSION IN HCT116 COLON CANCER CELLSENHANCES CETUXIMAB-MEDIATED ANTIBODY-DEPENDENT CELLULAR

CYTOTOXICITY LEADING TO INCREASED APOPTOSIS”

MiRNAs have been widely studied in human cancers as they elicit strong effects on cell growth,tumorigenesis and therapy response. miR-143 and miR-145 are tumor supressor miRNAs,downregulated in colon cancer. We have shown that miR-143 overexpression reduces tumorgrowth and proliferation, with increased apoptosis, while sensitizing to 5-fluoruracil. In thisstudy, we hypothesize that overexpression of miR-143 and miR-145 in colon cancer cells is ableto sensitize to cetuximab, promoting cetuximab-mediated antibody-dependent cellularcytotoxicity (ADCC) through modulation of signalling pathways involved in effector cell-mediated elimination of cancer cells.

We produced miR-143 or miR-145 overexpressing (and empty vector control) cell lines in KRASmutant HCT116 cells. We evaluated viability and proliferation of cells exposed to cetuximab.Alterations in protein expression patterns of HCT116 cell lines exposed to cetuximab or vehiclewere identified by proteomic analysis of two-dimensional gel electrophoresis. Cetuximab-mediated ADCC was evaluated using xCELLigence system and LDH assay, in cells exposed tocetuximab, and/or peripheral blood mononuclear cells (PBMCs) isolated from human healthydonors as effector cells.

Our results show that miR-143 and miR-145 overexpression increases the sensitivity of HCT116cells to cetuximab, resulting in 40% reduction of cetuximab IC50, compared to control (p<0.01).Importantly, overexpression of miR-143 and miR-145 triggered cetuximab-mediated ADCC byincreasing nuclear fragmentation and caspase-3/7 activity (p<0.05). Caspase inhibition reducedthe cytotoxic effect of PBMCs and cetuximab (p<0.05). In addition, inhibition of granzyme B, aserine protease involved in effector cell-mediated granule secretory pathway, abrogatedcetuximab-mediated ADCC, reducing caspase-3/7 activity in HCT116 cells overexpressing miR-143 or miR-145 (p<0.01). In addition, overexpression of miR-143 negatively regulated Bcl-2protein levels, which was further reduced upon treatment with cetuximab (p<0.01). Bcl-2silencing reduced HCT116 cell viability with a concomitant increase in cell death andcetuximab-mediated ADCC, compared to control siRNA (p<0.05). Moreover, proteomic analysisshowed that miR-143 or miR-145 overexpression altered the expression of proteins involved inseveral biological pathways/processes, including cell growth and proliferation, protein foldingand cell metabolism.

Collectively, restoration of miR-143 and/or miR-145 may provide a new approach fordeveloping a therapeutic strategy to re-sensitize colon cancer cells to cetuximab.

(PTDC/SAU-ORG/119842/2010, SFRH/BD/88619/2012, SFRH/BD/79356/2011, SFRH/BD/96517/2013, SPG)

2424



Marcelo Correia1,2,3, Tânia Perestrelo1,2,3, Ana S. Rodrigues1,3, Sandro L.Pereira3, Maria I. Sousa3,4, Marcelo F. Ribeiro3,4, João Ramalho-Santos3,4

1PhD Programme in Experimental Biology and Biomedicine (PDBEB), CNC - Center forNeuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Institute for

Interdisciplinary Research (IIIUC), University of Coimbra, Coimbra, Portugal; 3Biology ofReproduction and Stem Cell Group, CNC - Center for Neuroscience and Cell Biology, University

of Coimbra, Coimbra, Portugal; 4Department of Life Sciences, Faculty of Sciences andTechnology, University of Coimbra, Coimbra, Portugal

“HISTONE DEACETYLASE PROTEINS: A POSSIBLE GLYCOLYTIC METABOLICSHIFTER IN EMBRYONIC STEM CELLS”

Embryonic stem cells (ESCs) are a promising field in regenerative medicine due to theircapacity of self-renewal and differentiation in all cell types of an adult individual. ESCs, like theblastocyst’s inner cell mass and many cancer types, rely mostly in a glycolytic over oxidativemetabolism to supply their biosynthetic requirements to proliferate. When ESCs undergodifferentiation, a metabolic shift occurs from glycolysis to a predominant oxidative flux.

In this work, we have pharmacologically manipulated metabolism targeting differentmetabolic pathways in E14Tg2.a murine ESCs, namely using Antimycin A – an electrontransport chain complex III inhibitor – and Dichloroacetic acid – an PDHK inhibitor. Overallviability was not affected and both drugs are able to affect pluripotency positively andnegatively, respectively. Moreover, Antimycin A inhibits differentiation of ESCs intodopaminergic neurons.

Although metabolic manipulation is shown here to be important to pluripotency and/ordifferentiation, there are no clues about if epigenetic regulation is influencing the crosstalkbetween pluripotency and metabolism. In order to address this question, a knockout murineESCs line for a Histone Deacetylase class protein was used to unveil possible effects ofepigenetic alterations in the metabolic profile of our cells. In fact, our results show that thesilenced Histone Deacetylase leads to reinforcement of the referenced metabolic profile ofESCs, given that its absence induces a more pronounced glycolytic metabolism paralleled by adecrease in oxidative metabolism.

Overall our results suggest metabolism as a powerful tool to induce alterations inpluripotency/cellular fate in ESCs. Moreover, the results involving the epigenetic relatedenzyme, suggests that epigenetics in ESCs could be an important mechanism how metabolismcould be regulated.

Acknowledgements: FCT for funding [grants PTDC/EBB-EBI/101114/2008, PTDC/EBB-EBI/120634/2010 andPTDC/QUI-BIQ/120652/2010; PhD scholarships attributed to M.C. (SFRH/BD/51681/2011), T.P. (SFRH/BD/51684/2011)and M.S. (SFRH/BD/86260/2012)]. QREN for funding (CENTRO-01-0762-FEDER-00204). CNC funding is supported by

FCT (Pest-C/SAU/LA000172011).

2525


LIST OF POSTERSLIST OF POSTERS

Poster # Presenter Title

P1 Cláudia M. DeusCytotoxic Effects of Resveratrol in Breast Cancer Cells are Sirtuin 3-Independent

P2 André M. Ferreira

Doxorubicin decreases global DNA methylation levels, depletes mtDNA and causes down-regulation of transcripts for metabolic end epigenetic remodeling enzymes

P3 Nuno G. MachadoInfluence of Exogenous Lactate on H9c2 Cardiomyoblast Gene Expression and Proliferation

P4Sílvia Magalhães Novais

Cardiolipin Synthesis and Modifications During P19 Embryonic Carcinoma Cell Differentiation

P5 Vera M. RibeiroLipidomics approach of B-cell chronic lymphocytic leukemia: a study to identify new biomarkers for disease diagnosis

P6Vera F. Monteiro-Cardoso

Adaptation and survival of Caco-2 cells to glucosedeprivation involve and interconnected remodeling of bioenergetics and lipid metabolisms

P7Albert A. Rizvanov

Effect of Pluronic P85 Block Copolymer on transcriptome of human tooth germ stem cells in vitro

P8Albert A. Rizvanov

Application of anticancer drug based on recombinant histone protein as inhibitor of adenoviral infection in vitro

P9 Vilena V. Ivanova

Serum of patients with auto-immune diseases demonstrates higher affinity toward genomic DNA, RNA and mitochondrial DNA from immortalized HEK 293FT cells

2626



POSTER POSTER

COMMUNICATIONSCOMMUNICATIONS

27


P1

Cláudia M. Deus1, Teresa L. Serafim1, Andreia Vilaça1,2, Susana M. Cardoso3,Paulo J. Oliveira1

1CNC- Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal;2Department of Life Sciences, University of Coimbra, Coimbra, Portugal; 3CERNAS- Department

of Environment, Agricultural College of Coimbra, Coimbra, Portugal

“CYTOTOXIC EFFECTS OF RESVERATROL IN BREAST CANCER CELLS ARESIRTUIN 3-INDEPENDENT”

Introduction: Resveratrol is a polyphenol, identified as chemopreventive or cytotoxic agentagainst various types of cancer. Resveratrol has been described to stimulate the activity ofsirtuins, which have an important role in cancer progression and therapy. The objective of thisstudy was to investigate the cytotoxicity of resveratrol on human breast cancer cells andidentify whether this cytotoxicity is related with modulation of Sirtuins1 and 3 protein content.

Materials and methods: Two human breast cancer cell lines (MDA-MB-231 and MCF-7) werecultured in high glucose and glucose-free/glutamine/pyruvate-containing media (OXPHOSmedia). To silence SIRT1 and SIRT3, cells were transfected with small interfering RNA (siRNA)specific for hSIRT1 and hSIRT3. Cells were treated with 10, 25 and 50 µM resveratrol during 24hand 48h and viability, cell death, mitochondrial physiology, oxygen consumption and sirtuincontent were determined using standard methods.

Results: Cell proliferation studies showed that 50 μM resveratrol during 48h induced a decreasein MDA-MB-231 and MCF-7 cell mass when cells were cultured in OXPHOS medium. Also,resveratrol treatment led to morphological changes in the mitochondrial network andchromatin condensation, suggesting apoptotic cell death. Furthermore, live/dead assay showedthat 25 and 50 μM resveratrol treatment during 24h promoted MDA-MB-231 and MCF-7 celldeath when cells were cultured in glucose or OXPHOS medium, respectively. Resveratrolpromoted differentiation of MDA-MB-231 cells. When incubated in the OXPHOS mediumincreased ATP production was measured in MCF-7. However, resveratrol treatment decreasedand increased this parameter in MCF-7 and MDA-MB-231, respectively. Moreover, glycolyticreserve and capacity in MCF-7 was decreased after resveratrol treatment. The two cell linescultured in glucose medium showed that Sirtuin1 content was increased after resveratroltreatment, while the amount of Sirtuin3 was decreased in MCF-7. Transfected cells showed thatresveratrol effects in both breast cancer cell mass and cell death are independent of SIRT3 butappear to be related with SIRT1.

Conclusions: Our data suggests that SIRT3 are not involved in resveratrol cytotoxic effects inbreast cancer cells and the modulation of Sirtuins content have different degrees of resveratroltoxicity on distinct cell lines.

Acknowledgements: Supported by FCT-Portugal (PTDC/SAU-TOX/110952/2009, PEst-C/SAU/LA0001/2013-2014, co-funded by FEDER/ COMPETE/National Funds).

2828


P2

André M. Ferreira, Andreia Vilaça, Filipa S. Carvalho, Rita Garcia, AnaBurgeiro, Teresa Cunha-Oliveira, Paulo J. Oliveira

CNC- Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

“DOXORUBICIN DECREASES GLOBAL DNA METHYLATION LEVELS,DEPLETES MTDNA AND CAUSES DOWN-REGULATION OF TRANSCRIPTS FOR

METABOLIC END EPIGENETIC REMODELING ENZYMES”

Doxorubicin (DOX), a potent and broad-spectrum antineoplastic agent, causes a cumulativedose-dependent cardiomyopathy of late-onset that may ultimately lead to congestive heartfailure. The mechanisms responsible for the delayed nature of this pathology remain poorlyunderstood. Since DOX induces a persistent depression in cardiomyocyte gene expressionpatterns, it is reasonable to hypothesize that a “toxic memory” is being formed by means ofepigenetic modulation. To test this hypothesis, eight week-old male Wistar rats (n = 6 pergroup) were administered 7 weekly injections with DOX (2 mg/kg) or equivalent saline solutionand sacrificed one week after the last injection. We assessed gene expression patterns andmtDNA copy number by qRT-PCR and global DNA methylation by ELISA in cardiac tissue. DOX-treated animals demonstrated a trend towards depressed transcripts for various functionalgene groups in cardiac tissue. Regarding genes related to mitochondrial biogenesis, weobserved a significant decrease in PGC-1α and TFAM transcripts, as well as a global trendtowards a decrease in the various oxidative phosphorylation subunits encoded by nDNA andmtDNA. A concomitant decrease in mtDNA copy number was also observed. The masterregulator of lipid metabolism, PPARα, as well all assessed transcripts involved in lipidmetabolism (ACOX1, ACAA2, CACT, ACC, TAZ), were also found to be down-regulated. Regardingepigenetic modulation, we observed a decrease in transcripts involved in histone acetylation(CBP, EP300) and histone deacetylation (HDACs 3-10). Regarding DNA methylation, we observeda global decrease in 5-mC content in DNA from cardiac tissue from DOX-treated animals.Interestingly, this was accompanied by a decrease in the gene expression of MAT2A andDNMT1. These results are consistent with the interplay between mitochondrial dysfunctionand epigenetic alterations in the persistent nature of DOX-induced cardiotoxicity. Onepossibility is that DOX may modulate gene expression patterns via alterations to the epigeneticlandscape, which could be related to a disturbance in metabolite availability.

Acknowledgements: Supported by Foundation for Science and Technology, Portugal, grants PTDC/DTP-FTO/1180/2012, PEst-C/SAU/LA0001/2013-2014 and co-funded by FEDER/Compete and National Budget, CENTRO-07-

ST24-FEDER-002008, as well as personal fellowships (SFRH/BD/64694/2009 to F.C.).

2929


P3

Machado NG1, Cunha-Oliveira T1, Burgeiro A1, Oliveira PJ1

1CNC, Center for Neuroscience and Cell Biology, UC-Biotech, Biocant Park, University ofCoimbra, Portugal

“INFLUENCE OF EXOGENOUS LACTATE ON H9C2 CARDIOMYOBLAST GENEEXPRESSION AND PROLIFERATION”

During physical activity, lactate is not a waste metabolite but a fuel source and “good” signalingmolecule instead, and was shown to up-regulate reactive oxygen species (ROS)-responsiveantioxidants genes, activating mitochondrial biogenesis and inhibiting cell proliferation. Thus,the impact of lactate in muscle cells may be important for many physiological and pathologicalconditions. In fact, physical activity prevents chemotherapy-induced cardiac toxicity, e.g. thecardiovascular toxicity associated with anthracycline treatment. Since anthracycline toxicity ishigher in the differentiated H9C2 cardiomyoblast population, as previously demonstrated byus, we are interested in finding out how lactate orchestrates the cell fate between proliferationand differentiation states in that cell line and how that effect can explain the potentialprotective role of physical exercise during cancer treatment-associated toxicity. We incubatedundifferentiated H9C2 cells (UNDIF) with several concentrations of lactate ([lactate]) during48h to test the hypothesis that lactate regulates transcripts related to cell proliferation, lactatemetabolism, mitochondrial biogenesis, antioxidant defenses and sirtuins. By using thesulphorodamine B method (SRB), treatment with exogenous lactate (10, 20 and 30 mM) for 48hpromoted cell growth inhibition in UNDIF, showing a small dose-dependent effect and beingtoxic for supra-physiological levels (100 mM). Inhibition of H9c2 proliferation appeared to beindependent of medium acidification. qRT-PCR analysis showed that lactate treatment had littleeffect on transcripts measured with exception for sirtuin 3 (SIRT3, p<0.05) and cardiac troponinc (cTNT, p<0.01 and p<0.001).

We conclude that the inhibition seen on UNDIF proliferation appears to be independent of thegenes investigated here. Besides SIRT3 and cTNT, other inhibitory factors are probablyresponsible for lactate-induced cellular adaptations.

Supported by Foundation for Science and Technology, Portugal, grants PTDC/DTP-FTO/1180/2012, PEst-C/SAU/LA0001/2013-2014 and co-funded by FEDER/Compete and National Budget, CENTRO-07-ST24-FEDER-002008,

as well as personal fellowships (SFRH/BD/66178/2009- NGM)

3030


P4

Sílvia Magalhães Novais1,2; Maria Rosário Domingues3; Elisabete Maciel3;Tânia Melo3; Ignacio Vega-Naredo2; Paulo J. Oliveira2

1Department of Life Sciences, University of Coimbra, Coimbra, Portugal; 2CNC-Center forNeuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Cantanhede,

Portugal; 3Mass Spectrometry Centre, Chemistry Department, University of Aveiro, Aveiro,Portugal.

“CARDIOLIPIN SYNTHESIS AND MODIFICATIONS DURING P19 EMBRYONICCARCINOMA CELL DIFFERENTIATION”

The evolution of the carcinogenic process requires the knowledge of physiology andmetabolism of cancer stem cells (CSCs), which is crucial for the development of novel effectivetherapies. Mitochondria are unequivocally a target for cancer therapy and crucial in cell death.Given the relevance of mitochondrial metabolism for stemness maintenance andmitochondrial control of apoptosis, as well as the importance of cardiolipin (CL) formitochondrial metabolism, we propose here the study CL alterations during P19 embryonalcarcinoma cells (CSCs) differentiation. Our results indicate that, although the total content of CLdid not significantly change, CL remodeling, possible mediated by increased of taffazin andcardiolipin synthase proteins, may lead to a metabolic transformation towards the typicaloxidative metabolism that occurs during the differentiation of P19 CSCs. Differences in CLmolecular species were observed during differentiation of P19 CSCs. Moreover, thedifferentiation of P19 CSCs increased the peroxidation of CL whereby the P19 CSCs present alower amount of non-oxidized CL. We conclude that the metabolic remodeling during P19 CSCsdifferentiation probably also involves alterations in CL metabolism that probably are involvedin converting P19 differentiated cells more susceptible to mitochondrial-targeted antitumoragents.

Acknowledgements: Supported by the Portuguese Foundation for Science and Technology (FCT), co-funded byCOMPETE/FEDER/National budget (PTDC/QUI-BIQ/101052/2008, PTDC-SAU-TOX-117912-2010, CENTRO-07-ST24-

FEDER-002008 and PEst-C/SAU/LA0001/2013-2014).

3131


P5

Vera M. Ribeiro1, Rosário P. Leite2 and Romeu A. Videira1

1Chemistry Center – Vila Real (CQ-VR), University of Trás-os-Montes e Alto Douro (UTAD), VilaReal, Portugal; 2Genetic Service, Cytogenetic Laboratory, Hospital Center of Trás-os-Montes and

Alto Douro, Vila Real, Portugal

“LIPIDOMICS APPROACH OF B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA: ASTUDY TO IDENTIFY NEW BIOMARKERS FOR DISEASE DIAGNOSIS”

B-cell chronic lymphocytic leukemia (B-CLL), the most common leukemic disorder in thewestern hemisphere, is characterized by an abnormal accumulation of mature B lymphocytesin the blood, bone marrow and secondary lymphoid organs. Although apoptosis dysregulationconnected with characteristics genetic alterations and with dynamic changes of membrane B-cell phenotypes are recognized as pathological hallmarks of disease, the molecularmechanisms underlying B-CLL still uncertain in their nature and progression. Additionally,new diagnostic tools are need since the traditional tests that combine fluorescence in situhybridization (FISH) with cytogenetic analysis are not able to identify at least 20% of B-CLLcases. In the present work, a lipidomics approach were used to assess the blood lipid profiles ofsix human patients diagnosed with B-CLL, three of which exhibiting abnormal karyotype andpositive result on FISH analysis and others three without the genetic abnormalities detected byFISH and cytogenetic analyses, with the goal to detect new molecular markers with potential tobe used in the diagnosis of B-CLL. HPLC-MS/MS analyses of lipid extracts revealed significantchanges in phospholipid profile of blood samples from patients with normal karyotype andnegative FISH when compared with samples from patients with abnormal karyotype andpositive FISH. Increased levels of phosphatidylethanolamines (PE) associated with a decreaseof both phosphatidylinositol (PI) and cardiolipins (CL) content were detected in blood samplesof patients group with abnormal karyotype and positive FISH. Since the results werenormalized by the number of lymphocytes, the decreased CL levels indicate a lowermitochondrial mass and/or activity suggesting that the bioenergetics metabolism oflymphocytes is affected in B-CLL. Although the relative abundance of choline-containing-phospholipids (SM, PC) is similar in both groups, significant differences are detected in therelative abundance of the most representative molecular species of these phospholipid classes.Despite preliminary, our data suggest that lipidomics strategy can be used to identify lipidbiomarkers to improve diagnosis of B-CLL.

Acknowledgments: This work was supported by Foundation for Science and Technology (FCT), and EuropeanUnion Funds (FEDER/COMPETE) [Project Grants: PEst-OE/QUI/UI0616/2014]. Romeu A. Videira was supported by

BPD/ENOEXEL/UTAD/269/2014.

3232


P6

Vera F. Monteiro-Cardoso1, Amélia M. Silva2, Maria M. Oliveira1, FranciscoPeixoto2 and Romeu A. Videira1

1Chemistry Center – Vila Real (CQ-VR), University of Trás-os-Montes e Alto Douro (UTAD), VilaReal, Portugal; 2Centre for the Research and Technology of Agro-Environmental and Biological

Sciences (CITAB), UTAD, Vila Real , Portugal

ADAPTATION AND SURVIVAL OF CACO-2 CELLS TO GLUCOSE DEPRIVATIONINVOLVE AND INTERCONNECTED REMODELING OF BIOENERGETICS AND

LIPID METABOLISMS

Cancer cells can adapt their metabolic activity under nutritional hostile conditions in order toensure both bioenergetics and biosynthetic requirements to survive. In this study, the effect ofglucose deprivation on Caco-2 cells bioenergetics activity and putative relationship withmembrane lipid changes were investigated. Glucose deprivation induces a metabolicremodeling characterized at mitochondrial level by an increase of oxygen consumption, arisingfrom an improvement of complex II and complex IV activities and an inhibition of complex Iactivity. This effect is accompanied by changes in cellular membrane phospholipid profile.Caco-2 cells grown under glucose deprivation show higher phosphatidylethanolamine contentand decreased phosphatidic acid content. Considering fatty acid profile of all cellphospholipids, glucose deprivation induces a decrease of monounsaturated fatty acid (MUFA)and n-3 polyunsaturated fatty acids (PUFA) simultaneously with an increase of n-6 PUFA, withconsequent drop of n-3/n-6 ratio. Additionally, glucose deprivation affects significantly the fattyacid profile of all individual phospholipid classes, reflected by an increase of peroxidabilityindex in zwitterionic phospholipids and a decrease in all anionic phospholipids, includingmitochondrial cardiolipin. These data indicate that Caco-2 cells metabolic remodeling inducedby glucose deprivation actively involves membrane lipid changes associated with a specificbioenergetics profile which ensure cell survival.

Acknowledgments : This work was supported by Foundation for Science and Technology (FCT), and EuropeanUnion Funds (FEDER/COMPETE) [Grants: PTDC/SAU-NMC/115865/2009; FCOMP-01-0124-FEDER-022696:PEst-

C/AGR/UI4033/2011, PEst-OE/AGR/UI0772/ 2011 and PEst-C/QUI/UI0616/2011]. Vera F. Monteiro-Cardoso issupported by FCT grant BI/PTDC/SAU NMC/115865/2009

3333


P7

Atousa Ataei1, Nataliya L. Blatt1, Mansour Poorebrahim2, Mehmet E.Yalvaç3, Albert A. Rizvanov1

1Kazan Federal University, Kazan, Russia; 2Tehran University of Medical Science, Tehran, Iran;3Children's Hospital Columbus, Center for Gene Therapy, Columbus, United States

“EFFECT OF PLURONIC P85 BLOCK COPOLYMER ON TRANSCRIPTOME OFHUMAN TOOTH GERM STEM CELLS IN VITRO”

Pluronics blocking copolymers, such as P85, are currently considered as a drug deliverycarriers in various disorders and cancers. These compounds can impact many biologicalprocesses but there are few reports explaining their molecular function in the cell. Theinhibiting effect of Pluronics on cancer cells depends on their chemical structure andhydrophobic properties. Encapsulation of anti-cancer drugs in the Pluronic micelles alters theirdistribution in vivo leading to higher rate of accumulation inside the tumor when compared tofree drugs. Although Pluronics are extensively studied using tumor cell lines, there is littleknown about the effect of Pluronics on normal stem cells.

Here we present our data on genome-wide gene expression in human tooth stem cells (hTGSCs)treated with Pluronic P85 Block Copolymer (P58). Illumina microarray method was used in thisstudy. Substantial changes in the 252 genes were detected in hTGSCs exposed to P85 treatment.The gene enrichment approach was carried out using database for annotation, visualizationand integrated discovery (DAVID) and results were classified in several biologically meaningfulclusters. Finally, we constructed a global regulatory network of stem cell differentiationpathways and multi-drug resistance (MDR) process using available bioinformatics databases todemonstrate association of P85-modulated genes with stem cells differentiation and MDRprocesses as well as cross-talk between stem cell differentiation pathways with MDR associatedgenes. In conclusion, our results were in line with many of P85-mediated biological processespublished previously and can help us to gain better molecular perception of P85 biologicaleffects.

3434


P8

Valeriya V. Solovyeva, Albert A. Rizvanov

Kazan Federal University, Russia

“APPLICATION OF ANTICANCER DRUG BASED ON RECOMBINANT HISTONEPROTEIN AS INHIBITOR OF ADENOVIRAL INFECTION IN VITRO”

Histones are a family of basic proteins that associate with DNA in the nucleus and helpcondense it into chromatin. The balance of histone acetylation and deacetylation is anepigenetic layer with a critical role in the regulation of gene expression. Anticancer drugOncoHist based on recombinant histone H1.3 has strong anti-proliferative properties againstleukemic blast cells. OncoHist has been studied extensively as potential treatment forhematologic malignancies, such as leukemias, lymphomas and myelomas. Here we present ourdata on analysis of antiviral properties of the recombinant histone H1.3 in in vitro usingreplication-deficient recombinant adenovirus serotype 5 encoding green fluorescent protein.Recombinant adenovirus was generated using ViraPower Adenoviral Expression System(Invitrogen). Recombinant adenovirus was pre-incubated with recombinant histone H1.3 toassess its effect on adenoviral infection of HeLa cells. The efficiency of adenoviral infection wasdetermined by number of GFP+ cells using flow cytometry. To determine the effect ofrecombinant histone H1.3 on the ability of adenoviral particles to form plaques we usedHEK239A cells (Invitrogen). To investigate the effect of recombinant histone H1.3 on adenoviralinfection we used its maximum non-toxic dose — 250 ug/ml. In our work we demonstrated forthe first time that recombinant histone H1.3 significantly reduces the infectivity ofrecombinant adenovirus. Using of a mixture of recombinant histone H1.3 and recombinantadenovirus lead to decreases the fluorescence intensity (2% GFP-positive cells) in contrast tostandard adenovirus infection (14% GFP-positive cells). Also, recombinant histone H1.3 exertsan inhibitory effect on plaque formation on HEK293A cells, infected with recombinantadenovirus. Adding of histone H1.3 to the recombinant adenovirus at a dilution 10-6 lead to areduction of plaque formation in 7,7 times, which confirms antiviral properties of histone H1.3towards adenoviruses. Further experiments are needed to determine mechanism ofinteraction of recombinant adenoviral particles with histone H1.3. Our study provides newdirection for clinical application of recombinant of histone H1.3 as an antiviral drug for thetreatment of adenoviral infections.

3535


P9

Vilena V. Ivanova1, Svetlana F. Khaiboullina1,2, Irina G. Starostina1, AlbertA. Rizvanov1

1Kazan Federal University, Kazan, Russia; 2University of Nevada, Reno, NV, USA

“SERUM OF PATIENTS WITH AUTO-IMMUNE DISEASES DEMONSTRATESHIGHER AFFINITY TOWARD GENOMIC DNA, RNA AND MITOCHONDRIAL

DNA FROM IMMORTALIZED HEK 293FT CELLS”

Systemic lupus erythematosus (SLE) is the auto-immune disease characterized by a plethora ofclinical presentation and cycles of remission and reactivation. Circulating anti-dsDNAantibodies are hallmark features of SLE and detected in the serum as a reliable marker for SLEdiagnosis and are used as a key criterion for disease classification. Recently, potentialimmunogenicity of peroxy-nitrite modified mitochondrial DNA (mtDNA) has been shown inSLE patients. This suggests that circulating mtDNA can act as a trigger point for thedevelopment of autoimmune disorders.

Our data demonstrate that the sera from SLE patients display higher immunoreactivity towardtotal RNA and DNA from immortalized HEK 293FT cells when compared to healthy controls.Additionally, significantly higher levels of immunoreactivity to mtDNA were observed for thesera of SLE patients when compared to healthy controls. Finally, the sera from more than halfof all SLE cases (58.3%) demonstrated immunoreactivity to all types of nucleic acidpreparations as compared to only one serum sample (12.5%) from healthy donor.

Since a greater number of SLE patients displayed immunoreactivity toward mtDNA whencompared to healthy controls, we conclude that mtDNA from cells with high potential tomalignant transformation may play a role in pathogenesis of SLE. This assumption iscorroborated by the recent report of plasmacytoid dendritic cell activation by mtDNA leadingto the production of IFNα.

3636


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cancer epigenetics and metabolism: connecting the dots€¦ · cancer epigenetics and metabolism:...

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