LAAS 20
 
27-29 March 2014

Hadath, Lebanon

20th LAAS International Science Conference
    Advanced Research for Better Tomorrow

the Lebanese Association for the Advancement of Sience
Doctoral school of sciences and technology
Lebanese university

Keynote speaker in Biological, Medical, Pharmaceutical, Health Sciences

Genetic and epigenetic modifications in cancer result from stochastically occurring anomalies and selective pressure for cell survival http://www.edas.info/icon/favorite.gif

Charles Theillet

Prof. Charles Theillet
(the Institut de Recherche en Cancérologie de Montpellier, France)

 

Genetic and epigenetic modifications in cancer result from stochastically occurring anomalies and selective pressure for cell survival. We noted that breast tumors belonging to different molecular subgroups showed different profiles of genetic anomalies (Guedj et al., 2011) and questioned whether these differences could be due to the activation of distinct oncogenic cascades? Furthermore, we wanted to get better insight on the nature and the sequence of genetic and epigenetic alterations accompanying the transition between the normal and transformed state. To this aim, we devised a step wise transformation models allowing us to monitor the dynamics of genetic and DNA methylation changes. We opted for normal human mammary epithelial cells (hMEC) sequentially transduced with genetically defined elements. Step 1 aimed at bypassing the senescence block by means of p53 or p16 inactivation by shRNA expression, step 2 at transforming the cells. To assess the impact of the oncogenic cascade on the genetic and epigenetic modifications we transduced 3 oncogenes, WNT1, RASv12 and CCNE1, which activate distinct signaling pathways. Independent biological replicates were established for each genetic variant. Genetically modified cells were all thoroughly characterized at the phenotypic, genetic (CNC, mRNA and miRNA expression) and epigenetic (whole genome DNA methylation) levels for changes at different steps of progression in the immortalization and transformation process. Inactivation of p53 resulted in increased proliferation, Bgalactosidase expression peaked transiently in early passages. This faded concomitantly to the spontaneous reactivation of the endogeneous hTERT gene. At the genetic level, shp53 cells underwent early tetraploidy, showed few copy number changes and, noticeably, a gradual increase in DNA methylation along passages. Remarkably, transformed variants differed at the genetic and epigenetic levels according to the transduced oncogene. Indeed, while shp53-WNT and shp53-CCNE1 cells presented multiple CNC and no change in DNA methylation, shp53-RAS presented very distinct patterns of CNC and important modifications at the DNA methylation level. Our data show for the first time to our knowledge that: (1) inactivation of p53 does not result in gross structural anomalies, but rather in a drift in DNA methylation; (2) gross structural changes occurred upon oncogene activation. The pattern of genetic anomalies was determined by the oncogenic pathway that was activated; (3) structural rearrangements were more severe in variants showing no DNA methylation changes and vice versa, suggesting an inverse balance between CNC and DNA methylation modifications in this system.

Presenter bio: Prof Theillet is the reasearch director ar DR1 INSERM. Present situation : Head of Tumor Identity and Plasticity Group at the Institut de Recherche en Cancérologie de Montpellier (IRCM) and Deputy-Director of IRCM ; INSERM U896 ; CRLC Val d’Aurelle ; UMSF ; 34298 Montpellier cedex 5, France • Member of several selection comitees in France (ARC, Ministry of Research, INSERM) Member of Cancérropole Evaluation Board (2007). Chairman and member of several AERES evaluation committees, 2009 and 2011, 2013 Full member of the Scientific Steering Comitee of the Cancéropole Grand-Sud-Ouest and Coordinator of the Genetic Instability in Cancer Theme 2004-2009. Coordinator of the "Resistance to Treatment" theme of the Montpellier-Cancer Comprehensive Cancer Center (SIRIC) and member of the SIRIC Scientific Counsel. Member of the comité cancer of the Fondation de France 2013-2016 Coordinator of two research programs involving respectively 4 and 20 research groups with funds from the Ministry of Research. Coordinator of INTAS project # 1551-96: Dec 1, 1997 - Nov 30, 1999 Organisor or co-organisor of several workshops and practical courses in France Associate member of AACR External referee for AICR, Cancer Research, Clinical Cancer Research, PNAS, Gene Chromosomes and Cancer He has over 100 publications in peer reviewed articles.

Abstract

Claire Fonti1, Anne Saumet1, Béatrice Orsetti1, Laurence Lasorsa1, Michael Weber2, Charles Theillet1
1 Institut de Recherche en Cancérologie de Montpellier, INSERM U896/Université Montpellier 1, 2 Ecole Supérieure de Biotechnologie de Strasbourg, Biotechnology and cell signalling Lab UMR 7242 CNRS/University of Strasbourg

Genetic and epigenetic modifications in cancer result from stochastically occurring anomalies and selective pressure for cell survival. We noted that breast tumors belonging to different molecular subgroups showed different profiles of genetic anomalies (Guedj et al., 2011) and questioned whether these differences could be due to the activation of distinct oncogenic cascades? Furthermore, we wanted to get better insight on the nature and the sequence of genetic and epigenetic alterations accompanying the transition between the normal and transformed state.
To this aim, we devised a step wise transformation models allowing us to monitor the dynamics of genetic and DNA methylation changes. We opted for normal human mammary epithelial cells (hMEC) sequentially transduced with genetically defined elements. Step 1 aimed at bypassing the senescence block by means of p53 or p16 inactivation by shRNA expression, step 2 at transforming the cells. To assess the impact of the oncogenic cascade on the genetic and epigenetic modifications we transduced 3 oncogenes, WNT1, RASv12 and CCNE1, which activate distinct signaling pathways. Independent biological replicates were established for each genetic variant. Genetically modified cells were all thoroughly characterized at the phenotypic, genetic (CNC, mRNA and miRNA expression) and epigenetic (whole genome DNA methylation) levels for changes at different steps of progression in the immortalization and transformation process.
Inactivation of p53 resulted in increased proliferation, Bgalactosidase expression peaked transiently in early passages. This faded concomitantly to the spontaneous reactivation of the endogeneous hTERT gene. At the genetic level, shp53 cells underwent early tetraploidy, showed few copy number changes and, noticeably, a gradual increase in DNA methylation along passages. Remarkably, transformed variants differed at the genetic and epigenetic levels according to the transduced oncogene. Indeed, while shp53-WNT and shp53-CCNE1 cells presented multiple CNC and no change in DNA methylation, shp53-RAS presented very distinct patterns of CNC and important modifications at the DNA methylation level. Our data show for the first time to our knowledge that: (1) inactivation of p53 does not result in gross structural anomalies, but rather in a drift in DNA methylation; (2) gross structural changes occurred upon oncogene activation. The pattern of genetic anomalies was determined by the oncogenic pathway that was activated; (3) structural rearrangements were more severe in variants showing no DNA methylation changes and vice versa, suggesting an inverse balance between CNC and DNA methylation modifications in this system.