Thèses sur le sujet « Epigenetic Modulations »

Le carcinome hépatocellulaire (CHC) est le plus fréquent des cancers du foie et une importante cause de mortalité. Les modifications épigénétiques jouent un rôle essentiel dans ce cancer et sont des cibles thérapeutiques intéressantes. Au cours de ma thèse, je me suis intéressée à l‘histone méthyltransférase (HMT) EZH2, qui augmente dans le CHC et est liée à la résistance au traitement. En tant que sous-unité catalytique du complexe PRC2, EZH2 est responsable de la marque H3K27me3 et la répression de gènes (rôle canonique). Dans le cancer, EZH2 a d’autres fonctions indépendantes de PRC2 et peut contribuer à l’expression de gènes (rôle non canonique). Les inhibiteurs enzymatiques d’EZH2 ne sont pas efficaces sur les tumeurs solides, suggérant que l’activité non catalytique d’EZH2 joue un rôle dans ces cancers. EZH2 est régulé par des modifications post-traductionnelles, telle la O-GlcNAcylation par l’O-GlcNAc transférase (OGT) qui augmente dans le CHC. Nous avons montré que dans le CHC la fraction d’EZH2 associée à OGT est surtout impliquée dans l’expression de gènes. Notre but était de caractériser les mécanismes sous-tendant l’activation de gènes par EZH2/OGT et de déterminer le rôle du complexe PRC2 et c-Myc, qui peut être modulé par OGT et joue un rôle important dans le CHC, dans ces processus. Nous avons montré que EZH2 et c-Myc sont O-GlcNAcylés dans les cellules hépatocytaires et que l’O-GlcNAcylation de EZH2 est importante pour son recrutement aux gènes cibles. Nos données indiquent que c-Myc joue un rôle important dans la régulation de gènes par EZH2/OGT. De manière intéressante, nos résultats suggèrent qu’une partie des fonctions non canoniques d’EZH2 dans les cellules hépatocytaires humaines pourrait être dépendante du complexe PRC2. L’ensemble de nos résultats indiquent qu’OGT et c-Myc contribuent aux fonctions non canoniques d’EZH2 dans des cellules hépatocytaires humaines et apportent de nouvelles connaissances quant au potentiel des régulations épigénétiques comme cible thérapeutique dans le CHC. Une meilleure connaissance des mécanismes moléculaires sous-tendant les dérégulations de gènes dans le CHC ouvrira des perspectives pour de nouvelles stratégies thérapeutiques
Hepatocellular carcinoma (HCC), the most common form of liver cancer and leading cause of death, is a heterogeneous disease with no unique driver mutation. Up to 50% of HCCs harbor alterations in epigenetic machineries that represent promising therapeutic targets. During my PhD, I focused on the histone methyltransferase (HMT) EZH2 that is upregulated in HCC and related to therapy resistance. EZH2 is the catalytic subunit of the PRC2 complex responsible for H3K27me3, a repressive epigenetic mark (canonical function). In cancer, EZH2/PRC2 represses the expression of tumor suppressor genes but EZH2 can also activate oncogenes and cell cycle genes in a mostly PRC2-independent manner (non-canonical function). EZH2 HMT inhibitors have demonstrated low efficacy in solid tumors suggesting that HMT independent functions of EZH2 are key in these cancers. EZH2 can be regulated by post-translational modifications, including O-GlcNAcylation by O-GlcNAc transferase (OGT) whose expression is increased in HCC. Our data show that EZH2 and OGT are co-recruited to defined gene promoters in HCC and predominantly promote gene expression. To decipher the molecular mechanisms underlying EZH2/OGT-mediated gene activation in HCC, we assessed the roles of PRC2 and c-Myc that plays an important role in HCC and can be modulated by OGT. We showed that EZH2 and c-Myc are O-GlcNAcylated by OGT in human hepatoma cells and that EZH2 O-GlcNAcylation plays a role in EZH2 target promoter recruitment. Our data also indicate that c-Myc plays an important role in EZH2/OGT-mediated gene regulation. Interestingly, our results suggest that part of the non-canonical functions of EZH2 in human hepatoma cells may be PRC2 dependent. Collectively, our data uncover that OGT and c-Myc promote non-canonical functions of EZH2 in transformed liver cells and provide important insights for epigenetic strategies as potential future anti-HCC therapies. A better understanding of the regulatory networks controlling gene expression in HCC will open perspectives for the design of novel therapeutic strategies for HCC

In autonomous robotics, there is still a trend to develop and tune controllers with highly explicit goals and environments in mind. However, this tuning means that these robotic models often lack the developmental and behavioral flexibility seen in biological organisms. The lack of flexibility in these controllers leaves the robot vulnerable to changes in environmental condition. Whereby any environmental change may lead to the behaviors of the robots becoming unsuitable or even dangerous. In this manuscript we look at a potential biologically plausible mechanism which may be used in robotic controllers in order to allow them to adapt to different environments. This mechanism consists of a hormone driven epigenetic mechanism which regulates a robot's internal environment in relation to its current environmental conditions. As we will show in our early chapters, this epigenetic mechanism allows an autonomous robot to rapidly adapt to a range of different environmental conditions. This adaption is achieved without the need for any explicit knowledge of the environment. Allowing a single architecture to adapt to a range of challenges and develop unique behaviors. In later chapters however, we find that this mechanism not only allows for regulation of short term behavior, but also long development. Here we show how this system permits a robot to develop in a way that is suitable for its current environment. Further during this developmental process we notice similarities to infant development, along with acquisition of unplanned skills and abilities. The unplanned developments appears to leads to the emergence of unplanned potential cognitive abilities such as object permanence, which we assess using a range of different real world tests.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007.
Additional advisors: Vithal K. Ghanta, J. Michael Ruppert, Theresa V. Strong, R. Douglas Watson. Description based on contents viewed Oct. 3, 2008; title from PDF t.p. Includes bibliographical references.

Thesis (Honors)--Ohio State University, 2005.
Title from first page of PDF file. Document formattted into pages: contains 51 p.; also includes graphics. Includes bibliographical references (p. 29-33). Available online via Ohio State University's Knowledge Bank.

PTSD is a devastating disorder that affects 7-12% of individuals who experience a traumatic event in their lives. Individuals suffering from the disorder show a dysfunctional HPA axis with decreased basal cortisol release and increased glucocorticoid receptor sensitivity. Recently, epigenetic studies have shown associations between trauma and the subsequent effects on the HPA axis in abused participants who committed suicide. Importantly, findings also showed child trauma as being a significant factor in DNA methylation levels in promoter region 1F in the hippocampus. This thesis shows the possible role of epigenetic programming of glucocorticoid receptors in PTSD while examining the effects of different timing of trauma. Methods included the measurements of salivary basal cortisol levels, the expression of glucocorticoid receptors in whole blood and the DNA methylation levels in two promoter regions of the glucocorticoid receptors by sequenome epityper. Results showed that individuals suffering from PTSD have decreased salivary cortisol release in the morning, increased glucocorticoid receptors and increased total methylation levels in promoter region 1C in whole blood. Additionally, adulthood trauma seemed to be of greater importance with regards to HPA axis activity while childhood trauma showed to be more significant with regards to epigenetics. These results indicate that 1) individuals suffering from a lifetime PTSD have a dampened HPA axis activity, 2) epigenetics play a different role in PTSD compared to abused individuals who have committed suicide and 3) timing of trauma has a significant effect on both the HPA axis activity and the epigenetic modifications.
Le TSPT est un syndrome dévastateur qui touche entre 7 et 12% de gens qui vivent un événement traumatique. Les personnes qui souffrent du syndrome ont un axe HPA dysfonctionnel avec moins de cortisol salivaire et une augmentation de sensitivité des récepteurs glucocorticoïdes. Il y a récemment eu des études épigénétiques qui ont montré une association entre les traumas et les effets sur l'axe HPA chez des patients abusés qui ont commis un suicide. De plus, les résultats indiquent qu'un trauma durant l'enfance est un facteur significatif pour la méthylation d'ADN dans la région promoteur 1F de l'hippocampe. Cette thèse présente le possible rôle modulateur de l'épigénétique des récepteurs glucocorticoïdes chez les personnes touchées du TSPT tout en examinant les effets du temps du trauma. Les résultats montrent que les individus qui souffrent du TSPT ont moins de cortisol salivaire le matin, une augmentation des récepteurs glucocorticoïdes et une augmentation de niveaux de méthylation totale dans la région promoteur 1C du sang. Il semblerait aussi qu'un trauma vécu durant l'âge adulte soit plus significatif pour l'axe HPA, alors qu'un trauma durant l'enfance semblerait prendre le dessus pour les modifications épigénétiques. Ces résultats montrent que 1) les personnes souffrant du TSPT ont une activité HPA atténuée, 2) l'épigénétique joue un rôle différent chez les TSPT comparés aux patients abusés qui ont commis un suicide et 3) le temps du trauma a des effets significatifs pour l'axe HPA et les modifications épigénétiques.

SWI/SNF chromatin remodelling complexes are one of the well-characterized cellular machineries capable of regulation of gene expression. Numerous lines of evidence indicate that SWI/SNF complexes are involved in a wide range of cellular processes and the maintenance of homeostasis whereas aberrant expression of those proteins contributes towards cancer development. Colorectal cancer remains one of the most clinically significant cancers due to its high incidence in developed countries and previous studies have demonstrated that SWI/SNF complexes are aberrantly regulated in a significant proportion of patients with this disease. However, whilst the sequence of molecular events leading to CRC has been well-established, the role of SWI/SNF chromatin remodelling complex ATPase subunits Brm and its paralogue Brg1 in the colorectal tumorigenesis remains elusive. The chromatin remodelling catalytic subunit Brm has been found to interact with the Notch pathway effectors ICD-22 and CBF-1 and also to be necessary for expression of the Wnt target gene CD44 and for Rb-mediated cell cycle arrest. In this PhD thesis, the potential of Brm to modulate Wnt-driven intestinal tumorigenesis was addressed. Initially, a murine model carrying constitutively deleted Brm was used to assess the consequences of this loss on homeostasis of the small intestinal and colonic epithelia. The effects of Brm deficiency were also examined in the context of Wnt-activated epithelium via conditional loss of Apc. Additionally, the effect of concomitant loss of Brm and Brg1 was addressed in the contexts of both normal homeostasis and aberrant Wnt signalling. The results presented here demonstrate that Brm plays an important role in the small intestine by regulating the distribution of proliferating cells and cell fate decisions mediated through Notch pathway effectors. Furthermore, Brm deficiency was found to modulate intestinal phenotype of Wnt activation through the attenuation of the Wnt transcriptional programme and the suppressed expression of the intestinal stem cell marker Olfm4. Thus while Brg1 has been widely characterized as a bone fide tumour suppressor, the function of Brm continues to remain elusive especially in the light of contrasting effects co-mediated by Brm on proliferation, differentiation and gene expression. Taken together, these results elucidate the tissue-specific role of Brm, the catalytic subunit of SWI/SNF chromatin remodelling complex, on both normal intestinal homeostasis and acute activation of Wnt pathway while the extent of these Brm-dependent effects depend upon the gradient of Wnt signalling throughout the epithelium of small and large intestine.

PART I: Expression analysis of maternal and zygotic steroid hormone receptor mRNAs during zebrafish (Danio rerio) embryogenesis. I have analyzed by qRT-PCR and/or RT-PCR the abundance and degradation rates of maternal mRNAs for nine steroid hormone receptors and their possible replacement by corresponding embryonic transcripts in both ovulated oocytes and embryos of zebrafish collected at 0, 1, 2, 4, 8, 12, 24 and 48 h post-fertilization (hpf). The mRNAs encoded the nuclear receptors for progesterone (Pr), androgen (Ar), estrogen (Erα, Erβ1 and Erβ2), glucocorticoids (Gr), mineralocorticoids (Mr) and the membrane progestin receptor-α and β (mPrα and β). Gr mRNA was the most abundant maternal transcript in oocytes and early embryos, followed by erβ2 and ar mRNAs. They declined during the first 8 hpf, being replaced, thereafter, by the embryonic messengers. Erβ1 and mr transcript levels were low until 8 hpf, but increased steadily during embryonic transcription from 24 to 48 hpf. Pr transcripts were detectable only in ovulated oocytes and at 24 and 48 hpf. At these stages, there was a slight increase of erα mRNA that initially was very low. Mprα and β mRNAs were expressed in ovulated oocytes and faintly persisted during the first 4 hpf. There was no subsequent embryonic expression of these transcripts. The possible involvement of maternal mRNAs for glucocorticoid and sex hormone receptors in the programming of early zebrafish development is intriguing, since they mainly occur at stages in which gene replication predominates over transcription. PART II: Morpholino knockdown of glucocorticoid receptor in zebrafish during embryogenesis The glucocorticoid receptor (Gr) is involved in various physiological processes, including growth, osmoregulation and reproduction in various species of vertebrates. In this study, I have investigated the function of Gr during zebrafish embryogenesis using antisense Morpholino (MO) technology approach in order to block gr mRNA translation. I observed that Gr-morphants were characterized by small head and eyes, short body, pericardial edema, disrupted melanophore patterning, failure to inflate the swim bladder, tail curling, and abnormal swimming behaviour. The effectiveness of Gr knockdown was further verified by Western blotting and in vitro transcription and translation system, which showed marked reduction of the Gr protein in morphant embryos. Microarray analysis revealed that Gr functions prevalently as a negative regulator of gene transcription. Three genes found to be up-regulated following MO knockdown by microarray analysis, including caspase-8, igf2α and centaurin-1α, were evaluated by semiquantitative RT-PCR throughout the embryonic development. Furthermore, whole mount in situ hybridizations indicated that, whereas marker expression pattern was mostly unchanged in morphant embryos, three markers involved in neurogenesis, egr2b, emx1 and six3.1, were up-regulated. In conclusion, the knockdown of Gr in zebrafish revealed a requirement of both maternal and zygotic gr in multiple developmental processes involved in neurogenesis and gut and accessory organs formation. There are indications that the maternal receptor is critical in early embryogenesis, but more work is needed to elucidate the functional transition from the maternal to the zygotic gr and whether they affect distinctive components of the genetic machinery.
PARTE I: Analisi dell’espressione degli mRNA materni e zigotici per recettori di ormoni steroidei durante lo sviluppo nel pesce zebrato (Danio rerio). Ho analizzato mediante qRT-PCR e/o RT-PCR la presenza ed il grado di degradazione degli mRNA materni codificanti per nove recettori di ormoni steroidei, e la loro possibile sostituzione da parte dei trascritti embrionali corrispondenti, sia negli ovociti ovulati che in embrioni di pesce zebrato raccolti a 0, 1, 2, 4, 8, 12, 24 e 48 ore dopo la fecondazione (hpf). Gli mRNA studiati sono quelli codificanti i recettori nucleari per il progesterone (Pr), gli androgeni (Ar), gli estrogeni (Erα, Erβ1 ed Erβ2), i glucocorticoidi (Gr), i mineralcorticoidi (Mr) ed i recettori di membrana α e β per i progestinici (mPrα e mPrβ). L’mRNA codificante la proteina Gr costituisce il trascritto materno maggiormente presente negli ovociti e nei primi stadi di sviluppo embrionale, seguito dagli mRNA codificanti i recettori Erβ2 ed Ar. Tutti questi trascritti diminuiscono durante le prime 8 ore di sviluppo, e vengono poi sostituiti dai rispettivi messaggeri embrionali. I livelli dei trascritti per erβ1 e mr sono bassi prima delle 8 hpf, ed aumentano costantemente, dopo l’inizio della trascrizione embrionale, passando dalle 24 alle 48 hpf. L’mRNA codificante il recettore Pr è stato rilevato soltanto negli ovociti ovulati e nei campioni corrispondenti a 24 e 48 hpf. A questi stadi di sviluppo c’è anche un leggero aumento dell’mRNA codificante la proteina Erα. I trascritti per i recettori mPrα e mPrβ sono presenti negli ovociti ovulati e persistono, a basse concentrazioni, fino alle 4 hpf. Non è stata evidenziata la presenza di trascritti embrionali codificanti per questi due geni. E’ interessante il possibile coinvolgimento degli mRNA codificanti Gr e i recettori degli ormoni sessuali nella programmazione dello sviluppo embrionale precoce del pesce zebrato, dal momento che sono presenti principalmente negli stadi di sviluppo in cui la replicazione genica predomina sulla trascrizione. PARTE II: Inattivazione genica del recettore dei glucocorticoidi di pesce zebrato mediante morfolino Il recettore dei glucocorticoidi (Gr) è coinvolto in numerosi processi fisiologici, tra cui la crescita, l’osmoregolazione e la riproduzione in molte specie di vertebrati. In questo studio, ho analizzato la funzione del Gr durante l’embriogenesi di pesce zebrato utilizzando la tecnologia degli oligonucleotidi antisenso morfolino per inibire la traduzione dell’mRNA codificante Gr. Ho osservato che i morfanti di Gr presentano occhi e testa di dimensioni ridotte, corpo corto, edema pericardico, riduzione della pigmentazione, mancata insufflazione della vescica natatoria, coda incurvata e comportamento natatorio anormale. L’efficacia dell’inattivazione genica del Gr sono stati verificati mediante Western blotting ed utilizzando un sistema di trascrizione e traduzione in vitro, che hanno dimostrato una riduzione della traduzione del Gr negli embrioni morfanti. Analisi di microarray hanno messo in evidenza che questa proteina funziona prevalentemente come repressore della trascrizione genica. I geni caspase-8, igf2α e centaurina-1α risultati sovraespressi mediante microarray dopo trattamento con morfolino, sono stati analizzati mediante RT-PCR semiquantitativa durante tutto il corso dello sviluppo embrionale. Inoltre, esperimenti di ibridazione in situ in toto indicano che, mentre in generale il modello di espressione dei marcatori utilizzati non cambia nei morfanti, tre marcatori coinvolti nel processo di neurogenesi, e cioè egr2b, emx1 e six3.1, sono risultati sovraespressi. In conclusione, i risultati ottenuti dall’inattivazione genica del gr di pesce zebrato rivelano la necessità dei trascritti di origine materna in molteplici processi di sviluppo, quali neurogenesi e formazione di intestino ed organi accessori. Si dovrà comunque analizzare più in dettaglio la transizione funzionale dai trascritti materni a quelli zigotici e stabilire se essi influenzino componenti distinti dei meccanismi a livello genomico.

Accumulation of damage in DNA is a characteristic feature of ageing which may result from a decline in DNA repair efficiency. Base excision repair (BER) is the primary mechanism used to repair small-scale DNA damage such as that caused by oxidation. I hypothesised that epigenetic events contribute to the ageing process through deregulation of BER gene expression and that these adverse effects of ageing may be modulated by dietary restriction (DR). To test these hypotheses, I quantified DNA methylation and histone post-translational modifications at BER-gene (Ogg1 and Apex) promoters, together with expression of these genes, in tissues from ageing mice and from mice exposed to DR. Phenotypic changes in DNA incision activity and oxidative damage were also quantified. Cytosine methylation was measured by pyrosequencing at the Ogg1 promoter in brain and livers from ad libitum (AL) and 40% DR mice at 3, 12, 24 and 30 months of age (n=5/group). Ogg1 promoter methylation decreased with age in the liver (p=0.018) and brain (p=0.016) and DR significantly reduced Ogg1 promoter methylation (p=0.014) in the brain. Additionally, in the brain there was a 2 fold enrichment in histone 4 acetylation (H4Ac) and histone 3 lysine 27 tri-methylation (H3K27Me3) as measured by the ChIP assay at Ogg1 and Apex promoters in 30 month old AL animals compared with 3 month old animals (p<0.05). H4Ac was 2.5-fold higher in the Ogg1 promoter in liver in 30 month old DR versus 30 month old AL animals (p=0.004). Furthermore H4K27Me3 was significantly (p=0.023) lower at the liver Ogg1 promoter in 30 month old compared with 3 month old animals. Ogg1 gene expression decreased with age in the brain (n.s.) and liver (p=0.005). Perhaps surprisingly, Ogg1 and Apex expression levels were higher in the brain (p=0.034) but lower in the liver (p=0.003) of DR compared with AL animals. I used a comet-based in vitro assay to quantify BER-related incision activity but did not observe any significant changes in the liver or in whole brain in response to ageing or to DR. However DNA incision activity varied considerably between different brain regions and DR enhanced incision 2 fold in the cortex (p=0.031) and subcortical regions (p=0.019). 8-oxoguanine lesions measured by HPLC-ECD decreased with age (AL and DR) (p<0.001) in the liver but no effect of age was detected in the brain. ii This study revealed for the first time that tissue-specific epigenetic changes at BER genes occur during ageing and the data presented here suggest that epigenetic changes at BER-related gene promoters may affect BER activity in some tissues. Furthermore, I have shown that DR influences the epigenetic and transcription changes in BER-related genes observed during ageing.

Drug addiction manifests clinically as compulsive drug seeking, and cravings that can persist and recur even after extended periods of abstinence. The fundamental principle that unites addictive drugs is that each one enhances synaptic DA by means that dissociate it from normal behavioral control, so that they act to reinforce their own acquisition. Our attention has focused on the study of phenomena associated with the consumption of alcohol and heroin. Alcohol has long been considered an unspecific pharmacological agent, recent molecular pharmacology studies have shown that acts on different primary targets. Through gene expression studies conducted recently it has been shown that the classical opioid receptors are differently involved in the consumption of ethanol and, furthermore, the system nociceptin / NOP, included in the family of endogenous opioid system, and both appear able to play a key role in the initiation of alcohol use in rodents. What emerges is that manipulation of the opioid system, nociceptin, may be useful in the treatment of addictions and there are several evidences that support the use of this strategy. The linkage between gene expression alterations and epigenetic modulation in PDYN and PNOC promoters following alcohol treatment confirm the possible chromatin remodeling mechanism already proposed for alcoholism. In the second part of present study, we also investigated alterations in signaling molecules directly associated with MAPK pathway in a unique collection of postmortem brains from heroin abusers. The interest was focused on understanding the effects that prolonged exposure of heroin can cause in an individual, over the entire MAPK cascade and consequently on the transcription factor ELK1, which is regulated by this pathway. We have shown that the activation of ERK1/2 resulting in Elk-1 phosphorylation in striatal neurons supporting the hypothesis that prolonged exposure to substance abuse causes a dysregulation of MAPK pathway.

Drug addiction manifests clinically as compulsive drug seeking, and cravings that can persist and recur even after extended periods of abstinence. The fundamental principle that unites addictive drugs is that each one enhances synaptic DA by means that dissociate it from normal behavioral control, so that they act to reinforce their own acquisition. Our attention has focused on the study of phenomena associated with the consumption of alcohol and heroin. Alcohol has long been considered an unspecific pharmacological agent, recent molecular pharmacology studies have shown that acts on different primary targets. Through gene expression studies conducted recently it has been shown that the classical opioid receptors are differently involved in the consumption of ethanol and, furthermore, the system nociceptin / NOP, included in the family of endogenous opioid system, and both appear able to play a key role in the initiation of alcohol use in rodents. What emerges is that manipulation of the opioid system, nociceptin, may be useful in the treatment of addictions and there are several evidences that support the use of this strategy. The linkage between gene expression alterations and epigenetic modulation in PDYN and PNOC promoters following alcohol treatment confirm the possible chromatin remodeling mechanism already proposed for alcoholism. In the second part of present study, we also investigated alterations in signaling molecules directly associated with MAPK pathway in a unique collection of postmortem brains from heroin abusers. The interest was focused on understanding the effects that prolonged exposure of heroin can cause in an individual, over the entire MAPK cascade and consequently on the transcription factor ELK1, which is regulated by this pathway. We have shown that the activation of ERK1/2 resulting in Elk-1 phosphorylation in striatal neurons supporting the hypothesis that prolonged exposure to substance abuse causes a dysregulation of MAPK pathway.

Triple negative breast cancers (TNBCs) are high-grade invasive ductal carcinoma which lack expression of hormone receptors and which do not benefit from either endocrine or anti-human epidermal growth factor receptor 2 therapies leading to poor outcome. This heterogeneous group of cancers encompass different molecular subtypes reflecting treatment responsiveness. Among others, mesenchymal-like TNBC subtype is characterized by the enrichment of proteins involved in cell motility and Epithelial to Mesenchymal transition (EMT). High Mobility Group A1 (HMGA1) is a family of oncofetal proteins identified as inducers of this phenotypic transition that have been extensively associated to cancer onset and progression. Despite their well-described role in chromatin architecture and gene expression regulation, their potential role in setting histones post-translational dynamics has never been taken into account. In this thesis, we provide evidences of HMGA1 involvement in histone post-translational modifications modulation by sustaining the RAS/RAF/MEK/ERK/RSK2 pathway in mesenchymal-like TNBC cells. In particular, we suggest that HMGA1 proteins make chromatin prone to epigenetic modifiers actions sustaining both H3 serine 10 phosphorylation by RSK2 and H2B lysine 5 acetylation by CBP and modulating the expression of genes involved in tumor progression and EMT. We suggest that aside being considered as drug target, HMGA1 could be used as biomarker predicting responsiveness to epigenetic therapies in triple negative breast cancers.

2012/2013
HMGA1 is an oncogene encoding for an architectural transcription factor that affects fundamental cell processes, leading to neoplastic transformation. The two main mechanisms by which HMGA1 protein is known to be involved in cancer concern the regulation of gene expression by altering DNA structure and interacting with a conspicuous number of transcription factors. Here we provide evidence of an additional level of gene expression regulation exploited by HMGA1 to exert its oncogenic activity. Starting from protein-protein interaction data showing that HMGA1 interacts with histones, we show that HMGA1 regulates gene expression by affecting the epigenetic status of cancer cells. In particular, it modulates the signalling cascade mediated by the RAS/RAF/MEKK/ERK/RSK2 pathway regulating the levels of histone H3 phosphorylation at Serine 10 and Serine 28. We demonstrate that the down-regulation of these two H3 post-translational modifications by HMGA1 silencing and by inhibitors of the RAS/RAF/MEKK/ERK pathway is linked to cell migration decrease and morphological changes resembling the mesenchymal to epithelial transition.
HMGA1 è un oncogene codificante per un fattore trascrizionale architetturale che influenza fondamentali processi cellulari, portando alla trasformazione neoplastica. I due principali meccanismi tramite cui la proteina HMGA1 è nota essere coinvolta nel cancro riguardano la regolazione dell’espressione genica tramite l’alterazione della struttura del DNA e l’interazione con un cospicuo numero di fattori di trascrizione. Qui forniamo la prova di un addizionale livello di regolazione dell’espressione genica sfruttato da HMGA1 per esercitare la sua attività oncogenica. Partendo da dati d’interazione proteina-proteina che mostrano che HMGA1 interagisce con gli istoni, mostriamo che HMGA1 regola l’espressione genica influenzando lo stato epigenetico delle cellule cancerose. In particolare, essa modula la cascata di segnalazione mediata dalla via di RAS/RAF/MEKK/ERK/RSK2 regolando i livelli di fosforilazione dell’istone H3 sulla Serina 10 e sulla Serina 28. Noi dimostriamo che la down-regolazione di queste due modificazioni post-traduzionali di H3 tramite il silenziamento di HMGA1 e l’utilizzo di inibitori della via di RAS/RAF/MEKK/ERK/RSK2 è correlata alla diminuzione della migrazione cellulare e a cambiamenti morfologici che ricordano la transizione mesenchimo-epiteliale.
XXV Ciclo
1983

In this thesis, I examine the contribution of cigarette smoking and human papillomavirus infection, two independent risk factors for cervical neoplasia, to the epigenetic modulation of cellular genes in cervical epithelium. To determine the temporal relationship between cigarette smoking and the detection of CDKN2A methylation in cervical cytological samples, I used a unique cohort of 2011 women aged 15-19 who were recruited soon after they first had sexual intercourse. I have shown that compared with never-smokers, women who first started to smoke during follow-up had an increased risk of acquiring methylation of the tumour suppressor gene (TSG), CDKN2A (odds ratio=3.67; 95% confidence interval: 1.09 to 12.33; p=0.04). I was also able to show that this epigenetic change was often reversible following smoking cessation. To determine the spectrum of epigenetic changes associated with HPV infection I performed CpG island and gene expression arrays on two in vitro models, a HPV replication model and a cervical disease progression model. I went on to show that HPV infection is followed by the up-regulation of the DNA methyltransferase DNMT1 which binds to the TSG, RARB, resulting in its de novo methylation. Specific CpG loci in the RARB promoter appeared to be targeted for de novo methylation, rather than methylation of all the CpGs, and may represent the methylation pattern seen at the earliest stages of HPV-induced carcinogenesis.

La truite arc-en-ciel carnivore (Oncorhynchus mykiss) est considérée comme une espèce pauvre utilisatrice de glucides alimentaires. Des études récentes ont montré qu'une hypométhylation globale de l'ADN hépatique induite par un régime alimentaire riche en glucides et pauvre en protéines pourrait être impliquée dans l'établissement / le maintien du de ce phénotype chez la truite, mais le détail des mécanismes sous-jacents reste inconnu. La thèse vise à étudier les mécanismes épigénétiques sous-jacents à ce phénotype de faible utilisation des glucides alimentaire chez la truite et à examiner si le métabolisme du glucose et l’épigénome chez les juvéniles peuvent être programmés par un stimulus hypoxique précoce. Nous avons d’abord identifié tous les gènes paralogues liés aux voies de méthylation / déméthylation de l’ADN (dnmt, tet et tdg) dans le génome de la truite, clarifié leurs histoires évolutives et analysé leurs profils d’expression au cours de la gamétogenèse et de l’embryogenèse chez la truite. Nous avons ensuite étudiés plus en détail les processus et les mécanismes potentiellement à l’origine de l'hypométhylation de l'ADN hépatique global constatée chez la truite après un régime riche en glucides et pauvre en protéines. Les résultats ont montré pour la première fois qu'une diminution du taux deprotéines et une augmentation du taux de glucides dans l’aliment induisent de manière indépendante et en interaction une hypométhylation hépatique globale chez la truite, qui semble établie par le biais d'une voie de déméthylation active. Nous avons également constaté qu’une forte hyperglycémie induite par une injection de glucose induit une hypométhylation globale de l’ADN au niveau des sites CmCGG dans le foie de la truite. Les mécanismes détaillés de ces processus de déméthylation restent à élucider. Enfin, grâce à la stratégie de programmation métabolique, nous avons pour la première fois confirmé que l’utilisation d’un stimulus non nutritionnel au début de la vie, l’hypoxie, pouvait moduler de façon persistante la transcription des gènes liés au métabolisme du glucose chez la truite juvénile sans nuire aux performances de croissance. De plus, selon sa nature chronique ou aigue, l’hypoxie, a tendance à induire des effets de programmation opposés sur les gènes codants pour les transporteurs au glucose notamment dans le foie et le muscle de la truite juvénile. Dans son ensemble, la thèse met en avant notre compréhension du rôle du méthylome dans la contribution à la faible capacité d'utilisation des glucides alimentaires chez la truiteLa thèse met aussi en lumière le potentiel d'utilisation de l'hypoxie comme stimulus pour programmer le métabolisme du glucose, l'épigénome et l'utilisation des glucides alimentaires chez la truite arc-en-ciel
The carnivorous rainbow trout (Oncorhynchus mykiss) is considered as a poor user of dietary carbohydrates. Recent studies showed that a high-carbohydrate/low protein diet inducing hepatic global DNA hypomethylation could be involved in the establishment/maintenance of the poor dietary carbohydrates utilisation phenotype in trout, but the detail mechanisms remain unclear. The present thesis aimed at investigating the epigenetic mechanisms underlying this poor dietary carbohydrate utilisation phenotype in trout, and exploring if the glucose metabolism and the epigenome in juveniles can be programmed through a hypoxic stimulus during early life. We first identified all the paralogous genes related to DNA methylation/demethylation pathways (dnmt, tet and tdg) in trout genome, clarified their molecular evolution histories and monitored their transcriptional expression patterns during gametogenesis and embryogenesis in trout. Besides, we investigate further the causes, processes and potential mechanisms about the hepatic global DNA hypomethylation in trout after feeding a high carbohydrate/low protein diet. Results for the first time demonstrated that a decrease in protein content and an increase in carbohydrate content in the diet can independently as well as interactively induce hepatic global hypomethylation in trout. This global loss of methylation is probably established through an active demethylation pathway. We also found that a strong hyperglycaemia induced by glucose injection induces global CmCGG hypomethylation in the liver of trout. The detailed mechanisms of these demethylation processes remain to be elucidated. Finally, through metabolic programming strategy, we confirmed for the first time that using a non-nutritional stimulus, hypoxia, during early life stage persistently modulates the transcription of glucose metabolism-related genes in juvenile trout without negative effects on growth performance. Moreover, acute and chronic hypoxia tended to induce opposite programming effects on glucose-transporter encoding genes in both liver and muscle of juvenile trout. Together, the present thesis brings forward our understandings about the roles of epigenetics in contributing to the low ability to use dietary carbohydrates in trout, and sheds light on the potential of using hypoxia as the stimulus in metabolic programming strategy to tailor the glucose metabolism, the epigenome and dietary carbohydrate utilisation in rainbow trout

L'épissage alternatif de l'ARN précurseur est un processus co-transcriptionnel qui affecte la grande majorité des gènes humains et contribue à la diversité des protéines. Le dérèglement d'un tel processus est impliqué dans diverses maladies, y compris la tumorigènes. Cependant, les mécanismes qui régulent ces processus restaient à caractériser. Dans cette étude, nous avons montré que les perturbations de l'épissage alternatif étaient corrélées aux dérèglements de la transcription convergente et de la méthylation de l'ADN. Une transcription convergente peut être générée entre des paires de gènes voisins en en orientation opposée, ou entre des amplificateurs intragéniques et leur gène hôte. CENPO et ADCY3 ont été identifiés comme une paire de gènes de transcription convergentes. Nous avons constaté, dans un modèle de progression tumorale du cancer du sein, que le changement d'épissage de l'exon22 variant d'ADCY3 était corrélé à une augmentation de sa transcription qui allait contre celle de CENPO. En utilisant le système de répression ciblée de la transcription CRISPRi, nous avons démontré que l’inhibition de la transcription de CENPO ne pouvait pas inverser l'altération d'épissage alternatif d'ADCY3 dans les cellules cancéreuses (DCIS). Un activateur intragénique actif a été identifié dans l'intron16 du gène CD44, en aval de ses exons alternatifs. En utilisant le système d'activation ciblée de transcription CRISPRa, nous avons montré que l’augmentation de la transcription de CD44 ne pouvait pas modifier l'épissage alternatif de CD44 dans les cellules DCIS. Ces résultats suggèrent que la modification de transcription convergente par des changements d’activité des promoteurs ne permet pas d’altérer l'épissage alternatif de ADCY3 et CD44 dans les cellules DCIS. Cependant, en remplaçant l'amplificateur intragénique par un promoteur inductible, nous avons constaté que l'activation de la transcription intragénique augmentait le niveau d'inclusion de plusieurs exons alternatifs de CD44 dans les cellules HCT116. Ce résultat suggère que la transcription convergente local pourrait avoir un impact direct sur l'épissage alternatif de CD44. De plus, en utilisant le système de méthylation de l'ADN ciblée CRISPR/dCas9-DNMT3b, nous avons démontré que la méthylation de l'ADN au niveau des exons variants pouvait modifier l'épissage alternatif de CD44. Ce travail de thèse a également exploré les limites et la faisabilité de l'étude de l'épissage alternatif avec des outils moléculaires basés sur le système CRISPR
Alternative splicing of precursor RNA is a co-transcriptional process that affects the vast majority of human genes and contributes to protein diversity. Dysregulation of such process is implicated in various diseases, including tumorigenesis. However, the mechanisms regulating these processes were still to be characterized. In this study, we showed that perturbations of alternative splicing correlated with dysregulations of convergent transcription and DNA methylation. Convergent transcription could be generated between pairs of neighboring genes in opposite orientation, or between intragenic enhancers and their host gene. CENPO and ADCY3 was identified as a convergent transcription gene pair. We found, in a tumor progression model of breast cancer, that the splicing change of the ADCY3 variant exon22 correlated with an increase of its transcription that went against that of CENPO. By using targeted transcription repression system CRISPRi, we demonstrated that downregulating the transcription of CENPO could not reverse the alternative splicing alteration of ADCY3 in cancer cells (DCIS). An active intragenic enhancer was identified in the intron16 of CD44, at the downstream of its alternative exons. By using targeted transcription activation system CRISPRa, we showed that upregulating the transcription of CD44 could not alter the alternative splicing of CD44 in DCIS cells. These results suggest that convergent transcription modulation through changes of promoter activity does not alter the alternative splicing of ADCY3 and CD44 in DCIS cells. However, through replacing the intragenic enhancer by an inducible promoter, we found that intragenic transcription activation increased the inclusion level of several alternative exons of CD44 in HCT116 cells. This result suggested that local convergent transcription could have a direct impact on the alternative splicing of CD44. Furthermore, by using targeted DNA methylation system CRISPR/dCas9-DNMT3b, we showed that DNA methylation at variant exons could directly modify CD44 alternative splicing. This thesis work also explored the limitation and feasibility of studying alternative splicing with repurposed CRISPR systems

The cancer stem cell (CSC) concept is still very controversial; therefore identification and isolation of this specific population remain challenging. A variety of putative markers have been described and measurement of high aldehyde dehydrogenase (ALDH) activity has been defined as a characteristic of stem cells (SCs). In this study, a library of novel small molecules (1,4-disubstituted acetalanthraquinones, AAQs), containing an acetal group as protected aldehyde functionality, was designed with the aim of probing affinity for ALDH metabolism and demonstrating their potential as molecular fluorescent probes to identify CSCs. The AAQs were shown to be subjective to acidic hydrolysis using 2M HCl at 37ºC; however compounds containing secondary or tertiary amine functionalities in their sidechain were only partly hydrolysed at 70 ºC. Metabolism studies were conducted using cytosolic fractions from rat liver enriched in ALDHs, yeast ALDH and human recombinant ALDH1A1. Some evidence was demonstrated which linked ALDH metabolism with aldehyde functionalities of hydrolysed AAQs (HAAQs). The AAQs were shown to emit far-red fluorescence (600-750 nm). A close relationship between structure modifications and alteration of cellular localisation, with gained specificity for selected sub-cellular compartments were achieved when assessed in A549 and U-2 OS cell lines. Thermal DNA denaturation and chemosensitivity assays were used to obtain information about DNA binding properties and cytotoxicity of AAQs and HAAQ congeners. All compounds were shown to be weak-to-moderately binding to DNA, and symmetrical 1,4-disubstituted compounds were shown to be non-toxic (IC50 = 100 :M) with nonsymmetrical analogues generating IC50 values in the 1-100 :M range. No fundamental variation in the biological activity was observed when comparing AAQs with HAAQs in the A549 (+ALDH) and MCF7 (-ALDH) cell lines. A pilot investigation revealed that aberrant gene methylation was cell-type dependent for three ALDH isoforms (1A1, 2, 3A1). Decitabine treatment led to enhanced protein expression for ALDH1A1 (A549), ALDH2 (MCF7) and ALDH3A1 (A549). In contrast, the protein level was reduced for ALDH1A1 in HT29 cells after decitabine treatment. ALDH1A1, ALDH2 and ALDH3A1 were highly expressed in prostate cell lines, with expression linked to promoter methylation. In contrast, low levels of DNA methylation were found in primary prostate cancer cells and benign prostatic hyperplasia. Interestingly, ALDH1A1, considered a SC marker, was found to be expressed at low levels in CD133⁺/ α₂β₁ hi stem cell fraction and upregulated in CD133⁻/ α₂β₁ lo differentiated prostate cancer cells. In summary, the results in this thesis demonstrate the complexity and tumour type specificity of ALDH expression. This creates challenges for the development of selective probes for CSC isolation, such as the AAQs discussed in this thesis. Although inconclusive results were obtained in regard to AAQs and their potential in targeting ALDHs, selected AAQs were shown to reveal interesting biological features highlighting them as potential non-invasive cytometric probes for tracking molecular interactions in live cells.

Les lymphocytes T CD4 (LT CD4) sont nécessaires à l'établissement d'une réponse anticancéreuse efficace en contribuant à l’induction des LT cytotoxiques spécifiques de la tumeur et à leur persistance. Cependant, le pronostic des patients peut être affecté différemment selon le type de LT CD4 infiltrant la tumeur (TIL). L'épigénétique joue un rôle important dans la régulation de la polarisation des LT CD4, de leur plasticité vers d'autres sous-ensembles et de leur maturation. Il a été démontré que l'utilisation de modulateurs épigénétiques pouvait réguler la différenciation des LT CD4. Ainsi, la manipulation des LT CD4 par des thérapies épigénétiques peut être utilisée comme une stratégie pour améliorer l'immunothérapie. Nos résultats ont confirmé la présence de LTh17 parmi les LT expandus provenant de métastases hépatiques du cancer colorectal. Le nombre des LTh17 présents dans les TIL expandus était corrélé à la survie des patients. Nous avons ensuite tenté d'évaluer l'effet des régulateurs épigénétiques sur la différenciation et la fonction des LT CD4. Le criblage d'une banque de régulateurs épigénétiques nous a permis d'identifier un activateur de Sirtuine 1, l’Agrimol B, capable de réduire la prolifération, la sécrétion de cytokines et l'expression de CCR6 à la surface des LT CD4. L’Agrimol B a ainsi permis de réguler la migration et la fonctionnalité des LTh17 provenant de donneurs sains. L'inhibition des caspases par notre molécule a empêché le clivage de Sirtuine 1, permettant ainsi le maintien de son activité dans les LT CD4 activés. Dans l'ensemble, ces travaux visent à mettre en évidence le rôle de l'épigénétique dans la régulation des différentes populations de LT CD4 afin de potentialiser l'efficacité de l'immunothérapie anticancéreuse
CD4 T-cells are necessary for the establishment of an efficient anti-cancer response by providing help for the priming and persistence of tumor-specific cytotoxic T lymphocytes. However, the prognosis of patients can be differently affected, depending on CD4 T-cell subtypes infiltrating the tumor (TIL). Epigenetics take an important part in : i) the regulation of CD4 T-cells polarization, ii) plasticity towards other subsets and iii) maturation. It has been shown before that epigenetic modulators could regulate CD4 T-cells differentiation. Thus, the treatment of CD4 T-cells by epigenetic therapies can be used as a strategy to improve immunotherapy. Our results confirmed the presence of Th17-cells in expanded T-cells issued from liver metastases of colorectal cancer. Moreover, the number of expanded Th17-cells among total TIL was inversely correlated with patients' survival. We then attempt to evaluate the effect of epigenetic regulators on both CD4 T-cells differentiation and function. A screening of a bank of epidrugs allowed us to identify a Sirtuin 1 activator, Agrimol B, that might downregulate proliferation, cytokine secretion and CCR6 expression on CD4 T-cells. Our results thus indicated that Agrimol B might regulate the migration and the functionality of Th17-cells from healthy donors. Mechanistically, the inhibition of caspases by our molecule could prevent the cleavage of Sirtuin 1 and thus maintain its activity compared to control conditions. Together, this work aims to uncover the role of epigenetics in the regulation of CD4 T-cell subsets in order to potentiate the effectiveness of cancer immunotherapy

La maladie de Huntington (MH) est une maladie neurodégénérative génétique caractérisée par des symptômes moteurs, cognitifs et psychiatriques causés par une atteinte primaire du striatum. Le mécanisme pathogénique implique une dérégulation épigénétique et transcriptionnelle à l’origine d’une perte d’identité et de fonction des neurones. Cette thèse a consisté en la caractérisation épigénétique du striatum de modèles murins à une résolution type cellulaire-spécifique et à différent stades de la MH. Nous avons observé que les neurones striataux qui expriment le gène muté dans la MH présentent une érosion épigénétique traduisant un vieillissement accéléré qui implique une altération des complexes polycomb. Les régulations épigénétiques étant sensibles à l’environnement, nous avons caractérisé le phénotype comportemental et moléculaire de modèles murins de la MH hébergés en environnement enrichi (EE) afin de décrypter l’effet de l’EE sur les régulations épigénétiques et transcriptionnelles. Nos résultats permettent une meilleure compréhension des mécanismes pathogéniques de la MH, et offrent de nouvelles perspectives thérapeutiques
Huntington's disease (HD) is a neurodegenerative genetic disease characterized by motor, cognitive, and psychiatric disorders caused by primary damage to the striatum. The pathogenic mechanism is complex and involve epigenetic and transcriptional dysregulations leading to a loss of neuronal identity and cell function. This thesis aimed to characterize the striatal epigenetic signature in mouse models with a celltype-specific resolution at different stages of HD. We observed that striatal neurons expressing the HD mutation undergo epigenetic erosion, reflecting accelerated aging in HD, induced by alterations in polycomb complexes. As epigenetic regulations are sensitive to the environment, we characterized the behavioral phenotype and molecular alterations of HD mouse model after housing in an enriched environment (EE) to decipher the epigenetic and transcriptomic effects induced by EE. Our findings thus provide a better understanding of early pathogenic mechanisms in HD, opening new therapeutic perspectives

The epidermal differentiation complex (EDC) encodes co-ordinately regulated genes critically involved in epidermal differentiation, however knowledge of the molecular mechanisms involved in co-ordinating EDC gene expression is limited. Recent findings indicate p63 dependent changes in the nuclear localisation and higher-order chromatin folding the EDC coincide with the onset of epidermal stratification during embryonic development. Here it is demonstrated that a direct transcription target of p63, the chromatin-remodelling enzyme Brg1, modulates the specific nuclear positioning of the EDC and transcription of differentiation-specific gene encoded at the EDC. In addition, the results of high-resolution 5C-based analyses of the spatial chromatin interactome at a 5.3Mb region spanning the murine EDC in epidermal keratinocytes, and the silenced EDC in thymocytes, are presented. Chromatin interactions at the EDC region in keratinocytes include long-range interactions between multiple proximal and distal candidate gene regulatory regions. Many candidate regulatory elements involved in looping chromatin interactions at the EDC region are enriched for both active (H3K4me1, H3K27ac) and repressive (H3K27me3) chromatin marks and are bound by Sin3a and RBP2 co-repressor complexes. The chromatin interactome at the EDC in epidermal progenitor cells is enriched for bound chromatin architectural proteins Satb1, Satb2, and the cohesin subunit Rad21. Further, a substantial degree of co-localisation is observed between these chromatin architectural proteins, transcription factors and co-factors. Findings presented here suggest that a functional chromatin interactome, mediated by Satb proteins and cohesin, acts in conjunction with transcriptional repressor complexes to facilitate co-ordinated gene expression at the EDC in epidermal progenitor cells upon differentiation. These results provide a foundation for further study of the mechanisms controlling EDC gene expression in health and disease.

The epidermal differentiation complex (EDC) encodes co-ordinately regulated genes critically involved in epidermal differentiation, however knowledge of the molecular mechanisms involved in co-ordinating EDC gene expression is limited. Recent findings indicate p63 dependent changes in the nuclear localisation and higher-order chromatin folding the EDC coincide with the onset of epidermal stratification during embryonic development. Here it is demonstrated that a direct transcription target of p63, the chromatin-remodelling enzyme Brg1, modulates the specific nuclear positioning of the EDC and transcription of differentiation-specific gene encoded at the EDC. In addition, the results of high-resolution 5C-based analyses of the spatial chromatin interactome at a 5.3Mb region spanning the murine EDC in epidermal keratinocytes, and the silenced EDC in thymocytes, are presented. Chromatin interactions at the EDC region in keratinocytes include long-range interactions between multiple proximal and distal candidate gene regulatory regions. Many candidate regulatory elements involved in looping chromatin interactions at the EDC region are enriched for both active (H3K4me1, H3K27ac) and repressive (H3K27me3) chromatin marks and are bound by Sin3a and RBP2 co-repressor complexes. The chromatin interactome at the EDC in epidermal progenitor cells is enriched for bound chromatin architectural proteins Satb1, Satb2, and the cohesin subunit Rad21. Further, a substantial degree of co-localisation is observed between these chromatin architectural proteins, transcription factors and co-factors. Findings presented here suggest that a functional chromatin interactome, mediated by Satb proteins and cohesin, acts in conjunction with transcriptional repressor complexes to facilitate co-ordinated gene expression at the EDC in epidermal progenitor cells upon differentiation. These results provide a foundation for further study of the mechanisms controlling EDC gene expression in health and disease.

Les travaux de recherche que présente cette thèse sont le fruit de collaborations fructueuses entre mon entreprise d’accueil, l’Institut de Recherches SERVIER, mon laboratoire d’accueil, la plateforme BioPhenics de l’Institut Curie, et moi-même préparant mon doctorat à l’Ecole Doctorale CBMS de l’Université Paris-Saclay. Des partenariats internationaux ont également permis de générer et d’enrichir de multiples données dans un même but : identifier de nouvelles cibles thérapeutiques pour le traitement du cancer du sein triple-négatif (TNBC, Triple-negative breast cancer). Le cancer TNBC est une forme de cancer mammaire caractérisée par l’absence des récepteurs aux œstrogènes (ER, Estrogen Receptor), à la progestérone (PR, Progesterone receptor), et du récepteur de facteur de croissance épidermique humain 2 (HER2, Human epidermal growth factor receptor 2), touchant près de 20% des femmes diagnostiquées avec un cancer mammaire. Par l’absence de ces récepteurs, les patientes ne sont éligibles ni aux thérapies hormonales ni aux thérapies ciblées anti-HER2. Alors que le cancer TNBC est enrichi en cellule-souches cancéreuses (CSC) et que des dérégulations épigénétiques ont été identifiées dans les voies de signalisation des CSC des TNBC, nous avons émis l'hypothèse que les mécanismes épigénétiques pourraient être modulés et aboutir à deux phénotypes : un impact sur la viabilité des cellules d'une part, et un effet sur l'expression de HER2 de façon à sensibiliser les cellules aux thérapies anti-HER2 existantes d'autre part. Afin de vérifier ces hypothèses, nous avons réalisé des cribles de génomique fonctionnelle en siRNA ciblant 863 modulateurs épigénétiques par des approches à haut débit et haut contenu. Bien que l’utilisation de siRNA représente une approche puissante, le risque d’effets hors cible est important. Afin de renforcer la découverte de hits spécifiques et de limiter l’identification de hits non-spécifiques, différentes stratégies ont été mises en place pour les deux études. Alors que l’identification de gènes impliqués dans l’expression de HER2 est toujours à l’étude, nous avons identifié 3 gènes clés pour la viabilité des cellules TNBC, parmi lesquels CHAF1A, dont le rôle dans la viabilité des cellules TNBC est identifié pour la première fois. Aussi, suite à des analyses bioinformatiques réalisées à partir des résultats générés en viabilité, les effets non spécifiques à la cible initiale ont été considérés comme sources de hits potentiels, permettant d’envisager de nouvelles approches de génomique fonctionnelle
Research presented in this thesis manuscript is the result of a fruitful collaboration between my host company, Institut de Recherches SERVIER, my host laboratory, BioPhenics Laboratory at Institut Curie, and I, preparing my PhD at the doctoral school CBMS at Université Paris-Saclay. International partnerships also led to the generation of numerous data towards the same purpose: identifying novel therapeutic targets in triple-negative breast cancer (TNBC) treatment. TNBC is a breast cancer subtype characterized by its ER(Estrogen receptor)-, PR(Progesterone receptor)- and HER2(Human epidermal growth factor receptor 2)-negative phenotype, affecting almost 20% of breast cancer diagnosed women. In the absence of these receptors, patients cannot respond neither to hormone therapy nor anti-HER2 targeted therapies. While TNBC is enriched in cancer-stem cells (CSC) and epigenetic deregulations were identified in TNBC CSC signaling pathways, we supposed that epigenetic mechanisms could be modulated to result in two phenotypes : an impact on TNBC cell viability, and an impact on HER2 expression in order to sensitize cells to existing anti-HER2 therapies. To investigate these hypotheses, we performed siRNA functional genomics screening targeting 863 epigenetic modulators through high-throughput and high-content approaches. Although using siRNA represents a powerful approach, the risk of off-target effects is important. In order to reinforce on-target hits discovery and to prevent the identification of non-specific hits, various strategies were used for the two studies. While the identification of genes involved in HER2 expression is currently in progress, we identified 3 key genes for TNBC cell viability, including CHAF1A for which the role in TNBC cell viability was never revealed. Also, following bioinformatic analyses performed from viability data, off-target effects were considered as sources of potential hits, highlighting the potential of a new functional genomics screening approach

Recentes estudos genômicos têm mostrado que vários fungos e bactérias possuem um potencial biossintético superior à quantidade de me tabólitos secundários já isolados desses micro-organismos. A descoberta de produtos naturais inéditos e bioativos é limitada pela impossibilidade dos micro-organismos expressarem to das as suas rotas biossintéticas em laboratório. Assim, estratégias alternativas para i nduzir a produção de produtos naturais microbianos são necessárias. A utilização de cultur as mistas de micro-organismos é uma estratégia que vem sendo recentemente utilizada, na tentativa de mimetizar condições mais naturais de crescimento. Além disso, a adição de mo duladores químicos e epigenéticos às culturas microbianas também pode potencialmente est imular a produção de compostos de interesse, seja por ativar mecanismos celulares em resposta à condição de estresse, ou por alterar a taxa de transcrição de certos genes, em f unção de mudanças no grau de enovelamento da cromatina. Alternativamente, a indu ção de certos genes, e até mesmo a diversificação do metabolismo secundário, podem ser conseguidos através de engenharia genética, pela manipulação direta de genes de inter esse. A linhagem endofítica Alternaria tenuissima SS77, selecionada para os experimentos de modulaçã o química e epigenética, teve seu metabolismo secundário alterado após o tra tamento com diferentes moduladores. Provavelmente, o efeito observado ocorreu em função de uma eliciação inespecífica dos diferentes moduladores. Além disso, o cultivo misto desse fungo com o fungo endofítico Nigrospora sphaerica SS67 , isolada da mesma planta hospedeira ( Smallanthus sonchifolius ), levou ao isolamento de dois novos policetídeos, da classe das perilenequinonas, juntamente com um já relatado na literatura científica. Para r ealizar os cultivos microbianos mistos, envolvendo uma linhagem bacteriana e uma fúngica, t rês linhagens de actinobactérias e cinco de fungos, todos endofíticos da planta Lychnophora ericoides , foram selecionadas. Alterações no perfil metabólico da cultura mista de Phomopsis sp FLe6 com Streptomyces albospinus RLe7 foram as mais evidentes e por isso a maioria das investigações foram focadas nessa cultura mista. Várias condições de cu ltivo foram testadas e diferentes resultados foram obtidos. Em alguns casos, o desenv olvimento da linhagem fúngica foi inibido pela bacteriana, e em outros, foi observado o inverso. Da mesma forma, houve acentuada inibição da produção de alguns metabólito s secundários na presença da linhagem desafiadora, mas também foi verificada a eliciação de outros. Os extratos das culturas simples desses micro-organismos também apresentaram relativas alterações nos perfis metabólicos em função das condições de cultivo. Os metabólitos produzidos pelo fungo Phomopsis sp FLe6 e pela actinobactéria Streptomyces albospinus RLe7 foram isolados e caracterizados. Os resultados mostram que as intera ções entre os micro-organismos endofíticos são bastante complexas, estando sujeita s a ação de diversos fatores externos que muitas vezes não podem ser pré-determinados. Po r isso, estabelecer um cultivo misto adequado, do ponto de vista da eliciação da produçã o de metabólitos secundários, pode requerer uma série de tentativas. Ainda assim, os r esultados almejados podem ser conseguidos utilizando essa estratégia. Diferenteme nte das linhagens endofíticas, manipuladas quimicamente através de diferentes estr atégias, a linhagem sequenciada de Fusarium heterosporum ATCC 74349, foi manipulada geneticamente para a co nstrução de um gene biossintético híbrido pks-nrps , contendo a porção nrps do gene híbrido da equisetina e um pks críptico de Aspergillus fumigatus . Era esperado que a linhagem hibridizada fosse capaz de produzir o metabólito se cundário geneticamente planejado, entretanto, após seu cultivo, esse produto não foi detectado nos extratos, e as possíveis razões são discutidas. Ainda que os resultados espe rados não tenham sido obtidos, estudos que contribuam para a ampliação do entendimento das megassintases fúngicas são de extrema valia.
Recently, genetic studies have shown that several b acteria and fungi hold a greater biosynthetic potential than the amount of secondary metabolites isolated from these microorganisms. The discovery of novel bioactive na tural products is limited by the inability of microorganisms to express all their biosynthetic pa thways in laboratory conditions. Therefore, alternative strategies to induce the production of microbial natural products are required. Mixed cultures of microorganisms are a strategy tha t has been used to mimic more natural conditions of growth. Furthermore, the addition of chemical and epigenetic modulators to the microbial cultures can also stimulate the productio n of compounds by activating cellular mechanisms in response to stress conditions or by c hanging the transcription rate of certain genes, due to changes in the chromatin folding. Alt ernatively, the induction of some genes, and even the diversification of secondary metabolis m, can be achieved by genetic engineering, by manipulating genes of interest. The endophytic strain Alternaria tenuissima SS77, which was selected for the experiments of che mical and epigenetic modulation, had changed its secondary metabolism after treatment wi th different modulators. Probably, the observed effect was due to a nonspecific elicitatio n of those modulators. Moreover, the mixed cultures of this fungus with the endophytic fungus Nigrospora sphaerica SS67, isolated from the same host plant ( Smallanthus sonchifolius ), led to the isolation of two new polyketides, belonging to perylene quinone class, along with ano ther one already reported in the scientific literature. Three strains of actinobacteria and fiv e fungi, all endophytes of Lychnophora ericoides , were selected to grow in microbial mixed cultures comprising one bacteria and one fungus. Changes in the metabolic profile of the mix ed culture of Phomopsis sp. FLe6 with Streptomyces albospinus RLe7 were the most obvious, and then further studi es were focused on this mixed culture. Many culture conditions were analyzed and different results were obtained. In some cases, the development of the fun gal strain was inhibited by bacteria, and in other cases was observed the opposite. Similarly , there was a remarkable inhibition of the production of certain secondary metabolites in the presence of the challenging strain, but the eliciting of others was also observed. The extracts of the single cultures of these microorganisms also showed changes in metabolic pro files due to culture conditions. The metabolites produced by the fungus Phomopsis sp. FLe6 and the actinobacteria S. albospinus RLe7 were isolated and characterized. The results show that interactions between endophytic microorganisms are quite complex and are influenced by various external factors that often can not be previously determined. Theref ore, establishing a suitable mixed culture to elicit the production of secondary metabolites m ay require some attempts. Still, the expected results can be achieved using this strateg y. Unlike the endophytic strains, that was chemically manipulated by different strategies, the sequenced strain Fusarium heterosporum ATCC 74349 was genetically manipulated to construct a hybrid PKS-NRPS biosynthetic gene containing the NRPS portion of the hybrid gene of e quisetin and a cryptic PKS gene of Aspergillus fumigatus . It was expected that hybridized strain could be a ble to produce the secondary metabolite genetically planned, however, after its cultivation, this product was not detected in any extracts, and some possible reasons are discussed. Although the expected results have not been obtained, studies that contri bute to increasing the understanding of fungal megasynthases are extremely valuable

Foamy macrophages or Foam cells are a critical cellular component of the granuloma formed during pulmonary infection. These lipid rich cells generally contain neutral lipids, Cholesteryl Esters (CE) and/or Triglycerides (TAGs) which not only serve as the carbon source for the pathogen to thrive in the hostile environment of host, but also act as a substrate for various immunomodulatory enzymes such as COX-2 etc. Lipid accretion is a result of various cellular processes involved in lipid uptake and synthesis that are fine-tuned by various transcription factors and chromatin modifiers. The thesis submitted is focused around the epigenetics of foam cell formation during pulmonary infection stemming from Mycobacterium tuberculosis and Cryptococcus neoformans. Mycobacterium tuberculosis, which is the causative agent of tuberculosis induces the formation of foamy macrophages in the host to permit its own growth. In the present context, we have highlighted the role of WNT-responsive epigenetic modifiers, G9a (H3K9 methyltransferase) and SIRT6 (H3K9 deacetylase) in fine-tuning the expression level of genes involved in cholesterol biosynthesis and efflux. Moreover, augmented levels of cholesterol was observed to fuel anti-oxidative response as depletion of cholesterol or G9a/SIRT6 elevated the oxidative response and eventually reduced bacterial survival. Similarly, LncRNAs have recently been showed to play a cardinal role in regulating the gene expression via modulating the activity of various transcription factors and chromatin remodellers. In the present study, we have attempted to underpin the role of Malat1 in governing Mtb pathogenesis. High throughput analysis including RNA sequencing and ATAC sequencing revealed Malat1-dependent global change in transcriptome and chromatin accessibility, respectively. Furthermore, In vivo study utilising both WT and Malat1 KO mice study ascertained the pivotal role of Malat1 in regulating Mtb burden as Malat1 KO mice showed reduced bacillary burden and improved lung pathology. Moreover, macrophages devoid of Malat1 showed reduced lipid content and enhanced necroptosis and elevated extracellular burden. In addition to above, thesis submitted also elucidates the molecular players involved in perturbing lipid content during C. neoformans infection. Enhanced lipid content has been demonstrated to support intracellular growth of C. neoformans. In this respect, Pyk2- cRaf axis-driven WNT signalling was found to be critical in regulating the levels of Lysine Specific Demethylase-1 (LSD1). LSD1 owing to its demethylase activity removes H3K9me2 repressive marks over the promoters of the genes involved in lipid homeostasis, leading to their enhanced transcription. Moreover, LSD1 sustains elevated lipid levels by inhibiting host lipophagy and hence preventing lipid turnover.

Notch signaling is one of the pleotropic signaling pathway that plays key role in development as it promotes differentiation and tissue morphogenesis. In recent years it has been observed that this pathway along with many other developmental pathways is seen to be deregulated in different cancers. The role that is played by this pathway is highly context dependent in cancers that is in some cases it act as a tumor suppressor while as oncogenic in others. It is proved with its oncogenic role in all forms of breast cancer. We tried to assess the effects on this pathway in MDA MB-231 breast cancer cells after treatment with epigenetic modulators S-Adenosyl Methionine (SAM) and S-Adenosyl Homocysteine (SAH) in a time dependent manner for 0-48 hr. The expression of important notch receptor Notch-1 and downstream effector Hes-1 were shown to be down regulated after SAM treatment but the SAH treatment upregulated them. This was accompanied by apoptotic induction in SAM treated cells in a more aggravated manner as compared to the SAH treated cells. From all these results we tentatively conclude that SAM treatment of invasive breast cancer cell line induces cell death in a notch dependent manner.

Colorectal cancer has a 9.8% cumulative incidence rate, making it the third most common cancer in the Western world. Despite a 50-60% response rate in patients to current cancer therapies, drug resistance and tumour relapse remain a concern. While current therapies reduce the tumour mass, they possibly fail to eradicate a unique population of pluripotent tumour resident cells. These cells, known as cancer stem cells, may have similar properties of self-renewal and proliferation to embryonic and adult stem cells, as they also express a number of key pluripotent transcription factors, including amongst others, NANOG, OCT3/4 and SOX2. Furthermore, since discreet groups of such stem cells are proposed to essentially drive tumourigenesis, they present as potential novel targets for cancer therapy. This study aimed to isolate a putative CSC population from the advanced colon adenocarcinoma cell lines HT29 and DLD1 and to assess the therapeutic effects of the epigenetic drugs Valproic acid and Zebularine on pluripotent gene expression.

博士
臺北醫學大學
臨床醫學研究所
104
Triple negative breast cancers (TNBC) possess cell dedifferentiation characteristics, carry out activities connate to those of cancer stem cells (CSCs) and are associated with increased metastasis, as well as, poor clinical prognosis. The regulatory mechanism of this highly malignant phenotype is still poorly characterized. Accruing evidence support the role of non-coding RNAs (ncRNAs) as potent regulators of CSC and metastatic gene expression, with their dysregulation implicated in tumorigenesis and disease progression. In this study, we investigated TNBC metastasis, metastasis-associated genes and potential inhibitory mechanisms. Compared with other breast cancer types, the highly metastatic MDA-MB-231 cells concurrently exhibited increased expression levels of Lysine-specific demethylase 5B protein (KDM5B) and long non-coding RNA (lncRNA), MALAT1, suggesting their functional association. KDM5B-silencing in the TNBC cells correlated with the upregulation of hsa-miR-448 and led to suppression of MALAT1 expression with decreased migration, invasion and clonogenic capacity in vitro, as well as, poor survival in vivo. This projects MALAT1 as a mediator of KDM5B oncogenic potential and highlights the critical role of this microRNA, lncRNA and histone demethylase in cancer cell motility and metastatic colonization. Increased expression of KDM5B correlating with disease progression and poor clinical outcome in breast cancer was reversed by hsa-miR-448. In parallel experiments, Pre-treatment of the TNBC cells with 10μM Ovatodiolide (Ova) 24 h before Doxorubicin (Doxo) administration increased the Doxo anticancer effect (IC50 1.3μM) compared to simultaneous treatment with Doxo (IC50 3.4μM), or Doxo alone (IC50 5.0μM). The intracellular accumulation of Doxo was lowest in the Ova pre-treated cells at all Doxo concentrations compared to Doxo alone or simultaneously treated cells. The cell cycle analysis of MDA-MB-231 cells treated concurrently with 2.5μM Ova and 1.25μM Doxo, showed increased percentage of cells arrested at G0/G1 compared to the Doxo-only group; however, pre-treatment with same concentration of Ova 24 h before Doxo treatment showed greater tumor growth inhibition with a 2.4-fold increased percentage of cells in G0/G1 arrest, greater Doxo-induced apoptosis, and significantly reduced intracellular Doxo accumulation. Additionally, Ova-sensitized TNBC cells lost their cancer stem cell-like phenotype, as evidenced by significant dissolution, necrosis or terminal differentiation of formed mammospheres. Taken together, our findings demonstrate the critical role of KDM5B and its negative regulator hsa-miR-448 in TNBC metastasis and progression. The disruption of KDM5B-MALAT1 signalling axis and associated activities in TNBC cells after Ova sensitization and upregulation of hsa-miR-448, projects them as putative therapeutic factor for selective eradication of TNBC cells.

The development and progression of cancer is predominantly regulated by both tumor suppressor genes as well as oncogenes. Either gene mutation causes hyper-activation of ‘oncogenes’ and suppression of ‘cell cycle control and tumor suppressor genes’; or alleles of these sets of genes expression profiles are modulated by epigenetic mechanisms. Hence, cancer is the manifestation of both genetic and epigenetic alternations modulating the metabolic profile and cellular homeostasis. Since cellular metabolism is mostly controlled by enzymatic activities and enhanced during the disease progression, chemo-preventive bioactive molecules from edible and medicinal plants are being increasingly evaluated which could be one of the desired ‘toxicity free’ therapeutic strategies against cancer. This thesis attempts to investigate the anticancer and epigenetic regulatory activity of Paederia foetida (P. foetida) and its active constituents; lupeol and β-sitosterol. The regulation mechanism(s) of tumor suppressor gene E-cadherin (CDH1) is tested considering multiple modalities of epigenetic modulations; like, DNA methylation, histone methylation, acetylation and phosphorylation. The cellular environment(s)/metabolism(s) were experimentally modulated by ectopic application of drugs and/or H2O2 with the cell culture media/buffer executing in vitro cell cultures. Crude methanolic leaf extract of P. foetida (MEPL) and two of the identified products, lupeol and β-sitosterol exhibit anticancer activity through inhibition of ‘cellular viability and migration’ by up regulation of CDH1 gene expression. Along with this, it also induces apoptosis through up regulation of pro-apoptotic gene Bax and down regulation of the anti-apoptotic gene Bcl2 in human prostate (PC-3 and DU-145) and breast cancer (MCF-7 and MDA-MB-231) cell lines. Ectopic applications of MEPL, lupeol and β-sitosterol also significantly down regulated the expression of DNMTs and HDACs as well as reduced the total DNMT and HDAC activity. Loss of CDH1 protein and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) within cells are the products of metabolic reactions and play some crucial roles in cellular metabolism, physiological functions and homeostasis. Roles of E-cadherin, EMT and ROS are discretely and intriguingly illustrated in many cancers focusing their collective concert during cancer progression. We report that hydrogen peroxide (H2O2) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H2O2 treatment decrease of E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H2O2 was attenuated by ROS scavengers; NAC, lupeol and β-sitosterol. DNMT inhibitor, AZA prevented the H2O2induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and β-sitosterol may be tested for therapeutic intervention of breast cancer. In aggressive tumors, there is increase expression of enhancer of zeste homologue (EZH2) and silencing of CDH1 genes. EZH2 is histone-lysine N-methyltransferase enzyme encoded by EZH2 gene. It participates in histone methylation and, ultimately, transcriptional repression. EZH2 specifically catalyses the addition of methyl groups to histone H3 at lysine 27(H3K27). The role of oxidative stress upon EZH2 and EZH2 mediated CDH1 regulation is still not explored. To decipher this, this thesis aimed to investigate the effect of H2O2 induced ROS on EZH2 expression. Data obtained reveals the induced expression of EZH2 in H2O2 treated cells suggesting the role of oxidative stress in EZH2 expression. Again treatment with EZH2 siRNA results in enhanced expression of CDH1 and reduced expression of EZH2 suggesting the role of EZH2 in negative regulation of CDH1 (CDH1) expression. Simultaneous treatment with EZH2 siRNA and H2O2 results in enhanced CDH1 (CDH1) expression and decrease expression of EZH2 which reveals the involvement of oxidative stress in enhanced expression of EZH2 and thus reduced the expression of CDH1 in breast cancer. Moreover, other studies also demonstrate the regulation of CDH1 and EZH2 by DNA methylation and histone modifications. DNMT1 is overexpressed and inversely correlated with CDH1 expression in breast cancer. Our thesis here demonstrates the negative regulation of CDH1 by EZH2. Most importantly it was found that, by inhibiting DNMT1 with 5-AZA-cytidine, it induced the expression of CDH1 indicating DNA methylation as well as EZH2 methylation is responsible for CDH1 down regulation in breast cancer. H3K27me3 also helps in the methylation of EZH2 and EZH2 mediated trimethylation of H3K27 is a prerequisite for promoter DNA methylation. This thesis demonstrates the expression of EZH2 and occupancy H3K27me3 is notably higher in untreated control cells as compared to EZH2 siRNA treated cells suggesting that CDH1 expression is regulated by higher expression of EZH2 thus results in H3K27 hyper-methylation. Inhibition of histone deacetylation by TSA (HDAC inhibitor) also restores the expression of CDH1 by inhibiting the EZH2 expression. This suggests that, histone deacetylation also plays an important role in regulation of EZH2 expression. Collectively, this work broadens the amplitude of the epigenome, enhance the understanding of the modulation of the epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations of genes during tumor development and cancer progression.

abstract: Gene delivery is a broadly applicable tool that has applications in gene therapy, production of therapeutic proteins, and as a study tool to understand biological pathways. However, for successful gene delivery, the gene and its carrier must bypass or traverse a number of formidable obstacles before successfully entering the cell’s nucleus where the host cell’s machinery can be utilized to express a protein encoded by the gene of interest. The vast majority of work in the gene delivery field focuses on overcoming these barriers by creative synthesis of nanoparticle delivery vehicles or conjugation of targeting moieties to the nucleic acid or delivery vehicle, but little work focuses on modifying the target cell’s behavior to make it more amenable to transfection. In this work, a number of kinase enzymes have been identified by inhibition to be targets for enhancing polymer-mediated transgene expression (chapter 2), including the lead target which appears to affect intracellular trafficking of delivered nucleic acid cargo. The subsequent sections (chapters 3 and 4) of this work focus on targeting epigenetic modifying enzymes to enhance polymer-mediated transgene expression, and a number of candidate enzymes have been identified. Some mechanistic evaluation of these targets have been carried out and discussion of ongoing experiments and future directions to better understand the mechanistic descriptions behind the phenomena are discussed. The overall goal is to enhance non-viral (polymer-mediated) transgene expression by modulating cellular behavior for general gene delivery applications.
Dissertation/Thesis
Doctoral Dissertation Chemical Engineering 2016

Il metabolismo delle unità monocarboniose (o mono carbonioso) rappresenta un pathway complesso coinvolto sia nella sintesi dei nucleotidi che nelle reazioni di metilazione biologica inclusa quella del DNA, il principale meccanismo epigenetico negli esseri umani. Uno stato di aberrante metilazione del DNA è una caratteristica comune nel tumore. Numerose varianti polimorfiche di geni all’interno del metabolismo mono-carbonioso sono state correlate al rischio di cancro, ma se il legame tra queste varianti geniche e il rischio/tempo di sopravvivenza legati al cancro possa realizzarsi attraverso un’alterata metilazione del DNA è una ipotesi fino ad ora scarsamente studiata e ancora non chiarita. Oltre alla metilazione, l’idrossimetilazione del DNA è stata recentemente descritta come un nuovo, potenzialmente utile marker epigenetico. I tumori primitivi del fegato, in particolare l’epatocarcinoma (HCC) e il colangiocarcinoma (CC), e il cancro del colon, nonostante l’alta incidenza, sono stati scarsamente caratterizzati dal punto di vista epigenetico. Gli scopi del presente progetto sono stati definire il possibile ruolo della metilazione ed idrossimetilazione del DNA nel tumore ed in particolare: 1. Determinare se le varianti polimorfiche dei geni del metabolismo mono-carbonioso possano influenzare il rischio di tumore primitivo del fegato e di tumore del colon attraverso la metilazione del DNA, definita come livelli di metilcitosina (mCyt) misurata nel DNA delle cellule periferiche mononucleate del sangue (PBMCs). 2. Verificare se lo stato di metilazione del DNA misurato nei PBMCs dei soggetti affetti da HCC, CC e tumore del colon possa correlare con l’andamento clinico e il tempo di sopravvivenza, definendo pertanto un possibile marcatore epigenetico di malattia. 3. Caratterizzare ulteriormente lo status epigenetico di HCC e CC valutando il contenuto globale di mCyt e hmCyt nel DNA del tessuto neoplastico e nel tessuto omologo non neoplastico. Materiali e metodi: La genotipizzazione di BHMT 716A>G, DHFR 19bp ins/del, MTHFD1 1958G>A, MTHFR 677C>T, MTR 2756A>G, MTRR 66A>G, RFC1 80G>A, SHMT1 1420C>T, TCII 776C>G e TS 2rpt-3rpt è stata eseguita in 102 pazienti neoplastici e 363 soggetti liberi da tumore. La metilcitosina e l’idrossimetilcitosina sono state misurate attraverso un metodo LC/MS/MS nel DNA dei PBMCs di tutti i soggetti e nel tessuto neoplastico e nel tessuto omologo non neoplastico di 47 patienti affetti da tumore primitivo del fegato sottoposti a intervento chirurgico. Risultati: 1. Il genotipo MTHFD1 1958AA era significativamente meno frequente nei soggetti neoplastici rispetto ai controlli (p=0.007) e la sua presenza si associava ad una riduzione del 63% di rischio generale di tumore (OR=0.37, p=0.003) e del 75% di rischio di tumore del colon (OR=0.25, p=0.006). Il genotipo MTHFD1 1958GG era significativamente più frequente tra i pazienti neoplastici (p=0.007) ed era associato ad un più basso livello di metilazione del DNA se comparato ai soggetti portatori dell’allele A (p=0.048). 2. La variante polimorfica RFC1 80AA era associata ad una sopravvivenza significativamente ridotta nei pazienti con tumore primitivo del fegato, rispetto ai portatori dei genotipi GG e GA (p=0.005) in un periodo di follow-up di 60 mesi. Quando i livelli di mCyt sono stati stratificati come alti (> 5.34%) o bassi (≤ 5.34%), in accordo con il valore medio di mCyt, la concomitante presenza del genotipo AA e di bassa mCyt comportava una peggiore prognosi, nel confronto con i soggetti portatori dell’allele G, sia con alti che con bassi valori di mCyt (p<0.0001); non vi erano differenze di sopravvivenza tra i portatori dell’allele G e i soggetti AA con alti livelli di mCyt (p=0.919). Inoltre, un più alto rischio di mortalità era associato con la concomitante presenza del genotipo AA e di bassi livelli di mCyt se comparato ai soggetti portatori dell’allele G (OR=8.35, p=0.001). 3. Livelli di mCyt ≥5.59% nei PBMCs erano associati ad una significativa migliore aspettativa di vita se comparati a valori di mCyt < 5.59% (p=0.034) in un follow-up di 48 mesi nei pazienti affetti da tumore primitivo del fegato. Il contenuto di mCyt nel tessuto neoplastico di HCC era notevolmente più basso che nel tessuto di CC (rispettivamente 3.97% vs 5.26%, p<0.0001). Nell’HCC sono stati osservati livelli di mCyt significativamente ridotti nel tessuto neoplastico rispetto al tessuto non neoplastico (3.97% vs. 4.82% mCyt, p<0.0001), ma questa differenza non era presente nel tessuto epatico dei pazienti affetti da CC. L’idrossimetilazione era significativamente ridotta nel tessuto neoplastico degli HCC vs il tessuto epatico non neoplastico (0.044 vs. 0.128, p<0.0001), così come nel tessuto neoplastico del CC confrontato sia con il tessuto epatico non neoplastico, sia con la colecisti (rispettivamente 0.030 vs. 0.124, p=0.026; 0.030 vs. 0.123, p=0.006). Conclusioni: i nostri risultati suggeriscono una possibile associazione tra le varianti polimorfiche dei geni del metabolismo mono-carbonioso e la metilazione del DNA. Questa associazione suggerisce che la metilazione del DNA potrebbe rappresentare il legame importante tra varianti geniche polimorfiche e il tumore primitivo del fegato e il tumore del colon, sia in termini di rischio che di aspettativa di vita. Questo riscontro potrebbe collocare la metilazione del DNA nei PBMCs come possibile biomarcatore per questi tipi di tumore. Inoltre, considerato che la metilazione del DNA è un fenomeno potenzialmente reversibile che dipende dal metabolismo mono-carbonioso per il rifornimento di gruppi metilici, si possono immaginare possibili strategie indirizzate ad ottimizzare l’apporto di questi componenti allo scopo di mantenere la metilazione del DNA all’interno di valori adeguati per la prevenzione di malattia. I risultati di questo studio dimostrano, inoltre, che un significativo ridotto grado di metilazione del DNA caratterizza il tessuto neoplastico dell’HCC rispetto al CC, mentre bassi livelli di idrossimetilazione caratterizzano sia il tessuto neoplastico dell’HCC che del CC in confronto al tessuto non neoplastico. Ulteriori conferme dei presenti dati saranno opportune e necessarie per verificare l’utilità e l’affidabilità nella pratica clinica dei biomarcatori genetici-epigenetici come indici prognostici per i pazienti affetti da tumore primitivo del fegato e da tumore del colon.
One-carbon metabolism is a complex pathway involved both in nucleotide synthesis and in biological methylation reactions including that of DNA methylation, the main epigenetic mechanism in humans. An aberrant DNA methylation is a common feature in cancer disease. Several polymorphic gene variants within one-carbon metabolism have been related to cancer risk, but whether the link between one-carbon metabolism variants and cancer risk/survival rate may occur through an altered DNA methylation is still poorly investigated and yet unclear. In addition to DNA methylation, hydroxymethylation of DNA has been recently described as a novel, potentially useful epigenetic mark. Primary liver cancers, i.e. hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) as well as colon cancer are highly prevalent but epigenetically poorly characterized, so far. The main aims of the present project were to define a possible role for DNA methylation in cancer, and specifically: 1. to determine whether polymorphic variants of one- carbon metabolism genes may influence the risk of human primary liver cancers and colon cancer through DNA methylation, defined as methylcytosine (mCyt) levels measured in peripheral blood mononuclear cells (PBMCs) DNA; 2. to verify whether DNA methylation status measured in PBMCs of subjects affected by HCC, CC and colon cancer may correlate with clinical outcomes and survival rate, therefore defining a possible epigenetic marker of disease; 3. to further characterize the epigenetic layout of HCC and CC by evaluating the global DNA mCyt and hydroxymethylcytosine (hmCyt) content in neoplastic and homologous non-neoplastic tissues. Material and Methods: Genotyping for BHMT 716A>G, DHFR 19bp ins/del, MTHFD1 1958G>A, MTHFR 677C>T, MTR 2756A>G, MTRR 66A>G, RFC1 80G>A, SHMT1 1420C>T, TCII 776C>G and TS 2rpt-3rpt was performed in 102 cancer patients and 363 cancer-free subjects. Methylcytosine and hmCyt were measured by an LC/MS/MS method in PBMCs DNA of all subjects and in neoplastic and homologous non-neoplastic tissues of 47 primary liver cancer patients undergoing curative surgery. A follow-up examination was performed on all the patients 48-60 months after the surgical procedure. Results: 1. The MTHFD1 1958AA genotype was significantly less frequent among cancer patients as compared to controls (p=0.007) and related to 63% reduction of the overall cancer risk (OR=0.37, p=0.003) and 75% of risk for colon cancer (OR=0.25, p=0.006). The MTHFD1 1958GG was significantly more frequent among cancer patients (p=0.007) and associated to lower DNA methylation as compared to MTHFD1 1958 allele A carriers (p=0.048). 2. The RFC1 80AA polymorphic variant was associated in primary liver cancer with a significantly reduced survival rate as compared to GG and GA (p=0.005) at a follow-up period of 60 months. When the mCyt levels were stratified as either high (>5.34%) or low (≤5.34%) according to the mCyt median value, the combination of AA genotype and low mCyt led to a significantly worse survival as compared to the two genotype groups carrying the G allele considered as a whole (p<0.0001); no difference in survival was found between G carriers and AA in association with high mCyt (p=0.919). Moreover, a higher mortality risk was associated with the concomitant presence of the AA genotype and low mCyt, as compared to G carrier subjects (OR=8.35, p=0.001). 3. In primary liver cancer, levels of mCyt ≥5.59% in PBMCs were associated to a significantly higher life expectancy as compared to mCyt<5.59% (p=0.034) at a follow-up period of 48 months. Methylcytosine content in HCC neoplastic tissue was notably lower than in CC tissues (3.97% vs. 5.26% respectively, p<0.0001). Significantly reduced mCyt levels were observed in HCC neoplastic as compared to non-neoplastic tissue (3.97% vs. 4.82% mCyt, p<0.0001) but such difference was not found in liver tissue of patients affected by CC. Hydroxymethylation was significantly decreased in HCC neoplastic vs. non-neoplastic liver tissue (0.044 vs. 0.128, p<0.0001), as well as in CC neoplastic tissue vs. both non-neoplastic liver and gall bladder (0.030 vs. 0.124, p=0.026; 0.030 vs. 0.123, p=0.006, respectively). Conclusions: Our results suggest a possible association between one-carbon metabolism genes polymorphic variants and DNA methylation. This association suggests that DNA methylation may be the underlying link between polymorphic gene variants and primary liver and colon cancers, both in terms of risk and life expectancy. This finding may situate PBMCs DNA methylation as a possible biomarker for those types of cancer disease. Moreover, since DNA methylation is a reversible phenomenon that relies on one-carbon metabolism for provision of methyl groups, adequate strategies to optimize sources of such compounds to maintain DNA methylation within adequate levels for disease prevention may be considered in the framework to modulate this epigenetic mechanism. The results of this study demonstrate, furthermore, that a significantly lower degree of DNA hypomethylation characterize HCC from CC tissue, whereas DNA hypo-hydroxymethylation characterizes both HCC and CC neoplastic tissues as compared to the non-neoplastic. Further validation of the present novel data is required to assess whether this genetic-epigenetic biomarkers may indeed be a reliable and useful for clinical application as a prognostic tool for patients with primary liver and colon cancers.