Pre-eclampsia and intrauterine growth restriction (IUGR) are common complications of pregnancy with significant implications for mother and fetus. The exact cause is unknown, but current theories suggest that abnormal trophoblast invasion leads to placental under perfusion and hypoxia with the generation of reactive oxygen species (ROS). A result of this insult is increased trophoblast apoptosis. The cell cycle regulator p53 has been identified in increased amounts in the trophoblast in pre-eclampsia and IUGR. It is possible that modulation of this potent apoptotic signal could improve placental function, potentially offering a therapy for these presently untreatable conditions.In these studies, trophoblast apoptosis was induced with Nutlin-3, a synthetic compound which inhibits the binding of p53 to its inhibitor, Mdm2, therefore acting as a p53 activator. Treatment with Nutlin-3 induced apoptotic cell death in BeWo choriocarcinoma cells, primary trophoblast cells and placental villous explants, suggesting that p53 is intrinsic to trophoblast survival. This increase in apoptosis was associated with an increase in p53, Mdm2, p21 and Puma protein expression in BeWo cells and p21 and Puma mRNA in placental villous explants. No effect was observed in Bax expression. Co-treatment with Nutlin-3 and the p53 inhibitor Pifithrin-α reduced apoptosis to the level of control tissue in BeWo cells, primary trophoblast cells and villous explants, suggesting that reducing p53 activity improves trophoblast function.The use of hypoxia and ROS was associated with an increase in apoptosis and p21 and Puma in placental villous explants. Furthermore, the extent of apoptosis and p21 and Puma expression was reduced by co-treatment with Pifithrin-α, suggesting that the ameliorating effects of Pifithrin-α on trophoblast apoptosis are maintained following an insult from a less specific p53 activating mechanism, further supporting the continued investigation of p53 modulation as a therapeutic manipulation of placental function.Pregnant mice were exposed to Nutlin-3 to ascertain the effect of p53 over-activity on pregnancy outcome. The establishment of a model of placental dysfunction in the mouse would have allowed investigation into the effect of p53 inhibition with Pifithrin-α. Despite clear evidence of p53 modulation in maternal tissues, the decidua, no effect was observed upon pregnancy outcome or upon fetal or placental tissue. These findings suggest that the mouse placenta may not be a good model of p53 over-activity, although the exact mechanism of resistance remains unclear.In conclusion, p53 exaggeration is inducible in trophoblast with Nutlin-3, hypoxia and ROS. Furthermore, Pifithrin-α is able to reduce this effect, suggesting that the p53 pathway may be a realistic target for modulation in the placenta.

Puisque l'utilisation des pesticides augmente dans la pratique agricole (Gorse, 2015), il est inévitable que nous allons retrouver ces contaminants dans l'eau de surface qui se trouve à proximité des zones d'épandage. Il est certain que les pesticides ont plusieurs manières d'affecter notre organisme. Cependant, l'effet de ces pesticides sur les différents mécanismes cellulaires est peu étudié. En effet, il est connu que certains pesticides permettent l'activation de gènes régulés par le récepteur à l'hydrocarbure d’aryle (AhR), mais il n'y a peu de documentation par rapport à leur effet sur les dommages à l'ADN. De plus, ces pesticides sont la plupart du temps utilisés en combinaison lors de leur utilisation agricole, mais il n'y a quasiment aucune étude qui est faite sur leurs effets lorsqu'ils sont en combinaison. Ainsi, mon projet de maîtrise se base sur l'hypothèse que certains contaminants présents dans l’environnement ainsi que dans les eaux de surface peuvent, à court et à long terme, perturber deux voies cellulaires, soit celle du récepteur à l'hydrocarbure d’aryle et celle de p53. Pour la première partie de mon projet, je me suis intéressée principalement aux gènes CYP1A1 et CYP1B1, deux enzymes impliquées dans le métabolisme de ces contaminants environnementaux dont l'expression est régulée par AhR. L'activation de ces deux gènes a été mesurée suite à un traitement avec différents ingrédients actifs seuls à haute concentration ou en combinaison à faible concentration. Nous avons démontré que chacun des ingrédients actifs séparément permettent l'activation d’AhR. En plus, lorsque nous les combinons, nous observons une activation synergique d’AhR pour la plupart des combinaisons. Ainsi, leur effet en combinaison est beaucoup plus grand que celui qu'ils ont lorsqu'ils sont seuls. Nous avons aussi déterminé que les pesticides seuls ainsi que les combinaisons permettaient l'observation de l’interaction croisée entre AhR et le récepteur à l'oestrogène (ER[alpha]). Pour la deuxième partie de mon projet, j'ai vérifié si les pesticides utilisés à haute concentration ainsi que les différentes combinaisons à faible concentration entraînent l’activation du point de contrôle des dommages à l’ADN. Le carbaryl, le linuron et le bromoxynil sont les trois seuls pesticides permettant l’activation de ce point de contrôle à haute concentration. Toutes les combinaisons de pesticides à faibles concentrations ne permettent pas l’activation de ce point de contrôle. Ainsi, tous les pesticides testés à haute concentration permettent l’activation d’AhR, mais seulement certain d’entre eux permettent l’activation du point de contrôle des dommages à l’ADN. Cela montre donc que les pesticides ont plusieurs cibles dans la cellule et que selon le pesticide étudié, les cibles changent. La dernière partie de mon projet consistait à vérifier l’impact sur AhR des contaminants environnementaux présent dans la rivière Saint-François le 26 septembre 2016 sur AhR. Les contaminants ont été extraits et concentrés avant d'être testés sur des cellules en culture. Les contaminants présents dans l'eau de rivière permettent l'activation d’AhR. En conclusion, mes études permettent de mieux comprendre l'effet de ces cinq pesticides sur ces deux mécanismes cellulaires. Des études plus approfondies sont nécessaires afin de comprendre l'ensemble des mécanismes affectés par ces pesticides

The cellular inhibitor of apoptosis protein (cIAP)-2 plays an important role in the protection against apoptosis by inhibiting the endogenous IAP inhibitor Smac, thus allowing other members of the IAP family, such as XIAP to block caspases. Additionally, cIAP2 functions as a ubiquitin ligase and mediates survival/proliferative signaling through NF-κB. cIAP2 is overexpressed in many human cancers and is believed to play an oncogenic role. This led to the development of small molecule IAP antagonists aimed at eliciting apoptosis in cancer cells. However, the loss of cIAP2 is also associated with multiple myeloma, in which constitutively active NF-κB signaling contributes to pathogenesis of the disease and suggests that cIAP2 may also perform a tumour suppressive function. We demonstrate a novel role for cIAP2 in maintaining p53 levels in mammary epithelial cells that express wildtype p53. Downregulation of cIAP2 resulted in activation of IKKs, which led to increased Mdm2-mediated degradation of p53. cIAP2 depletion also led to increased phosphorylation of ERK1/2. Reduction of p53 levels, in combination with survival signaling provided by NF-κB and MEK-ERK pathways were associated with increased colony formation in vitro and increased DMBA-induced adenocarcinomas in cIAP2-null mice. Treatment of cells with IAP antagonists resulted in significant cytotoxicity only in p53-mutant MDA-MB-231 cells, which was associated with autocrine production of TNF-α. We propose that the transcription of TNF-α is potentiated by gain-of-function mutation in p53 since downregulation of mutant p53 in MDA-MB-231 cells decreased TNF-α mRNA. Downregulation of cIAPs in p53-mutant cells resulted in a decrease in nuclear IKK-α, which may result in decreased IKK-α-mediated survival signaling. In contrast, cIAP downregulation in p53-wildtype cells resulted in no change in nuclear IKK-α, degradation of the corepressor SMRT and cell survival. We show that the effects of cIAP2 downregulation are context-dependent. Downregulation of cIAP2 in p53-wildtype cells results in a decrease in p53 and an increase in survival and proliferative signaling. These results suggest a tumour suppressor function for cIAPs that may account for cIAP mutation-associated cancers such as multiple myeloma. Moreover, our data also defines gain-of-function p53 mutation as a possible contributor to IAP antagonist sensitivity.

Cisplatin (CDDP; cis-diamminedichloroplatinum) resistance presents a major impediment in the treatment of several gynecologic solid tumors, including ovarian and cervical tumors. p53, a critical regulator of cellular apoptosis, is a determinant of CDDP sensitivity. In our study, we have observed that the dysregulation of p53 regulators, checkpoint kinase 1 (Chk1) and protein phosphatase magnesium-dependent 1 (PPM1D), significantly reduced CDDP responsiveness in human cancer cells. Isogenic wt-p53 CDDP-sensitive (OV2008) and -resistant (C13*) cervical cancer cells, and isogenic wt-p53 CDDP-sensitive (A2780s) and p53 mutant resistant (A2780cp) ovarian cancer cells, along with CDDP-resistant ovarian cancer cell lines (OCC-1 and OVCAR-3, mutant p53; SKOV-3, p53 null) were used to elucidate the mechanisms of p53 regulation in human gynecologic cancer cells. We have complemented our study with a xenograft model (A2780s) and a tissue microarray of human ovarian tumors to validate our in vitro observations. We have demonstrated that CDDP differentially regulated the p53 activator Chk1 in sensitive and resistant cancer cells; it enhances Chk1 activation in sensitive but not resistant cells. This differential regulation also extended to PPM1D, whereby CDDP enhanced PPM1D content in resistant but not sensitive cells. PPM1D knockdown sensitized resistant cells to CDDP, which was associated with up-regulation of Chk1 and p53 activations, while PPM1D over-expression had the opposite effect. We have also shown that CDDP sensitivity in response to PPM1D down-regulation was p53-dependent. Moreover, CDDP promotes PPM1D nuclear localization in resistant cells and nuclear exclusion in sensitive cells and xenograft tumors. Enhanced PPM1D expression in human ovarian tumors is significantly associated with tumor aggression. Dysregulation of the oncogene Akt has been implicated in a variety of human malignancies, including ovarian cancer. We have demonstrated that Akt regulates PPM1D stability, since activated Akt over-expression in sensitive cells rescued PPM1D from CDDP-induced proteasomal degradation and Akt down-regulation in resistant cells lead to PPM1D de-stabilization and down-regulation. We have shown for the first time that PPM1D is downstream of Akt through which it can modulate CDDP sensitivity in human cancer cells. These findings extend the current knowledge on the molecular basis of CDDP resistance in gynecological cancers and may help in developing effective therapeutic strategies.

L'inactivation des gènes p53 et pten présente un facteur de mauvais pronostic et de résistance à différents traitements anticancéreux incluant les thérapies ciblées. Le cétuximab (Erbitux®) est un anticorps monoclonal chimérique dirigé contre le domaine extracellulaire du récepteur à l'EGF. Son mécanisme d'action est fortement lié à la fonctionnalité des voies de signalisation de PI3KJAKT et des MAPK. Nous avons évalué l'influence de la réintroduction des gènes p53 et pten par vectorisation non viral à base de polyéthylènimine, couplée ou non à l'internalisation photochimique sur l'induction de l'apoptose et l'inhibition de la croissance cellulaire dans des cellules P53 et pten mutés d'une part, et sur l'impact de ce transfert de gène sur la fonctionnalité des voies de signalisation impliquées dans la réponse cellulaire au cétuximab. Nous avons montré que la transfection des gènes suppresseurs de tumeurs p53 et pten par polyétylènimine couplé à la PCI rétablit l'expression de P53 et de PTEN et permet de restaurer l'inhibition de croissance et l'induction de l'apoptose. Associée au traitement par cétuximab La restauration de PTEN et P53 réprime l'activation constitutive des voies de signalisation PI3K et MAPK et potentialise l'inhibition de la croissance et l'induction de l'apoptose induites par le cétuximab. Ces résultats montrent que l'inactivation de p53 et pten pourrait être prédictive de la résistance au cétuximab à travers l'activation constitutive de la voie de signalisation PI3/AKT et confirme que la détennination de la fonctionnalité des voies de signalisation des rtiveaux d'expression des phosphoprotéines de signalisation permettrait de prédire la réponse au cétuximab et de proposer des options thérapeutiques originales. / P53 and PTEN abnormalities are early events in carcinogenesis and are associated with poor prognosis and resistance to cancer therapies including targeted therapy. Cetuxirnab (Erbitnx®) is a IgG chimeric monoclonal antibody, directed against the extracellular dornain of EGFR. !ts activity has been shown to be dependent on the functionality of PI3KJAKT and MAPK signaling pathways as weil as the apoptosis induction capacity of the cells . The present study was designed to evaluate the influence of pten and p53 gene transfer using polyethylenimine with or without photochemical internalisation on cell apoptosis induction and cell growth in cells bearing p53 and pten mutations and on the response to cetuximab. The results presented show that pten and p53 gene transfer using polyethertimine and PCI restored P53 and PTEN expression, cell growth inhibition and apoptosis induction. Associated with cetuximab treatrnent, pten and p53 reintroduction, restored the functionality of AKTIPI3K and MAPK signaling pathways and increased cell growth inhibition and apoptosis induction by cetuximab. The results achieved in the present study show that pten and p53 mutations could be predictive of cell response to cetuximab through the functional impact of these mutations on cell signaling. The data presented put forward the interest of the analysis of EGFR phosphoproteins downstream signalling to evaluate the functionality of the signaling pathways implicated in the cell response to cetuximab and could be used to propose the original targeted therapies.

Chemotherapy is commonly used in cancer treatments, however only 25% of cancers are responsive and a significant proportion develops resistance. The p53 tumour suppressor is crucial for cancer development and therapy, but has been less amenable to therapeutic applications due to the complexity of its action reflected in 67,000 papers describing its function. Here we provide a systematic approach to integrate this information by constructing large-scale logical models of the p53 interactome using extensive database and literature integration. Initially we generated models using manual curation to demonstrate the feasibility of the approach. This was followed by creation of the next generation models by automatic text mining results retrieval. Final model PKT205/G3 was generated by choosing the size of the interactome that could be analysed with current available computing power and by linking upstream nodes to input environmental signals such as DNA damage and downstream nodes to output signal such as apoptosis. This final version of the PKT205/G3 model contains 205 nodes representing genes or proteins, DNA damage input and apoptosis output, and 677 logical interactions. Predictions from in silico knock-outs and steady state model analysis were validated using literature searches and in vitro experiments. We identify an up regulation of Chk1, ATM and ATR pathways in p53 negative cells and 58 other predictions obtained by knockout tests mimicking mutations. The comparison of model simulations with microarray data demonstrated a significant rate of successful predictions ranging between 52 % and 71 % depending on the cancer type. Growth factors and receptors FGF2, IGF1R, PDGFRB and TGFA were identified as factors contributing selectively to the control of U2OS osteosarcoma and HCT116 colon cancer cell growth. In summary, we provide the proof of principle that this versatile and predictive model has vast potential for use in cancer treatment by identifying pathways in individual patients that contribute to tumour growth, defining a sub population of “high” responders and identification of shifts in pathways leading to chemotherapy resistance.

Transcription factor AP2C (TFAP2C) is believed to be involved in breast cancer carcinogenesis. However, the molecular mechanisms of regulating its trans-activation activity are not well understood. One of the potential mechanisms is through p53-mediated regulation. Using ChIP-seq analysis to map the TFAP2C occupancy across the genome, we found that the introduction of p53 to HCT 116 p53 -/- colon cancer cell line significantly augments TFAP2C occupancy on the promoter regions of a group of genes. Of these, six genes were further investigated. First, TFAP2C binding sites were identified in the center of ChIP-seq peaks on the promoters of the six genes and these were verified by gel shift assays. One of these genes, MUC1, was then determined to be activated by TFAP2C in MCF-7 breast cancer cell line. Subsequently, MUC1 was selected as the model target gene to elucidate the mechanism for p53-mediated enhancement of TFAP2C occupancy. We hypothesized that DNA methylation of the MUC1 promoter is altered by p53, leading to the increased TFAP2C occupancy to its TFBS on MUC1 promoter. To examine this, CpG methylation assay was performed. The result showed the DNA methylation of MUC1 promoter region remains identical with or without over-expression of p53 in HCT 116 p53 -/- cell line. From these studies, I conclude that 1) introduction of p53 augments TFAP2C binding on specific gene targets; 2) MUC1 gene is activated by TFAP2C and two TFAP2C binding sites were verified; 3) promoter DNA methylation does not explain the increased occupancy of TFAP2C on MUC1 promoter.

Cancer is the second leading cause of death in the United States, and it results from genetic alterations that promote the survival and proliferation of neoplastic cells. One of the most commonly disrupted cancer gene networks is the ARF-Mdm2-Tip60-p53 pathway. Inactivation of the ARF, Tip60 and p53 tumor suppressors and/or overexpression of the Mdm2 oncogene occurs in most, if not all, human cancers. An improved molecular understanding of that pathway, especially how it becomes activated, is expected to advance the development of innovative therapeutics aimed at restoring its function in tumors. Our group originally discovered NIAM (Nuclear Interactor of ARF and Mdm2) as a novel binding partner of ARF that has several functional ties to Mdm2 and p53. Early studies showed that NIAM is negatively regulated by Mdm2, can collaborate with ARF to block cell proliferation, and is a new activator of p53-mediated transcription. NIAM could also act independent of those factors to suppress cell proliferation and promote chromosomal stability, and microarray studies suggested its expression is significantly reduced in many human cancers. Those findings led us to speculate that NIAM is a tumor suppressor that functions in both the ARF-Mdm2-p53 pathway as well as other undefined anti-cancer pathways. My thesis research explored two different aspects of that hypothesis: 1) how does NIAM activate p53, and 2) is NIAM a tumor suppressor? Initial work showed that NIAM could stimulate p53 independently of ARF, the major activator of p53, indicating that other factors must be required. My studies revealed that NIAM indirectly promotes p53 activation through functional interactions with two other p53 regulators, Tip60 and Mdm2. Tip60 is an acetyltransferase that activates p53 through direct association on p53 target promoters as well as acetylation of p53 at lysine 120 (K120). I found that NIAM can induce K120 acetylation of p53; however, NIAM's association with Tip60 (not the acetylation of p53) was essential for maximal p53 transcriptional activation. Mdm2, the major antagonist of p53, is an E3 ubiquitin ligase that promotes p53 ubiquitylation and degradation. I found that NIAM disrupts Mdm2-p53 complexes and blocks p53 ubiquitylation, thereby interfering with p53 inhibition by Mdm2. Thus, NIAM regulates two critical pathways that control p53 function and are altered in human cancers, implying an important role for NIAM in tumorigenesis. To test that idea directly, we generated NIAM gene-trap knockout mice (hypomorphs) that expressed greatly reduced yet detectable levels of NIAM in all tissues. Fifty percent of the NIAM knockout mice developed benign and early stage cancers, including B-cell lymphoma, whereas all age-matched control animals were tumor-free. These results showed that NIAM loss causes increased susceptibility to tumorigenesis. In sum, my work suggests a significant role for NIAM in p53 control and tumor biology. Additional studies will be needed to determine which physiological or pathological signals normally engage NIAM to promote p53 function and suppress tumor development. It is anticipated that the NIAM knockout mice will provide an outstanding platform to interrogate NIAM's biological role in cancer, particularly in ARF-Mdm2-Tip60-p53 signaling as well as other pathways affecting genome maintenance. Ultimately, insights gained from such studies may justify novel therapies that seek to restore NIAM activity in tumors.

No description available.

In cells undergoing stress, the p53 transcription factor is stabilized and activates the expression of numerous genes contributing to p53-mediated tumor suppression. One p53 target gene is Mdm2, which encodes an oncoprotein that binds and ubiquitinates p53 for proteasomal degradation, thus limiting the amplitude and duration of the p53-mediated stress response. Our lab recently discovered that Mdm2 phosphorylation by ATM and c-Abl regulates the DNA damage response and tumorigenesis in mice. AKT has also been found in transfection studies to phosphorylate Mdm2 at serine residues 166 and 186 (mouse S163 and S183) to alter p53 activity. However, the physiological significance of Mdm2 phosphorylation by Akt remains unknown. Therefore, I generated Mdm2S163A or Mdm2S183A mice expressing mutant Mdm2 incapable of being phosphorylated by Akt. In contrast with our previous studies, Akt phosphorylation of Mdm2 does not alter spontaneous tumorigenesis or the DNA damage response to ionizing radiation. However, Akt phosphorylation of Mdm2-S183 (but not -S163) upregulates nuclear localization of Mdm2, destabilizes p53, and reduces p53-mediated senescence in response to elevated levels of reactive oxygen species (ROS). To examine the effects of Mdm2-S183 phosphorylation on p53 tumor suppression, I utilized three different mouse models of ROS-induced cancer. Increased levels of p53 and senescence in Mdm2S183A mice yielded reduced tumorigenesis in an activated Ras model of lung cancer, a phorbal ester-induced skin cancer model, and a diethylnitrosamine-induced model of hepatocellular carcinoma. Since AKT is also important regulator of cell metabolism, I explored the impact of the Mdm2-S183 allele on metabolic functions. Mdm2 phosphorylation by Akt reduced glucose metabolism via glycolysis in vitro, and reduced insulin tolerance in mice, without altering glucose tolerance and glucose-stimulated insulin secretion. Collectively, these findings document a unique physiologic role for the AKT-Mdm2-p53 signaling axis in regulating cell growth and tumorigenesis.