Investigating the Regulation and Function of the NR4A Nuclear Receptors in Cancer

The nuclear receptor (NR) superfamily represents a structurally-conserved group of ligand-regulated transcription factors. These proteins have critical roles in various physiological and pathological processes, including cancer, and have been targets of drug therapy. The orphan NR subfamily 4A (NR4A), which includes the NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (Nor-1) genes, has been implicated in adult solid tumors and has been characterized as pro-tumorigenic mediator of cell proliferation, transformation, migration, and drug resistance. Alternatively, in leukemia, NR4A1 and NR4A3 have been described as tumor suppressors in hematologic malignancies. Members of the NR4A family are commonly overexpressed in cancer and this has been attributed to their regulation by other oncogenic signaling pathways. Despite the understanding of signaling cascades that lead to overexpression of the NR4A members, little is known about their regulation by microRNAs (miRNAs). miRNAs are small, non-coding, endogenous RNAs that are transcribed, processed, and used to direct cellular proteins that destabilize or block translation of target mRNA. In this study, we first sought to determine the miRNAs that are responsible for regulating NR4A2. Using a 3ʹ UTR reporter assay, we identified miR-34 as a regulator of the NR4A2 through its 3ʹ UTR, which was confirmed using mutagenesis of the predicted binding region of the miR-34 seed region to its target site. We demonstrated that overexpression of exogenous or induction of endogenous miR-34 expression downstream of p53 activation by Nutlin-3a was associated with decreased endogenous NR4A2. Additionally, overexpression of NR4A2 was capable of suppressing the activation of p53 target genes, and was also able to attenuate the sensitivity of cells to the anti-proliferative effect of Nutlin-3a. We further explored the roles of the NR4A family in pediatric cancer, an area that has not been fully investigated. We first determined that the members of the NR4A family are overexpressed in rhabdomyosarcoma (RMS) cell lines compared to normal muscle cells. Knockdown of NR4A1 or NR4A2 led to a reduction in cell proliferation and transformation, while knockdown of NR4A2 could also affect cell migration. Using a microarray approach, we sought to investigate the transcriptome-level changes in response to NR4A knockdown, and determined that knockdown of NR4A2 led to a unique gene signature, while NR4A1 and NR4A3 knockdown had large overlaps in expression changes. These unique gene expression changes in response to NR4A2 knockdown could explain the unique effects that NR4A2 has on migration. Overall, this study has discovered miR-34 as a novel regulator of NR4A2, and places NR4A2 in a potential feedback mechanism involving p53, miR-34, and NR4A2. This could indicate that NR4A2 mediates at least some of its pro-oncogenic effects through the inhibition of p53, which is relieved by p53 itself upon activation. Alternatively, NR4A2, is shown to have other roles in cancer progression, potentially through novel downstream target genes. These data may be used in understanding the effects of miR-34 replacement therapy, as this method of treatment is progressing through clinical trials, allowing us to understand the diverse regulator cascades being modulated

Diindolylmethane Analogs as Novel NR4A1 Antagonists and as a Novel Class of Anticancer Agents and Sp Transcription Factors as Nononcogene Addiction Genes That Are Targets of ROS Inducing Agents

The orphan nuclear receptor 4A1 (NR4A1) and specificity protein (Sp) transcription factors (TFs) are both overexpressed in the majority of solid tumors. Our laboratory has researched the molecular mechanisms of a novel class of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) as NR4A1 antagonists and Sp proteins as non-oncogene addiction genes (NOA) that are targets of reactive oxygen species (ROS) inducing agents. Nr4A1 antagonists (DIM-C-pPhOH) (C-DIM 8) and (DIM-C-pPhCO2Me) (C-DIM 14) inhibited cancer cell proliferation, induced apoptosis, and inhibited migration. The NR4A1 antagonists inhibited constitutive and TGFβ-induced migration in Triple Negative Breast Cancer (TNBC) cells. We also demonstrate that p38α is necessary and sufficient for TGFβ-mediated migration and NR4A1 nuclear export in triple negative breast cancer (TNBC) which was attenuated with NR4A1 antagonists (C-DIMs), leptomycin B, and the p38 inhibitor SB202190. We also demonstrate that NR4A1 is essential for TGFβ-induced EMT and NR4A1 antagonists promoted cytosolic sequestration of the transcription factor β-catenin and its proteasome-dependent degradation in a time dependent manner. β-catenin, along with TCF-3, TCF-4, and LEF-1 binds to TCF/LEF response elements in the NR4A1 promoter, regulating its expression. RNA interference (RNAi) demonstrates that Sp1, 3, and 4 TFs individually play a role in cancer cell growth, survival and migration/invasion in cancer cell lines. Individual knockdown of Sp1, Sp3, or Sp4, resulted in inhibition of cell growth, migration, and induction of apoptosis, with no compensation. Moreover, tumor growth in athymic nude mice bearing pancreatic cancer xenografts was significantly attenuated in cells depleted of Sp1, Sp3, and Sp4 in combination or Sp1 alone. Ingenuity Pathway Analysis (IPA) of changes in gene expression in Panc1 pancreatic cancer cells after individual knockdown of Sp TFs demonstrates that Sp1-, Sp3- and Sp4-regulated genes were associated with pro-oncogenic activity. C-DIMs are promising anticancer agents in NR4A1-overexpressing solid tumors and represent a novel class of mechanistic-based drugs that target TGFβ/NR4A1-dependent inducible migration in TNBC. The functional and genomic results coupled with overexpression of Sp transcription factors in tumor vs. non-tumor tissues and decreased Sp1 expression with age indicate that Sp1, Sp3 and Sp4 are non-oncogene addiction (NOA) genes and are attractive drug targets for individual and combined cancer chemotherapies

Caractérisation de la modulation de l’activité du récepteur nucléaire orphelin NUR77 (NR4A1) par ses modifications post-traductionnelles et son interactome

NUR77 est un récepteur nucléaire (RN) orphelin impliqué dans la régulation de processus biologiques dont la mort cellulaire, notamment dans la maladie de Parkinson (MP), découlant de la perte de neurones dopaminergiques, et dans le cancer du sein, résultant de la prolifération de cellules mammaires. NUR77 est impliqué dans le déclenchement et la protection de la mort cellulaire et son activité serait indépendante de la liaison d’un ligand. Nous avons émis l’hypothèse que l’activité de NUR77 est influencée par ses modifications post-traductionnelles (MPTs) et ses partenaires d’interactions. L’objectif général de cette thèse était de caractériser les MPTs et les partenaires d’interaction modulant l’activité de NUR77, dans des modèles de cellules en culture, afin de mieux comprendre ses fonctions biologiques - notamment dans la mort cellulaire. Le premier objectif de ce doctorat était de caractériser le rôle de la SUMOylation, une modification modulant l’activité des RN, chez NUR77, par des essais rapporteurs dans les cellules Human Embryonic Kidney 293 (HEK293). La surexpression de la E3 SUMO ligase PIASγ et/ou de l’isoforme 2 de la SUMO, protéines importantes dans la régulation de la SUMOylation chez les RN, a engendré un effet répresseur sur l’activité transcriptionnelle de NUR77. L’effet de PIASγ sur l’activité de NUR77 est modulé par la Sentrin SUMO-specific protease 1, qui hydrolyse la liaison des SUMO. Les mutations des résidus lysine dans des sites consensus de SUMOylation, de NUR77 (K102 et K577), empêchant cette MPT, ont causé des effets opposés sur son activité transcriptionnelle, suggérant le recrutement différent de corégulateurs de la transcription. Ces résultats combinés indiquent que la SUMOylation et les PIASγ et SUMO2 sont, respectivement, une MPT et des corégulateurs importants dans l’activité de NUR77. Le deuxième objectif de cette thèse était de caractériser l’interactome de NUR77 dans des HEK293 vivantes afin d’identifier les interacteurs pouvant moduler son activité, à l’aide d’une méthode de marquage des protéines proximales avec la biotine basée sur la peroxydase APEX2, combinée à la spectrométrie de masse. Ce procédé a identifié 336 potentiels interacteurs de NUR77, dont plusieurs connus. Des essais de coimmunoprécipitation et de coimmunofluorescence menés dans les HEK293 et dans les cellules du cancer du sein MCF-7 ont montré, respectivement, que la protéine régulatrice de l’apoptose Apoptosis Inhibitor 5 (API5), interagissait et colocalisait avec NUR77. La privation de sérum dans le milieu de culture des cellules et la diminution de l’expression de API5 a conduit à une augmentation des niveaux protéiques et de l’activité de NUR77 et à une diminution de la survie cellulaire. Ces données suggèrent que API5 constitue un régulateur de NUR77 dans les voies de signalisation associées à la mort cellulaire et que cette interaction pourrait constituer une cible pour moduler l’apoptose. Elles valident également l’approche d’identification d’interacteurs de NUR77. Les travaux de cette thèse ont donc permis de générer des outils pour caractériser l’activité de NUR77 et ont révélé des corégulateurs de cette activité. La poursuite de ces projets pourrait révéler le caractère opportun de cibler NUR77 comme modulateur de la mort cellulaire, notamment dans la MP et le cancer du sein.NUR77 is an orphan nuclear receptor (NR) involved in the regulation of multiple cell biology processes including cell death, in particular in Parkinson's disease (PD), which results of the loss of dopaminergic neurons, and in breast cancer (BC), which is caused by the proliferation of mammary epithelial cells. NUR77 is involved in triggering and inhibiting cell death and its activity is believed to be independent of a ligand binding. We hypothesized that the regulation of NUR77 activity does not occur through a ligand, but through the influence of its post-translational modifications (PTMs) and its interaction partners. The general objective of this PhD project was to characterize the PTMs and the interacting partners that modulate the activity of NUR77 in cultured cell models, to better understand its physiological roles, in particular in the regulation of cell death. The first objective of this thesis was to characterize the role of SUMOylation, a modification that regulates NR activity, in regulating NUR77 transcriptional activity in reporter assays in Human Embryonic Kidney (HEK293) cells. Overexpression of the E3 SUMO ligase PIASγ or/and the isoform 2 of SUMO, both important regulators in SUMOylation of the NUR77 homolog NURR1, produced a repressive effect on the transcriptional activity of NUR77. The effect of PIASγ on the activity of NUR77 was shown to be modulated by the Sentrin SUMO-specific protease 1 protein, which removes SUMO tags on target proteins. In addition, mutations of lysine residues in SUMO consensus sites in NUR77 (K102 and K577) had opposite effects on its transcriptional activity, suggesting different recruitment of coregulators of transcription in the regions. The combination of these results indicates that SUMOylation is an important PTM for the regulation of NUR77 activity and that PIASγ and SUMO2 proteins are important transcriptional coregulators of NUR77. The second objective of this thesis was to evaluate NUR77 interactome in HEK293 living cells to identify the interactors that can modulate its activity, using a biotin-labelling method for proximal proteins based on the APEX2 peroxidase combined with mass spectrometry. This approach identified 336 potential interactors of NUR77, some that are consistent with the literature. Coimmunoprecipitation and coimmunofluorescence assays carried out in HEK293 cells and in MCF-7 breast cancer cell line have shown that the regulator of apoptosis Apoptosis Inhibitor 5 vi (API5), interacted and colocalized with NUR77. By depriving cells of serum and decreasing API5 expression, increased protein levels and activity of NUR77 was observed, as well as a decrease in cell viability. These data support that API5 is a regulator of NUR77 in its involvement in signalling pathways associated with cell death and that this interaction could be a target for modulating apoptosis. More generally, they validate the APEX2 tool which can be used to identify novel NUR77 interactors. In conclusion, the work of this thesis resulted in the generation of tools to better understand the activity of NUR77 and revealed important coregulators in this activity. The continued characterization of these interactors may provide opportunities to target NUR77 as a regulator of cell death, particularly in PD and in breast cancer