Decreased expression of the tumor suppressor gene BRCA1 (breast cancer 1, early onset) is frequently observed in sporadic breast tumors, with the decrease not attributed to mutations or hypermethylation of the promoter. A critical regulator of the BRCA1 promoter is the GA-Binding Protein (GABP), a heterotetramer of GABPα and GABPβ. Previous analysis of the GABPβ promoter revealed a regulatory multi-protein complex containing Nuclear Respiratory Factor 1 (NRF-1), which was aberrant in SK-BR-3 cells, resulting in decreased GABPβ and BRCA1 expression. To identify the unknown co-regulators of the NRF-1-containing complex at the GABPβ promoter, an immobilized-template assay containing the complex binding site was utilized. Despite optimization of binding and elution conditions through variations in salt content, Mg2+ concentration and pH, non-specific DNA-binding proteins were present in the column eluate. Further experimentation is therefore required to distinguish between non-specific proteins and complex co-activators. As this complex containing NRF-1 is also able to modulate BRCA1, a regulator of luminal progenitor differentiation, a defect in NRF-1 or complex co-activators could contribute to the abnormal morphogenesis and differentiation of BRCA1-deficient tumors. We therefore investigated the role of NRF-1 in mammary cell morphogenesis and its link to the GABP/BRCA1 pathway. As both GABPβ and NRF-1 are known regulators of nuclear-encoded mitochondrial proteins, this association also provides a link between mitochondrial metabolism and breast differentiation, both of which are frequently disrupted in breast cancer. An inducible lentiviral system was used to generate NRF-1 knockdown cell lines to examine its effect on morphogenesis. Growth in 3D culture resulted in abnormal cell structures having impaired cell polarization and lumen formation. In monolayer culture, prolonged NRF-1 knockdown did not result in decreased BRCA1 or GABP expression. However, these cells did display notable mitochondrial dysfunction, accompanied by the downregulation of several NRF-1 target genes involved in mitochondrial biogenesis including Tfam and cytochrome c. These results suggest a role for NRF-1 in mediating mammary morphogenesis through maintenance of functional mitochondria. Further investigation into the role of the NRF-1-containing complex at the GABPβ promoter during differentiation might also provide insight into the altered cell metabolism and differentiation observed in cancer cells. / Thesis (Master, Biochemistry) -- Queen's University, 2012-09-06 14:39:04.852

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20977号 / 医博第4323号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 小川 誠司, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Breast cancer is a disease associated with excess exposures to estrogens. While the mode of cancer causation is unknown, others have shown that oxidative stress induced by prolonged exposure to estrogens mediates renal, liver, endometrial and mammary tumorigenesis though the mechanism(s) underling this process is unknown. In this study, we show that 4-hydroxyl 17β-estradiol (4-OHE2), a catechol metabolite of estrogen, induces mammary tumorigenesis in a redox dependent manner. We found that the mechanism of tumorigenesis involves redox activations of nuclear respiratory factor-1 (NRF1); a transcriptions factor associated with regulation of mitochondria biogenesis and oxidative phosphorylation (OXPHOS), as well as mediation of cell survival and growth of cells during periods of oxidative stress. Key findings from our study are as follows: (i) Prolonged treatments of normal mammary epithelial cells with 4-OHE2, increased the formation of intracellular reactive oxygen species (ROS). (ii) Estrogen-induced ROS activates redox sensitive transcription factors NRF1. (iii) 4-OHE2 through activation of serine-threonine kinase and histone acetyl transferase, phosphorylates and acetylate NRF1 respectively. (iv) Redox mediated epigenetic modifications of NRF1 facilitates mammary tumorigenesis and invasive phenotypes of breast cancer cells via modulations of genes involved in proliferation, growth and metastasis of exposed cells. (v) Animal engraftment of transformed clones formed invasive tumors. (vi) Treatment of cells or tumors with biological or chemical antioxidants, as well as silencing of NRF1 expressions, prevented 4-OHE2 induced mammary tumorigenesis and invasive phenotypes of MCF-10A cells. Based on these observations, we hypothesize that 4-OHE2 induced ROS epigenetically activate NRF1 through its phosphorylation and acylation. This, in turn, through NRF1-mediated transcriptional activation of the cell cycle genes, controls 4-OHE2 induced cell transformation and tumorigenesis.

La NADH:ubiquinone oxydoréductase (complexe I) est le plus gros complexe enzymatique du système mitochondrial d'oxydation phosphorylante (43 sous-unités chez l'homme). Très peu de données sont disponibles concernant les mécanismes régulant l'expression de ces protéines. <br />Cette étude a été initiée par l'étude des promoteurs de deux gènes du complexe I mitochondrial humain. Les résultats montrent que le gène NDUFS8 qui code pour la sous-unité 23 kDa (TYKY) est transcrit sous le contrôle des facteurs de transcription YY1 et Sp1 tandis que gène NDUFS7 codant pour la sous-unité 20 kDa (PSST) est régulé par NRF-1 et Sp1. <br />Dans la deuxième partie de ce travail, une méthode d'analyse du protéome mitochondrial humain par électrophorèse bidimensionnelle a été développée. Le but est d'aborder de manière globale et sans a priori l'expression des protéines du complexe I : déterminer qui est régulé et comment en réponse à un stimulus déterminé? <br />Des cartes de référence ont été développées à partir de mitochondries extraites de placenta humain en utilisant deux types de gradient de pH : l'un est adapté aux protéines acides et neutres, l'autre aux protéines basiques. Sur ces cartes, 85 protéines mitochondriales ont été identifiées par spectrométrie de masse dont 17 sous-unités du complexe I. Cette technique d'analyse protéomique a ensuite été utilisée pour étudier la régulation de l'expression des protéines mitochondriales par le fer. Sur le plan technique, les premiers résultats sont encourageants : les gels d'électrophorèse bidimensionnelle préparés avec des mitochondries extraites de cellules en culture sont de bonne qualité et des variations reproductibles de l'expression de sous-unités du complexe I et d'autres protéines mitochondriales ont pu être détectées. Sur le plan fondamental, les données obtenues sont préliminaires. Il sera nécessaire de réaliser de nouvelles expériences pour confirmer les premières observations et analyser la cinétique des variations détectées.