Each year over 260,000 new cases of breast cancer will be diagnosed in the U.S. and approximately 40,000 women will die of metastatic breast cancer. The etiology of breast cancer is poorly understood and only 5 -10% of cases can be attributed to genetic factors. This suggests that the development of breast cancer may involve environmental factors including diet, lifestyle, and exposure to chemicals. Several lines of experimental and epidemiological evidence have highlighted COX-2 as a potential target for breast cancer prevention. The central hypothesis of this proposal is that activation of COX-2 transcription by epigenetic effectors can be prevented by dietary agents that target the activator protein-1 (AP-1) transcription factor and the aromatic hydrocarbon receptor (AhR). The first specific aim was to determine the mechanism through which conjugated linoleic acid (CLA) and rosmarinic acid (RA) inhibit TPA-induced COX-2 trancription. These studies documented that CLA and RA repressed COX-2 transcription by antagonizing the AP-1 transcription factor. The second specific aim was to investigate whether or not the AhR plays a role in TCDD-induced COX-2 transcription and effects of chemopreventive agents. Results indicated that AhR agonists induced the binding of the AhR to COX-2 and was prevented by CLA and the AhR antagonist, resveratrol (RES) and 3-methoxy-4-nitroflavone (3M4NF). The third specific aim was to examine the effects of AhR agonists and dietary selective AhR modulators on chromatin modifications associated with the COX-2 promoter. Chromatin immunoprecipitation (ChIP) assays revealed that the AhR is recruited to the region of the COX-2 promoter containing a xenobiotic response element and accompanied by recruitment of p300 and acetylation of histone H4. Transcriptional regulation of COX-2 by AhR agonists and dietary antagonists may also involve other post-transcriptional modifications of histones, which along with chromatin remodeling factors modulate the structure of chromatin and recruitment of RNA polymerase II. Overall, the results demonstrated that COX-2 transcription can be targeted by a variety of dietary agents that act through different mechanisms. Therefore, inhibition of transcriptional regulation of COX-2 by selected dietary factors may be a breast cancer preventive strategy that bypasses the side effects of drugs that target COX-2.

Breast cancer is the second leading cause of cancer-related death in women. Mutations in the tumor suppressor gene BRCA-1 confer a high risk of breast tumor development. However, in sporadic breast cancers, which represent 90-95% of breast cancer cases, BRCA-1 expression is downregulated in the absence of mutations in the BRCA-1 gene. This suggests that epigenetic effectors may contribute to disruption of BRCA-1 expression and the onset of mammary tumors.Prototypical environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to alter mammary gland development, act as endocrine disruptors and tumor promoters. Population studies detected accumulation of TCDD in women's adipose tissue and breast milk. Moreover, sporadic breast tissue exhibited statistically significant higher levels of PAH-DNA adducts. Based on this information, we examined the effect of B[a]P on the tumor suppressor BRCA-1and observed that exposure to B[a]P led to repression of BRCA-1 transcription through a p53-dependent mechanism. We have also demonstrated that 17β-estradiol (E2) stimulated the recruitment of ERα and AP-1 family members to a region of the BRCA-1 promoter flanking an AP-1-like site. However, accumulation of p53 prevented E2-mediated BRCA-1 transcription and recruitment of ERα, potentially providing one mechanism of B[a]P-mediated repression.In addition, the effects of B[a]P and TCDD are mediated through binding of the liganded aromatic hydrocarbon receptor (AhR) to dioxin or xenobiotic-responsive elements (XRE). We have evidence that suggests B[a]P and TCDD may modulate repression of E2-stimulated BRCA-1 expression through 1) binding of the liganded AhR to XREs on the BRCA-1 promoter and 2) preventing promoter occupancy by p300 and SRC-1.Taken together, the data presented here suggest that the transcriptional regulation of BRCA-1 is complex and involves modulation of the recruitment of ERα, AhR, p53, and their cofactors. An important implication of these findings is a greater understanding of the role of ERα, AhR, and p53 in regulation of BRCA-1 which could lead to the development of therapeutic strategies that target these interactions to enhance upregulation of BRCA-1 expression in sporadic breast tumors.

Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorobenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XRE=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XRE in the proximal BRCA-1 promoter, and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen receptor-alpha-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17-beta estradiol (E2)-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR, DNA methyl transferases (DNMT)1, DNMT3a, and DNMT3b; methyl binding protein (MBD)2; and tri-methylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation, and the recruitment of the AhR, MBD2, H3K9me3, and DNMTs (1, 3a, and 3b). Taken together, these observations provide evidence for a mechanistic role for AhR-agonists in establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists.

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La stéatose est la pathologie hépatique la plus répandue dans le monde, touchant environ 25 % de la population générale et jusqu’à 80 % des personnes en surpoids ou obèses. Cette maladie se traduisant par l’enrichissement des hépatocytes en triglycérides, est considérée comme bénigne. Cependant, environ 20 % des personnes atteintes de stéatose développent une stéatohépatite, pathologie caractérisée par une mort cellulaire et une inflammation et représentant un stade favorable au développement du carcinome hépatocellulaire. Les causes et mécanismes impliqués dans la progression de la stéatose vers la stéatohépatite sont encore à préciser. L’obésité, l’exposition aux contaminants environnementaux et la consommation d’alcool sont trois facteurs participant au développement des pathologies hépatiques. Pourtant, l’effet de l’interaction entre ces différents facteurs sur les pathologies hépatiques n’est pas connu. Dans ce contexte, cette thèse a eu pour objectif d’étudier l’impact d’une co-exposition au benzo[a]pyrène (B[a]P), un contaminant carcinogène environnemental, et à l’éthanol, utilisé à de faibles doses, sur les cellules hépatiques WIF-B9, présentant une stéatose préalable. Les résultats obtenus ont permis de démontrer qu’une telle exposition induit une progression de la stéatose hépatique vers un stade apparenté à une stéatohépatite, marquée par une augmentation de la mort cellulaire et de l’inflammation. Nous avons mis en évidence que la mort cellulaire résulterait de l’activation de p53 et d’une peroxydation lipidique. Une activation du récepteur aux hydrocarbures aromatiques et une production de monoxyde d’azote sont à l’origine de ces évènements via une modification du métabolisme de l’éthanol et du B[a]P conduisant à des dommages à l’ADN dépendants de la formation d’anion peroxynitrite. / Steatosis is the most common form of liver disease in the world, affecting around 25 % of the general population and up to 80 % of obese people. The disease is defined by the accumulation of triglycerides in hepatocytes and is generally considered as a benign condition. However, around 20 % of people with steatosis develop steatohepatitis, a disease characterized by cell death and inflammation, a condition that favors the development of hepatocellular carcinoma. The causes and mechanisms involved in the progression from steatosis to steatohepatitis are yet not fully understood. Obesity, exposure to environmental contaminants and alcohol consumption are three major factors contributing to the development of liver diseases. However, is still not yet clear what is the effect of the interaction between these different factors on liver diseases. In this context, the aim of this study was to evaluate the impact of a co-exposure to benzo[a]pyrene (B[a]P), an environmental carcinogenic contaminant, and ethanol, used at low doses, in WIF-B9 hepatic cell line, with a prior steatosis . The results demonstrate that this type of co-exposition induces a progression of hepatic steatosis to a steatohepatitis-like stage, marked by an increased cell death and an inflammation. We have shown that in this condition, cell death results from the activation of p53 and lipid peroxidation. Activation of the aromatic hydrocarbon receptor and production of nitric oxide are the origin of these events by a modification of both ethanol and B[a]P metabolism leading to peroxynitrite-dependent DNA damage.