- Romagnolo, Donato; Burdn, Randy
- University of Arizona
- Start date
- End date
- 1. Determine the mechanisms by which dietary bioactive compounds protect against human diseases. 2. Elucidate mechanisms of action of dietary toxicants and develop biomarkers for human risk assessment and disease prevention. 3. Discover and characterize novel bioactive dietary compounds that have beneficial or adverse effects on human health.
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- Non-Technical Summary:
The loss of BRCA-1 protein is a causative factor in the etiology of breast cancer in BRCA-1 mutation carriers. BRCA-1 exerts pleiotropic effects including maintenance of genomic integrity through homologous recombination, cell cycle control, and transcriptional regulation. One of the puzzles in breast carcinogenesis is that the vast majority (90-95%) of breast cancer cases are sporadic and occur in the absence of mutations in the BRCA-1 gene. On the other hand, sporadic breast tumors have absent or markedly reduced levels of BRCA-1. This raises the possibility that loss or reduced DNA repair functions controlled by BRCA-1 due to non-genetic influences may increase the risk of developing sporadic breast cancer. Epigenetic mechanisms involve the complex orchestration of DNA methylation enzymes, post-translational modifications of histones, alterations in the recruitment and functions of non-histone proteins, and changes in expression of non-coding RNAs. The extent of BRCA-1 promoter methylation in sporadic breast tumors may depend on the clinical subtype and varies from ~10% up to 85% in ductal-infiltrating tumors. Hypermethylation at CpG islands neighboring the transcription initiation sites of the BRCA-1 gene may contribute to organization of a compact heterochromatin and silencing of BRCA-1 expression. Therefore, the impact of this project is that it will help identifying the mechanisms responsible for the reduced expression of BRCA-1 mammary tumors and may offer new insight for preventing through nutrition the silencing of BRCA-1 expression or re-establishment of normal expression in breast tissue.
Previous studies reported that aromatic hydrocarbon receptor (AhR) activation during pregnancy or in nulliparous mice slowed the promotion of preneoplastic mammary lesions. These cumulative data suggest that timing of AhR activation differentially influences the risk of breast tumorigenesis with possible promoting effects in-utero and pre-puberty and protective effects on more differentiated mammary tissue (i.e. postpuberty, pregnancy). The biochemical mechanisms of this dichotomy are largely unknown. In this study, we will investigate the influence of gestational activation of the AhR on the epigenetic regulation of BRCA-1 in mammary tissue of female offspring and the preventative effects of resveratrol, an antagonist of the AhR. In addition, the influence of AhR activation on BRCA-1 in pubertal female rats will be examined. Results may suggest that in utero activation of the AhR increases methylation levels in the rat BRCA-1 promoter in mammary tissue of female offspring. These results may be coupled with reduced BRCA-1 mRNA and protein, and increased proliferation. Conversely, if the AhR antagonist resveratrol were to exert protective effects, the findings of this project will bring attention to the potential impact of maternal nutrition on epigenetic imprinting in the offspring. Efforts that will be implemented to cause a change in knowledge and evaluate impact include publication in scientific journals, presentations at national and international meetings, and citations of published work.
2012/01 TO 2012/12
OUTPUTS: Activities: 1. Does exposure to ligands of the aromatic hydrocarbon receptor influence the risk of disease Epigenetic mechanisms 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. We conducted experiments to investigate if XREs present in the proximal BRCA-1 promoter the expression of endogenous AhR mediated silencing of BRCA-1 expression by TCDD. These activities have been used to mentor Ph.D. students (Andreas Papoutsis) and train staff (Jamie Borg); data were presented at the 2012 Experimental Biology Meetings in the form of a symposia in San Diego, CA. Dissemination of this information occurred through seminars presented by Andreas Papoutsis on ther Campus of the University of Arizona; class presentations by Dr. Romagnolo, specifically to students enrolled in Nutritional Biology NSC408 and Metabolic Integration NSC602; and publication in scientific journals. Products include one Ph.D. student (Dr. Papoutsis)graduated in nutritional sciences and development of laboratory skills. 2. Are dietary flavonoids protective against cancer The objective of this work was to review data from epidemiological and preclinical studies addressing the potential benefits of diets based on flavonoids for cancer prevention. Flavonoids are subdivided into subclasses including flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones. Epidemiological studies suggest dietary intake of flavonoids may reduce the risk of tumors of the breast, colon, lung, prostate, and pancreas. However, some studies have reported inconclusive or even harmful associations. 3. Are proteomic tools useful for studies of cancer prevention through dietKnowledge gaps persist about the efficacy of cancer prevention strategies based on dietary food components. Adaptations to nutrient supply are executed through tuning of multiple protein networks that include transcription factors, histones, modifying enzymes, translation factors, membrane and nuclear receptors, and secreted proteins. However, the simultaneous quantitative and qualitative measurement of all proteins that regulate cancer processes is not practical using traditional protein methodologies.
PARTICIPANTS: Individuals from the University of Arizona included Dr. Donato Romagnolo, principal investigator; Dr. Ornella Selmin, collaborator; Andreas Papoutsis, who received his Ph.D. under supersivion of Dr. Romagnolo; and Jamie Borg. Collaborators were: Dr. Dashwood R., from Oregon State University; Dr. Stover PJ, from Cornell University; Dr. Waterland RA, from Baylor College of Medicine; Dr Ziegler TR, from Emory University; and Dr. DeRoos B, from the University of Aberdeen, UK.
TARGET AUDIENCES: Target audiences for this work included undergraduate and graduate students attending the University of Arizona; principal investigators and scientists in training on the Campus of the University of Arizona; nutrition and clinical scientists in the US, and the general public at large in the State of Arizona and the US.
PROJECT MODIFICATIONS: There were no major changes in approach.
Change in knowledge: ACTIVITY 1: We found that in estrogen receptor alpha (ERalpha)-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17beta 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 (1 micromol/L) 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). These observations provide mechanistic evidence for AhR-agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of preventive actions based on AhR antagonists.
ACTIVITY 2: A major challenge in the interpretation of epidemiological studies is that most of the data originate from case-control studies and retrospective acquisition of flavonoid intake. Differences in agricultural, socio-demographics, and lifestyle factors contribute to the heterogeneity in the intake of flavonoids among populations residing in the U.S., Europe, and Asia. Dose and timing of exposure may influence the anticancer response to flavonoid-rich diets. A limited number of intervention trials of flavonoids have documented cancer preventative effects. Proposed anticancer mechanisms for flavonoids are inhibition of proliferation, inflammation, invasion, metastasis, and activation of apoptosis. Prospective studies with larger sample sizes are needed to develop biomarkers of flavonoid intake and effect. Mechanistic studies are needed to ascertain how flavonoid-rich diets influence gene regulation for cancer prevention.
ACTIVITY 3. Proteomics offers an attractive opportunity to fill a knowledge gap and unravel the effects of dietary components on protein networks that impinge on cancer. Recent advances in MS technologies suggest that studies in nutrition and cancer prevention may benefit from the adoption of proteomic tools to elucidate the impact on biological processes that govern the transition from normal to malignant phenotype; to identify protein changes that determine both positive and negative responses to food components; to assess how protein networks mediate dose-, time-, and tissue-dependent responses to food components; and, finally, for predicting responders and nonresponders.
- Funding Source
- Nat'l. Inst. of Food and Agriculture
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- Prevention and Control
- Risk Assessment, Management, and Communication