Cell bioassays for detection of aromatase gene activators

Institution: University of California, Davis
Investigator(s): Michael  Denison , Ph.D. -
Award Cycle: 2011 (Cycle 17) Grant #: 17UB-8703 Award: $421,680
Award Type: SRI Request for Proposal (RFP)
Research Priorities
Etiology and Prevention>Etiology: the role of environment and lifestyle

Initial Award Abstract (2011)

The incidence of breast cancer in the United States is on the rise. Most breast cancers are initially hormone-dependent and require estrogens for growth. Estrogens are produced by the enzyme aromatase which is active in various tissues including the breast. Aromatase activity is regulated differently in different tissues and in mammary tissue containing a tumor this enzyme is overexpressed, leading to localized overproduction of growth-stimulatory estrogens. Elevated aromatase gene expression in breast cancer is caused by the increased activity of the specific aromatase promoters pII, I.3, I.4 and I.7. Determining the effects of chemical exposures on the activity of these aromatase promoters in humans is not possible and specific screening tools do not yet exist. In this research application, we propose to construct breast cancer cells that contain the above 4 promoters of aromatase coupled to a luciferase gene. These cell constructs will be able to produce light upon exposure to chemicals that stimulate the activity of one or more of these promoters. Increased aromatase promoter activity results in increased expression of mammary aromatase, production of estrogens and ultimately stimulation of hormone-dependent mammary tumors.

Research questions:
Are chemicals capable of activating the breast cancer-relevant aromatase promoters PII, I.3, I.4 and I.7, thus increasing the risk of developing hormone-dependent mammary tumors? How can we detect this?

General methodology:
Aromatase promoter gene sequences for pII, I.3, I.4 and I.7 will be coupled to the gene for the enzyme luciferase which upon stimulation produces light. These constructs will be incorporated into the DNA of commercially available breast cancer cell lines. These stably transfected cells will be characterized and validated by determining their response to compounds known to stimulate the activity of each of the inserted aromatase promoters. Cell constructs that respond to these compounds as would endogenous aromatase will be selected for the screening of various chemicals known or suspected to cause endocrine disruption, including pesticides, environmentally persistent chemicals (e.g. flame retardants), and chemicals used in large quantities in industrial processes or consumer products (e.g. phthalate plasticizers, bisphenol A, etc).

Innovative elements:
This research project would provide a number of screening tools to identify chemicals that may increase the risk of breast cancer by interfering with the regulation of aromatase via promoters that are specifically active in the presence of breast tumors. These screening tools would be relatively cheap to use and have the capacity to evaluate large numbers of chemicals many of which that have not been assessed for potential disruption of mammary-specific expression of aromatase. So far, breast cancer-relevant screening tools for aromatase disruption do not exist.

Advocacy involvement and relevance to the human issues associated with breast cancer:
Better understanding of the risks posed by chemicals to which women are exposed either in daily life or under certain exceptional circumstances for the development of hormone-dependent breast cancer. The proposed screening tools will provide scientifically based information that will influence decisions on acceptable exposure levels to certain chemicals or whether or not to avoid use of certain chemicals in consumer products.

Final Report (2015)

Aromatase (CYP19) is the final enzyme in the biosynthesis pathway of estrogen and it is critically important in breast cancer development. The regulation of aromatase expression is very complicated and in humans it is regulated in a tissue-specific manner through the use of alternate gene promoters. In hormone-dependent breast cancer, aromatase is over-expressed via several normally inactive gene promoters (PII, I.3, I.7), in addition to its expression from the normal breast adipose tissue aromatase gene promoter (I.4). While most breast cancers are estrogen-dependent, relatively little is known about what chemicals (natural or synthetic) can increase or decrease aromatase gene expression and estrogen production in this tissue and its subsequent effects on breast cancer growth and proliferation. Since there were no bioassays that directly examine breast cancer aromatase gene expression, we proposed to develop cell-based screening bioassays to identify chemicals that increase the risk of development or progression of breast cancer via their ability to increase aromatase promoter-specific gene expression in cancer associated cells. In this project, we completed the construction of five aromatase promoter-specific firefly luciferase reporter gene plasmids and confirmed their functional activity and response to known aromatase inducers in human cancer cell lines (Aim 1). These vectors were then permanently inserted into cancer cells in culture with the goal of generating recombinant cell lines that can respond to aromatase inducing chemicals with an increase in expression of firefly luciferase and luciferase-dependent light production. We developed one stable cell line (SKBR3I.4) that contained a pI.4 aromatase gene promoter-luciferase reporter gene and showed that it could be used as a relatively rapid screening bioassay to identify both natural (flavones) and synthetic (pesticides) chemicals that stimulate expression from the I.4 aromatase gene promoter and thus may stimulate estrogen production in humans (Aims 2 and 3). We also developed a sensitive and robust RT-qPCR method to detect promoter-specific expression of aromatase in human adrenocortical carcinoma (H295R) cells (which express the breast cancer-relevant promoters PII and I.3). Using this method, in combination with that of the new SKBR3I.4 cell line, we were the first to show that atrazine and several commonly used neonicotinoid insecticides can increase the expression of aromatase in a promoter specific manner. Given the importance of the promoter-specific expression of aromatase in breast cancer, is it important to identify new classes of endocrine disrupting chemicals (such as the neonicotinoids and other chemicals) that can work by this mechanism. There is also a critical need to understand how such chemicals can activate individual aromatase promoters in order to better understand and predict the potential risk of these chemicals to exposed women. These will be the focus of future studies in our laboratories.

Effects of Neonicotinoids on Promoter-Specific Expression and Activity of Aromatase (CYP19) in Human Adrenocortical Carcinoma (H295R) and Primary Umbilical Vein Endothelial (HUVEC) Cells
Periodical:Toxicological Sciences
Index Medicus: Toxicol Sci
Authors: Elyse Caron-Beaudoin, Michael S. Denison, and J. Thomas Sanderson
Yr: 2016 Vol: 149 Nbr: 1 Abs: Pg:134-144