Hormonal Control of HER2neu and BRCA1 in Breast Cancer

Institution: Sidney Kimmel Cancer Center
Investigator(s): Wanda Reynolds, Ph.D. -
Award Cycle: 1997 (Cycle III) Grant #: 3IB-0181 Award: $286,676
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1997)
The BRCA1 and the HER2neu genes are known to be important in the pathogenesis of breast cancer, but despite the efforts of many researchers, little is definitively known about how the control of these genes breaks down, or the mechanism by which estrogen might turn these genes on and off. The absence of BRCA1 protein in breast cancer cells is strongly linked to some familial breast cancers. An increased amount of HER2neu protein is associated with 30% of breast cancers, and correlates with poor prognosis and aggressive tumor growth. Considering the significance of these genes to breast cancer, there is a clear need to understand the mechanisms underlying this dysregulation, especially in regards to hormonal regulation of the amounts of HER2neu and BRCA1 protein.

There is evidence that estrogen influences the amounts of both HER2neu and BRCA1 protein in breast epithelial cells. Retinoic acid, a vitamin A derivative, and thyroid hormone are also implicated in the regulation of HER2neu. Our laboratory has obtained evidence that Alu elements associated with both HER2neu and BRCA1 genes may be responsible for this hormonal regulation. Alu elements are very abundant DNA elements, derived from a normal cellular gene termed 7SL. During the preceding 50 million years, the Alu elements have been replicated much like a virus and reinserted throughout the genome. During this time, for reasons as yet unknown, the Alu elements evolved to contain receptor binding sites. We found that the Alu elements associated with HER2neu and BRCA1 contain binding sites for receptors for retinoic acid, thyroid hormone, and estrogen receptor. We hypothesize that these hormones/vitamins associate with their receptors, which then bind to the DNA promoter sequences upstream of HER2neu and BRCA1 genes, thereby increasing or decreasing the amounts of HER2neu and BRCA1 that are made. We propose to determine whether these receptor binding sites are of key importance to the hormonally controlled dysregulation of HER2neu and BRCA1 in breast cancer.

Final Report (1999)
The objective of this project was to understand how the control of the HER2neu and BRCA1 genes is lost in breast cancer cells -- a process that leads to unrestrained cell growth. The specific aims were to (1) determine if the control of the HER2neu and BRCA1 genes is due in part to hormone receptor binding sites in upstream Alu elements (AluHRE), and (2) to determine if these elements are preferential sites of DNA mutations or breakpoints which occur in breast cancer.

To investigate these possibilities, we assayed the effects of estrogen, retinoic acid (RA) and thyroid hormone (T3) on the levels of expression of HER2neu mRNA in breast cancer cell lines. Estrogen was found to increase HER2neu mRNA levels by four fold in MCF7 breast cancer cells, while T3 and RA decreased levels by three fold. We further demonstrated specific binding by estrogen, retinoic acid, and thyroid hormone receptors to the HER2neu and BRCAI Alu elements. Extracts of breast cancer cells were also found to contain endogenous receptors which bound to these sites. The receptor binding region in the Alu overlap with a synergistic SPI transcription factor binding site, and we detected much higher levels of SPI transcription factor in MCF7 breast cancer cells than in control cells, suggesting that elevated SPI levels in breast cancer cells might contribute to an AluHRE mediated over expression of HER2neu. Cellular transfection assays provided further evidence that the AluHRE modulate transcription of HER2neu in breast cancer cells. The AluHRE were linked to a reporter gene and transferred to MCF7 breast cancer cells Endogenous receptors in MCF7 cells activated reporter gene expression by ten fold in the presence of estrogen, and four fold in the presence of either retinoic acid or thyroid hormone, demonstrating receptors for these ligands are present in MCF7 cells and that these are able to bind and trans activate a nearby gene through the AluHREs. These findings clearly indicate that the AluHRE cassette can contribute to the upregulation of HER2neu, as well as the downregulation of BRCA I in breast cancer cells. We also looked for evidence of breakpoints or mutations in the AluHRE associated with HER2neu and BRCAI in breast cancer cells, but found no such evidence.

The demonstration that estrogen and retinoic acid receptors bind to the upstream AluHRE of the HER2neu and BRCAI genes, and modulate expression of these genes in breast cancer cells, may lead to the development and usage of novel estrogens or retinoids (or antagonists thereof) to control the expression of these genes.

Symposium Abstract (2003)
Tumor associated macrophages participate in the immune response against the tumor through release of cytokines (molecules that allow communication between white blood cells and other cells) and reactive oxidizing chemicals. Recent findings suggest that the enzyme myeloperoxidase (MPO) is present in some reactive macrophages and thus may contribute to the immune response to the tumor. MPO reacts with hydrogen peroxide and chloride to produce hypochlorous acid (HOCl)(bleach), a potent killing agent. A polymorphism (change in the genetic code) in the portion of the MPO gene that turns protein production on, -463G/A, is associated with risk for lung cancer and myeloid leukemia. This G/A base substitution changes the proteins that are able to turn MPO on. The presence of the G base pair ( 463G allele) allows binding of Sp1 transcription factor and the presence of the A base pair (463A allele) allows control by the estrogen receptor. The MPOG allele is associated with 32 fold higher expression than the A allele in macrophages, and is most often associated with increased disease risk. To investigate the possibility that MPO plays a role in the immune response against breast tumors, we analyzed the MPO genotype of 214 Austrian breast cancer cases and 122 controls. The GG genotype was associated with smaller breast tumor size (p=0.0035), lower tumor grade, and with negative lymph node status (p=0.05). Immunohistochemical analysis confirms the presence of abundant MPO protein in breast tumors within invading macroph-ages. These findings provide evidence that MPO plays a protective role in the immune response to breast cancer.