Role of Estrogen-modulated Protein AGR2 in Breast Cancer

Institution: University of California, Irvine
Investigator(s): Mikhail Geyfman, B.S. -
Award Cycle: 2008 (Cycle 14) Grant #: 14GB-0163 Award: $71,491
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2008)

Over half of all breast cancers contain the estrogen receptor (ER-positive cancers). While such tumors tend to have a relatively favorable prognosis, it is clear that estrogens stimulate the growth of these tumors. In fact the growth of these tumors can be suppressed with drugs such as tamoxifen that block estrogen action. It is thought that estrogens stimulate cancer growth by turning on the expression of other molecules that in turn mediate their effects. One such candidate mediator is a poorly characterized gene that encodes a secreted protein referred to as Anterior Gradient-2 (AGR2). This gene is strikingly upregulated in breast cancers, especially ER-positive tumors. While the expression of AGR2 has been well characterized in human breast cancer, its potential mechanism of action remains unclear.

First, we plan to test the role of the AGR2 gene in mice, since this system allows the study of AGR2 in the context of a whole organism. We have already created a mouse that allows us to delete the AGR2 gene either in all cells of the body or specifically in breast epithelial cells. I have also created transgenic mice that overexpress the AGR2 gene in mammary glands of mice, thus simulating the situation in human breast cancer. We will use histology, as well as immunohistochemistry to analyze the effect of AGR2 gene on mammary gland development and homeostasis. Specifically, we will examine pro-tumorigenic effects, such as hyperplasia and tumor formation. Secondly, we plan to interfere with the production of the AGR2 protein in human breast cancer cell lines that are known to express the AGR2 at high levels. These experiments will employ an RNAi system to knock down AGR2 in estrogen-responsive cells. We then can test whether lack of AGR2 alters the response of these cells to estradiol in terms of cellular proliferation (division), adhesion, and invasion. Taken together, these studies will allow us to directly test whether AGR2 is required for estrogen actions in human breast cancer cells.

While the expression of AGR2 in human breast cancer has been extensively characterized, there is only a single study on its potential function. Our project is designed to test the role of AGR2 in multiple phases of cancer via its modulation of the estrogen receptor.

Final Report (2010)

AGR2 is a secreted protein overexpressed in many estrogen-positive mammary tumors. The role of this protein in tumorigenesis is not clear. AGR2 is thought to be a disulfide isomerase participating in protein folding. In intestinal epithelium it was demonstrated to play a role in modulating stress response. Data in cancer cell lines shows that it also plays a role in proliferation and programmed cell death.

I chose primarily an in vivo (mouse model) approach to study AGR2 function in normal mammary gland development and cancer. First, we sought to determine the role of AGR2 in normal mammary gland development and physiology. Then, we wanted to decipher the molecular basis for positive correlation between AGR2 expression and estrogen receptor.

In order to accomplish our first goal we generated a “genetically engineered” mouse (GEM) line overexpressing AGR2 specifically in the mammary epithelium and second line where AGR2 is deleted in the mammary epithelium. We also generated a GEM where AGR2 is globally deleted as well as deleted in the keratin 14 expressing epithelia.

The GEMs with mammary specific overexpression and deletion of AGR2 yielded important insight about the normal function of AGR2 in the mammary gland as well as provided clues to its role in tumorigenesis such as regulation of proliferation, epithelial differentiation, and apoptosis. To date, the results of these studies have involved visual (phenotypic) comparisons of mammary grant development in mice with and without AGR2, and tracking these mammary changes though the mammary stages in the adult animal (i.e., virgin, pregnant, and lactating stages). Overall, AGR2 amounts are highest in lactating animals (luminal and myoepithelial cells). Increased AGR2 amounts are associated with to hyperplastic mammary alveolar development, while AGR2 removal results in endoplasmic reticulum “stress” along with programmed cell death (apoptosis) and decreased cell proliferation during pregnancy.

Since the in vitro (cell line) work we proposed initially as a second goal was published by Vanderlaag et al. in Breast Cancer Research in 2010, our focus shifted to an in vivo (mouse studies using the MMTV-NeuNT model) approach to study AGR2 involvement in breast cancer. We are in the process of evaluating mouse breast cancer models that overexpress AGR2. This will entail crossing our AGR2 overexpressing GEM to this breast cancer mouse model in order to determine the impact of AGR2 on the etiology of breast cancer as well as its effect on estrogen receptor signaling and other molecular pathways.

Symposium Abstract (2010)

AGR2 is a protein disulfide isomarase that is expressed significantly higher in breast tumors compared to normal mammary epithelium. The severity of breast cancer prognosis also correlates positively with AGR2 expression. AGR2 is also present selectively in breast tumors that express estrogen receptor.

We analyzed expression of AGR2 in normal mice through post-pubertal mammary gland development. I have generated and validated a trangenic mouse line that overexpresses AGR2 in mammary epithelium after doxycycline administration. These experiments showed that AGR2 is highly expressed during pregnancy and lactation when alveolar development is at its highest. The AGR2 overexpression line was generated and initial results promise to provide valuable information about the role of AGR2 in tumor formation. Overexpression of AGR2 leads to increased ductal branching and alveolar development that can be a pre-cancerous phenotype. Results obtained with this line as well as analysis of normal mice provide important clues about potential biological role of AGR2 in normal mammary gland biology and cancer.

Role of Estrogen-modulated Protein AGR2 in Breast Cancer
Periodical:Developmental Biology
Index Medicus: Dev Biol
Authors: S. Verma, M. Salmans, M. Geyfman, H. Wang, Z. Yu, F. Zhao, S. Lipkin, B. Andersen
Yr: 2012 Vol: 369 Nbr: 2 Abs: Pg:249-260