Coactivators in Mammary Gland Development and Tumorigenesis

Institution: Salk Institute for Biological Studies
Investigator(s): Zhiyong Wang, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7FB-0040 Award: $86,400
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2001)
The underlying molecular mechanisms causing breast cancer are not fully understood despite intensive studies. Estrogen and progesterone, ovary hormones, mediate epithelial cell proliferation by binding to estrogen receptor (ER) and progesterone receptor (PR) in mammary gland, respectively. They are important factors for both normal breast development and breast cancer formation. Additional proliferative and tumorigenic pathways for both mammary gland development and tumorigenesis include other agents of cell division including IGF1 and prolactin pathways. My initial genetic studies have identified that p/CIP is critical for IGF1 proliferative signaling, besides the well-known effects of p/CIP on enhancing activity of estrogen and progesterone receptors. p/CIP/A1B1 is amplified and present at elevated levels in 64% of human breast cancers. Excessive amounts of p/CIP therefore might activate these cell division pathways simultaneously to induce tumors.

The central hypothesis of my research is that p/CIP induces mammary tumors by activating synergistic proliferative pathways, when p/CIP is present at high levels. I will use available technology to create two kinds of mice, one kind has no endogenous p/CIP, the other has excessive levels of p/CIP in mammary gland specifically. I will then study mammary gland development and tumorigenesis in these two kinds of mouse models, respectively.

As far as I know, there is no available mouse model overexpressing the potent transcriptional coactivators. This is a significant void. My research will try to answer whether the coactivator, p/CIP, is a proto-oncogene (cancer causing gene), and how it causes tumors in mouse models. Since the pathways such as steroids and IGF synergize for initial and early steps of human breast cancer formation, the proposed studies directly relate with breast cancer. The knowledge generated from my research will provide novel intervention strategies and therapeutic approaches targeting p/CIP activity during early stages of feast cancer development.

Final Report (2003)
The p160 coactivators of p/CIP, SRC1, and TIF2 increase the action of nuclear hormone receptors such as the estrogen receptor (ER) and progesterone receptor (PR) in vitro by enhancing their transcriptional activities. One p160 family member, p/CIP/AIB1, is present at high levels in human breast and ovarian cancers. However, whether these coactivators play any physiological role in nuclear hormone target organs, such as mammary glands, remains unknown.

To investigate their potential roles in mammary gland organogenesis, we have generated single knockout mice lacking p/CIP and SRC1, and double knockouts (DKO) of p/CIP and SRC1. We have observed no discernible defect during different stages of mammary gland development in either p/CIP or SRC1 single knockout mice. We suspect that there is compensation by the other two other family members in the single knockouts. When we tried to generate p/CIP and SRC1 DKO mice by crossing the single knockouts, we found that more than 70% of the DKO mutants died within three weeks of birth, demonstrating a gene-dosage dependent effect on postnatal survival of p/CIP/SRC1 DKO. We examined mammary gland development from puberty onwards in surviving DKO mice, and found that in contrast to wild type there was minimal epithelial cell proliferation and penetration into the mammary fat pad, even in mature adult DKO females. To exclude the possibility that this was due to a lack of systemic hormones, such as estrogen, in the DKO mutants, we transplanted mammary epithelium from DKO mutants into cleared fat pads (rid of endogenous mammary epithelium) of 3 week old nude mice, which have full complement of systemic hormones. Six weeks after transplantation, we still observed very little penetrance of mammary epithelium from DKO mutants into cleared fat pads, whereas control wild type mammary epithelium penetrated extensively into cleared fat pads based on whole mount studies. These results demonstrate that the mammary development defect in p/CIP/SRC1 DKO mutants is cell autonomous. We performed in vivo BrdU labeling experiments on transplant mice, and found minimal cell proliferation in DKO mammary epithelium, compared to extensive proliferation in control epithelial transplants. The phenotype we observed in DKO mammary gland is similar to that of ER? knockouts. We are currently studying target gene expression of the ER pathway, such as PR, in the DKO null mammary glands to determine which pathways are involved in the phenotype. To complement our loss-of-function studies, we have expressed a mammary-specific p/CIP transgene, expressed from the WAP promoter, and have observed mammary tumors in aging transgenic positive mice, not found in their wild type littermates.

In summary, we have identified an obligatory role for p/CIP and SRC1 in mammary gland development, and conclude that tightly regulated expression levels of these p160 coactivators are critical for mammary development and tumorigenesis.

Symposium Abstract (2003)
Breast cancer is the most commonly diagnosed cancer in women, and one of eight women will develop breast cancer in their lifetime. The underlying molecular mechanisms causing breast cancer are not fully understood despite intensive studies. Estrogen and progesterone, ovarian hormones, mediate epithelial cell proliferation by binding to estrogen receptor (ER) and progesterone receptor (PR) respectively in mammary gland. These receptors, when activated, turn on other genes, which are critical for both normal breast development and breast cancer formation. The p160 coactivators p/ CIP and SRC1 increase these activities of ER and PR. In addition, p/CIP is amplified and overexpressed in human breast and ovarian cancers. Therefore, p/CIP and SRC-1 are potential novel factors involved in breast cancer formation.

The goal of our research is to determine physiological roles of these coactivators and whether they can induce tumor directly in genetically modified mouse models. In the mutant mice with both p/CIP and SRC1 genes deleted (double knockout), we have observed severe proliferation defects in the mammary gland after puberty. We have determined the proliferation defects are epithelial cell autonomous with mammary epithelial transplantation experiments. We are investigating potential pathways, such as estrogen receptor pathway, which are involved in the defects. These loss-of-function experiments suggest that p/CIP and SRC1 are required for normal proliferation and development of mammary gland in the animal. We also address another critical question about the potential roles of p/CIP in mammary tumor formation with a transgenic approach. We have established transgenic mouse lines overexpressing p/CIP only in mammary gland. We have found mammary tumor formation in the aging transgenic mice. We are examining these mice before tumor formation to determine the cause of the tumor. In summary, we have determined critical roles of p160 coactivators during mammary gland development and tumor formation. The knowledge and mouse models from our research will contribute to mammary tumor prevention and therapy.