Mechanism of Estrogen Receptor Loss in Breast Cancer

Institution: University of California, Irvine
Investigator(s): Keon Wook Kang, Ph.D. -
Award Cycle: 2003 (Cycle IX) Grant #: 9FB-0135 Award: $17,298
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2003)
Estrogen is a critical factor for the proliferation of early stage breast cancer. But, paradoxically one-third human breast cancers are estrogen receptor alpha-negative (ERα-) tumors. There breast cancers are generally refractory to anti-hormone therapy and have a lower success rate in overall treatment. The biological origin of the ERα- breast cancers is still unclear. Recently, a new mammary-specific tumor model with conditional mutation of the tumor suppressor gene p53 (strain = WAP-Cre;p53exon5&6) was established in our lab. Surprisingly, in contrast to most mammary tumors developed in transgenic mice that are ERα-, we found that about 30% of tumors in our mice are ERα+. Thus, our new mouse model provides a unique system to address origin of ERα- and ERα+ mammary tumors.

In this project we propose to explore the underlying cell origins, timeframe, and specific genetic changes for ERα- and ERα+ mammary tumors. We will use molecular markers to facilitate the isolation of live ERα mammary tumor cells. These cells will be transplanted into the fat pad of recipient mice to study whether the tumor cells will progress to the ERα- phenotype. We will also treat the tumors with different anti-hormone agents to study whether this will alter the frequency of ERα- conversion. Similar approaches will be used to isolate ERα- and ERα+ pre-malignant mammary epithelial cells for transplantation into mouse mammary fat pads. Then we can study the neoplastic development of ERα- and ERα+ mammary tumors. Next, we want to study the relationship of Her2/neu in ERα- and ERα+ mammary tumors. It has been reported that increased levels Her-2/neu in breast cancer correlate with loss of ERα. Lastly, we will apply molecular techniques to identify "biomarkers" or genes that control the development of ERα- and ERα+ mammary tumors. For this aim we will use microarray or SAGE (serial analysis of gene expression) to quantitate all cellular RNA to detect those changes associated with ERα.

We hope that these studies will clarify the long existing questions in breast cancer field and provide new markers for breast cancers useful in clinical diagnosis and decision-making.

Final Report (2005)
The p53 tumor suppressor is a transcription factor that controls cellular stress response. Deregulation of p53 activities and mutations of p53 are frequent events in human tumors. Approximately 30-50% of human breast carcinomas harbor a mutant p53 gene. Heritable mutations of p53 in the Li-Fraumeni syndrome leads to breast cancer predisposition. Somatic mutations of p53 in mammary glands lead to mammary tumor with high frequency. On the other hand, ovarian steroid hormones including estrogen are critical for mammary gland development and breast carcinogenesis. We examined the impact of p53 on estrogen signaling on the proliferation of primary mammary epithelial cells (MECs).

Somatic mutations of p53 in mouse mammary epithelial cells using the Cre/loxP system lead to mammary tumors with long latency. Exposure to steroid hormones enhances tumorigenesis in p53-null mammary cells. However, the mechanisms are not well understood. We found that both genomic and non-genomic actions of estrogen were altered in mammary epithelial cells (MECs) harboring deletion of exons 5 and 6 of the p53 gene (p53D5,6). For the non-genomic actions in response to estrogen, we observed a rapid activation of extracellular signal-regulated kinase (ERK) was in MECs, which was substantially elevated and sustained in p53D5,6 MECs. In contrast, the activities of p38 kinase and c-Jun N-terminal kinase were not affected by p53 mutation. The genomic actions of estrogen were assessed by luciferase reporter assays using five-repeated ERE (estrogen response element) sequences and by measuring the expression level of pS2, a downstream target gene of estrogen receptor. Mutations of p53 increased the basal luciferase reporter gene activity and pS2 levels, which were abolished by treatment with an ERK specific inhibitor, U0126. Furthermore, we identified a significant role of ERK-specific phosphatase, dual specific phosphatase (Dusp)-3 in the regulation of ERK activities by p53. Expression levels of Dusp3 in p53D5,6 MECs decreased substantially, but not Dusp2. Overexpression of Dusp3 in p53D5,6 MECs resulted in decreased cell proliferation. These cells also had limited proliferation potential when transplanted into the fat pads of Rag-1 mice.

Our results support a notion that p53 inhibits genomic and non-genomic action of estrogen in MECs through regulation of specific mechanisms. These findings provide novel pathways linking p53 tumor suppressor to estrogen-mediated cellular response in mammary epithelial cells.