Novel Regulation of the Rb Pathway in Breast Epithelium

Institution: Stanford University
Investigator(s): Deborah Burkhart, Ph.D. -
Award Cycle: 2007 (Cycle 13) Grant #: 13GB-0150 Award: $74,808
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (2007)

The development of breast cancer from normal cells in the mammary epithelium requires multiple mutations in cellular genes. In particular, members of a class of genes called tumor suppressors are often lost in breast cancer; these genes usually act as cellular brakes, preventing breast cells from abnormally multiplying. Our research focuses on the pRB family of tumor suppressors, comprising pRB (retinoblastoma protein) itself, and the associated proteins p107 and p130. Normally pRB prevents the cell from replicating damaged DNA by blocking its progression through the cell cycle. Thus, when pRB becomes ineffective, mutated cells can continue to divide and this can lead to cancer initiation and progression. In fact, the function of pRB family proteins is altered in more than 80% of breast cancers, and our goal is to understand how this family of proteins normally prevents cells in the mammary gland from becoming tumor cells.

In the breast, the function of the RB-related proteins, p107 and p130, need to be inactivated in addition to that of pRB before cancer can occur. Thus, the conventional thinking is that p107 and p130 display compensatory, although not well understood functions that prevent pRB-deficient mammary cells from becoming tumor cells. This project focuses on the regulation and the function of p107 in the mammary gland in mice, with the specific goal of understanding how p107 can block cancer in pRB-deficient breast cells. We propose to study p107 in the mammary epithelium for: (1) expression pattern in various cells; (2) its functional role to prevent proliferation and control apoptosis, and (3) the molecular mechanisms controlling its transcription (i.e., regulation of gene expression). We will use single and double knockout mice for RB and p107 to investigate the functional role of p107 in the presence and in the absence of pRB. These studies will allow us to investigate any unique or overlapping functions that p107 and pRB may have in controlling proliferation and apoptosis in the mammary gland. In addition, we have created mouse embryonic stem (ES) cells with point mutations in the E2F transcriptional control binding sites in the p107 promoter in order to dissect gene regulation.

Throughout a woman’s life, the breast undergoes a series of hormonally driven developmental changes that involve signaling pathways to prevent abnormal expansion of mammary cells. The proposed experiments will uncover important insights into key nodes in these pathways, such as pRB and p107. A greater understanding of the normal functions of these proteins will allow the further investigation of targeted therapeutic strategies to replace these functions in breast cancer. These molecular therapies may have the potential improve the quality of life of a patient during and after treatment.

Final Report (2009)

Note: the PI graduated from Stanford and is now a postdoctoral fellow in the lab of Gerard Evan, UCSF

The retinoblastoma gene was the first tumor suppressor to be identified, through its association with hereditary retinoblastoma. The Rb family, which includes p107 and p130, mediate numerous cellular functions critical for normal development and for tumor prevention, including regulation of the cell cycle and cell cycle exit, differentiation, maintenance of quiescence, and apoptosis. While some tumors develop upon loss of pRb alone, in breast cancer, loss of pRb is most often accompanied by loss of the functions of p107 and p130, suggesting that p107 and/or p130 may serve as a backup to pRb in suppressing tumor development. We initially set out to understand the expression of p107 in wild-type and Rb-deficient mouse mammary glands, and to understand the role of E2F in controlling these expression patterns.

We successfully developed and published a novel p107-GFP reporter transgenic mouse line and spent a great deal of time adapting techniques that worked well on other tissues to also work in the mammary gland. While technical obstacles and quantities of research animals limited the amount of data we accumulated to address our original aims in the time period covered by this grant, we did find that in wild-type animals p107 levels surprisingly appear to decrease over the course of development, even during mid-pregnancy when the ducts are expanding. Furthermore we found that in some cells in the mammary gland, p107 expression may increase in the absence of Rb. The relatively high levels of p107 throughout mammary gland development may explain the need for developing tumor cells to inactivate multiple Rb-family members, without necessarily requiring additional transcriptional compensation by p107.

In spite of the series obstacles preventing more significant progress made toward completing the stated aims, a great deal of progress has been made to increase our understanding of the transcriptional networks encompassing the Rb-family more broadly. We further developed and validated other tools to understand the Rb-family transcription, including Rb-GFP transgenic mice and cells that carry homozygous point mutations in the promoter of p107. Together our novel tools have allowed us to begin to explore the molecular mechanisms which control the genetic circuitry that exists connecting the Rb-family members to each other, and what role this potential transcriptional regulation may have on compensation within the family, in wild-type tissues and in tumor development.

The most significant barriers that were not overcome:
- germline transmission of an incorrectly targeted allele for the E2F binding site mutations in the p107 promoter
- prohibitive numbers of mice needed to obtain correctly timed mammary glands of all genotypes simultaneously
- inability of Rb-deficient mice to become pregnant and undergo normal mammary gland development through pregnancy, potentially due to rapid development of pituitary tumors

The most significant advances:
- eventually optimized protocols for examining GFP expression in single cells through FACs and immunostaining
- validated and published the p107-GFP transgenic mice
- validated and made efforts toward publishing Rb-GFP transgenic mice
- examined the functional importance of the E2F binding sites in the p107 promoter in mouse cells

GFP reporter mice for the retinoblastoma-related cell cycle regulator p107.
Periodical:Cell Cycle
Index Medicus: Cell Cycle
Authors: Burkhart DL, Viatour P, Ho VM, Sage J
Yr: 2008 Vol: 7 Nbr: 16 Abs: Pg:2544-52

Cellular mechanisms of tumour suppression by the retinoblastoma gene.
Periodical:Nature Reviews. Cancer
Index Medicus: Nat Rev Cancer
Authors: Burkhart DL, Sage J
Yr: 2008 Vol: 8 Nbr: 9 Abs: Pg:671-82