Proteolysis of Cyclin E in Normal and Malignant Breast Cells

Institution: Scripps Research Institute
Investigator(s): Heimo Strohmaier, Ph.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5FB-0171 Award: $31,582
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (1999)
In order for a breast cell to divide, it must pass through an ordered progression of control, check-points, called the cell cycle. This cell division cycle is composed of growth phases (G1 and G2), chromosome duplication phase (S), and a cell division phase (M), which are temporally G1, S, G2, and M. Unfortunately, in breast cancer cells, the control mechanisms can become defective, which cause cells to constantly proliferate.

The key checkpoints are regulated by proteins called cyclins, and an associated group of regulatory proteins called cyclin-dependent kinases (CDKs). In normal cells, periodic synthesis and destruction of cyclins is essential for ordered cell cycle passage. In normal cells, the decision is often to leave G1 and the move to a quiescent state, called G0. But in breast cancer, the decision of the cell, which might be based on defects in cyclins and CDKs, is to go directly into S (DNA replication) and proceed through cell division. A main feature of cyclins is their temporal nature in the cell. Our interest is in two elements of this process, cyclin E and its partner CDK2. Previous work has indicated that defects in synthesis or regulation (degradation) in these proteins are key to unwanted breast cancer cell division. Yeast is an excellent model to study the cyclins and CDKs, since their function has been conserved in evolution.

Our goal is to identify the cellular factors that are involved in cyclin E degradation. Recently, the components that target yeast G1 cyclins for ubiquitin-dependent degradation were identified. Ubiquitin is a common method used by cells to tag proteins, so that they can be removed by proteolysis. Human cyclin E is functional as a G1 cyclin in yeast where it is also ubiquitinated and degraded. Thus, we will employ the yeast model system to identify the elements of the pathway that are responsible for cyclin E degradation. In breast cancer cells we will study a panel of 40 samples to determine the amount and activity of cyclin E. Next, using SSCP analysis, we will test whether cyclin E might be mutated in breast cancer to alter its rate of degradation. Finally, we will examine the degradation pathway of ubiquination to determine if there are defects in breast cancer.

This knowledge will help us to understand the role of cyclin E and other cell cycle regulators. Ultimately, this will potentially offer new approaches for diagnosis, therapy, and prevention of breast cancer.

Final Report (2000)
Note: PI resigned after one year to accept another grant offer.

A breast cell must pass through an ordered series of sequential phases, termed the cell cycle, before it divides into two daughter cells. In breast cancer, the regulatory mechanisms that operate to control proper progression through each cell cycle are perturbed, leading to uncontrolled cell division. Key regulators of the cell cycle are proteins, called cyclins that are synthesized and destroyed periodically. Abnormally high levels of cyclin E are frequently observed in breast cancer and have been shown to correlate with a poor prognosis.

We first used the power of the yeast genetic system to identify the components of the cell cycle pathway that controls cyclin E degradation in yeast. We have identified a protein, named Cdc4 that belongs to the family of F-box proteins and specifically partners with cyclin E. Due to the high degree of evolutionary conservation of proteolytic pathways from yeast to humans we were able to identify the respective human proteins. Thus, we have identified a potential human counterpart of yeast Cdc4 that binds to cyclin E when it is phosphorylated and induces the attachment of a small molecule, named ubiquitin, which in turn acts a signal for the removal of cyclin E from the cell.

Having established the mechanism of the degradation of cyclin E, our next step will be to find out whether excess amounts of cyclin E in breast cancer cells can be accounted for by defects in the ubiquitin process. We have developed an ubiquitination assay, which will enable us to assay breast cancer cell lines for any loss of cyclin E degradation activity. Cell lines that prove to be devoid of cyclin E ubiquitination activity will be subject to careful mutational analysis. It will be of particular interest to look for deletions or mutations in the gene encoding the receptor protein for cyclin E.

Ultimately, this information will potentially help reduce human and economic costs by offering new measures for determination of genetic predisposition, diagnosis, and treatment of breast cancer.