Identifying Sources of Genomic Instability in Breast Cancer

Institution: Stanford University
Investigator(s): Karlene Cimprich, Ph.D. -
Award Cycle: 2002 (Cycle VIII) Grant #: 8IB-0090 Award: $119,059
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2002)
Human cancers at diagnosis have dramatic changes in their chromosomes, including large deletions, duplications, and rearrangements of DNA segments. The DNA damage checkpoint is a cellular surveillance system that helps to maintain genomic stability by sensing the presence of DNA damage. When this system works properly, it coordinates an appropriate and effective cellular response to that damage. The basic molecular mechanisms that underlie DNA checkpoints and damage control/repair are well conserved in evolution. Thus, it makes sense to explore these processes in animal models, especially those where the genes and proteins of interest are better characterized and might be studied more effectively. In our laboratory, we use the eggs from Xenopus, a frog, as a model system. These eggs can be used to make a protein extract in which we can manipulate the proteins of interest and perform larger-scale biochemical studies. Using this model system we can induce DNA damage with UV light and determine how DNA damage response proteins interact with each other.

Recent research has led to the hypothesis that checkpoint signaling requires assembly of a protein complex at sites of DNA damage. In our previous work we showed that two proteins, called ATR (ataxia telangiectasia related) and Rad1, bind to chromatin and are needed to phosphorylate several other proteins that are critical to the damage response. One of these key proteins is BRCA1, which when mutated leads to hereditary breast cancer. Thus, our approach is to identify other key proteins in Xenopus egg extracts, by detecting the proteins that become phosphorylated in response to UV damage. We plan to isolate and partially sequence these DNA damage repair genes with a colleague at Harvard University. Our next aim is to translate this information to breast epithelial cells. This will involve determining the human counterparts of our Xenopus genes using published gene/protein databases. Then, we can use a new approach, called "small interfering RNA" (siRNA) to dissect out which human DNA damage repair genes are operative in breast cells, and identify the ones that might be worthy of future study in the context of breast cancer progression.

Our CBCRP-funded project is logical, because mutations in checkpoint proteins and related targets have already been shown to predispose individuals to breast and other cancers. It seems likely that identification of these phosphorylated proteins could lead to an understanding of genomic stability in breast epithelial cells. With a complete cataloguing and detailed information on the full spectrum of DNA repair proteins and checkpoint control in breast epithelial cells, we would be in position to evaluate them for potential diagnostic/detection uses and therapeutic targets.


Final Report (2004)
Human cancers at diagnosis have dramatic changes in their chromosomes, including large deletions, duplications, and rearrangements of DNA segments. These defects arise because cancer cells are defective in detecting and responding to DNA damage. Cells have a complex surveillance system that helps to maintain genomic stability by sensing the presence of DNA damage. The basic molecular mechanisms that underlie DNA checkpoints and damage control/repair are well conserved in evolution. Thus, it makes sense to explore these processes in animal models, especially those where the genes and proteins of interest are better characterized and might be studied more effectively. In our laboratory, we use the eggs from Xenopus, a frog, as a model system. The large frog eggs can be used to make a protein extract in which we can manipulate the proteins of interest and perform larger-scale biochemical studies. Using this model system we can induce DNA damage with UV light and determine how DNA damage response proteins interact with each other.

Recent research has led to the hypothesis that checkpoint signaling initially requires assembly of a protein complex at sites of DNA damage. In our previous work we showed that two proteins, called ATR (ataxia telangiectasia-related) and Rad1, move within the cellís nucleus to chromatin where they phosphorylate several other proteins that are critical to the damage response. One of these key proteins is BRCA1, which when mutated leads to hereditary breast cancer. We are interested in cataloging all the proteins involved in DNA surveillance, since these proteins are likely to be important for maintaining genomic stability in breast epithelial cells. One of our approaches is to identify other key proteins in Xenopus egg extracts is by detecting the proteins that become phosphorylated in response to UV damage. We have found several new proteins on chromatin that become phosphorylated in an ATR-dependent manner and we are now characterizing these proteins. In addition, we are using a "small interfering RNA" (siRNA) approach in mammalian cells to identify other proteins that are needed for ATR to translocate to sites of DNA damage. Once these targets and proteins are better characterized, we will attempt to translate this information to breast epithelial cells. This will involve determining the human counterparts of our Xenopus genes and using "small interfering RNA" (siRNA) to determine those human DNA damage repair genes are operative in breast cells.

It seems likely that identification of new protein targets of ATR and other proteins involved in activating ATR could lead to an understanding of genomic stability processes and defects in breast epithelial cells. With a complete cataloguing and detailed information on the full spectrum of DNA repair proteins, we would be in position to evaluate them for potential diagnostic/detection uses and therapeutic targets.