Regulation of the ATR Checkpoint Response in Breast Cancer

Institution: Stanford University
Investigator(s): Dawn Yean, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7FB-0157 Award: $86,297
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2001)
By the time a cancer cell becomes malignant and gains the ability to metastasize, many changes in the original genetic programming for the cell have accumulated. Genetic mutations constantly occur in normal cells, but they usually become detected and corrected by special genes that work in processes known as the DNA damage checkpoints. These checkpoints act as the quality control machinery for the cell. Importantly, cancer cells have defects in the checkpoint processes that detect and correct DNA damage. An example in a failure of this surveillance system is the breast cancer gene, BRCA1, which is mutated in 50% of familial breast and ovarian cancers. BRCA1 is involved in this quality control process, but the mechanism of the process and all of the other proteins involved are not known.

Our interest is a gene, named ATR, which produces a protein that has been shown to regulate the function of BRCA1. ATR is one of the first proteins that recognizes damaged DNA, but the mechanism by which it does so is unknown. We believe that there are additional proteins that serve as either a bridge or accessory component of the ATR-DNA recognition process. We have developed two systems to study this interaction. First, we will study how this interaction occurs outside of cells using a special biochemical test. This method will use extracts from normal cells, which have normal DNA repair functions. Using these extracts we will purify and clone the proteins that help ATR recognize damaged DNA. Our second goal is to study the ATR-DNA interactions in normal cells, then determine if breast cancer cells exhibit changes in this response. The absence or mutation in a DNA damage surveillance protein could be an important biomarker for detection or treatment.

The development of an effective method to fight against breast cancer requires a thorough understanding of the processes that fail when a normal cell develops into a cancer cell. A key element in the development of a cancer cell involves the accumulation of DNA mutations. We have developed a novel test to identify proteins that play a role in allowing the cell to sense when DNA damage has occurred. Our hope is that if any of these new proteins are involved in breast cancer, it will provide new clues to prevent the disease.

Final Report (2003)
Introduction and Topic:
The link between permanent damage to DNA and cancer formation is well established. Genetic changes constantly occur in normal cells, but they usually become detected and corrected by a network of genes that function together in processes known as the DNA damage checkpoints. Many cancer cells have defects in the checkpoint process and hence fail to detect and repair damaged DNA. An integral part of this surveillance system is the breast cancer gene, BRCA1, which is mutated in 50% of familial breast and ovarian cancers. To better understand breast cancer and the role of the BRCA and other genes, researchers are working to identify the key genes/proteins and how they are organized into regulatory pathways.

Our studies are focused on a pair of large protein kinases, termed ATM (ataxia-telangiectasia-mutated) and ATR (ATM-Rad3-related) that belong to the phosphoinositide 3-kinase-related kinase (PIK) gene superfamily. ATM and ATR function as DNA damage-signaling kinases. In this capacity they regulate diverse cellular processes including cell cycle checkpoint activation, DNA repair, gene transcription, and apoptosis. ATM and ATR have both been implicated as tumor suppressor genes that are mutated in human cancer. In addition, mutations in ATM are causally associated with an inherited disease, called ataxia-telangiectasia (A-T), an invariably fatal syndrome of cancer, neurodegeneration and premature aging that affects approximately 1 in 40,000 births. Our lab studies ATM/ATR in the frog (Xenopus) with the aim of translating this information into the genetic changes that lead to breast cancer.

We have characterized a partially purified complex containing ATR and its partner protein, called ATRIP, that can associate with DNA in a test tube. Furthermore, we found evidence that ATR/ATRIP complex can associate with DNA in at least two different modes. We found that neither ATR nor ATRIP can bind to DNA (chromatin) independently, but rather need to be associated in order to retain DNA-binding function. In addition, our work indicates that there exists an additional component of the ATR/ATRIP complex that facilitates DNA binding, but has not yet been identified. Thus, our study provides some new details of the DNA binding properties of ATR and ATRIP. We have submitted a manuscript for publication to describe this research.

Future direction:
Our findings indicate that association of ATR/ATRIP with DNA depends on an unidentified protein factor. This is a complex process and requires further investigation as to what this factor may be.

Understanding how DNA damage checkpoint pathway is activated can contribute the development of novel cancer treatment drug and therefore is of clinical importance. Our studies here were performed with greater attention to the biochemical nature of this activity. We have recently summarized and submitted our result for review. Although it is unclear at this point what this activity represents, it is of interest to the field and should invite more study addressing this critical issue.

Symposium Abstract (2003)
There is a well-established link between environmental factors, damage to DNA, and cancer incidence. In order to maintain genomic integrity and normal function, cells have evolved surveillance systems (dubbed “DNA damage checkpoints”), which can detect the damaged DNA and initiate repair processes before the”“bad copy” can do any harm. The DNA damage checkpoints are signaling pathways that link DNA damage to the repair and cell cycle machinery. Central to these signaling pathways are human proteins known as ATR and ATM. Their targets include the tumor suppressor protein p53, and the checkpoint kinases, Chk1 and Chk2. The two breast cancer hereditary genes, BRCA1 and BRCA2, are also associated with DNA repair processes.

Activation of the DNA damage response requires detection of the initial lesion in DNA, and this recognition event must be coupled to the activation of the signaling pathway that mediates the damage response. ATR has been shown to play a major role in cells in the response to various genotoxic agents, including the UV light. Because its essential role in damage checkpoint response, we have focused our study on how ATR-mediated checkpoint pathways is regulated. We reason that in order to sense damaged DNA, ATR needs to interact with insulted DNA and upon doing so, ATR’s enzymatic activity is activated, which allows it to initiate the checkpoint response through activating downstream substrates including Chk1, BRCA1, and p53.

To address this, we have developed a DNA-binding assay to explore how ATR-containing complex isolated from mammalian cells interacts with DNA. We have analyzed the enzymatic activity of ATR before and after treatment of cells with UV light as well. In our studies, we will present data, which show that ATR binds to DNA in an indirect manner. This implies that ATR plays a role in’“sensing” damaged DNA. We will also present results that show the activity of ATR is increased in response to DNA damage, suggesting DNA-binding may be a way that ATR’s activity is regulated, which in turns initiates the checkpoint response. Our ultimate goal is to identify the mechanism by which ATR binds to damaged DNA and becomes activated.