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Regulation of Genomic Stability Through Cell Cycle Checkpoint Signaling
Background: Breast cancers, like many other tumor types, are characterized by a high degree of genomic instability. The heightened molecular evolution that can arise from this instability is thought to be important in the initiation and progression of the tumors. In addition to DNA repair mechanisms, DNA damage checkpoints, the signaling pathways that pause cell cycle progression to allow time for DNA repair, are important determinants of genome stability. G1/S checkpoints are almost ubiquitously inactivated in tumors, with lack of p53 function accounting for a large proportion of these defects. DNA damage responsive checkpoints in the G2 phase of the cell cycle, which act independently of p53, are rarely defective in tumors, suggesting they be important for tumor cell viability. The G2 checkpoint is under the control of a phosphorylation cascade, culminating in the activation of the Chk1 protein kinase. In this study, I propose to characterize the relationship between G1/S and G2 checkpoints in breast cancer cells. I will further characterize the regulation of Chk1, with the aim of identifying additional molecules important for this kinase, which will represent novel targets for the treatment of breast cancer, and other cancer types, characterized by G1 checkpoint defects.
Objective/Hypothesis: The objective of this study is to characterize the relationship between G1/S and G2 checkpoints in breast cancer cells. The central hypothesis is that G1 checkpoint defects render a cell dependent on the Chk1-dependent G2 checkpoint for viability. This assertion predicts that inhibition of Chk1, either directly or through its regulators, should selectively target cells with defective G1 checkpoints. I will therefore further characterize the regulation of Chk1, in the context of cells that are apparently normal or defective in G1 checkpoints, and then isolate additional molecules important for this kinase. Such proteins would represent additional targets for the treatment of breast cancer, and other cancer types.
Specific aims: The proposal is structured over three integrated specific aims:
1. To characterize Chk1 expression and activation state in a panel of breast cancer cell lines, and to compare these data to the status of key G1/S cell cycle regulators, especially p53;
2. Assess the utility of ablation of Chk1 signaling in the context of defective and proficient p53 signaling;
3. Purify Chk1 and associated proteins using iTAP technology in breast tumor cells in which Chk1 is either inducible or constitutively active.
Study Design: We have antibodies that detect total or activated Chk1 by western blotting. We have a small collection of breast tumor cell lines that we have begun to analyze, and access to a larger collection from colleagues. These will be assessed for Chk1 expression and activation status, and analyzed against published p53 status and G1 checkpoint proficiency in response to genotoxins by FACS. We have constructed two siRNA vectors for Chk1, each of which significantly knocks down Chk1 expression, and are particularly potent when used in combination. These will be used to abolish Chk1 signaling in a sub-panel of breast tumor lines characterized in Aim 1. The host laboratory has extensive evidence from both yeast and mammalian systems that as yet unidentified trans-acting regulators interact with the C-terminal regulatory domain of Chk1 in both inhibitory and stimulatory ways. Using iTAP technology, in which the only Chk1 expressed contains dual affinity tags, I will purify Chk1 from this sub-panel, and characterize stoichiometric interacting proteins by mass spectrometry. Ongoing work will involve further characterization of these proteins.
Potential Outcomes and Benefits: I will establish a functional relationship between Chk1 signaling and the status of G1/S checkpoint and cell cycle defects in breast cancer cells. Further, there is the potential to identify regulators of Chk1. These proteins, together with Chk1, represent potential targets for anti-cancer therapy, which when combined with the information from my functional studies, can become tailored therapies designed specifically against tumor genotype/phenotype.
Breast cancers, like many other cancer types, show highly disorganized chromosomes. Our genes are located on these chromosomes, and so this rearrangement can scramble the important genetic information they hold. The alterations this brings to cells can lead to the formation of a cancer, and also to its development into a life threatening malignancy. Cells have many defense mechanisms to protect themselves from chromosomal damage. These include various “molecular toolkits” to repair breaks in chromosomes, and also security systems, known as checkpoints, that ensure damaged cells are repaired before they can divide. Cancer cells are frequently defective in both repair and checkpoint processes, and it is widely believed that these processes are absolutely crucial in the development of cancer. However, nature may have left a catch, in which the very defects in the cancer can be used to successfully kill these cells, leaving normal tissues relatively untouched. Two main checkpoints exist in our cells. One acts before chromosomes are copied, called the G1 checkpoint. If chromosomal damage is detected, it will prevent a damaged chromosome from being copied. A second checkpoint, called the G2 checkpoint, functions after the chromosomes are copied, but before they are separated as the mother cell divides into two daughters. The G1 checkpoint is almost always nonfunctional in cancer, leaving the G2 checkpoint in control. As the G2 checkpoint is only used in rapidly dividing cells, temporally switching it off may kill cancer cells with a faulty G1 checkpoint. As we move into the age of the human genome, we need to learn the precise genetic defects of cancers in different patients, and then use this information to tailor a therapy specifically for them, rather than one designed on the laws of averages. In my research, I aim to test this idea by studying a checkpoint function that is never defective in cancer, and for which there is a lot of evidence that the cancer cells need to stay alive. I will determine molecular details controlling this checkpoint, pinpointing targets for future tailored therapies.
The objective of this study is to characterize the relationship between different checkpoints that protect the chromosomes in breast cancer cells. The central hypothesis is that cancer cells need the G2 checkpoint to stay alive to a far greater degree than normal healthy cells. This assertion predicts that switching off the G2 checkpoint, either directly through it’s master regulator, a protein called Chk1, or through other proteins that help Chk1, should selectively kill cancer cells. I will therefore further characterize how Chk1 is switched on and off as cells divide, in the context of different types of breast cancer cells. Also, using very recently developed technologies, I will identify the proteins that collaborate with Chk1. Such collaborators would represent additional drug targets for the treatment of breast cancer.
Different cancers are caused by a variety of defects in the genetic code. There are several different genes that are important in many cancer types, whilst others seem to be important in particular cancers. An example of this is the Brca1 gene, which is defective in inherited breast cancer, and in some cases, ovarian cancer. Currently, patients receive therapy based on criteria defined from the population of patients as a whole. As we decipher the signals in the human genome, we should be able to design therapies that are specific to a particular patient as a well-tailored suite. To do this, we need to define intelligent strategies in the laboratory, through studies such as this, and then through collaboration with our clinical colleagues, move such findings to the bedside.